Thermo Fisher Scientific Surveyor Plus Getting Started

Surveyor Plus
Getting Started with
ChromQuest 4.2
Guide
60053-97102 Revision D
January 2009
© 2009 Thermo Fisher Scientific Inc. All rights reserved.
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Contents
Preface ............................................................................................. xi
About This Guide ......................................................................xi
Related Documentation .............................................................xi
Safety and Special Notices.........................................................xii
Contacting Us...........................................................................xii
Changes to the Manual and Help.............................................xiii
Thermo Scientific
Chapter 1
Introduction to the Surveyor Plus LC System.................................1
Surveyor PDA Plus Detector.......................................................2
Surveyor UV/Vis Plus Detector...................................................5
Surveyor FL Plus Detector ..........................................................6
Surveyor RI Plus Detector...........................................................7
Surveyor LC Pump Plus..............................................................8
Surveyor Autosampler Plus........................................................11
Tray Compartment ................................................................11
Injection System.....................................................................12
Injection Modes .....................................................................21
Temperature Control .............................................................24
Status LEDs ..............................................................................25
Power LED ............................................................................25
Comm LED...........................................................................26
Run LED ...............................................................................26
Lamp(s) LED .........................................................................27
Temp LED ............................................................................27
Degas LED ............................................................................27
System Interconnect Cable........................................................28
Communication with ChromQuest ..........................................31
Solvent Path..............................................................................33
Navigation in ChromQuest.......................................................36
The Tutorials ............................................................................38
Chapter 2
Administrating the Enterprise.........................................................41
Defining the Enterprise.............................................................42
Adding a Location/Group to the Enterprise ...........................42
Adding an Instrument to the Enterprise .................................43
Surveyor Plus Getting Started with the ChromQuest 4.2 Chromatography Data System
vii
Contents
Enabling Instrument Login and Project Management ...............45
Obtaining User Lists from the Data System Computer .............47
Obtaining User Lists from a Domain Controller.......................49
Creating a Project .....................................................................51
Assigning Privileges to Users .....................................................61
viii
Chapter 3
Configuring Your Instrument .......................................................... 69
Adding an Instrument to the Enterprise ....................................70
Configuring Your Instrument ...................................................74
Opening the Surveyor Modules Dialog Box ...........................74
Adding Modules to the Instrument Configuration.................76
Completing the Configuration of Each Module .....................77
Selecting the Baseline Check Option......................................85
Returning to the Main Menu Window ..................................86
Chapter 4
Creating Methods............................................................................. 87
Description of ChromQuest Methods.......................................88
Creating an Acquisition Method ...............................................91
Opening the Offline Instrument Window..............................91
Activating the Method Wizard...............................................93
Entering the Instrument Parameters.......................................95
Triggering Data Acquisition.................................................105
Entering the Column Parameters .........................................106
Saving the Method...............................................................108
Creating a Shutdown Method.................................................110
Chapter 5
Preparing Your Instrument for a Run .......................................... 115
Setting Up the System.............................................................116
Turning On the Power............................................................118
Opening the Online Instrument Window...............................120
Checking the Status of the Instrument Modules .....................124
Removing Air from the Solvent Lines......................................126
Purging the Pump................................................................126
Flushing the Syringe.............................................................130
Purging the Flow Cell of the RI Detector................................134
Downloading the Method.......................................................137
Checking the Stability of the Baseline .....................................140
Previewing the Baseline ........................................................140
Performing a Manual Baseline Check...................................141
Surveyor Plus Getting Started with the ChromQuest 4.2 Chromatography Data System
Thermo Scientific
Contents
Chapter 6
Making Your First Injection ..........................................................145
Loading the Sample into the Tray Compartment ....................146
Starting, Extending, and Stopping a Single Run......................149
Viewing the Progress of Your Run...........................................153
Viewing Your Chromatograms.............................................153
Viewing Your Spectral Data .................................................154
Checking the Purity of the Toluene Peak ................................158
Enabling Spectrum Calculations ..........................................158
Performing a Manual Peak Purity Check .............................159
Adding Annotations to Your Chromatograms .........................161
Chapter 7
Adding Integration Events Graphically.......................................165
Opening a Stored Data File.....................................................166
Adding Integration Events to the Method...............................168
Adding Integration Events to the 230 nm Chromatogram ...168
Adding Integration Events to the 260 nm Chromatogram ...172
Adding a Manual Integration Fix to a Data File ......................175
Chapter 8
Specifying the Calibration Curve Parameters ...........................181
Adding a Peak Table Graphically ............................................182
Using the Define Single Peak Dialog Box ............................182
Using the Define Peaks Dialog Box......................................185
Modifying the Properties of the Peak Table ............................191
Performing Multi-Wavelength Analyses ..................................194
Modifying the Analysis Channels in the Peak Table.............194
Modifying the Annotations Listed on the Screen..................195
Chapter 9
Adding a Custom Report to the Method ......................................197
Adding a Standard Custom Report to the Method..................198
Creating a Custom Report Template ......................................201
Adding Text to a Custom Report Template .........................201
Modifying Data Graph Annotations ....................................202
Changing the Appearance of the Data Graph.......................204
Modifying the Run Report...................................................205
Chapter 10 Creating a Sequence Table...........................................................209
Using the Sequence Wizard to Create a Sequence Table .........210
Modifying the Sequence Table................................................217
Deleting Unused Columns from the Sequence Table ...........217
Changing the Vial Locations ................................................219
Changing the Injection Volume ...........................................221
Adding a Shutdown Line .....................................................222
Adding an Action.................................................................224
Thermo Scientific
Surveyor Plus Getting Started with the ChromQuest 4.2 Chromatography Data System
ix
Contents
Creating a New Sequence Summary Template........................225
Opening the Sequence Editor ..............................................225
Using the Table Wizard .......................................................227
Selecting a Sequence Summary Template.............................234
Chapter 11 Running and Reprocessing a Sequence .................................... 237
Starting a Sequence Run .........................................................238
Reviewing the Peak Calibration...............................................242
Reprocessing a Sequence Run..................................................244
Chapter 12 Diluting Samples with a Pretreatment Method........................ 247
Creating a Pretreatment Method.............................................248
Opening the Pretreatment Window .....................................248
Adding Functions to the Pretreatment Table .......................248
Saving the Pretreatment Method..........................................254
Creating a Prep Only Method.................................................256
Using a Pretreatment Method .................................................259
Pretreatment Rules..................................................................263
Appendix A Calibration Procedures ................................................................. 265
Verifying the Performance of the PDA Detector .....................266
Adjusting the Light Intensity................................................266
Performing a Wavelength Calibration ..................................272
Performing an Array Calibration ..........................................279
Checking the Status of the Lamps ........................................280
Calibrating the Autosampler ...................................................288
Column Oven Calibration ...................................................288
Tray Calibration ..................................................................292
Bottom Distance Calibration ...............................................295
Calibrating the LC Pump........................................................298
Calibration Options .............................................................298
Calibration Procedures.........................................................302
Calibrating the RI Detector.....................................................308
Preparing to Perform the Validation Procedure....................308
Performing the Validation Procedure ...................................309
Restoring the RI Detector to Normal Operation..................316
Index ................................................................................................. 317
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Surveyor Plus Getting Started with the ChromQuest 4.2 Chromatography Data System
Thermo Scientific
Preface
About This Guide
This Surveyor Plus Getting Started with ChromQuest 4.2 Guide contains an
introduction to the modules of the Surveyor Plus LC system, as well as
eleven tutorial chapters. The tutorials guide you through the process of
setting up your system, creating data acquisition and shutdown methods,
making your first injection, running multiple samples using a sequence
table, and performing automated sample dilutions with a pretreatment
method.
Related
Documentation
In addition to this guide, Thermo Fisher Scientific provides the following
documents for the Surveyor Plus LC instruments and the ChromQuest
chromatography data system:
•
ChromQuest 4.2 Installation Guide
•
ChromQuest 4.2 Administrator’s Guide
•
ChromQuest 4.2 User’s Guide
•
ChromQuest 4.2 Reference Guide
•
Surveyor Plus Preinstallation Requirements Guide
•
Surveyor Plus Getting Connected Guide
•
Surveyor Autosampler Plus Hardware Manual
•
Surveyor LC Pump Plus Hardware Manual
•
Surveyor UV/Vis Plus Detector Hardware Manual
•
Surveyor PDA Plus Detector Hardware Manual
•
Surveyor FL Plus Detector Hardware Manual
•
Surveyor RI Plus Detector Hardware Manual
The ChromQuest data system manuals are provided on the ChromQuest
software CD. The Surveyor Plus Preinstallation Requirements Guide, the
Surveyor Plus Getting Connected Guide, and the hardware manuals for the
modules of the Surveyor Plus LC system are provided on the Surveyor Plus
documentation CD (P/N 60053-64200).
Thermo Scientific
Surveyor Plus Getting Started with the ChromQuest 4.2 Chromatography Data System
xi
Preface
Safety and Special Notices
Safety and Special
Notices
Make sure you follow the precautionary statements presented in this guide.
The safety and other special notices appear in boxes.
Safety and special notices include the following:
CAUTION Highlights hazards to humans, property, or the environment.
Each CAUTION notice is accompanied by an appropriate CAUTION
symbol.
IMPORTANT Highlights information necessary to avoid damage to
software, loss of data, invalid test results, or information critical for
optimal performance of the system.
Note Highlights information of general interest.
Tip Highlights helpful information that can make a task easier.
Contacting Us
There are several ways to contact Thermo Fisher Scientific for the
information you need.
• To contact Technical Support
Phone
800-532-4752
Fax
561-688-8736
E-mail
[email protected]
Knowledge base
www.thermokb.com
Find software updates and utilities to download at
mssupport.thermo.com.
• To contact Customer Service for ordering information
Phone
800-532-4752
Fax
561-688-8731
E-mail
[email protected]
Web site
www.thermo.com/ms
• To copy manuals from the Internet
Go to mssupport.thermo.com and click Customer Manuals in the left
margin of the window.
xii
Surveyor Plus Getting Started with the ChromQuest 4.2 Chromatography Data System
Thermo Scientific
Preface
Changes to the Manual and Help
Changes to the
Manual and Help
To suggest changes to this guide or to Help, use either of the following
methods:
• Complete a brief survey about this document by clicking the link below.
Thank you in advance for your help.
• Send an e-mail message to the Technical Publications Editor at
[email protected]
Thermo Scientific
Surveyor Plus Getting Started with the ChromQuest 4.2 Chromatography Data System
xiii
Chapter 1
Introduction to the Surveyor
Plus LC System
The Surveyor Plus™ modular LC system and the ChromQuest™ data system
are products of Thermo Fisher Scientific San Jose.
The Surveyor Plus modular LC system consists of the Surveyor LC Pump
Plus, the Surveyor Autosampler Plus, and one or more of the following
detectors: the Surveyor UV/Vis Plus Detector, the Surveyor PDA Plus
Detector, the Surveyor FL Plus Detector, or the Surveyor RI Plus Detector.
This chapter contains a brief description of each module, as well as an
overview of the system connections, the ChromQuest chromatography data
system, and the tutorials provided in this manual.
This chapter contains the following sections:
• Surveyor PDA Plus Detector
• Surveyor UV/Vis Plus Detector
• Surveyor FL Plus Detector
• Surveyor RI Plus Detector
• Surveyor LC Pump Plus
• Surveyor Autosampler Plus
• Status LEDs
• System Interconnect Cable
• Communication with ChromQuest
• Solvent Path
• Navigation in ChromQuest
• The Tutorials
Thermo Scientific
Surveyor Plus Getting Started with the ChromQuest 4.2 Chromatography Data System
1
1
Introduction to the Surveyor Plus LC System
Surveyor PDA Plus Detector
Surveyor PDA Plus
Detector
The Surveyor PDA Plus Detector is a full-featured, photodiode array
detector. The detector contains a dual-light source: a deuterium lamp for
detection in the ultraviolet wavelength range (190 to 360 nm) and a
tungsten-halogen lamp for detection in the visible wavelength range (360 to
800 nm).
The settings for the scan data include the following: scan range, step size,
scan rate, and bandwidth.
The maximum scan range is from 190 to 800 nm. You can collect
absorbance data for every wavelength in the scan range or in wavelength
steps as large as 20 nm. You can collect data points for each wavelength in
the scan range at scan rates from 0.5 to 20 Hz (1 Hz = 1 data point per
second). Decreasing the scan rate for scans significantly reduces the size of a
data file. Because scan data is typically acquired for qualitative purposes, a
scan rate of 1 Hz is generally adequate.
The bandwidth setting is an electronic filter that specifies the range of
wavelengths used to determine the absorbance for the central wavelength.
Therefore, the maximum allowable bandwidth setting depends on the scan
range. For the full scan range of the detector from 190 to 800 nm, the
maximum allowable bandwidth for each wavelength is 1 nm because the
detector cannot monitor wavelengths below 190 nm or above 800 nm. For a
scan range from 214 to 776 nm, the maximum allowable bandwidth is
49 nm. At a bandwidth setting of 49 nm, the reported absorbance value for
214 nm is a weighted average of the absorbance values from 190 to 238 nm.
And likewise, the reported absorbance value for 776 nm is a weighted
average of the absorbance values from 752 to 800 nm.
In addition to scan data, the detector can simultaneously collect up to three
independent discrete channels. The data acquisition options for the discrete
channels are independent of the scan data, therefore, you can collect the
discrete channel data at a higher data rate and a wider bandwidth than
appropriate for scan data.
The appropriate data rate setting for the discrete channels depends on the
width of the narrowest peak that is integrated. For optimal performance, the
integration algorithm in ChromQuest requires twenty points across a peak.
Therefore, if the narrowest peak in your chromatogram is 20 seconds wide,
set the data rate for the discrete channels to a minimum of 1 Hz (1 point per
second).
Increasing the bandwidth for scans decreases the spectral resolution of the
scan data. This is an important factor to consider for compounds that
exhibit fine spectral features such as toluene.
2
Surveyor Plus Getting Started with the ChromQuest 4.2 Chromatography Data System
Thermo Scientific
1
Introduction to the Surveyor Plus LC System
Surveyor PDA Plus Detector
Figure 1 shows the effect of bandwidth on spectral resolution. Increasing the
bandwidth for the discrete channel data decreases the baseline noise.
Therefore, you might choose to use a lower bandwidth setting for the scan
data than for the discrete channels.
1 nm Bandwidth
11 nm Bandwidth
Figure 1.
Effect of bandwidth on resolution
In addition to the bandwidth filter, the detector also contains a computer
generated rise-time filter. Increasing the rise time setting can increase the
signal to noise ratio for the chromatographic peaks. However, setting the rise
time too high can also result in band broadening. In general, a rise time
setting that is greater than one-tenth the width of the chromatographic peak
at half-height will cause band broadening. For most HPLC applications, a
rise time setting of 1 or 2 seconds is preferable.
Figure 2 shows the effect of rise time on peak broadening. The effect of rise
time on baseline noise is shown in Table 1.
Thermo Scientific
Surveyor Plus Getting Started with the ChromQuest 4.2 Chromatography Data System
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1
Introduction to the Surveyor Plus LC System
Surveyor PDA Plus Detector
Rise Time = 0.1 s
Rise Time = 1 s
Rise Time = 2 s
Rise Time = 5 s
Rise Time = 10 s
Figure 2.
Effect of Rise Time on peak broadening
Table 1.
4
Effect of rise time on the signal to noise ratio
Rise Time (s)
Signal to Noise Ratio (ASTM)
0.1
7543
1.0
18004
5.0
22654
10.0
72350
Surveyor Plus Getting Started with the ChromQuest 4.2 Chromatography Data System
Thermo Scientific
1
Surveyor UV/Vis Plus
Detector
Introduction to the Surveyor Plus LC System
Surveyor UV/Vis Plus Detector
The Surveyor UV/Vis Plus Detector is a full-featured, time-programmable,
variable-wavelength UV/Vis (ultraviolet / visible) absorbance detector. It
operates in either the single wavelength mode or the dual wavelength mode.
The wavelength range in the single wavelength mode is 190 nm to 800 nm.
In the dual wavelength UV mode, the range is 190 nm to 365 nm. In the
dual wavelength Visible mode, the range is 366 nm to 700 nm.
The wavelength time table is available in all three modes. The time table can
contain up to 10 lines. If the Zero On Wavelength Change feature is
enabled, the absorbance of the baseline is re-zeroed between each line in the
time table, even if the wavelengths remain the same. The absorbance of the
baseline is not zeroed between the last two lines in the table.
To provide a complete spectrum of ultraviolet and visible light, the detector
uses a deuterium lamp for the UV range (190-365 nm) and a tungsten lamp
for the visible range (366-800 nm). The lamps are protected by a cover with
a special safety interlock to reduce the possibility of human exposure to
harmful UV light.
Thermo Scientific
Surveyor Plus Getting Started with the ChromQuest 4.2 Chromatography Data System
5
1
Introduction to the Surveyor Plus LC System
Surveyor FL Plus Detector
Surveyor FL Plus
Detector
The Surveyor FL Plus Detector is a full-featured, time-programmable,
fluorescence detector that integrates with the Surveyor Plus LC system.
The Surveyor FL Plus Detector consists of a xenon (Xe) lamp light source,
dual gratings, a flow cell, and a mercury (Hg) lamp for wavelength
calibration. The Hg lamp is protected by a cover with a special safety
interlock to reduce the possibility of human exposure to harmful UV light.
CAUTION The glass bulb of the xenon lamp contains xenon gas under
high pressure. Because it can explode, take care when handling the
xenon lamp.
The xenon gas inside the glass bulb of the Xe lamp exerts a pressure of
4 MPa at normal operating temperatures, which means that the glass bulb of
the Xe lamp can explode. The xenon lamp is contained within the detector
housing, which ensures your safety during normal use of the detector.
However, the Xe lamp has a limited operating time and must be replaced
occasionally. Before you replace the Xe lamp, allow it to cool to room
temperature and put on extra personal protective gear, including a face
shield and puncture resistant gloves. See the booklet or the CD stored in the
left door of the FL detector or the Surveyor FL Plus Detector Hardware
Manual for information on installing or replacing the Xe lamp.
CAUTION Because the flow cell of the FL detector cannot withstand a
backpressure higher than 1MPa (10 bar, 145 psi), do not connect the
outlet of the flow cell to another detector.
To collect UV/Vis data in addition to fluorescence data, place the UV/Vis
detector on the top of the LC stack, and then connect the outlet line from
the UV/Vis detector to the inlet of the FL detector’s flow cell.
You can control the fluorescence detector from a keypad on its front panel
or from the ChromQuest chromatography data system. A USB
communications link connects the detector to the ChromQuest data
system. Light emitting electrodes (LEDs) on the front of the module keep
you informed of the power, communications, run, and lamp status.
The Surveyor FL Plus Detector has its own specialized maintenance
software program that you can access by choosing Start > All Programs >
Chromatography > Maintenance for the FL Plus. You can use the
maintenance software program to monitor the performance of the FL
detector and to optimize the emission and excitation wavelengths for your
analytes. For information on the maintenance software application, see the
Surveyor FL Plus Detector Hardware Manual.
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Surveyor Plus Getting Started with the ChromQuest 4.2 Chromatography Data System
Thermo Scientific
1
Surveyor RI Plus
Detector
Introduction to the Surveyor Plus LC System
Surveyor RI Plus Detector
The Surveyor RI Plus Detector is a full-featured, low-maintenance,
refractive index detector that integrates with the Surveyor Plus LC system.
With the addition of this detector to the Surveyor Plus product line, you can
detect carbohydrates, alcohols, fatty acids, and other compounds having
weak chromophores.
The thermostatically regulated optical bench provides stable baselines, with
a drift of 2 × 10-7 RIU/ h or less and a noise level of 2.5 × 10-9 RIU or less.
The tungsten lamp has an operating lifetime of 4.5 years, which means that
you can avoid lengthy warm-up periods by leaving the detector on and ready
for operation. The operating range of 1.00 to 1.75 RIU allows you to use a
broad selection of mobile phases. Table 2 lists the refractive indices of some
common mobile phase solvents.
Table 2.
Refractive Indices of some common mobile phase solvents
Solvent
RI
Acetonitrile
1.34
Water (20°C)
1.33
Methanol
1.329
Acetic Acid
1.372
Cyclohexane
1.427
Hexane
1.375
Benzene
1.501
Ethylene glycol
1.427
Carbon disulfide
1.626
Tetrahydrofuran
1.408
Chloroform
1.443
You control and validate the RI detector from the ChromQuest data system.
The method editor in ChromQuest provides control of the following
detector parameters: the operating temperature from 30 to 50°C, the rise
time from 0.1 to 6 seconds, the data rate from 0.5 to 10.0 Hz, and the
signal polarity of the chromatographic peaks. A built-in calibration
procedure allows you to validate the accuracy of the detector with a 0.35%
by weight sucrose in water solution.
CAUTION The flow cell in the Surveyor RI Plus Detector has a pressure
rating of 517 kPa (75 psi). Do not connect tubing with an inner
diameter of less than 0.060-in. to the OUT port of the RI detector.
Doing so can damage the flow cell.
Thermo Scientific
Surveyor Plus Getting Started with the ChromQuest 4.2 Chromatography Data System
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1
Introduction to the Surveyor Plus LC System
Surveyor LC Pump Plus
Surveyor LC Pump
Plus
The Surveyor LC Pump Plus is a dual-piston, quaternary, low-pressure
mixing pump with a built-in vacuum membrane degasser and pulse
dampener. The pumping system provides stable isocratic flow rates from
0.001 to 9.999 mL/min. You can run precise gradients at flow rates from
0.200 to 2.000 mL/min and your gradient table can contain up to forty
lines. The integral vacuum degasser offers efficient solvent degassing while
requiring only 500 µL of volume, and the pulse dampener produces stable
flow rates while adding only 400 µL of gradient delay volume to the system.
The Surveyor LC Pump Plus has five major components: the vacuum
membrane degassing assembly, the solvent proportioning assembly, the
pump head assemblies, the pulse dampening assembly, and the purge
manifold assembly (see Figure 3).
Pump Head
Assemblies
Solvent Lines Entering and Exiting
the Degassing Assembly
IN
OUT
Line Body
Containing
Filter Frit
A
N
PE
DRAIN
O
SE
Right
LO
Left
C
B
Purge
Manifold
Assembly
C
D
On/Off Switch
Figure 3.
8
Solvent
Proportioning
Assembly
Pulse Dampening Assembly
(Behind the Front Panel)
Front of Surveyor LC Pump Plus
Surveyor Plus Getting Started with the ChromQuest 4.2 Chromatography Data System
Thermo Scientific
1
Introduction to the Surveyor Plus LC System
Surveyor LC Pump Plus
You use the wash tube assembly and the rinse tube assembly to rinse the
pump head assemblies with distilled water when you are pumping buffered
mobile phases. Rinsing buffered solutions out of the pump heads extends
the lifespan of the seals and the pistons. You can rinse the pump heads at any
time: while the pump is running or while the pump is stopped. See Figure 4.
Waste
Syringe
Figure 4.
Set up for rinsing the pump head assemblies
The purge manifold assembly consists of the pressure sensor, the drain valve
knob, and the line filter body. The pressure sensor constantly monitors the
backpressure of your system. See Figure 5.
Thermo Scientific
Surveyor Plus Getting Started with the ChromQuest 4.2 Chromatography Data System
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1
Introduction to the Surveyor Plus LC System
Surveyor LC Pump Plus
To Autosampler
Filter
Frit
Drain
Valve
Knob
Pressure
Sensor
Pulse
Dampener
To Waste
Outlet Tube
Figure 5.
Purge manifold assembly
The line filter body screws into the top of the purge manifold assembly and
holds a replaceable filter frit that prevents particulate matter from entering
the injection valve of the autosampler. The particulate matter can come
from the mobile phase solvents or from the piston seals as they wear. The
backpressure of your system will rise as this filter becomes clogged.
Replace the frit when the system backpressure rises above the typical
operating range for your application. How frequently you will need to
replace the frit will depend on the purity of your mobile phase solvents and
the rate at which the piston seals in your pump wear.
The drain valve knob on the front of the purge manifold assembly can be
opened or closed. In the open position, mobile phase flows out the left side
of the purge manifold assembly to waste. In the closed position, mobile
phase flows out the top of the purge manifold assembly to the autosampler.
To open the drain valve, turn the knob 180° counter-clockwise until the
word DRAIN is upside down. To close the drain valve, gently turn the knob
clockwise until you start to feel resistance. Do not overtighten the knob.
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Surveyor Autosampler
Plus
Introduction to the Surveyor Plus LC System
Surveyor Autosampler Plus
The full-featured Surveyor Autosampler Plus includes a built-in column
oven (5 to 95 °C), a tray compartment with temperature control (0 to
60 °C), and automatic sample preparation.
The tray compartment, the injection system, the injection modes, and the
temperature control features of the autosampler are described in the
following topics:
• Tray Compartment
• Injection System
• Injection Modes
• Temperature Control
Tray Compartment
The tray compartment can hold up to five conventional sample trays or one
microwell carrier. The microwell carrier holds up to three 96-well
microplates or up to three 384-well microplates. The tray compartment also
holds up to four-16 mL capacity reservoir vials The reservoir vials are
located behind the wash station and are designated RV1, RV2, RV3, and
RV4. See Figure 6.
Tray Runner
Slot for Tray
RV4
Reservoir Vial
16 mL capacity
RV3
RV2
RV1
Standard
1.8 mL vial
A
B
Figure 6.
Thermo Scientific
C
D
E
Tray
Handle
Tray Compartment
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Introduction to the Surveyor Plus LC System
Surveyor Autosampler Plus
The door to the tray compartment contains a magnetic switch. When you
open the door, the switch signals the autosampler that the door is open. If
you select the Verify Door is Closed check box when you configure the
autosampler, the XYZ arm of the autosampler moves to the back of the tray
compartment when you open the to the tray compartment. If you are
running a sequence of injections, the sequence pauses until you close the
tray compartment door.
Injection System
The major components of the injection system are as follows:
• Wash Bottle and Wash Bottle Tube
• XYZ Arm Mechanism, Needle Assembly, and Needle Tube Assembly
• Syringe Drive Assembly and Syringe Valve
• Concentric Syringe
• Wash Station
• Needle Port of Autosampler and Transfer Tube
• Injection Valve and Interchangeable Sample Loop
Wash Bottle and Wash Bottle
Tube
The wash bottle is located in the solvent platform mounted on the top of
the Surveyor Plus stack. Low pressure tubing connects the wash bottle to the
syringe valve. During an injection, the syringe draws solvent from the wash
bottle. If the wash bottle is empty, the autosampler cannot withdraw liquid
from a sample vial during an injection sequence. To avoid making blank
injections, always check the solvent volume in the wash bottle before you
begin a sequence of injections.
The following direct commands, Flush (from bottle) and Wash Needle
(from bottle), also draw solvent from the wash bottle.
Note If you are using a wash solvent of relatively high viscosity, such as
water, reduce the wash speed to 125 μL/s or lower. If you flush a viscous
solvent through the injection port at a high speed, the syringe will stall
and make a high-pitched screeching sound.
IMPORTANT Ensure that there is a sufficient volume of solvent in the
wash bottle before performing a sequence of injections.
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XYZ Arm Mechanism, Needle
Assembly, and Needle Tube
Assembly
Introduction to the Surveyor Plus LC System
Surveyor Autosampler Plus
The XYZ arm mechanism holds the sample needle and moves to the sample
location during an injection. The Home position of the XYZ arm is located
at the front right-side of the tray compartment, just behind the injection
port of autosampler. If you enable the Verify Door Is Closed feature (See
Chapter 3, “Configuring Your Instrument.” ), the XYZ arm moves to the
back of the tray compartment when you open the left-front door.
When you make an injection, the XYZ arm moves along the X-Y plane to
the requested vial or well location, and then lowers the needle to the height
requested in the injection method. The inner plunger of the concentric
syringe descends, drawing liquid out of the vial, through the needle into the
needle tubing. After the autosampler withdraws liquid from the requested
well or vial, the XYZ arm returns to the injection port. The autosampler
lowers the needle into the injection port, and then the inner plunger of the
syringe ascends, expelling the liquid into the injection port.
When you perform a Wash Needle command, the XYZ arm moves along
the X-Y plane to the wash station position, located behind the injection
port, where it lowers the needle. Confined within the wash station, the
needle expels enough wash solvent to clean both its inner and outer surfaces.
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Introduction to the Surveyor Plus LC System
Surveyor Autosampler Plus
Figure 7.
XYZ arm mechanism and needle tubing assembly
The needle assembly consists of a blunt-tip needle, a latch nut, a flag, a
compression spring, and a 10-32 fitting that connects to the needle tube
assembly (see Figure 8). The needle is inserted into the needle mount on the
XYZ arm.
The needle tube assembly (see Figure 8) consists of a piece of low-pressure
tubing, a PEEK fitting that connects to the needle assembly, a fitting and
flangeless ferrule that connects to the right side of the syringe valve, and a
needle tube guide that is inserted into the X-axis positioning frame.
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Introduction to the Surveyor Plus LC System
Surveyor Autosampler Plus
Needle
assembly
Fitting to
syringe valve
PEEK fitting to
needle assembly
Figure 8.
Needle
guide
Needle assembly and needle tubing assembly
To prevent the needle tubing from interfering with the movement of the
XYZ arm, the needle tubing is secured with a bracket on the Z-axis of the
XYZ arm and the needle guide on the X-axis positioning frame.
Syringe Drive Assembly and
Syringe Valve
Thermo Scientific
The syringe drive assembly consists of a stepper motor drive mechanism, a
syringe valve, and fittings that hold the interchangeable syringe. The syringe
drive assembly mounts to the front of the autosampler. The connection
between the syringe drive assembly and the body of the autosampler consists
of three mating holes with rubber grommets on the back of the syringe drive
assembly and three mounting studs on the body of the autosampler. While
minimizing vibration, the rubber grommets make the connection between
the syringe drive assembly and the autosampler feel loose. See Figure 9.
Surveyor Plus Getting Started with the ChromQuest 4.2 Chromatography Data System
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Introduction to the Surveyor Plus LC System
Surveyor Autosampler Plus
Needle
tubing
10-pin
connector
Wash bottle
tubing
Two-way
syringe valve
Syringe
fitting
Mating hole
(with grommet)
to vertical
mounting stud
Mating holes
(with grommets)
to horizontal
mounting studs
Front view
Figure 9.
Back view
Front and back of the syringe drive assembly
The syringe valve is a 2-position rotary valve. In the wash bottle position,
the syringe draws solvent from the wash bottle into its barrel as its plunger
descends. See Figure 10.
In the needle position, the syringe draws sample into the needle tubing as its
plunger descends. See Figure 11. Unlike the wash solvent, the syringe does
not draw liquid from a sample location into its barrel. As the syringe plunger
ascends, it pushes sample solution or solvent out of the needle tubing into
the injection port or wash station of the autosampler.
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1
Introduction to the Surveyor Plus LC System
Surveyor Autosampler Plus
Wash Bottle
Tubing
Syringe
Valve
Needle
Tubing
Wash
Bottle
Figure 10. Syringe valve in wash tubing position
Wash Bottle
Tubing
Wash
Bottle
Syringe
Valve
Needle
Tubing
p
Figure 11. Syringe valve in needle tubing position
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Introduction to the Surveyor Plus LC System
Surveyor Autosampler Plus
Concentric Syringe
The Surveyor Autosampler Plus uses a concentric syringe to deliver solvent
and sample. See Figure 12. The concentric syringe consists of a small, inner
plunger, and a larger, outer plunger. The inner plunger is used to draw
sample into the needle tubing assembly, and then deliver the sample to the
injection port. The outer plunger is used to draw and expel large volumes of
solvent. Liquid from a sample vial is never drawn into the syringe itself.
Wash Station
The wash station is a waste cup containing an inner sleeve that is slightly
larger than the syringe needle. See Figure 12. The waste cup drains into a
waste receptacle. The XYZ arm moves the needle to the wash station to
perform an external needle wash or to initialize the syringe. The needle is
inserted down into the sleeve and solvent is flushed through the needle. This
process washes the needle exterior as expelled solvent flows up past the
exterior surface of the needle and into the waste cup.
Waste Cup
Concentric
Syringe
Outer
Plunger
Inner
Plunger
Needle
Sleeve
To Waste
Figure 12. Wash station
Needle Port of Autosampler and
Transfer Tube
The injection port of the autosampler is located behind the syringe drive
assembly. The injection port is connected to the 6-port Valco injection valve
by way of the transfer tube. The Surveyor Autosampler Plus has a a
0.012-in. ID transfer tubing. The volume of the transfer tube assembly is
specified on its attached label. See Figure 13 and Figure 14.
IMPORTANT The volume of each transfer tube is individually
calibrated. Enter the volume specified on the label attached to the
transfer tube assembly when you configure the autosampler. See
“Configuring the Autosampler” on page 78.
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Introduction to the Surveyor Plus LC System
Surveyor Autosampler Plus
Injection Port
Transfer Tube
Figure 13. Needle port and transfer tube
Wash
Station
Transfer Tube Assembly Label
(specifying the Dead Volume)
Injection
Port
22µL
Transfer
Tube
Figure 14. Injection port and transfer tube
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Introduction to the Surveyor Plus LC System
Surveyor Autosampler Plus
Injection Valve and
Interchangeable Sample Loop
The injection valve is a six-port rotary valve that introduces sample onto the
column by way of the sample loop. See Figure 15.
Note Because the rotor portion of the valve contains narrow passages, it
is important that you remove particulate matter from your samples
before you load them into the autosampler.
Port 2
Connection to Transfer Tube
Port 3
Connection to Waste
Port 1
Connection to Front
of Sample Loop
Port 6
Connection to
Column
Port 4
Connection to Back of
Sample Loop
Port 5
Return From Behind Column Oven
Figure 15. Valco C2 type six-port injection valve
The sample loop is an interchangeable part that is attached to the six-port
injection valve. It is a stainless steel tube that holds the sample prior to its
introduction onto the column. The Surveyor Autosampler Plus has a 25 µL
sample loop.
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Injection Modes
Introduction to the Surveyor Plus LC System
Surveyor Autosampler Plus
The Surveyor Autosampler Plus can operate in any of the following three
injection modes:
• No Waste Injection Mode
• Partial Loop Injection Mode
• Full Loop Injection Mode
The optimum injection mode depends upon the amount of sample that you
have and the degree of precision that your application requires.
No Waste Injection Mode
The no waste injection mode is a technique that withdraws only the exact
amount of sample requested from the sample vial. Of the three injection
modes, the no waste injection mode uses the least amount of sample, but it
is also the least precise. Use this injection mode to conserve sample.
IMPORTANT For no waste injections, do the following:
• Use a sample loop that is at least 5 μL larger than the injection volume.
Because the accuracy of the nominal size is ± 20%, use an estimate of 80%
for the actual size. For example, use 20 μL as an estimate for the actual
volume of a 25 μL loop, and inject no more than 15 μL with this loop size.
• Consider matching the chemistry of the sample matrix, the flush solution,
and the mobile phase. For no waste injections, the autosampler loads
approximately 2 μL of flush solvent and 3 μL of air into the sample loop,
regardless of the requested injection volume.
• Inject at least 1.0 μL of sample.
Approximately 0.25 μL of the sample is lost as it travels from the injection
port through the transfer tubing and into the injection valve. Because of this
loss, inject at least 1.0 μL of sample with the no waste injection mode.
In addition to the sample bolus, the no waste injection mode loads
approximately 2 μL of wash solvent and 3 μL of air into the sample loop.
These values are independent of the injection volume. Therefore, the
chemistry of the wash solvent can affect your chromatographic results. For
the best chromatographic results, consider matching the chemistry of the
sample matrix, the wash solvent, and the mobile phase. See Figure 16.
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Introduction to the Surveyor Plus LC System
Surveyor Autosampler Plus
Air Bubbles
From
Injection Port
To
Column
1
2
6
3
Mobile Phase
Wash Solvent
Sample
5
4
From
Pump
Waste Line
Figure 16. No Waste Injection mode, showing the valve in the fill position
Partial Loop Injection Mode
The partial loop injection mode is a technique that withdraws 22 μL of
excess sample from the vial, in addition to the requested injection volume.
Approximately one-half of the excess volume is expelled to waste before the
center of the sample bolus is metered into the front of the sample loop. The
second portion of excess sample is expelled to waste after the sample bolus is
backflushed onto the column. Partial loop injections are useful when you
have a limited volume of sample. Using the partial loop injection mode, you
can inject variable amounts of sample, ranging from a minimum of 0.1 μL
to a practical maximum of one-half the volume of your sample loop. This
maximum volume limitation is a consequence of the laminar flow of fluid
within the stainless steel sample loop. See Figure 17.
Note The actual size of a sample loop can be ±20% of its nominal size.
The actual size of the 25 μL sample loop attached to the Surveyor
Autosampler Plus is anywhere from 20 to 30 μL. Therefore, limit the
maximum injection volume to 10 μL.
From
Injection Port
To
Column
1
2
6
Air Bubbles
Mobile Phase
Wash Solvent
Sample
3
4
To Waste
5
From
Pump
Figure 17. Partial loop injection mode, showing the valve in the fill position
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Full Loop Injection Mode
Introduction to the Surveyor Plus LC System
Surveyor Autosampler Plus
The full loop injection mode is a technique that withdraws a sample volume
from the vial that is sufficient to overfill the loop by a minimum factor of
two. Because the actual injection volume is determined by the size of the
loop, not the metering action of the stepper motor, a full loop injection is
very reproducible. However, because the intent of the full loop injection
mode is to completely fill the sample loop, you cannot inject variable
amounts of sample.
Full loop injection is useful when you want maximum precision and have
unlimited sample. If you want to change the injection volume, you must
change the sample loop size.
Note The value you enter in the Loop Size box when you configure your
autosampler must match the sample loop size. The injection volume box
in the Single Run Acquisition dialog box is disabled for Full loop
injections.
In the full loop injection mode, the autosampler withdraws a large excess of
solution from the sample vial according to the following equation:
Amount Withdrawn = 3 × Injection Volume + Dead Volume + 7.5 μL
where:
Dead Volume =Volume of Transfer Tube + Volume of Injection Port and
Rotor Slot
This equation is valid until the maximum capacity of the syringe is reached,
at which point only the maximum capacity of the syringe is withdrawn. The
maximum capacity of the 250 μL concentric syringe is 265 μL. See
Figure 18.
From
Injection Port
To
Column
1
2
6
Air Bubbles
Mobile Phase
Wash Solvent
Sample
3
4
To Waste
5
From
Pump
Figure 18. Full loop injection mode, showing the valve in the fill position
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Introduction to the Surveyor Plus LC System
Surveyor Autosampler Plus
Temperature Control
The full-featured Surveyor Autosampler Plus has two built-in temperature
control features:
• Tray Temperature Control
• Column Oven Control
Tray Temperature Control
The built-in tray temperature control feature provides temperature control
of the samples in the range from 0 to 60 °C. A Peltier device maintains the
tray temperature.
Column Oven Control
The built-in column oven controls the temperature of the air surrounding
the chromatographic column. Isothermal temperature control is achieved
using a Peltier device. The Peltier device is a solid-state, heat-transferring
assembly used to heat or cool the column oven. The range of temperature
control is 5 to 95 °C.
Between the pump and the autosampler injection valve, the mobile phase is
diverted through approximately 122 cm of 0.020-in. ID stainless steel
high-pressure tubing that is located behind the column oven. This tubing
holds approximately 250 μL of mobile phase, which allows the mobile phase
ample time to equilibrate to the temperature of the column oven before it
reaches the injection valve. This additional tubing also adds 250 μL of
gradient delay volume to the Surveyor Plus System.
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Status LEDs
Introduction to the Surveyor Plus LC System
Status LEDs
Each of the Surveyor Plus LC modules has a panel of four status LEDs
(light-emitting diodes) located on its front-right door.
All the modules have a Power, Comm, and Run LED. In addition, the PDA,
UV/Vis, and FL detectors have a Lamp(s) LED; the autosampler and RI
detector have a Temp LED; and the pump has a Degas LED.
The states of the LEDs are described in the following topics:
• Power LED
• Comm LED
• Run LED
• Lamp(s) LED
• Temp LED
• Degas LED
Power LED
The Power LED informs you whether the module is turned on or off. The
Power LED is unlit before you turn on the module’s power switch and
remains a steady green after you turn on the power switch. See Figure 19.
Power
Comm
Run
Lamps
UV/Vis or PDA
Detector LEDs
Power
Switches
Power
Power
Comm
Comm
Run
Run
Temp
Degas
Autosampler LEDs
Pump LEDs
Power
Comm
Run
Temp
FL Detector LEDs
Figure 19. Location of power switches and LEDs
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Introduction to the Surveyor Plus LC System
Status LEDs
Comm LED
The Comm LED informs you whether the module is communicating with
the data system. The Comm LED has the states described in Table 3.
Table 3.
Run LED
Comm LED states
State
Module
Meaning
Steady green
All modules
The module is communicating with the data system.
When you open the online Instrument window in
ChromQuest, the Comm LEDs for the configured modules
of the instrument should turn green.
Steady amber
All modules
The module is not communicating with the data system.
To communicate with the data system, the
communication cable for the module must be connected
and the online Instrument window in ChromQuest must
be open.
Flashing amber
UV/Vis and
PDA detector
The rotary switches on the back panel of the detector are
set to 0 for a firmware download.
The Run LED has the states described in Table 4.
Table 4.
Run LED states
State
Module
Meaning
Steady green
All modules
The power to the module is turned on.
Pump
A run is not in progress.
Detector
The detector is not sending data to the data system PC.
Detectors
The detector is sending data to the data system PC.
Autosampler
An injection or a timed event is in progress.
Pump
A run is in progress.
PDA detector
The PDA detector is not ready to start a run. This
condition occurs if both lamps are off, the lamp specified
in the method fails to turn on, or the wavelength
calibration is not valid.
RI detector
The RI detector is not ready to start a run. This condition
occurs when the operating temperature has not reached
the set temperature.
Pump
The pump is not communicating with the data system,
the pump’s motor is stopped, the pump’s pistons are
homing, the pump is stabilizing, or the pump is in the
purge mode.
All modules
An error condition has occured.
Flashing green
Steady amber
Flashing amber
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Lamp(s) LED
The Lamp(s) LED informs you of the status of the detector’s lamps. The
Lamp(s) LED has the states described in Table 5.
Table 5.
Lamp(s) LED states
State
Module
Meaning
Steady green
PDA and
UV/Vis detector
The deuterium, tungsten, or both lamps are on.
FL detector
The xenon lamp is on.
PDA and
UV/Vis detector
The lamp specified in the method (deuterium for the
UV range or tungsten for the visible range) is not on.
FL detector
The xenon lamp is off.
Steady amber
Temp LED
Introduction to the Surveyor Plus LC System
Status LEDs
The autosampler and the RI detector have a Temp LED with the states
described in Table 6.
Table 6.
Temp LED states
State
Module
Description
Steady green
Autosampler
The Wait for temperature ready check box in the Surveyor
AS Configuration dialog box is not selected. See Figure 63
on page 79.
Or this check box has been selected and the column oven
and tray temperature zones are in equilibrium at the set
temperatures.
RI detector
The operating temperature of the detector is in
equilibrium at the set temperature.
Or the detector is under direct control and the On check
box in the Temperature control area of the RI Diagnostic
page is not selected. See Figure 248 on page 309.
Steady amber
Degas LED
Autosampler
A temperature change is in progress.
RI detector
A temperature change is in progress.
The pump has a Degas LED that informs you whether the built-in
degassing unit has developed sufficient vacuum to for chromatography to be
performed. The states of the Degas LED are as follows:
• Steady green—sufficient vacuum has developed for chromatography to
be performed.
• Steady amber—the degassing unit is building vacuum.
• Flashing amber—the vacuum has dropped below an acceptable limit of
the degasser is unable to build vacuum.
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Introduction to the Surveyor Plus LC System
System Interconnect Cable
System Interconnect
Cable
A system interconnect cable assembly coordinates the run control signals
between the Surveyor Plus modules. There are two versions of this
cable.The older version of the cable has five combicon connectors labeled as
follows: LC Pump, MS Pump, A/S, Det, and M/S detector. See Figure 20.
The newer version of the cable has seven combicon connectors: two
connectors are labeled PUMP; three connectors are labeled DETECTOR;
the connector for the autosampler has a A/S tag on its adjacent cable; the
unused connector for a mass spectrometer has a M/S tag on its adjacent
cable. See Figure 21.
To
MS Detector
F5049-010
M/S
To
Autosampler
A/S
To
LC Pump
MS PUMP
LC PUMP
To
MS Pump
To
Detector
DET
Figure 20. System interconnect cable with 5-combicon connectors
DETECTOR
To
Detector
To
Pump
To
Pump
PUMP
A/S
To
Detector
PUMP
To
Autosampler
To
Detector
DETECTOR
M/S
DETECTOR
To
MS Detector
Figure 21. System interconnect cable with 7-combicon connectors
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Introduction to the Surveyor Plus LC System
System Interconnect Cable
These connectors attach to an 8-pin connection on the back panel of each
module. The MS Pump and the MS detector connectors are not used for
the Surveyor Plus modular HPLC system. To connect the Surveyor FL Plus
Detector, you need an additional adapter cable. SeeFigure 22 and Figure 23.
See the Surveyor Plus Getting Connected Guide for more information on
connecting the interconnect cable.
Solvent Platform
Detector
(back panel)
To
MS detector
(unused)
Autosampler
(back panel)
Adapter cable for Surveyor FL Plus Detector
Solvent Platform
To
FL detector
8-Pin
connector
Autosampler
(back panel)
LC PUMP
LC pump
(back panel)
USE ONLY WITH A
250V FUSE
EMPOYER
UNIQUEMENT
AVEC UN FUSIBLE
DE 250V
FL detector
(back panel)
LC pump
(back panel)
MS PUMP
MS pump
(Unused)
Not Drawn to Scale
Figure 22. Surveyor Plus modular HPLC system with the 5-connector system interconnect cable attached
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Introduction to the Surveyor Plus LC System
System Interconnect Cable
Solvent Platform
To
MS detector
(unused)
Autosampler
(back panel)
USE ONLY WITH A
250V FUSE
FL detector
(back panel)
EMPOYER
UNIQUEMENT
AVEC UN FUSIBLE
DE 250V
Detector
(back panel)
Adapter cable for Surveyor FL Plus Detector
(P/N 60153-63002)
To
FL detector
LC pump
(back panel)
8-Pin
connector
Figure 23. Surveyor Plus modular HPLC system with the 7-connector system
interconnect cable attached
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Communication with
ChromQuest
Introduction to the Surveyor Plus LC System
Communication with ChromQuest
With the exception of the Surveyor FL Plus Detector, the Surveyor Plus
modules communicate with the ChromQuest data system through an
Ethernet connection (see Figure 24).
Solvent Platform
OR
PDA or UV/Vis
detector
RI detector
Autosampler
Computer
USB
ENET
LC pump
Ethernet Box
CAT5 shielded
Ethernet cables
Figure 24. Ethernet connections between the Surveyor Plus modules and the data system computer
The Surveyor FL Plus Detector communicates with the ChromQuest data
system through a USB connection (see Figure 25).
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Introduction to the Surveyor Plus LC System
Communication with ChromQuest
Computer
FL Detector
USE ONLY WITH A
250V FUSE
EMPOYER
UNIQUEMENT
AVEC UN FUSIBLE
DE 250V
USB
Figure 25. USB connection between the FL detector and the data system
computer
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Solvent Path
A
B
Introduction to the Surveyor Plus LC System
Solvent Path
Figure 26 shows the solvent path of the mobile phase. The mobile phase
solvent path begins at the top of the system stack in the solvent reservoir
bottles and ends in the waste container as used eluent.
C
D
Solvent reservoir bottles
IN
OUT
A
B
C
Injection port
D
Degassing
assembly
Solvent
proportioning
assembly
1
Pump
head
2
1
4
5
6
3
2
Pump
head
Pressure
sensor
Pulse
dampening
assembly
Waste
Figure 26. Solvent path of the mobile phase
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Introduction to the Surveyor Plus LC System
Solvent Path
The solvent platform at the top of the Surveyor Plus System stack holds four
1-L solvent reservoir bottles and a 1-L wash bottle. Four low-pressure Teflon
solvent lines connect the solvent reservoir bottles to the vacuum degassing
assembly. Each of the degassing chambers contains permeable Teflon® AF
tubing with a capacity of 500 μL. Four short low-pressure Teflon solvent
lines connect the outlet ports of the degassing chambers to the solvent
proportioning assembly.
Each stroke of the pump’s pistons draws the solvents that are selected in the
method into the solvent proportioning assembly. If you are pumping
multiple solvents, mixing is performed in the low-pressure line that connects
the solvent proportioning assembly to the inlet check valve. At least one of
the four valves in the solvent proportioning assembly is open at all times.
Note When you perform routine maintenance tasks, such as replacing
the pump seals, you must switch off the power to the pump to close the
valves in the solvent proportioning assembly.
As the mobile phase exits the primary pump head, it is under high-pressure.
The crossover tube connects the primary pump head to the secondary pump
head. The outlet tube connects the secondary pump head to the purge
manifold assembly. Both the crossover tube and the outlet tube are
constructed of 1/16-in. ID high-pressure stainless steel tubing.
As the mobile phase enters the purge manifold assembly, it passes through a
pressure sensor that monitors the backpressure of the system. Then it passes
through the pulse dampening assembly, where its pressure pulsations are
reduced. As the mobile phase exits the pulse dampening assembly it
re-enters the purge manifold assembly.
When the drain valve knob is in the open position, the mobile phase is
directed out the left side of the purge manifold assembly to waste. When the
drain valve knob is in the closed position, the mobile phase is directed out
the top of the purge manifold assembly towards the autosampler. through a
stainless steel tube that passes behind the column oven where it loops
around the heat exchanger. This tube has a 250 μL volume, which allows
the mobile phase to equilibrate to the set temperature of the column oven
before it reaches the injection valve.
When the injection valve is in the inject position, mobile phase enters the
sample loop from the back, sweeping the liquid in its path out the front of
the loop and into the tubing that is connected to the head of the column.
When the injection valve is in the fill position, mobile phase bypasses the
sample loop.
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Solvent Path
Red, insulated PEEK tubing connects the end of the column to the inlet
port of the flow cell. The insulation on this tubing helps to reduce
temperature fluctuations. Blue, PEEK tubing connects the outlet port of the
LightPipe flow cell to waste.
CAUTION Do not connect tubing with an inner diameter of less than
0.060-in. to the OUT port of the RI detector. Doing so can damage the
flow cell. The outlet tubing supplied with the Surveyor RI Plus Detector
is 0.060-in. ID × 1/16-in., Teflon® tubing.
If you are running the pump in the gradient mode, the gradient delay
volume of the system begins at the point the mobile phase exits the solvent
proportioning assembly. It consists of all the wetted components that lie in
the flow path between the solvent proportioning valve of the pump and the
inlet of your chromatographic column. This volume is an important factor
to consider when developing gradient methods that will be transferred to
other instruments. For the Surveyor Plus system, the primary contributors
to the gradient delay volume are the pulse dampening assembly of the LC
pump (approximately 400 μL) and the tubing that lies behind the built-in
column oven of the Surveyor Autosampler Plus (approximately 250 μL).
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Introduction to the Surveyor Plus LC System
Navigation in ChromQuest
Navigation in
ChromQuest
The ChromQuest chromatography data system contains two primary
windows: the Main Menu window and the Instrument window. You
administrate and configure your instrument in the Main Menu window.
You control your instrument and process your data in the Instrument
window.
Figure 27 shows the Main Menu window, which appears when you launch
ChromQuest from the desktop. Right-clicking the icon for your instrument
opens a shortcut menu with access to the Configuration dialog box.
Double-clicking the icon for your instrument opens the Instrument
window, where you perform instrument control and data processing.
Instrument Icon
Instrument
Shortcut Menu
Figure 27. Main Menu window
The Instrument window shown in Figure 28 contains a chromatogram from
a stored data file (LabTest2004.dat) supplied with ChromQuest. The title
bar for the Instrument window lists the instrument name, the active
method, the active data file, the active pretreatment method, and
the project. The Channel Selector box lists the discrete and
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Navigation in ChromQuest
multi-chromatogram wavelengths contained in the instrument setup section
of the method, as well as the scan wavelength that is currently selected, and
the spectrum max plot. The command toolbar contains shortcuts to the
frequently used features of ChromQuest.
Clicking the Help button, which is located on the far right-side of the
command toolbar, opens the online Help for the current window or
dialog box.
Channel
Selector
Title Bar
Menu Bar
Command
Toolbar
Integration Events
Toolbar
Status Bar
Figure 28. Instrument window, showing the Chromatogram window
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Introduction to the Surveyor Plus LC System
The Tutorials
The Tutorials
In addition to the appendix, which contains calibration procedures for the
modules of the Surveyor LC system, this manual contains eleven tutorials as
follows:
Chapter 2, “Administrating the Enterprise.” shows you how to enable
instrument log ins and project management, create users, and create
projects.
Chapter 3, “Configuring Your Instrument.” shows you how to add
instruments to the Enterprise and configure the modules of your Surveyor
Plus system to communicate with the ChromQuest data system.
Chapter 4, “Creating Methods.” shows you how to create acquisition
methods for collecting chromatographic and spectral data and shutdown
methods for automatically turning off the pump flow and the lamps of the
detector.
Chapter 5, “Preparing Your Instrument for a Run.” shows you how to
prepare the LC system for a run, which includes removing air from the
solvent lines and checking the stability of the chromatographic baseline.
Chapter 6, “Making Your First Injection.” shows you how to load a vial into
the sample tray, inject a single sample, and view the results of your injection
on the screen.
Chapter 7, “Adding Integration Events Graphically.” shows you how to add
integration events to a method and manual integration fixes to a data file.
Chapter 8, “Specifying the Calibration Curve Parameters.” shows you how
to add a peak table to the method for the identification and quantitation of
your analytes.
Chapter 9, “Adding a Custom Report to the Method.” shows you how to
add a custom report to your method so that you can print the results of each
injection.
Chapter 10, “Creating a Sequence Table.” shows you how to use the
Sequence wizard to create a sequence table for injecting multiple samples. In
addition, this chapter shows you how to modify the sequence table by
adding a shutdown run or using a different sequence summary report.
Chapter 11, “Running and Reprocessing a Sequence.” shows you how to
start and reprocess sequences.
Chapter 12, “Diluting Samples with a Pretreatment Method.” shows you
how to create a pretreatment method for diluting samples.
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Introduction to the Surveyor Plus LC System
The Tutorials
If your system administrator has established the security features for
ChromQuest on your computer, verify the privileges that have been
assigned to you. If you do not have Administrative privileges, skip
Chapter 2, “Administrating the Enterprise.” If you do not have Instrument
privileges, skip Chapter 3, “Configuring Your Instrument.”
The tutorials contained in chapters 2 through 5 and chapters 7 through 10
can be performed without access to a Surveyor Plus LC system. In addition,
the topics “Viewing Your Chromatograms” on page 153, “Viewing Your
Spectral Data” on page 154, “Performing a Manual Peak Purity Check” on
page 159, and “Reprocessing a Sequence Run” on page 244 can be
performed without access to an instrument.
If you do not have a set of data files to work with, use the data files that are
supplied with the ChromQuest data system. You can find these tutorial
data files in the directory: Drive:\ChromQuest\Data. The data files named
multi calibration level 1.dat through multi calibration level 6.dat contain a
single chromatogram. The data files named LabTest2001.dat through
Labtest2007.dat contain scan data from 220 nm to 360 nm, as well as one
discrete channel at 254 nm. For instructions on how to open these data files
with their original acquisition method, see “Opening a Stored Data File” on
page 166.
In addition to these stored data files, ChromQuest contains the stored
sequence table named multilevel calibration.seq. You can find this sequence
file in the directory: Drive:\ChromQuest\Sequence.
In chapters 6, 11, and 12, you inject, analyze, and dilute the Autosampler
Test Mix (P/N A4991-010), which is a solution of toluene in methanol that
is shipped with the Surveyor Autosampler Plus. If you no longer have
ampules of the Autosampler Test Mix or you are performing this tutorial to
familiarize yourself with the features of the FL or RI detectors, substitute a
sample about which you have chromatographic knowledge and adjust the
suggested chromatographic parameters accordingly.
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Chapter 2
Administrating the
Enterprise
In this tutorial, you learn how to enable the security features provided by the
ChromQuest 4.2 chromatography data system. In addition, you learn how
to create projects to organize your data, methods, sequences, and templates.
Note ChromQuest contains a login security feature that allows the
system administrator to assign privileges to users and groups. If this
security feature is enabled, users can be denied administrative privileges,
instrument privileges, or both. You must have administrative privileges
to perform this tutorial. If you are responsible for administrating the data
system, make sure that you record your password.
The Enterprise in ChromQuest consists of the instruments controlled from
a standalone workstation or the networked instruments administrated from
a domain controller. After you define your Enterprise, you must
administrate it to enable the security features provided by the ChromQuest
chromatography data system.
In addition to the security features that allow you to restrict the access that a
user has to the data system, ChromQuest also contains a useful tool called
the Project Wizard, which enables you to automatically organize your data,
methods, sequences, and templates into project folders.
This chapter contains the following sections:
• Defining the Enterprise
• Enabling Instrument Login and Project Management
• Obtaining User Lists from the Data System Computer
• Obtaining User Lists from a Domain Controller
• Creating a Project
• Assigning Privileges to Users
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Administrating the Enterprise
Defining the Enterprise
Defining the
Enterprise
When you install the ChromQuest data system, the Main Menu window
has no defined Enterprise (see Figure 29). You or the ChromQuest system
administrator must define the Enterprise before instruments can be
configured and used. If your Enterprise includes more than one laboratory
or group, you might want to add locations to your Enterprise, and then
subordinate the instruments by location.
Defining the Enterprise involves these two tasks:
• Adding a Location/Group to the Enterprise
• Adding an Instrument to the Enterprise
Figure 29. Main Menu window, showing an undefined Enterprise
Adding a Location/Group
to the Enterprise
To add a Location or Group to your Enterprise
1. Right-click the Enterprise and, choose New > Location/Group from
the shortcut menu. See Figure 30.
A New Location subdirectory appears in the left panel of the Main
Menu window.
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Defining the Enterprise
Figure 30. Main Menu window, showing shortcut menu
2. Rename the New Location/Group:
a. Right-click the new location and choose Rename from the shortcut
menu.
b. Type an appropriate name for this Location or Group in the box
next to the Location icon, and then press ENTER.
Adding an Instrument to
the Enterprise
To add an instrument to the Enterprise
1. Right-click the Enterprise or a Location and choose New > Instrument
from the shortcut menu.
A new instrument appears in the right panel (see Figure 31).
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Defining the Enterprise
Figure 31. Main Menu window, showing a new instrument
2. Highlight the name in the box below the instrument icon to select it.
3. Type the name Surveyor System 1 (or your assigned instrument name)
in the box, and then press ENTER.
See Chapter 3, “Configuring Your Instrument,” for information on
configuring your instruments. Instruments that have not been configured
are labeled with a question mark.
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2 Administrating the Enterprise
Enabling Instrument Login and Project Management
Enabling Instrument
Login and Project
Management
When ChromQuest is initially installed, the security features are not
enabled, and everyone has access to all the pages of the Options dialog box
and the System Administration Wizard. Although everyone can create
projects, the project login feature is not available, which means that projects
are not automatically assigned to specific folders.
To automate project management and enable the security features available
in ChromQuest, you must enable instrument login and project
management.
To enable instrument login and project management
1. From the menu bar in the Main Menu window, choose Tools >
Options.
The Options dialog box appears.
2. Click the Enterprise tab
The Enterprise page appears (see Figure 32).
Figure 32. Options dialog box – Enterprise page
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Administrating the Enterprise
Enabling Instrument Login and Project Management
3. Select the Enable Instrument Login and Project Management
check box.
4. Select the source of the user lists for the Enterprise:
• If your Enterprise consists of a workstation that is not networked or
a workgroup that is not connected to a domain controller, select the
Data System option, and then continue at the section, “Obtaining
User Lists from the Data System Computer” on page 47.
• If your ChromQuest Enterprise consists of networked workstations
connected to a domain controller, select the Domain Controller
option, and then continue at the section, “Obtaining User Lists
from a Domain Controller” on page 49.
Note If you do not want to enable the login feature., clear the
Enable Instrument Login and Project Management check box
after you finish this tutorial
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2 Administrating the Enterprise
Obtaining User Lists from the Data System Computer
Obtaining User Lists
from the Data System
Computer
If your Enterprise consists of a workstation that is not networked or a
workgroup that is not connected to a domain controller, you obtain your
user list from your data system computer.
In this tutorial, you create a user list that contains two users.
To create the user list
1. In the Enterprise page of the Options dialog box (see Figure 32),
confirm that the Data System option is selected.
2. Click Add User.
The User Information dialog box appears (see Figure 33).
Figure 33. User Information dialog box
3. In the User Information dialog box, do the following:
a. Type All Privileges in the User Name box.
Note If you are following these instructions only to familiarize
yourself with the administrative features of ChromQuest, leave
the Password and Confirm Password boxes blank.
b. Click Save.
c. Repeat steps 3a and 3b to add yourself (your name) to the list of
Data System Users.
ChromQuest adds the user All Privileges and the name you gave
yourself to the Data System Users list (see Figure 34).
4. After you finish adding your users to the Data System Users lists, click
OK to exit the Options dialog box.
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Obtaining User Lists from the Data System Computer
Figure 34. Options dialog box – Enterprise page with a list of Data system users
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2 Administrating the Enterprise
Obtaining User Lists from a Domain Controller
Obtaining User Lists
from a Domain
Controller
If your ChromQuest Enterprise consists of networked workstations
connected to a domain controller, you obtain your user list from the domain
controller.
To select the domains to be scanned for user information
1. From the Enterprise page of the Options dialog box, confirm that the
Domain Controller option is selected, and then click Add Domain
The Domain Information dialog box appears (see Figure 35).
Figure 35. Domain Information dialog box
2. Type the domain name in the Domain Name box, and then click Save.
The domain name appears in the list of domains to be scanned (see
Figure 36).
3. Select the domains that you want to scan, and then click OK to exit the
Options dialog box.
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Figure 36. Options dialog box – Enterprise page with a list of domains to be
scanned
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Creating a Project
Administrating the Enterprise
Creating a Project
The system administrator has the choice to enable the Login and Project
Management feature:
• If the system administrator enables the Login and Project Management
feature, you must be a user with system administration privileges to
create projects.
• If the system administrator does not enable the Login and Project
Administration feature, all users can create and access projects. Users are
not required to login and the project management feature is not
automated. This lack of automation means users have to confirm that
they are storing data files, methods, sequences, templates, and advanced
reports in the appropriate directories.
To create a project to be used for the tutorials in this manual
1. If the System Administration Wizard is unlocked, go to step 2. If the
System Administration Wizard is locked (grayed out), log in:
Locked
UnLocked
Note If your user names are obtained from a domain controller, the
Enable Administration login box will contain a list from which you
select the domain name.
a. Click the Enterprise Login or Logout button
The Login dialog box appears (see Figure 37).
Figure 37. Login dialog box.
b. Type your user name in the User Name box and your password in
the Password box, and then click Login.
The System Administration Wizard becomes available.
2. Click the System Administration button.
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Creating a Project
The Select Administration page of the System Administration wizard
appears (see Figure 38).
Figure 38. System Administration Wizard – Select Administration Wizard page
3. Select the Project option, and then click Next.
The Select Project Action page of the Project Wizard appears (see
Figure 39).
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Creating a Project
Figure 39. Project Wizard – Select Project Action
4. Select the Create A New Project option, and then click Next.
The General Projects Settings page appears (see Figure 40). You name
the project and set up the file locations on this page.
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Creating a Project
Figure 40. Project wizard - General Settings
5. Give the project a name:
a. Type Tutorial in the Name box. As you type a name in the Name
box, the location of the project files updates (see Figure 41).
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Creating a Project
Figure 41. Project wizard – General Settings, showing the creation of the Tutorial project name
b. (Optional) Type a description of the project in the Description box.
c. Click Next.
The second page of the General Project Settings wizard appears (see
Figure 42). You select the auditing options on this page.
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Creating a Project
Figure 42. General Project Settings – audit trail options
6. Select the audit trail settings for the project:
a. Select the Automatically Enable Method Audit Trail check box,
and then select the Prompt For Reason When Saving Method
option.
b. Select the Automatically Enable Sequence Audit Trail check box,
and then select the Prompt For Reason When Saving Sequence
option.
c. Click Next.
The Define Electronic Signature Roles page of the Project wizard
appears (see Figure 43).
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Creating a Project
Figure 43. Project Wizard – Define Electronic Signature Roles
7. Define the electronic signature roles for the project:
a. Type 5 in the Number of Levels combo box.
b. (Optional) Modify the signature reasons by clicking Modify to open
the Modify Electronic Signature Reasons dialog box. Add, delete, or
modify the signature reasons.
c. Click Next.
The Select Users page appears.
8. Select the users for the Tutorial project that you are creating:
a. In the Available Users box, double-click each user that you want to
add to this project. Add All Privileges and yourself (your user name)
to the Selected Users box.
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Creating a Project
When you double-click an available user, the name of the user
moves to the Selected Users list. See Figure 44.
Figure 44. Project Wizard – Select Users page, showing users with access to the Tutorial project
b. Click Next.
The Set User Privileges page of the Project wizard appears (see
Figure 45).
9. Set the privileges for the users of the Tutorial project:
a. Select All Privileges and yourself (your user name) in the Selected
Users box, and then double-click all the privileges in the Unassigned
privileges box.
Note You can assign privileges by type, such as giving a user
privileges to all the method keys; or you can assign privileges
individually, such as letting a user open methods, but not save
methods.
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Creating a Project
When you double-click a privilege, it moves to the Assigned
Privilege box (see Figure 45).
Figure 45. Project Wizard – Set User Privileges page
b. Click Next.
The Set User Electronic Signature Role page appears (see Figure 46).
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Figure 46. Project Wizard - Set User Electronic Signature Role page
10. Select the electronic signature role for each user with privileges to the
Tutorial project, and then click Finish.
ChromQuest saves the project settings.
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Assigning Privileges
to Users
Administrating the Enterprise
Assigning Privileges to Users
After you define your Enterprise and create users, you are ready to assign
privileges to users.
In this tutorial, you assign instrument and system administration privileges
to users. You allow users access to every instrument in the Enterprise. You
assign access to the existing projects, as well as all privileges within each
project.
Users with system administration privileges can do the following:
• Access the Options dialog box - Enterprise page to enable login and
project management and create user lists.
• Access the Options dialog box – E-mail page to set up e-mail options.
• Access the Options dialog box - General page, where the Extended
Security, the Automatically Enable Method Audit Trail, the Save All
Analysis Results, and the Instrument Activity Log Purge Authorized
Only After Archive options are located.
• Access the System Administration wizard to create projects and assign
privileges to users.
Users with instrument administration privileges can do the following:
• Add and configure instruments.
• Access the Calibration and Maintenance pages of the Diagnostics dialog
box for the Surveyor LC Pump Plus.
• Access the Diagnostics dialog box for the Surveyor Autosampler Plus.
To assign privileges to users
1. Open the Select Administration page of the System Administration
wizard as described in steps 1 to 3 of “Creating a Project” on page 51.
2. In the Select Administration page, do the following:
a. Select the User option.
b. Select the Restart Selected Wizard When Finished check box.
You can assign user privileges to only one user at a time. Use the
Restart Selected Wizard When Finished option to enter privileges
for more than one user without having to restart the System
Administration wizard.
c. Click Next.
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The Select User page appears (see Figure 47).
Figure 47. User Wizard - Select User page
3. Select All Privileges or the user that you want to modify, and then click
Next.
Note If the User list is empty, see “Enabling Instrument Login and
Project Management” on page 45 for information on adding users.
The Administrative Privileges page appears (see Figure 48).
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Figure 48. User Wizard - Administrative Privileges page
4. Give the selected user instrument and system administration privileges
by selecting the System Administration and the Instrument
Administration check boxes, and then click Next.
The Select Instruments page appears (see Figure 49).
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Assigning Privileges to Users
Figure 49. User Wizard – Select Instruments page
5. Give the user privileges to all the instruments in the Enterprise by
double-clicking The Enterprise icon, and then click Next.
The Select Projects page appears (see Figure 50).
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Figure 50. User Wizard - Select Projects page
6. Double-click both the Default project and the Tutorial project in the
Available projects box, and then click Next.
The Set User Privileges page appears (see Figure 51).
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Figure 51. User Wizard – Set User Privileges page
7. Give the user every privilege in both the Default project and the Tutorial
project:
a. Select Default in the Selected projects box, and then double-click
the privileges in the Unassigned privileges box.
Note Privileges can be assigned one by one or by file type. Assign
privileges one by one by double-clicking the keys. Assign
privileges by file type by double-clicking a key set.
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b. Select Tutorial in Selected projects box, and then double-click the
privileges in the Unassigned privileges box.
c. Click Next.
The Set User Electronic Signature Role page appears (see Figure 52).
Figure 52. User Wizard – Set User Electronic Signature Role page
8. Select the electronic signature roles for the user, All Privileges, to both
the Default and Tutorial project, and then click Finish.
ChromQuest saves the project, user, and instrument information, and
then returns you to the Select User page of the User Wizard.
9. Assign yourself (your user name) Instrument Administration privileges,
System Administration privileges, access to the Enterprise, and every
privilege for the Default and Tutorial projects.
10. Close the System Administration Wizard.
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Assigning Privileges to Users
11. If you do not want to enable the security and administration features
offered in ChromQuest, do the following:
a. From the Main Menu bar, choose Tools > Options.
b. Click the Enterprise tab.
c. Clear the Enable instrument login and project management
check box.
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Chapter 3
Configuring Your Instrument
In this tutorial, you learn how to add an instrument to the Enterprise. The
Enterprise includes all instruments controlled within a domain or all
instruments controlled by a standalone workstation. In addition, you learn
how to configure your instrument so that the data system recognizes it.
Before you configure your instrument, look at the back panels of its
modules. On the back panel of each module, you will find a unit ID that
consists of two rotary switches. See Figure 53. Each rotary switch contains
ten positions. The arrow on the left switch points to the “tens” digit of the
unit ID. The arrow on the right switch points to the “ones” digit of the unit
ID. The unit ID can range from 01 to 99. The value 00 is reserved for
service functions. When you configure a module, you enter this unit ID
value in the Stack ID boxes. The unit ID value must match the Stack
address or the module will not communicate with the data system.
9
5 6
7 8
9
4
5 6
0 1
2 3
4
0 1
2 3
7 8
Figure 53. Unit ID, showing setting of 01
This chapter contains the following sections:
• Adding an Instrument to the Enterprise
• Configuring Your Instrument
Note ChromQuest contains a login security feature that allows the
system administrator to assign privileges to users and groups. If this
security feature has been enabled, you must have instrument privileges to
be able to add an instrument to the Enterprise or to configure an
instrument.
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Configuring Your Instrument
Adding an Instrument to the Enterprise
Adding an Instrument
to the Enterprise
The Enterprise consists of one or more networked instruments.
To add a new instrument to the Enterprise
1. Double-click the ChromQuest icon on the Windows desktop to open
the Main Menu window (Figure 54). Alternatively, choose Start >
Programs > Chromatography > ChromQuest to open ChromQuest.
Enterprise Login
Button
System Administration
Wizard Button
Figure 54. Main Menu window with System Administration locked
2. Look at the toolbar in the Main Menu window.
• If the System Administration Wizard button is unlocked, continue
at step 4.
Unlocked
Locked
• If the System Administration Wizard button is locked, the system
administrator has enabled the security feature, and you are required
to login. Continue at step 3.
3. To login to the ChromQuest chromatography data system:
a. Click the Enterprise Login or Logout button to
The Login dialog box appears (see Figure 55).
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3 Configuring Your Instrument
Adding an Instrument to the Enterprise
Figure 55. Enterprise Login dialog box
b. Type your user name in the User Name box.
c. Type your password in the Password box.
d. If available, select the appropriate domain from the Domain list.
Note If your workstation is not networked to a domain
controller, the Enable Administration login box does not
contain the Domain list.
e. Click Login.
4. Right-click The Enterprise icon, which is located on the left panel of
the Main Menu window, to open a shortcut menu.
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Adding an Instrument to the Enterprise
Figure 56. Enterprise shortcut menu
5. Choose New > Instrument from the shortcut menu.
A new instrument icon with a question mark appears in the right pane.
6. Type a new name for the instrument (for example, Surveyor Plus
System 1) in the box below the instrument button, and then press
ENTER.
Now that you have created a new instrument, you are ready to configure its
modules.
Note The Enterprise can be further subdivided into locations and
groups. If your Enterprise consists of locations and groups, add your new
instrument to the appropriate location or group.
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Adding an Instrument to the Enterprise
Figure 57. Main window for ChromQuest 4.2, showing the newly created
instrument
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Configuring Your Instrument
Configuring Your
Instrument
You must configure each module of the instrument. To configure the
instrument, perform the following procedures:
1. Opening the Surveyor Modules Dialog Box
2. Adding Modules to the Instrument Configuration
3. Completing the Configuration of Each Module
4. Selecting the Baseline Check Option
5. Returning to the Main Menu Window
Note If you are changing the configuration of an instrument, you
must exit the Instrument window before you open the Instrument
Configuration dialog box.
Opening the Surveyor
Modules Dialog Box
To open the Surveyor Modules dialog box
1. In the Main Menu window, right-click the icon of the instrument that
you want to configure, and then choose Configure > Instrument from
the shortcut menu.
The Instrument Configuration dialog box appears (see Figure 58).
Figure 58. Instrument Configuration dialog box
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Configuring Your Instrument
2. In the Instrument Configuration dialog box (Figure 58), do one of the
following:
• To erase the current configuration or change the instrument type,
select Surveyor from the Instrument type list, and then click OK in
the dialog box that appears. See Figure 59.
Figure 59. Warning message dialog box
• To modify your current instrument configuration, click Configure
in the Instrument Configuration dialog box (see Figure 58) to open
the Surveyor dialog box (see Figure 60).
Figure 60. Surveyor dialog box
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Configuring Your Instrument
Adding Modules to the
Instrument Configuration
Now that you have the Surveyor dialog box open (see Figure 60), you are
ready to add the modules of your instrument to its configuration. You can
add the modules in any order.
To add the instrument modules to the Configured Modules box
• Double-click the Surveyor LC Pump button to add the pump to the
Configured modules box.
• Double-click the Surveyor AS button to add the autosampler to the
Configured modules box.
• If you have a Surveyor UV/Vis Plus Detector, a Surveyor FL Plus
Detector, or a Surveyor RI Plus Detector, double-click its respective
button to add it to the Configured modules box.
• If you have a Surveyor PDA Plus Detector, double-click the Detector
button to begin the process of adding the Surveyor PDA Plus Detector
to the configuration.
When you double-click a button in the Available modules box, a copy of
the button appears in the Configured modules box (see Figure 61). The
Detector button defaults to the Analog icon in the Configured modules
box (see Figure 61).
Figure 61. Surveyor dialog box, showing added modules
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Completing the
Configuration of Each
Module
Configuring Your Instrument
Configuring Your Instrument
Now that you have added the modules of your instrument to the
Configured modules box, you are ready to complete their configuration.
This topic contains the following procedures. Perform the procedures that
pertain to your Surveyor Plus system.
• Configuring the LC Pump
• Configuring the Autosampler
• Configuring the PDA Detector
• Configuring the UV/Vis Detector
• Configuring the FL Detector
• Configuring the RI Detector
Configuring the LC Pump
To complete the configuration of the Surveyor LC Pump Plus
1. Double-click the Surveyor LC Pump icon in the Configured modules
box (see Figure 61).
The Surveyor LC Pump Configuration dialog box appears (see
Figure 62).
Figure 62. Surveyor LC Pump Configuration dialog box
2. From the Pressure list, select the Pressure units—MPa, Bar, or psi—that
you prefer to use to display the backpressure of your system.
1 MPa = 10 Bar = 145 psi.
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3. (Optional) Type an identification number for the pump in the ID
Number box.
4. In the Stack ID box, type or select the unit ID value for the pump. See
Figure 53 on page 69. Unless you are operating more than one Surveyor
LC Pump Plus from the same computer, leave the unit ID value (rotary
switch settings on the back panel of the pump) at its factory setting of 1.
5. If you want the injection sequence to pause if the built-in degasser loses
vacuum, select the Pause Sequence following Degasser Error
check box.
Configuring the Autosampler
To configure the Surveyor.Autosampler Plus
1. Double-click the Surveyor Autosampler icon in the Configured
modules box.
The Surveyor Autosampler Configuration dialog box appears.
2. Set the parameters in the dialog box to the settings that are shown below
and are reflected in Figure 63.
Parameter
Setting
Result
Stack
The value of the Unit ID on
the back panel of the
autosampler.
Must match the Unit ID on the back panel of the autosampler
Dead volume
The value specifed on the
transfer tube assembly label.
Specifies the volume of the transfer tubing that connects the injection port to the
Valco injection valve. See Figure 14 on page 19.
Communication
Options
Verify door is closed
Specifies that the autosampler can begin an injection or that direct control
commands can be performed whether the tray compartment door is open or closed
Loop size
Enter the size of the sample
loop.
Specifies the injection volume for the Full Loop injection mode.
Syringe type
Select the appropriate type
for your syringe.
Specifies the type of syringe. The autosampler ships with a 250 μL concentric
syringe.
Signal Polarities
Run starts when Pump
Ready
Specifies that the pump sends a signal to the autosampler while it detects a stable
backpressure.
Inject when Inject Hold
signal is Off
Specifies that the pump sends a signal to the autosampler when it reaches a
certain point in its piston cycle.
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3. Under Communication, click Detect.
There are two Surveyor Autosampler Plus models: the full-featured
Surveyor Autosampler Plus and the Surveyor Autosampler Lite Plus.
The Surveyor Autosampler Lite Plus does not come with a built-in
column oven or tray temperature control. Clicking the Detect button
downloads the autosampler model to the ChromQuest data system.
4. Click OK to exit the Surveyor Autosampler Configuration dialog box.
The appropriate
value is
specified on
the label
attached to the
transfer tube
assembly.
Figure 63. Surveyor AS Configuration dialog box, showing configuration settings
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Configuring Your Instrument
Configuring the PDA Detector
To complete the configuration of the Surveyor PDA Plus Detector
1. Double-click the Analog icon in the Configured modules box.
The Detector Configuration dialog box appears (see Figure 64).
Figure 64. Detector Configuration dialog box
2. In the Detector Model list, select PDA Plus.
.
Note After you select PDA Plus, the Acquisition Source list
automatically lists the PDA ACQ option. Leave the Y-axis Units set
to mAU and the Y-axis Multiplier set to 0.001. ChromQuest stores
the absorbance data from the PDA in microvolts. The Y-axis
Multiplier of 0.001 scales the display to mV (1 mV = 1 mAU).
3. Click the button to the right of the Detector Model list.
The PDA Plus dialog box appears (see Figure 65).
Figure 65. PDA Plus dialog box.
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4. In the Stack boxes, select or type the unit ID value for your Surveyor
PDA Plus Detector.
5. Click OK to exit the PDA Plus dialog box.
Configuring the UV/Vis Detector
To complete the configuration of the Surveyor UV/Vis Plus Detector
1. Double-click the Surveyor UV/VIS icon in the Configured modules
box.
The Surveyor UV/VIS Detector Configuration dialog box appears (see
Figure 66).
Figure 66. Surveyor UV/Vis Detector Configuration dialog box
2. In the Stack boxes, type or select the unit ID value for your detector.
The unit ID on the back panel of the detector consists of two rotary
switches. Each switch has ten positions. The arrow on the left switch
points to the “tens” digit of the unit ID. The arrow on the right switch
points to the “ones” digit of the unit ID.
3. (Optional) Type an identification number for your Surveyor UV/VIS
Detector, such as its license number, in the ID number box.
4. Click OK to exit the Surveyor UV/VIS Detector Configuration
dialog box.
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Configuring the FL Detector
To complete the configuration of the Surveyor FL Plus Detector
1. Double-click the Surveyor FL Plus Detector icon in the Configured
modules box
The Surveyor FL Plus Detector Configuration dialog box appears (see
Figure 67).
2. (Optional) Type an identifying name in the Detector Name box.
3. (Optional) Type an identifying label in the Flow Cell ID box.
Figure 67. Surveyor FL Plus Detector Configuration dialog box
Note The Surveyor FL Plus Detector uses the light spectrum
produced by a Hg lamp to calibrate its wavelength accuracy. This
lamp is turned on only during a wavelength calibration.
4. To set a usage limit for the Hg lamp:
a. Click Lamp Exchange.
The Set Usage Limit: Surveyor FL Plus Detector dialog box appears
(see Figure 68).
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Figure 68. Set Usage Limit: Surveyor FL Plus Detector dialog box
b. Type a value for the usage limit in the Usage limit box.
The allowable range of values is 1 to 9999 with a default of 100.
The FL detector turns on the mercury lamp on for the Wavelength
Check procedure performed by the Maintenance software. The
operating life of the mercury lamp is approximately 300 hours.
c. Click OK to exit the Set Usage Limit: Surveyor FL Plus Detector
dialog box.
d. Click OK to exit the Surveyor FL Plus Detector Configuration
dialog box.
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Configuring Your Instrument
Configuring the RI Detector
To complete the configuration of the Surveyor RI Plus Detector
1. Double-click the Surveyor RI Plus Detector button in the Configured
Modules box.
The Surveyor RI Plus Detector Configuration dialog box appears (see
Figure 69).
Figure 69. Surveyor RI Plus Detector Configuration dialog box
2. Unless you are controlling more than one Surveyor RI Plus Detector
from the same computer, leave the stack value at its default of 1.
Note The Surveyor RI Plus Detector stores its IP address electronically.
The detector does not have rotary switches on its back panel.
3. (Optional) Type an identification number in the ID number box.
4. Click OK.
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Selecting the Baseline
Check Option
Configuring Your Instrument
Configuring Your Instrument
ChromQuest 4.2 contains two features that provide information on the
stability of the chromatographic baseline: Preview Run and Baseline Check.
You can use the Preview Run feature to visually check the baseline. In
addition to a visual check, the Baseline Check feature gives you quantitative
results for the noise and drift levels of the baseline.
Note If you create a method for an instrument that has the Baseline
Check feature enabled, you will not be able to use the method for
instruments that do not have the Baseline Check feature enabled. The
reverse is also true.
By default, the Baseline Check feature is enabled.
To ensure that the Baseline Check feature is enabled
1. In the Surveyor dialog box (Figure 60 on page 75), click Options.
The Configuration Options dialog box appears (see Figure 70).
Figure 70. Configuration Options dialog box – General page
2. Verify that the Baseline Check check box is selected.
3. Click OK to exit the Configuration Options dialog box.
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Note If you have configured a PDA detector, the Spectral Analysis check
box is automatically selected after you exit the Surveyor dialog box.
Returning to the Main
Menu Window
After you finish configuring your instrument, return to the Main Menu
window.
To return to the Main Menu window
1. Click OK at the bottom of the Surveyor Modules dialog box (Figure 61
on page 76).
2. Click OK at the bottom of the Instrument Configuration dialog box
(Figure 58 on page 74) to return to the Main Menu of ChromQuest.
You have now successfully added and configured the modules of your
instrument. In addition, you have verified that the Baseline Check option is
selected.
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Chapter 4
Creating Methods
In this tutorial, you learn how to create an acquisition method containing
the instrument control parameters and column performance information
for your chromatographic method. In addition, you learn how to create a
shutdown method that turns off the lamps and the mobile phase flow at the
end of a sequence run.
Note To turn off the lamp in the Surveyor RI Plus Detector, you must
turn off the power to the detector.
Before you can perform this tutorial, your instrument must be configured. If
your instrument has not been configured, see Chapter 3, “Configuring Your
Instrument.”
This chapter contains the following sections:
• Description of ChromQuest Methods
• Creating an Acquisition Method
• Creating a Shutdown Method
Note ChromQuest contains a login security feature that allows the
system administrator to assign privileges to users and groups. If this
security feature is enabled, your system administrator can restrict your
access to specific tasks such as creating or modifying methods. To avoid
confusion, determine your user privileges before you begin this tutorial.
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Description of ChromQuest Methods
Description of
ChromQuest Methods
There are three types of methods in ChromQuest:
• Acquisition methods, which contain the information required to control
the instrument modules, acquire data, and process data files
• Shutdown methods, which turn off the pump flow, the detector lamps,
or both at the end of a sequence run
• Prep only methods, which contain the information to perform an
automated sample preparation routine and maintain a set of
chromatographic conditions, such as column temperature and mobile
phase parameters
In this tutorial, you create an acquisition method containing the instrument
control parameters required to create the specified chromatographic
conditions. In addition, you add the column parameters to the method,
which enables ChromQuest to calculate the performance parameters for
your chromatographic separation.
If your instrument includes a PDA or UV/Vis detector, use the Autosampler
Test Mix (P/N A4991-010) contained in the Surveyor Autosampler
accessory kit. This test mix consists of a solution of 0.5% toluene in
methanol.
If you do not have a vial of the Autosampler Test Mix or you are performing
the tutorial to familiarize yourself with the features of the RI or FL
detectors, plan to inject an analyte about which you have some
chromatographic knowledge and adjust the instrument parameters
accordingly.
Using the chromatographic conditions listed in Table 7, toluene elutes at
approximately 2 minutes.
Table 7.
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Chromatographic conditions for toluene
Parameter
Setting
Mobile phase
80:20 Methanol \ Water
Flow rate
1 mL/min
Column
100 x 4.6 mm, C-18 column, 5 micron particle size, or equivalent
Temperature
ambient
Discrete channel
wavelengths
230 nm, 1 nm bandwidth for the PDA detector
Scan range (PDA)
220 nm to 300 nm, 1 nm step, 1 nm bandwidth
260 nm, 1 nm bancdwidth for the PDA detector
Surveyor Plus Getting Started with the ChromQuest 4.2 Chromatography Data System
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4 Creating Methods
Description of ChromQuest Methods
A spectrum of toluene in 80:20 methanol / water is shown in Figure 71 on
page 89.
Figure 71. Spectrum of Toluene in 80:20 methanol / water
If your system contains a Surveyor FL Plus Detector or a Surveyor RI Plus
Detector, inject an analyte for which you have a proven chromatographic
method. In this tutorial, a solution of anthracene is used for systems
containing an FL detector, and a solution of sucrose in water is used for
systems contains an RI detector.
The sections of an acquisition method, as well as their descriptions are listed
in Table 8. The first section, Instrument Setup, is the only section that you
need to complete before you can make an injection. You can complete the
other sections of the method after you make an injection and acquire a
data file.
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Description of ChromQuest Methods
Table 8.
Description of the sections of an acquisition method
Section
Description
Instrument Setup
Use this window to do the following:
Integration Events
•
Enter the control parameters for each module in your instrument
•
Add an auxiliary trace to record the backpressure of your system during a run
•
Enter baseline check parameters if the Baseline Check option in the Configuration Options dialog box is
selected
•
Select a type of trigger
This window contains the integration table(s) for your method. There is one integration table for each discrete
wavelength and each multichromatogram wavelength in the method.
Use the integration tables to enter the integration parameters for each discrete or multichromatogram
wavelength specified in the method. To open a specifice integration table, select the wavelength in the
Analysis Channel list.
Spectral Options
This window is available if your system contains a configured PDA detector. This window contains the
following pages: Libray, Purity, Spectrum, Multi-Chromatogram, and Ratio.
Use this window to do the following:
•
Select the automated Library search parameters
•
Select the parameters for peak purity calculations
•
Select spectral filtering and spectral definition options
•
Select multi-chromatogram wavelengths
•
Set up ratio plots
Peak/Groups
Use the Peak table to enter the calibration curve parameters for your analytes. Use the Group tables to
combine peaks into groups and to calculate group totals. There is one Peak table for each configured detector
of your instrument.
Review Calibration
Use this window to review the calibration curve for your method. This window displays the calibration table, a
graphical display of the calibration points, the calibration parameters, and the regression statistics for each
named peak in the Peak table.
Advanced
This window contains the following pages: Export, Custom Parameters, Column / Performance, Files, and
Advanced Reports.
Use this window to calculate performance parameters such as capacity factor and resolution, column
parameters must be entered in the Column / Performance page.
Custom Report
Use this window to create a report for each data file.
System Suitability
Use this window to enter system suitability parameters for each named peak and to perform noise and drift
tests for the discrete and multi-chromatogram wavelengths in your method.
Properties
This dialog box contains the following pages: Description, options, Calibration, Autit Trail.
Use this dialog box to do the following:
• Enter a description of the method
• Select the analysis options: Analyze after acquisition (default setting) or Analyze during acquisition
• Select the Amount / Area or the Area / Amount response factor definition
• Select or clear the automatic averaging of standard replicates
• Specify the number of standard replicates in the rolling average
• Enable the method audit trail
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4 Creating Methods
Creating an Acquisition Method
Creating an
Acquisition Method
Methods can be created from either the offline Instrument window or the
online Instrument window. In this topic, you open the offline Instrument
window, and then use the Method Wizard to create a method. To create
your first method, perform the procedures in this section in the order listed:
1. Opening the Offline Instrument Window
2. Activating the Method Wizard
3. Entering the Instrument Parameters
4. Triggering Data Acquisition
5. Entering the Column Parameters
6. Saving the Method
Opening the Offline
Instrument Window
There is an Online Instrument window and an Offline Instrument window
for each instrument in the Enterprise. From the Online Instrument window,
you can perform both instrument control and data processing operations.
From the Offline Instrument window, you can perform only data processing
operations, such as creating methods and reprocessing data files.
To open the Offline Instrument window
1. In the Main Menu window (see Figure 27 on page 36), right-click the
icon that represents your instrument, and then choose Open Offline
from the shortcut menu.
2. Depending on which dialog box or window appears, do one of the
following:
• If the Login dialog box shown in Figure 72 appears, the Enable
Logins feature has been selected. Continue at step 3.
• If the Instrument Wizard dialog box shown in Figure 73 appears,
continue at step 5.
• If the Instrument window is activated as indicated by its title bar,
you have successfully opened the Instrument window and are ready
to go to the next topic, “Activating the Method Wizard” on page 93.
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Figure 72. Login dialog box
3. Log in:
a. Type your user name in the User name box
b. Type your password in the Password box
c. Select a project from the Project list. If you created the project,
Tutorial, as you performed the Creating a Project tutorial
beginning on page 51, select it from your project list.
d. Click Login.
4. Depending on whether the Instrument window appears, do one of the
following:
• If the Instrument Wizard dialog box appears, as shown in Figure 73,
continue at step 5.
• If the Instrument window is activated as indicated by its title bar,
you have successfully opened the Instrument window and are ready
to go to the next topic, “Activating the Method Wizard” on page 93.
5. Clear the Show at instrument startup check box at the bottom of the
Instrument Wizard dialog box.
You do not use the Instrument Wizard dialog box in this tutorial.
6. Close the Instrument Wizard dialog box to activate the Instrument
window.
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4 Creating Methods
Creating an Acquisition Method
Figure 73. Offline Instrument Wizard dialog box, showing the Show at
instrument startup check box cleared
Activating the Method
Wizard
Before you can inject a sample, you must create a method that contains your
instrument control parameters. In this tutorial, you use the Method Wizard
to create a new method.
The Method Wizard opens a method that contains default control
parameters for each module of the instrument. The Method Wizard also sets
up a navigation bar at the bottom of the Instrument window. Clicking the
blue arrows in the navigation bar opens the dialog boxes of the method
one-by-one.
To activate the Method Wizard
1. From the menu bar in the Instrument window, choose File > Method >
Method Wizard.
The Method Wizard appears (see Figure 74).
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Figure 74. Method Wizard dialog box
2. Click the Create a new method button.
3. If the dialog box shown in Figure 75 appears, click No.
You save your method after you enter your instrument setup parameters
and your column information.
The Instrument Setup window appears. The Method dialog buttons
appear at the bottom of the Instrument window. The active method is
the default method named “untitled” and the instrument setup
parameters are those contained in the default method.
Figure 75. Dialog box that appears when you attempt to open a New
Method
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4 Creating Methods
Creating an Acquisition Method
Entering the Instrument
Parameters
The Instrument Setup window contains a page for each configured module.
Enter the chromatographic conditions of your method by performing the
following procedures that apply to your Surveyor Plus system:
• Entering the Pump Parameters
• Entering the Autosampler Parameters
• Entering the PDA Detector Parameters
• Entering the UV/Vis Detector Parameters
• Entering the FL Detector Parameters
• Entering the RI Detector Parameters
Entering the Pump Parameters
The Surveyor LC Pump Plus is a quaternary gradient pump capable of
proportioning four solvents and performing gradient programs containing
up to 40 lines.
In this tutorial, you run the pump in the isocratic mode. This means that
the mobile phase composition remains constant during the run. You
program the pump to create a binary mobile phase consisting of 80%
methanol and 20% water (v/v). This saves you the time and effort of
preparing a pre-mixed mobile phase.
In addition to entering the parameters to create a binary mobile phase, you
enter the conditions under which the pump will continue to operate. You
use the minimum and maximum pressure boxes, shown in Figure 76, to
program the pump to stop if the backpressure falls below or rises above an
unacceptable level.
A drop in the system backpressure is typically caused by a leak in the solvent
lines. A rise in the system backpressure is typically caused by column
degradation or a clogged frit in the line body at the top of the purge
manifold (see Figure 5 on page 10) of the pump.
To enter the parameters for the Surveyor LC Pump Plus
1. In the Instrument Setup window, click the Surveyor LC Pump tab to
open the Surveyor LC Pump page. See Figure 76.
2. Keep all parameters in the Surveyor LC Pump page set to the default
settings except those that are shown in the following table and in
Figure 76.
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After you enter a value of 80 in the Conc box for solvent line A,
ChromQuest automatically adjusts the concentration of solvent line B
to 20.
Parameter
Setting
Result
Name
Methanol
Specifies Methanol as the name for solvent A. The solvent name appears in the Method
report.
Conc
80
Specifies that the initial composition of the mobile phase is 80% Methanol by volume
Name
Water
Specifies Water as the name for solvent B. The solvent name appears in the Method
report.
Conc
20
Specifies that initially the mobile phase will consist of 20% water by volume
Min Pressure
15 (psi) or 1 (bar) or 0.1 (MPa)
Specifies the minimum system backpressure at which the pump will continue to run. If
the backpressure falls below this limit, which happens when a leak occurs, the pump
pistons will stop.
Max Pressure
2900 (psi)
(20 MPa = 200 bar = 2900 psi)
Specifies the maximum backpressure at which the pump will continue to run.
Initial Settings
Pressure Limits
Figure 76. Surveyor LC Pump Instrument Setup window - Surveyor LC Pump page, showing the
parameters for the pump
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Creating an Acquisition Method
Entering the Autosampler
Parameters
In this tutorial, you use the partial loop injection mode because you want to
be able to inject variable amounts of sample.
To enter the parameters for the Surveyor Autosampler Plus
1. Click the Surveyor AS tab to open the Instrument Setup page for the
Surveyor Autosampler.
2. Leave the autosampler program at its default settings as shown in
Figure 77.
Note The default flush speed of 250 mL/s is only suitable for solvents
with a low viscosity. If you are using water or a water / methanol mixture
for the flush solvent, lower the flush speed to 100 μL/s or less. If you
hear the syringe drive rattle, the flush speed is too high.
Figure 77. Instrument Setup window – Surveyor AS page
Entering the PDA Detector
Parameters
Thermo Scientific
The Surveyor PDA Plus Detector is a photodiode array detector that is
capable of scanning the UV/Vis range from 190 to 800 nm. In addition to
collecting scan data, the detector is capable of simultaneously collecting up
to three discrete channels. You can use this capability to set the bandwidth
and data rate parameters for the discrete channels separately from those of
the scan data.
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In this tutorial, you program the PDA detector to collect scan data from 220
to 300 nm at a step size of 1 nm. In addition, you program the detector to
collect two discrete channels.
Note Because the spectral analysis program compares spectra on a
wavelength to wavelength basis, Set the step size to 1 nm when collecting
scan data to create spectrum files. If you collect one spectrum from 200
to 300 nm with a step size of 2 nm, and another spectrum from 201 to
301 nm with a step size of 2 nm; these spectra will have no wavelengths
in common. Therefore, even if the spectra are of the same analyte, the
comparison algorithm will report a match of 0.
To enter the parameters for the Surveyor PDA Plus Detector
1. Click the Surveyor PDA Plus tab to open the detector page.
2. Keep all parameters in the Surveyor PDA Plus dialog box set to the
default settings except those that are shown in the following table and in
Figure 78.
Parameter
Setting
Result
Run time
4
Specifies a data collection time of 4 min
Rise time
1
Specifiea a rise time of 1 second
Step
1
Specifies that a chromatogram will be
collected for every wavelength in the
scan range
Bandwidth
1
Specifies a spectral resolution of 1 nm
Channels
2
Specifies that two discrete
wavelengths will be collected
Wavelength A
230
Specifies that absorbance data for
230 nm will be collected
Wavelength B
260
Specifies that absorbance data for
260 nm will be collected
Scans
Discrete
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Figure 78. Instrument Setup window – Surveyor PDA Plus page, showing the
parameters for the Surveyor PDA Plus Detector
Entering the UV/Vis Detector
Parameters
The Surveyor UV/Vis Plus Detector is a programmable, dual-wavelength
detector. In this tutorial, you run the detector in the dual-wavelength mode
and collect absorbance data for a period of 4 minutes. At the end of your
first injection, you will have a data file containing two chromatograms
(230 nm and 260 nm).
To enter the parameters for the Surveyor UV/Vis Plus Detector
1. Click the Surveyor UV/Vis tab to open the detector page.
2. Keep all parameters in the Surveyor UV/Vis dialog box set to the default
settings except those that are shown in the following table and in
Figure 79.
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Parameter
Setting
Result
Program Type
Program Type
Dual wavelength 190 to 365 nm
Specifies that two chromatograms in the UV/Vis
range will be collected
Wavelength Table
Row 1
Time = 0, Wavelength 1 = 230 nm, Wavelength 2 = 260 nm
Row 2
Time = 4, Wavelength 1 = 230 nm, Wavelength 2 = 260 nm
Specifies that the wavelength program will collect
data at 230 nm and 260 nm for a period of 4 minutes
Figure 79. Instrument Setup window – Surveyor UV/Vis page, showing the parameters for UV/Vis detector
Entering the FL Detector
Parameters
To perform the tutorial, enter the chromatographic conditions for the
analyte that you plan to inject.
To specify the parameters for the FL detector
1. Click the Surveyor FL Plus tab to open the method page for the FL
detector.
Figure 80 shows the default parameters for the Surveyor FL Plus
Detector.
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Figure 80. Instrument Setup window – Surveyor FL Plus page
2. Enter the FL detector settings for your application as follows:
a. In the Run Time box, type the length of time that you want the
detector to acquire data.
Because the program cannot collect more than 72000 data points
per run, the allowable run time depends on the sampling period. For
a sampling period of 50 ms (20 Hz), the maximum allowable run
time is 60 minutes. For a sampling period of 800 ms (1.25 Hz), the
maximum allowable run time is 600 minutes.
b. In the Rise Time list, select a rise time that is appropriate for your
application.
Increasing the rise time filter decreases the baseline noise, but it can
also result in peak broadening. In general, select a rise time that is no
higher than one-tenth the base peak width for the narrowest peak of
interest.
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c. In the Sampling Period list, select a sampling period based the on
the run time of your analysis and the expected baseline peak width
of the narrowest peak in your chromatogram.
As you increase the run time beyond 60 minutes, you must increase
the sampling period above the minimum sampling period of 50 ms
(20 Hz) because the program cannot collect more than 72000 data
points per run.
To integrate a peak, you must collect a minimum of three points
across the peak. For optimal integration, collect at least 20 points
across the peak.
d. In the Offset box, leave the value at the default of 0 for most
applications.
e. In the Em bandwidth list, select Standard (15 nm) or Wide (30nm).
f. If necessary, select the Autozero Before Injection check box to
compensate for baseline drift during a sequence run.
3. In the Time Table, enter the initial conditions or the conditions for the
duration of the run:
a. Type an excitation wavelength from 200 nm to 800 nm in the
Ex WL column, and then press ENTER.
b. Type an emission wavelength from 250 nm to 900 nm in the
Em WL column, and then press ENTER.
c. Select a PMT voltage in the PMT Voltage column. Increasing the
PMT voltage increases the sensitivity of the analysis, but also
shortens the operating lifetime of the photomultiplier.
If the program contains one time line, the specified conditions
remain the same for the time period specified in the Run Time box.
4. To change the excitation wavelength, emission wavelength, or PMT
Voltage settings or to zero the baseline during the run, enter more time
lines in the time table:
a. In the Time column, type a time value from 0.1 to 600.0 minutes
for the second row onward.
b. Type appropriate excitation and emission wavelengths in their
respective columns.
c. In the Baseline column, select Autozero to zero the baseline at the
specified time point or Hold to leave the baseline unchanged.
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Entering the RI Detector
Parameters
To specify the parameters for the RI detector
1. Click the Surveyor RI Plus tab to open the Surveyor RI Plus method
parameters page (see Figure 81).
Figure 81. Surveyor RI Plus Detector method parameters page
2. Enter the instrument control parameters for the RI detector as follows:
a. Type a value in Run Duration box to specify the length of time in
minutes that data is to be acquired from the detector during the run.
b. Select a value from the Rise Time list to specify the response time of
the detector in seconds.
Increasing the rise time reduces the baseline noise. However, setting
the rise time to a value greater than one-tenth the width of the
chromatographic peak at half-height results in peak broadening.
The 2 second default value is appropriate for most LC applications.
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c. Select a value from the Data Rate list.
For optimal results, select a data rate that collects at least 20 points
across the peaks of interest.
d. Select the Zero on Start Run check box if you want to zero the
chromatographic baseline at the start of a run.
You can use this feature to compensate for baseline drift during a
sequence run.
e. Select the Temperature Control check box to turn on the
temperature control feature.
Because RI measurements are very sensitive to temperature, use this
feature to improve the reliability of your analyses.
f. Select the signal polarity option for your analysis that produces
chromatograms with positive peaks, rather than negative dips.
If the analyte has a higher refractive index than the mobile phase,
select the Positive option. If the analyte has a lower refractive index
than the mobile phase, select the Negative option.
The Baseline Offset, Integrator Output Range, and Recorder
Output Range parameters are used to scale the output to an
integrator or a recorder.
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Triggering Data
Acquisition
To specify the trigger type for your method
1. From the Instrument Setup window, click the Trigger tab to open the
Trigger page.
2. Select a trigger type in the Type list.
If your instrument contains a Surveyor Autosampler Plus, use the
External trigger mode to start your runs. See Figure 82.
Figure 82. Instrument Setup window - Trigger page
When you select External as the triggering mode, the autosampler triggers
the detector to begin data acquisition.
Note If your run remains in the Waiting For Trigger status mode, check
the system interconnect cable connections on the back of your
instrument and verify that the autosampler is configured properly.
If your instrument does not contain an autosampler, you might choose to
Trigger data acquisition by selecting None or Manual from the Type list. If
you select Manual, the Start Acquisition dialog box pops up between each
run in a sequence table. See Figure 83.
Figure 83. Start Acquisition dialog box
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Entering the Column
Parameters
Adding column parameters to the method enables ChromQuest to calculate
performance parameters for your chromatographic separation. You can add
the column parameters to your method either before or after data
acquisition.
Note ChromQuest calculates performance parameters on a per detector
basis.
To enter the parameters for your column to the method
1. Click the right blue navigation arrow at the bottom left of the
Instrument window. Continue to click the arrow until the Advanced
Method Options window appears.
On the first click, the blue arrow opens the Integration Events dialog
box. On the second click, the blue arrow opens the Peak / Group Tables
dialog box. On the third click, the blue arrow opens the Advanced
Method Options window. See Figure 84.
Figure 84. Instrument window, showing the Export page and the blue
navigation arrows at the bottom of the window
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2. From the Advanced Method Options window, click the
Column / Performance tab to open the Column / Performance page.
3. Enter your column information. If you are using a Hypersil C-18,
4.6 × 100 mm, 5 micron particle size column, make the entries that are
listed in the following table and shown in Figure 85.
4. Select the Calculate performance parameters for this channel
check box.
ChromQuest calculates performance parameters on a per detector basis.
Selecting the Calculate performance parameters for this channel
check box enables the calculation of performance parameters for all the
chromatograms produced by the detector listed in the Channel Selector
list and the title bar of the window.
5. If your instrument contains two detectors (for example, a UV/Vis
detector and an RI detector) and you want to report performance
parameters for the second detector as well, select the second detector in
the Channel Selector list, and then select the Calculate performance
parameters for this channel check box a second time.
Parameter
Setting
Result
1
Specifies the void volume of the LC column in
min [volume (mL) × flow rate (mL/min)]
Column Information
Unretained PeakTime
Column Length*
cm
Specifies the units of length as centimeters
Column Length
10
Specifies a length of 10 cm
Particle Diameter
5
Specifies that the column packing material
has an average diameter of 5 microns
Column Description
Descriptive information about your column
The information provided in this box can be
printed in the method report.
Calculate Performance Parameters For This Channel
Calculate Performance Parameters For This
Channel
Calculation Methods
Specifies that performance parameters will
be calculated for the detector listed in the
title bar of the window
USP
Specifies that the performance parameters
will be calculated using the USP equations
*Note. Select the centimeters option, and then type the length of your column in the column length box.
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Figure 85. Advanced Method Options window – Column/Performance page
ChromQuest requires your column information and a calculation method
to calculate parameters such as the capacity factor, asymmetry, and
resolution for a chromatographic peak. If you attempt to add annotations
for these parameters to your chromatograms before you add your column
information to the method, ChromQuest reports these values as zero.
Saving the Method
To save your new method
1. Click the Save Method As button at the bottom of the Instrument
window.
The Save Method As dialog box appears (see Figure 86).
2. In the Save Method As dialog box, browse to the appropriate directory
for your method file if you are not logged into a project.
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If you are logged into the Tutorial project, the method is saved in the
following folder: Drive:\ChromQuest\Projects\Tutorial\Methods.
3. Type the name Test Mix in the File Name box, and then click Save.
ChromQuest adds the .met file extension to method files.
4. Do not exit the Instrument Setup window. Proceed to the next topic,
Creating a Shutdown Method, which describes how to create a
shutdown method by modifying an acquisition method.
Figure 86. Save Method File As dialog box
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Creating a Shutdown
Method
To automatically turn off the pump flow, the detector lamps, or both at the
end of a sequence run, the last line of the sequence table must contain a
shutdown method. You cannot use a shutdown method to make injections.
Creating sequence tables is discussed in Chapter 10, “Creating a Sequence
Table.”
For a shutdown method to be valid, the Shutdown check boxes in the
method pages for the pump and the detectors must be selected, even if you
do not want to turn off the lamps or the solvent flow.
To create a shutdown method
1. If the Instrument Setup window is not open, open it by choosing
Method > Instrument Setup from the Instrument window menu bar.
2. If your method, Test Mix.met, is not open, open it:
a. From the Instrument window menu bar, choose File > Method >
Open.
The Open Method File dialog box appears.
b. Browse to the appropriate directory, and then select the Test
Mix.met method that you created while performing this tutorial.
3. To set the shutdown parameters for the detector:
a. In the Instrument Setup window, click the tab for your detector to
open its method parameters page.
b. Select the Shutdown Method check box.
c. To turn off the lamp(s) at the end of a sequence run, do the
following:
• For the Surveyor UV/Vis Plus Detector and the Surveyor PDA
Plus Detector, clear the check boxes for the lamps.
• For the Surveyor FL Plus Detector, select the Lamp Off
check box.
Note For the Surveyor RI Plus Detector, you can only turn off the lamp
by turning off the power to the detector. The operating lifetime of the
lamp is 4.5 years.
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d. Verify that you have entered the appropriate parameter settings:
• Figure 87 shows the settings for the Surveyor UV/Vis Detector.
• Figure 88 shows the settings for the Surveyor PDA Plus
Detector.
Figure 87. Instrument setup page for the Surveyor UV/Vis Plus Detector, showing a shutdown method
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Figure 88. Instrument Setup window – Surveyor PDA Plus page, showing the
parameters for a shutdown method
4. To turn off the pump flow at the end of a sequence run:
a. Click the Surveyor LC Pump tab to open the Surveyor LC Pump
page.
b. Leave all parameters in the pump page the same as the settings for
the acquisition method, Test Mix.met, except those that are shown
in the following table and in Figure 89.
Parameter
Setting
Result
Initial Settings
Shutdown Method
Total Flow
Specifies that this method is
a shutdown method
0
Pump flow will be turned Off
0
Allows a minimum
backpressure of 0
Pressure Limits
Min Pressure
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Figure 89. Instrument Setup page for the Surveyor LC Pump, showing shutdown method
5. Save the method as Shutdown.met:
a. From the Instrument window menu bar, choose File > Method >
Save As to open the Save Method As dialog box.
b. Type Shutdown in the Filename box.
c. Click Save.
ChromQuest saves methods with the .met file extension.
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Chapter 5
Preparing Your Instrument
for a Run
In this tutorial, you learn how to open the online Instrument window,
monitor the status of your instrument, remove air from the solvent lines,
download a method, and check the stability of the baseline.
Before you can perform this tutorial, you must configure your Surveyor Plus
system. If your instrument has not been configured, see Chapter 3,
“Configuring Your Instrument.” Check the configuration for the
autosampler, and ensure that the Verify Door Is Closed option is not
selected.
To perform this tutorial, you must have the following items:
• HPLC-grade water
• HPLC-grade methanol (not required for the RI detector)
• Hypersil, 100 × 4.6 mm, C-18 column, 5 micron particle size or
equivalent
To prepare your instrument for a run, perform the procedures that apply to
your instrument in the order listed:
1. Setting Up the System
2. Turning On the Power
3. Opening the Online Instrument Window
4. Checking the Status of the Instrument Modules
5. Removing Air from the Solvent Lines
6. Purging the Flow Cell of the RI Detector
7. Downloading the Method
8. Checking the Stability of the Baseline
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Setting Up the System
Setting Up the System
The following instructions describe how to set up the Surveyor LC system
so that you can perform the remaining tutorials contained in this manual.
These tutorials guide you through the process of injecting a sample and
reporting the results of your run.
To set up the LC system
1. Fill the wash bottle with 90:10 methanol / water (v/v) or 100%
methanol or an appropriate solvent for your chromatographic method.
2. Fill the solvent reservoir bottle A with HPLC-grade methanol or an
appropriate solvent for your chromatographic method.
3. Fill the solvent reservoir bottle B with HPLC-grade water or an
appropriate solvent for your chromatographic method.
4. Install a standard sample tray into the tray compartment of the
autosampler.
The Surveyor Autosampler is shipped with four standard sample trays in
the tray compartment. The standard sample tray holds forty, 1.8 mL
standard vials.
5. Connect the LC Column (see Figure 90):
a. If it has not already been done, connect high-pressure tubing to
port 6 of the Valco injection valve. Use a high-pressure fitting to
connect the other end of the tubing to the inlet of the LC Column.
b. Use a high-pressure fitting to connect the outlet of the LC column
to the tubing that is attached to the flow cell of the detector.
Note If you are using a LightPipe flowcell, make sure that you
are also using the insulated, red, PEEK tubing that is supplied
with the LightPipe. This insulated tubing minimizes temperature
fluctuations.
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Setting Up the System
Red, Insulated, PEEK
Tubing to Flow Cell
Hypersil Column
Connection to Port 6
of the Injection Valve
Figure 90. Valco injection valve and column connections
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Turning On the Power
Turning On the Power
Before you launch ChromQuest from the Windows desktop, turn on the
power to each module of your instrument. The power switch for each
module is located below its left door (see Figure 91).
Shortly after you switch on the power, the Power LEDs and the Run LEDs
turn green and the autosampler syringe performs its initialization process of
homing the syringe. The homing process takes approximately 20 seconds.
You can hear the movement of the syringe drive as the plungers are homed.
If the temperature control feature for the RI detector is On, the Temp and
Run LEDs for the detector do not turn green until the operating
temperature of the detector stabilizes to the set temperature.
As you power up your instrument, you might encounter the following
problems:
• The Power LEDs remain amber. If this happens, make sure that the
power line to the affected module(s) is firmly connected.
• The Degas LED on the pump flashes amber. If this happens, the degas
unit has failed to produce a vacuum, and you need to call your local
Thermo Fisher Scientific representative for repairs.
• The Lamp LED remains amber. If this happens, most likely the lamps
are turned off. Continue on with this tutorial. You will turn on the
lamps of the Surveyor PDA Plus Detector in the topic, “Checking the
Status of the Instrument Modules” on page 124. You will turn on the
lamps of the Surveyor UV/Vis Plus Detector in the topic,
“Downloading the Method” on page 137.
If your instrument modules powered up correctly, go to the next topic,
“Opening the Online Instrument Window” on page 120.
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Power
Comm
Run
Lamps
Preparing Your Instrument for a Run
Turning On the Power
UV/Vis or PDA
Detector LEDs
Power
Switches
Power
Power
Comm
Comm
Run
Run
Temp
Degas
Autosampler LEDs
Pump LEDs
Power
Comm
Run
Temp
FL Detector LEDs
Figure 91. Surveyor stack, showing power switches and LEDs
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Opening the Online Instrument Window
Opening the Online
Instrument Window
There is an online Instrument window and an offline Instrument window
for each instrument in The Enterprise. From the online Instrument
window, you can perform both instrument control and data processing
operations. In this tutorial you learn how to use some of the instrument
control features of ChromQuest available in the online Instrument window.
Before you open the online Instrument window, the Comm LEDs on the
modules of your instrument are amber. After you open the online
Instrument window, the Comm LEDs change from amber to green. In
addition, the autosampler syringe goes through its initialization process.
Note Before you open the Online Instrument window, wait for the
Comm LEDs to illuminate green and for the syringe to complete its
initialization process.
After you turn on the power to the modules of your instrument, launch
ChromQuest and open the online Instrument window.
To open the online Instrument window from the Windows desktop
1. Launch the ChromQuest Chromatography Data System by choosing
Start > All Programs > Chromatography > ChromQuest or by
double-clicking the ChromQuest application icon.
The Main Menu window of ChromQuest appears. See Figure 92.
Instrument and System Administration are performed from this
window.
Unlocked
120
Locked
In the Main Menu window toolbar, shown in Figure 92, notice that the
System Administration Wizard button is grayed out (Locked). This
means that the Enable Login and Project Management feature is
enabled. To open an instrument or perform any administrative
activities, you must log in.
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Opening the Online Instrument Window
System Administration Wizard Button
Figure 92. Main Menu window
Also notice that the right panel of the window in Figure 92 contains one
instrument: Surveyor Plus System 1. The Main Menu window displayed
on your computer screen contains the Enterprise that you or your
System Administrator created. If the right panel of the Main Menu
window does not contain any instruments, see Chapter 3, “Configuring
Your Instrument,” which contains instructions on how to add an
instrument to The Enterprise. In addition, Chapter 3 contains
instructions on how to configure your instrument for the tutorials
contained in this manual.
2. Right-click the icon that represents your instrument, and then choose
Open from the shortcut menu.
3. Depending on which dialog box or window appears, do one of the
following:
• If the Login dialog box appears, go to step 4. See Figure 93.
• If the Instrument Wizard appears, go to step 5. See Figure 94.
• If the Instrument window is activated as indicated by its title bar,
you have successfully opened the Instrument window and are ready
to go to the next topic, “Checking the Status of the Instrument
Modules” on page 124.
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Opening the Online Instrument Window
Figure 93. Login dialog box
4. Log in:
a. Type your user name in the User Name box
b. Type your password in the Password box
c. Select a project from the Project list.
d. Click Login:
• If the Instrument Wizard appears, go to step 3. See Figure 94.
• If the Instrument window is activated as indicated by its title
bar, you have successfully opened the Instrument window and
are ready to go “Checking the Status of the Instrument
Modules” on page 124.
5. Clear the Show at Instrument Startup check box at the bottom of the
Instrument Wizard dialog box. You do not use the Instrument Wizard
in this tutorial.
6. Close the Instrument Wizard to activate the Instrument window.
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Opening the Online Instrument Window
Figure 94. Instrument window at startup
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Checking the Status of the Instrument Modules
Checking the Status
of the Instrument
Modules
After you open the Instrument window, check the status of the instrument
modules.
To check the status of the modules
1. From the Instrument window menu bar, choose Control > Instrument
Status.
The Instrument Status window opens. See Figure 95. This dialog box
contains one page for each configured module.
Figure 95. Instrument Status window – Surveyor PDA Plus page
2. Click each page in the Instrument Status window and check the status
readouts.
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Checking the Status of the Instrument Modules
If you have just turned on the power and have not yet downloaded a
method, you see the following status readouts:
Module
Status
UV/Vis detector
Ready or Lamps Off.
PDA detector
Ready or Not Ready Waiting for Download
Pump
Idle
Autosampler
Waiting for Download
RI detector
Ready or Wait Temp
FL detector
Ready or Device Lamp Off
3. If the lamps are Off, do one of the following depending on which
detector you are running:
• For the Surveyor PDA Plus Detector, click the D2 Lamp On button
and the W Lamp On button. See Figure 95.
• For the Surveyor UV/Vis Plus Detector, the lamps turn on when
you download a method.
The deuterium lamp takes approximately 10 seconds to ignite. If
you have a PDA detector, ensure that the lamps are turned on and
ready.
• For the Surveyor FL Plus Detector, the xenon lamp turns on when
you download a method.
• For the Surveyor RI Plus Detector, the tungsten lamp should always
be On when the detector is turned on.
Go to the next topic, Removing Air from the Solvent Lines.
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Removing Air from the Solvent Lines
Removing Air from the
Solvent Lines
After you change the solvents in the solvent reservoir bottles or the wash
bottle, your system will have air in the solvent lines. The solvent lines
connecting the solvent reservoir bottles to the built-in vacuum degasser of
the pump hold approximately 3 mL of solvent each [5-ft. l × 0.0625-in. ID
(152 cm l × 0.159 cm ID)]. The wash bottle solvent line that connects the
wash bottle to the syringe valve holds a similar volume of solvent.
Air in the solvent reservoir lines causes excessive pump pulsation, as well as
non-reproducible retention times. Air in the wash bottle tubing or the
syringe or both causes non-reproducible injection volumes.
Even if you have not just changed the solvents in the solvent reservoir
bottles or the wash bottle, check the solvent lines of your system for air
before you begin making injections. The solvent lines are permeable to air,
which means that inevitably, over time, air bubbles form in the lines.
To remove air from your solvent lines, purge the pump. To remove air from
the syringe, flush it.
Note ChromQuest contains a login security feature that allows the
system administrator to assign privileges to users and groups. If this
security feature is enabled, users can be denied administrative and/or
instrument privileges. To access the direct commands menu for the
autosampler, which contains the Flush Syringe command, you must have
instrument privileges.
This section contains the following procedures:
• Purging the Pump
• Flushing the Syringe
• Purging the Flow Cell of the RI Detector
Purging the Pump
The purge command draws eluent from a solvent reservoir bottle at a rate of
approximately 10 mL/min. As the solvent is drawn into the pump, it pushes
air out of the solvent lines and the pump head assemblies.
To purge the solvent lines
1. Open the drain valve by turning it counter-clockwise 180° to the purge
position.
The word DRAIN on the knob appears upside down as shown on the
left in Figure 96.
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Closed Position
PE
N
LO
PE
N
C
SE
DRAIN
O
DRAIN
O
SE
LO
C
Open Position
Preparing Your Instrument for a Run
Removing Air from the Solvent Lines
Figure 96. Drain valve positions
2. Open the Diagnostics dialog box – Operation page for the LC pump:
a. From the Instrument window menu bar, choose Control >
Instrument Status to open the Instrument Status window.
b. Click the Surveyor LC Pump tab to open the Surveyor LC Pump
page. See Figure 97.
Figure 97. Instrument Status window – Surveyor LC Pump page
c. Click Diagnostics.
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Removing Air from the Solvent Lines
The Diagnostics dialog box – Operation page appears (see
Figure 98).
When the drain valve is open, the mobile phase flows to waste rather
than to the column. Therefore, the backpressure of your system will
be close to zero or zero.
Figure 98. Diagnostics dialog box – Operation page
3. Purge solvent lines A and B:
a. Under Purge, type 3 in the Purge time box.
b. In the Solvent valve list, select A.
c. Click Start to start purging the solvent line.
Each solvent line holds approximately 3 mL of solvent. In the purge
mode, the pump draws solvent through the lines at a rate of
approximately 10 mL/min. If there is a significant amount of air in a
solvent line, you might need to purge the system for a longer period
of time. Purge a solvent line until you flush all the air out of it.
Note If you generate a pump error (see Figure 99), ensure that
you opened the purge valve as directed in step 1 of this
procedure. To recover from the error, open the drain valve, close
the Diagnostics dialog box, and then click Reset Error on
Instrument Status window – Surveyor LC Pump page. See
Figure 100.
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Removing Air from the Solvent Lines
Figure 99. Error message dialog box
Figure 100. Error message read-only box
d. To purge solvent line B, repeat steps 3a to 3c, selecting B from the
solvent valve list.
Note If the replacement solvent is not miscible with the current
solvent in the line, flush the line with an intermediate solvent
that is miscible with both the replacement solvent and the
current solvent before flushing the line with the replacement
solvent. If the current solvent is a buffered solution, purge the
flow line with distilled water before you replace the solvent.
4. After you have finished purging the solvent lines of air, close the drain
valve by gently turning it clockwise as far as it will go.
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Note Ensure that the drain valve is closed. If you do not close the
drain valve, the mobile phase will continue to flow out through the
waste line rather than to the autosampler.
5. Click Close to exit the Diagnostics dialog box.
Flushing the Syringe
If the syringe or the wash bottle line contains a large quantity of air, the
autosampler will not be able to withdraw sample from the sample vial.
Therefore, before you attempt to make an injection, check the wash bottle
line and the syringe for air. If you see air in the wash bottle line or the
syringe, perform a Flush Syringe direct command to remove the air.
Note To flush the syringe, you must have Instrument privileges.
To flush the syringe
1. Ensure that the wash bottle contains solvent.
The wash bottle is contained in the solvent platform, located at the top
of the Surveyor Stack. The wash bottle tubing is connected to the left
side of the syringe valve.
2. Open the Direct Controls page of the Diagnostics dialog box:
a. From the Instrument window menu bar, choose Control >
Instrument Status.
b. Click the Surveyor AS tab to open the Surveyor AS Instrument
Status page (see Figure 101).
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Figure 101. Instrument Status – Surveyor AS page
c. Click Diagnostics.
The Diagnostics dialog box appears with the Direct Controls page
displayed (see Figure 102).
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Figure 102. Direct Controls page, showing the selection of the Flush
Syringe direct command
3. Perform a Flush Syringe command:
a. In the Direct Commands list, select the Flush Syringe command.
After you select the Flush Syringe command, the Parameters area
below the list box appears.
b. In the Parameters area, select Bottle from the Reservoir list, and
then type a value in the range from 100 to 6000 in the Volume box.
The wash line holds approximately 3 mL of solvent. To replace the
solvent in the line with new solvent, type a minimum value
of 3000 μL.
Note The default flush speed of 250 μL/s is only suitable for low
viscosity solvents, such as 100% methanol. If you are using water
or a water / methanol mixture for the flush solvent, lower the
flush speed to 100 μL/s or less. If the flush speed is set too high,
the syringe makes a grinding noise during the flush cycle and can
stall.
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Removing Air from the Solvent Lines
Note If the Verify Door is Closed feature is enabled, the
Surveyor AS will not execute this command while the tray
compartment door is open. To execute the command, close the
tray compartment door, and then resubmit the command.
c. Click Submit to execute the command.
d. Click Done to exit Diagnostics.
During a flush, the syringe needle is moved to the injection port of
the autosampler to dispense a specified volume of solvent from the
wash bottle or a reservoir vial. The injection valve is switched to the
Inject position to prevent solvent from entering the sample loop.
The flush solvent removes residual sample from the needle tubing
assembly and the transfer tubing.
The Flush With Injector in Fill Position direct command leaves
the sample loop in the path of the wash solvent as you perform a
flush.
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Purging the Flow Cell of the RI Detector
Purging the Flow Cell
of the RI Detector
Use the purge control feature to remove air bubbles from the flow cell and to
fill the reference compartment of the flow cell with the pre-mixed mobile
phase specified in the method.
To purge the flow cell of the RI detector
1. Start the pump flow:
a. Choose Method > Instrument Setup.
b. Click the Surveyor LC Pump tab.
c. Under Initial Settings, enter the appropriate flow rate and select the
solvent bottle that contains your pre-mixed mobile phase.
Your set up might differ from the one shown in Figure 103.
d. Choose Control > Download Tab to download these parameters to
the LC pump.
The pump flow starts. You can monitor a rise in the backpressure of
the system by viewing the pressure readout in the Status area.
Pressure Readout
Figure 103. Instrument Setup window – Surveyor LC Pump page, showing entries for pumping water
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Purging the Flow Cell of the RI Detector
2. Begin flushing the flow cell:
a. Choosing Control > Instrument Status.
b. Click the Surveyor RI Plus tab
c. Click the RI Diagnostic tab.
d. Under Purge Control, click On. See Figure 104.
Figure 104. RI Diagnostic page, showing the purge control buttons
3. Pump the pre-mixed mobile phase at the set flow rate through the
flowcell for 10 minutes.
4. To remove air bubbles, turn the purge control on for 10 seconds and
then off for 10 seconds a few times.
Purging is complete when the purge valve can be switched from On to
Off with minimal baseline disturbance.
5. To view the baseline stability, click the RI Status tab to open the status
page for the RI detector, and then monitor the rate of change of the
most recent data. See Figure 105.
Or, preview the baseline by choosing Control > Preview Run.
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Purging the Flow Cell of the RI Detector
Intensity
Readback
Figure 105. RI Status page, showing the Most Recent Data readback
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Downloading the
Method
Preparing Your Instrument for a Run
Downloading the Method
You added your instrument control parameters to the method when you
performed the procedure “Creating an Acquisition Method” on page 91.
For the new settings to take effect, you need to download the method to the
instrument. Starting a single run automatically downloads a method.
However, in general, you will probably want to download your method
before your start a run so that you can equilibrate the LC column.
Note It requires approximately 15 to 20 times the unretained peak time
of an LC column to equilibrate it. For reversed-phase chromatography,
you can determine the unretained peak time of an LC column by
injecting a non-retained analyte such as uracil or sodium nitrate, and
then monitoring the absorbance at 254 nm or 210 nm for uracil or
sodium nitrate, respectively.
After you download the method, the LC pump starts pumping mobile phase
through the LC column. At a flow rate of 1 mL/min, it takes approximately
15 to 20 minutes to equilibrate the 10 cm, 5 micron particle size, Hypersil,
C-18 column.
To download your method
1. If the name of your method is not listed in the title bar of the
Instrument window, open your method:
a. From the Instrument window menu bar, choose File > Method >
Open.
The Open Method dialog box appears (see Figure 106).
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Figure 106. Open Method File dialog box
b. Browse to the appropriate directory:
Drive:\ChromQuest\Projects\Tutorial\Methods.
c. Select your method and click Open.
d. Verify that your method is listed in the title bar of the Instrument
window.
2. Choose Control > Download Method from the Instrument window
menu bar to download your method.
The Analysis Channel list displays the analysis wavelengths contained in
the method. The LC pump begins pumping the specified conditions.
If your system contains a UV/Vis detector, the deuterium lamp ignites,
and the Lamps LED turns green within 10 seconds.
If your system contains an RI detector and the method specifies an
operating temperature, the RI detector equilibrates to the set
temperature. Before beginning an analysis that requires maximum
sensitivity, allow the RI detector to equilibrate at the set temperature for
24 hours.
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Downloading the Method
If you did not close the purge valve after you purged the solvent lines, a
below minimum pressure pump error appears when the pump attempts
to stabilize the solvent flow.
3. If the below minimum pump error message appears, do the following:
a. Close the purge valve of the pump.
b. Open the Instrument Status page for the Surveyor LC Pump.
c. Click Reset Error.
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Checking the Stability of the Baseline
Checking the Stability
of the Baseline
There are two features in ChromQuest that allow you to check the stability
of the baseline: Preview Run and Baseline Check. The Preview Run feature
allows you to visually check the stability of the baseline. In addition to a
visual check, the Baseline Check feature determines the noise and drift of
the baseline and allows you to delay a run until these parameters meet the
specified test criteria. To familiarize yourself with these features perform the
following procedures:
• Previewing the Baseline
• Performing a Manual Baseline Check
Previewing the Baseline
You can visually inspect the stability of the baseline by previewing the run.
To preview the baseline for both wavelengths
1. From the Command toolbar, click the Preview Run button.
After you initiate a preview run, the following messages appear
sequentially in the status bar at the bottom of the Instrument window:
Loading Method
Creating Data File
Downloading Method
Equilibrating Method
Running Sample <None>
The data file name for the preview run, Instrument # Preview.dat, is
listed in the Title bar of the Instrument window.
2. Choose View > Tile Data to tile the two chromatograms on your view
screen.
3. Choose Windows > Tile Horizontally to tile the chromatograms
horizontally on the view screen.
If you created the method, Test Mix.met, while performing the
procedure “Creating an Acquisition Method” on page 91, the two
chromatograms are for wavelengths 230 nm and 260 nm. See
Figure 107.
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4. Click the Stop Run button to stop the preview run, and then wait for
the run to end.
Figure 107. Preview run screen for Test Mix method
Performing a Manual
Baseline Check
Before you make your first injection, perform a manual baseline check to
confirm that the baseline is stable and to test the baseline for excessive drift.
To perform a manual baseline check
1. From the Instrument window, choose Control > Baseline Check to
open the Baseline check dialog box.
If the Baseline Check option is not available, the Baseline Check check
box was cleared when your instrument was configured. Skip this
procedure.
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Checking the Stability of the Baseline
2. Keep all parameters in the Baseline Check dialog box set to the default
settings except those that are shown in the following table and in
Figure 108.
Parameter
Setting
Result
To pass, the baseline must meet the
test criteria for
1
Specifies that the baseline must pass the test criteria for a time interval of 1 min
Stop checking baseline if conditions
are not met after
4
Specifies that if the baseline has not passed the test criteria for the length of
time specified (1 min), then the program will discontinue the test after a time
interval of 4 min and report a failing result
General
Test Criteria
PDA-230 nm Enable Noise Test
Threshold (Noise)
Specifies that a noise test will be performed for the 230 nm wavelength channel
50
PDA-230 nm Enable Drift Test
Threshold Drift
Specifies that a drift test will be performed for the 230 nm wavelength channel
5000
PDA-260 nm Enable Noise Test
Threshold (Noise)
The drift must be < 5 mAU/hr (< 83 μAU/min) to pass.
Specifies that a noise test will be performed for the 260 nm wavelength channel
50
PDA-260 nm Enable Drift Test
Threshold Drift
The rms noise must be < 50 μAU.
The rms noise must be < 50 μAU.
Specifies that a drift test will be performed for the 260 nm wavelength channel.
5000
The drift must be < 5 mAU/hr (< 83 μAU/min) to pass.
Wavelengths
Specified in the
Method
Figure 108. Baseline Check dialog box
3. Click Submit.
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The Baseline Check window appears.
4. If the Baseline Check window does not appear, from the Instrument
window menu bar, choose View > Baseline Check.
5. To print a Baseline Check report (see Figure 109) after the baseline
check is complete, choose Reports > View > Current Baseline Check.
Figure 109. Baseline Check Results
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Chapter 6
Making Your First Injection
In this tutorial, you learn how to load a sample into the tray compartment
of the autosampler; start, extend, and stop a single run; view the real-time
progress of a run; manually check the purity of your chromatographic peak;
and add annotations to your chromatograms.
Before you perform this tutorial, confirm that you have done the following:
• Configured your Surveyor Plus HPLC instrument
• Filled the solvent bottles for the mobile phase and the wash solvent
• Removed the air from the solvent lines
• Downloaded an acquisition method, warmed up the lamps of the
detector, and equilibrated the LC column
• Checked the stability of the baseline
If your instrument has not been configured, see Chapter 3, “Configuring
Your Instrument.” If you have not created an acquisition method, see
Chapter 4, “Creating Methods.” If your instrument is not ready to perform
a run, see Chapter 5, “Preparing Your Instrument for a Run.”
This chapter contains the following sections:
• Loading the Sample into the Tray Compartment
• Starting, Extending, and Stopping a Single Run
• Viewing the Progress of Your Run
• Checking the Purity of the Toluene Peak
• Adding Annotations to Your Chromatograms
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Making Your First Injection
Loading the Sample into the Tray Compartment
Loading the Sample
into the Tray
Compartment
A set of ampules labeled Autosampler Test mix is supplied in the accessory
kit for the autosampler. This test mix contains a solution of 0.5% toluene in
methanol. If your system contains a UV/Vis or PDA detector, use this test
mix to make your first injection. If you do not have an ampule of this test
mix solution or your system contains an RI or FL detector, inject an analyte*
for which you know the chromatographic conditions, and adjust the
instrument setup section of the method accordingly.
Note *Ensure that your sample(s) are completely soluble in the mobile
phase and that you have filtered your sample(s) and solvents through a
0.5 micron filter. These techniques minimize sample precipitation in the
lines and remove any particulate matter that could obstruct the flow
through the injection port of the autosampler, the transfer tube
connecting the injection port to the injection valve, and the injection
valve itself.
To load a sample vial into the tray compartment of the autosampler
1. Fill a standard 1.8 mL autosampler vial with the sample that you want
to inject.
2. If you are currently working from the Offline Instrument window, open
the Online Instrument window for your instrument:
a. In the Instrument window toolbar, click the Main Menu button.
The Main Menu window appears.
b. Double-click the icon for your instrument to open the Online
Instrument window.
c. If you have enabled the instrument login and project management
feature, (see Chapter 2, “Administrating the Enterprise.” ) log in to
the Tutorial project.
3. Open the left door of the autosampler.
If you performed the tutorial contained in Chapter 3, “Configuring
Your Instrument,” which instructed you to clear the Verify door is closed
option for the autosampler, the XYZ arm of the autosampler will not
move to the back of the tray compartment when you open the door.
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Loading the Sample into the Tray Compartment
Note ChromQuest contains a login security feature that allows the
system administrator to assign privileges to users and groups. If this
security feature is enabled, users can be denied administrative and/or
instrument privileges. To access the direct commands menu for the
autosampler, which contains the Position Arm to Remove Tray
command, you must have instrument privileges.
4. Perform the Position Arm to Access Tray direct command to move the
XYZ arm to the back of the tray compartment:
a. From the Online Instrument window, choose Control >
Instrument Status.
b. Click the Surveyor AS tab to open the Instrument Status window Surveyor AS page.
c. Click Diagnostics to open the Diagnostics dialog box for the
autosampler.
d. From the Direct Commands list, select Position Arm to
Access Tray.
e. Click Submit.
The XYZ arm moves to the back of the tray compartment.
5. Place the vial in position A1 of a standard tray. Then, load the tray into
the autosampler tray compartment. If the tray is already loaded in the
tray compartment, place the vial in position A1. See Figure 110.
Note If the autosampler configuration option, Verify Door is
Closed, is enabled the autosampler arm will not move to the
requested vial position until the tray compartment door is closed.
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Loading the Sample into the Tray Compartment
Slot
for Tray
Tray
Runner
Vial
Position
A:20
Vial
Position
E:40
RV4
RV3
RV2
RV1
Standard
1.8 mL vial
Vial
Position
E:21
Vial
Position
A:1
A
B
C
D
E
Tray
Handle
Figure 110. Tray compartment, showing five standard trays installed
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Starting, Extending, and Stopping a Single Run
Starting, Extending,
and Stopping a Single
Run
Before you can start a single run, you must create an acquisition method
containing the appropriate instrument control parameters. If you performed
the tutorial in Chapter 4, “Creating Methods,” you created the method,
Test Mix.met.
To start a single run
1. Ensure that the method, Test Mix.met, is listed in the title bar of the
Instrument window. If the appropriate method is not listed, open it:
a. From the menu bar, choose File > Method > Open.
b. Browse to the appropriate directory:
Drive:\ChromQuest\Projects\Tutorial\Method.
c. Select the method, Test Mix.met.
d. Click Open.
2. In the Instrument window toolbar, click the Single Run button to open
the Single Run Acquisition dialog box.
3. Keep all parameters in the Single Run Acquisition dialog box set to the
default settings except those that are shown in the following table and in
Figure 111. The active method will be listed in the Method box.
4. To create the data file name listed in the table below, type Preliminary
Run in the Data File box. Then click the blue arrow to the right of the
Data file box to open a shortcut menu and choose Increment Number.
Parameter
Setting
Result
Sample ID
0.5% Toluene in Methanol
Gives the data file a sample ID, which can be used in the
search feature within ChromQuest
Data Path
Drive:\ChromQuest\Projects\Tutorial
Stores the data file in this folder
Data File
Preliminary Run <001>
Names the data file as Preliminary Run 001.dat.*
Vial
A;1
Specifies that the autosampler withdraws sample from
vial location A1. You must type a semicolon between the
tay (A) and the tray location (1).
Injection Volume
If your detector contains a 5 cm LightPipe flowcell, type 1.
Specifies that the autosampler will inject 1 μL of sample
If your detector contains a standard 1 cm flowcell, type 5.
Specifies that the autosampler will inject 5 μL of sample
Run Information
Autosampler
* ChromQuest automatically adds the .dat file extension to your data files.
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Note The LightPipe flowcell has five times the sensitivity of a standard
1 cm flowcell. Therefore, if your detector contains a 5 cm LightPipe
flowcell, you can inject one-fifth the sample volume to achieve the
same absorbance level as you would achieve using a standard 1 cm
flowcell.
You must type a semicolon between the tray
(A) and the tray location (1).
Figure 111. Single Run Acquisition dialog box, containing your new entries
Note Ensure that the check box to the left of the Use Program box is
not selected. If you leave this check box selected, the autosampler
attempts to run a pretreatment method before it injects the sample.
5. Click Start.
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Starting, Extending, and Stopping a Single Run
The following steps are included in this procedure to introduce you to the
Extend Run and Stop Run features in ChromQuest.
6. After 3 minutes into the run, choose Control > Extend Run.
The Extend Run dialog box appears, with a default time of 5 minutes in
the Extend run by box.
Figure 112. Extend Run dialog box.
7. Click OK to extend the run by five minutes.
8. Before the additional time has elapsed, click the Stop Run button in the
Command toolbar to stop the run.
The Stop Run dialog box appears (see Figure 113).
Figure 113. Stop Run dialog box
9. Click OK to stop the run.
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Starting, Extending, and Stopping a Single Run
By default, the chromatograms are analyzed after the data file is
acquired. After you stop a run, ChromQuest stores the data file without
analyzing it. The frequency at which ChromQuest analyzes data can be
changed in the Method Properties dialog box – Options page.
10. Because you stopped the run, you must click the Analyze button in the
Command toolbar to integrate the chromatograms. The sequence of
events for a partial loop injection is listed in Table 9.
Table 9.
Sequence of events for a partial loop injection
Step
Events
Draw Transport
Solvent
The autosampler switches the two-way syringe valve to the wash line position.
Draw
Sample
The XYZ arm moves to the requested sample vial.
The inner plunger of the concentric syringe moves downward, drawing solvent from the wash bottle. The amount of
solvent drawn is equal to the dead volume of the autosampler plus 7.5 μL.
The autosampler switches the two-way syringe valve to the needle tube position.
The inner plunger of the concentric syringe moves downward, drawing 3 μL of air into the needle.
The XYZ arm lowers the needle into the sample vial.
The inner plunger of the concentric syringe moves further downward, drawing the requested sample volume of 1 μL plus
an additional 22 μL into the needle tubing.
The XYZ arm raises the needle out of the sample vial.
The inner plunger of the concentric syringe moves further downward, drawing an additional 3 μL of air into the needle,
effectively isolating the sample with air.
Push Transport
Solvent
The XYZ arm moves to the injection port, and then lowers the needle into the injection port.
Load
Sample
Into Loop
The autosampler switches the 6-port injection valve to the fill position, and then meters 1 μL of sample (requested
injection volume) into the front of the sample loop (through port 1).
Inject
Sample
The autosampler switches the injection valve to the inject position, allowing the mobile phase stream to backflush the
sample onto the column (mobile phase enters the sample loop through port 4).
The Inner plunger of the concentric syringe moves upward, pushing the sample out of the needle and into the transfer
tubing that connects the injection port of the autosampler to the injection port (port 2) of the injection valve. The inner
plunger of the concentric syringe continues to move upward until approximately half of the excess sample is pushed out
to waste through port 3 of the 6-port injection valve.
If you listen carefully, you can hear the valve switch to the fill position.
The inner plunger of the syringe continues to move upward, expelling excess sample to waste.
Again, if you listen, you can hear the valve switch. The injection valve remains in the inject position for the remainder of
the run, allowing the mobile phase to thoroughly flush the sample loop.
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Viewing the Progress of Your Run
Viewing the Progress
of Your Run
After you begin your first injection, you can view the real-time progress of
the run on your view screen. The viewing options that are available to you
depend on the type of data you are collecting. If you using the UV/Vis
detector to collect dual-wavelength data files, you are able to simultaneously
view the collection of both chromatograms. If you are collecting discrete
channels and scan data using the PDA detector, you are able to view the
discrete channel chromatograms as well as the spectral data.
This section contains the following topics:
• Viewing Your Chromatograms
• Viewing Your Spectral Data
Viewing Your
Chromatograms
The chromatograms can be viewed in separate windows or overlaid in one
window. If you are viewing the chromatograms in separate windows, the
windows can be tiled horizontally or vertically on the screen.
• To overlay the chromatograms, choose View > Overlay.
• To tile the Chromatogram windows, choose View > Tile Data.
• To tile the Chromatogram windows horizontally, choose Window >
Tile Horizontally. See Figure 114.
Figure 114. Horizontally tiled chromatograms
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Viewing the Progress of Your Run
• To tile the Chromatogram windows vertically, choose Window > Tile
Vertically. See Figure 115.
Figure 115. Vertically tiled chromatograms
Viewing Your Spectral
Data
154
There is a separate submenu for viewing scan data. If you are using the PDA
detector to collect a scan data file, choose View > Spectral View > Mixed
View to open the view shown in Figure 116. This view contains four tiled
window panes. The upper left quadrant contains the Contour Plot pane.
The lower left quadrant contains the Chromatogram pane. The upper right
quadrant contains the Spectrum pane. The lower right quadrant contains
the Peak Purity pane. Notice the two handles on the contour plot. The
handle pointing to the 220 nm wavelength is the Y-axis handle. The handle
pointing to the 0 min time point is the X-axis handle.
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Viewing the Progress of Your Run
Y-Axis Handle
X-Axis Handle
Figure 116. Instrument window, showing Mixed View
To view the chromatogram for a specific wavelength
1. Click the Y-axis handle on the contour plot.
The arrow at the left end of the handle points to the wavelengths on the
Y-axis of the contour plot.
2. Drag the handle down from the 220 nm wavelength to the 300 nm
wavelength. As you drag the handle down the Y-axis, notice how the
Chromatogram window changes to display the chromatogram for the
selected scan wavelength.
See Figure 117, which shows the Y-axis handle pointing to the 260 nm
scan wavelength.
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Viewing the Progress of Your Run
Y-Axis Handle
Dragged to 260 nm
Figure 117. Instrument window, showing the Y-axis handle pointing to the
260 nm scan wavelength
To view the spectrum for a specific time point
1. Click the X-axis handle on the contour plot.
The arrow at the bottom of the handle points to the time on the X-axis
of the contour plot.
2. Drag the handle across the peak for toluene and notice how the display
in the Spectrum window changes.
See Figure 118, which shows the X-axis handle pointing to the 2.20 min
time point.
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Viewing the Progress of Your Run
X-Axis Handle
Dragged to the 2.2 min Time Point
Figure 118. Instrument window, showing the X-axis handle pointing to the 2.20 min time point
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Checking the Purity of the Toluene Peak
Checking the Purity of
the Toluene Peak
If you have collected scan data with the PDA detector, you can determine
the purity of the chromatographic peaks.
ChromQuest determines the purity of a peak by calculating the similarity of
the spectra taken across the entire peak or by calculating the similarity of the
spectra from the beginning, peak apex, and end of the peak. In both cases,
peak purity is a measure of the homogeneity of the spectral slices that are
being compared. If two or more compounds perfectly co-elute,
ChromQuest reports a peak purity value of 1.0 (or close to 1.0) even though
the peak is impure.
After you acquire a data file, perform the following procedures to check the
purity of your toluene peak:
• Enabling Spectrum Calculations
• Performing a Manual Peak Purity Check
Enabling Spectrum
Calculations
To check the purity of your peaks, your must enable spectrum calculations
in the method.
To enable spectrum calculations
1. From the Instrument window, choose Method > Spectral Options to
open the Spectral Options window.
2. Click the Purity tab to open the Purity page.
3. Keep all parameters in the Purity page set to the default settings except
those that are shown in the following table and in Figure 119.
Parameter
Setting
Result
220 - 300
Specifies that the spectra are
taken from the wavelength
range of 220 to 300 nm
Purity Calculations
Wavelength Range
Per Peak Spectrum Calculations
158
Per Peak Spectrum
Calculations
Total Purity
Enables the total purity
calculation
Per Peak Spectrum
Calculations
3-Point Purity
Enables the 3-point purity
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Checking the Purity of the Toluene Peak
Figure 119. Spectral Options window – Purity page
4. Click Apply.
5. Close the Spectral Options window.
6. Save the method by choosing File > Method > Save.
Performing a Manual Peak
Purity Check
The purity of the chromatographic peaks can be checked manually or
automatically. For instructions on performing automated peak purity
checks, see the online Help in ChromQuest.
To manually check the purity of the toluene peak
1. Drag the Y-axis handle in the contour plot to the 260 nm wavelength.
The unintegrated chromatogram for the 260 nm scan wavelength
appears in the chromatogram window (Figure 117 on page 156). An
unintegrated chromatogram can be distinguished by its lack of a
baseline.
2. In the Instrument window toolbar, click the Analyze button to integrate
the chromatogram.
After you click the Analyze button, a chromatographic baseline appears.
3. In the Chromatogram window of the Mixed View, hold down the
CTRL key, and then click the toluene peak.
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Checking the Purity of the Toluene Peak
A peak purity plot appears in the Peak Purity pane of the Mixed View
window, as shown in Figure 120.
Analyze Button
Baseline
Figure 120. Instrument window, showing the purity plot for the toluene peak in the bottom-right quadrant
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Adding Annotations to Your Chromatograms
Adding Annotations to
Your Chromatograms
Now that you have collected your first data file, you are ready to add
annotations to the chromatograms. In this tutorial, you add the following
annotations: peak number, retention time, area, and capacity factor.
Capacity factor is a performance parameter that uses the column
information entered in the Advanced Method Options section of the
method. If your method does not contain column information,
ChromQuest reports a value of zero for any performance parameter
annotation. For instructions on entering the information for your column
to the method, see “Entering the Column Parameters” on page 106.
To add annotations to your chromatograms
1. Choose Window > Tile Vertically to tile the discrete channel
chromatograms on the view screen.
2. Right-click a chromatogram (alternatively referred to as a trace in
ChromQuest) and choose Annotations to display the Trace Annotation
Properties dialog box. See Figure 121.
3. In the Available Annotations list, double-click the following
annotations:
• Pk#
• Retention Time
• Area
• Capacity Factor
4. In the Show the Following Annotations list, select Retention Time.
The Decimals box is enabled and contains the default value of 3, as
shown in Figure 121.
5. In the Decimals box, change the number of decimals to 2:
a. Highlight the number 3.
b. Type 2 in the Decimals box.
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Figure 121. Trace Annotations Properties dialog box
6. Click Apply To All to apply these annotations to both the 230 nm and
the 260 nm discrete channel chromatograms.
7. Click OK to exit the dialog box.
Both of your chromatograms now contain annotations for Pk#,
retention time, area, and capacity factor. By default, the orientation of
the annotations is at a 90° angle to the baseline. See Figure 122.
Note If you attempted to add the Name annotation at this point,
you will notice that the peaks are not named. To identify the peaks
with names, the peaks must be identified in a peak table. You will
add a peak table to your method in Chapter 8, “Specifying the
Calibration Curve Parameters.”
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Adding Annotations to Your Chromatograms
Figure 122. Vertically tiled chromatograms, showing annotations
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Chapter 7
Adding Integration Events
Graphically
In this tutorial, you learn how to graphically add integration events to a
method. In addition, you learn how to graphically add manual integration
fixes to individual data files.
A method in ChromQuest contains an Integration Events table for
each analysis channel.
Depending on the detector you are using, your method should include the
following analysis channels:
• For the Surveyor UV/Vis Plus Detector, which is a dual-wavelength
detector, the analysis channels include the wavelengths you entered in
the Surveyor UV/Vis page of the Method – Instrument Setup window.
• For the Surveyor PDA Plus Detector, which is a photodiode detector
capable of scanning the UV/Vis range from 190 to 800 nm, the analysis
channels include the wavelengths you entered in the Surveyor PDA Plus
page of the Method – Instrument Setup window and the wavelengths
you entered in the Multi-Chromatogram page of the Method – Spectral
Options dialog box.
• For the Surveyor FL Plus Detector, which is a programmable,
single-wavelength detector, the method does not contain analysis
channels.
• For the Surveyor RI Plus Detector, which is a bulk properties detector,
the method does not contain analysis channels.
In addition to an Integration Events table associated with the method, the
integration of a chromatogram can be affected by a Manual Integration
Fixes table that is associated with the data file.
This chapter contains the following sections:
• Opening a Stored Data File
• Adding Integration Events to the Method
• Adding a Manual Integration Fix to a Data File
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Opening a Stored Data File
Opening a Stored
Data File
In Chapter 6, “Making Your First Injection.” , you acquired a data file
named Preliminary Run.dat. If you did not perform the tutorial in
Chapter 6, you can learn how to add integration events to a method by
using one of the stored data files supplied with ChromQuest.
To open a stored data file
1. Choose File > Data > Open from the menu bar.
The Open Data File dialog box appears.
2. Click the down arrow next to the Look In box and browse to the
appropriate directory:
• If you performed the tutorial in Chapter 6, “Making Your First
Injection,” you will find the data file Preliminary Run.dat in the
directory: Drive:\ChromQuest\Projects\Tutorial\Data.
• If you did not perform the tutorial in Chapter 6, “Making Your First
Injection,” use the stored data file Multi Calibration Level 6.dat
that is supplied with ChromQuest. You will find these data files in
the directory: Drive:\ChromQuest\Data.
3. From the list of data files, click a data file to select it.
Note If you do not see your file listed, try changing the Files of Type
selection to All Files (*.*) so that all the files in the Data directory are
shown including those without file extensions.
The [.dat] file extension is not automatically added to a data file
when it is acquired using the single run acquisition dialog box. The
[.dat] file extension must be added to the data file name as described
on page.
4. Under Options, select Original / Acquisition from the Method list.
5. Click the Preview button to see a preview of the chromatogram(s) in the
data file. See Figure 123.
6. Click the Description button to view a description of the data file. See
Figure 124.
7. Click Open to open the selected data file.
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Opening a Stored Data File
Figure 123. Open Data File dialog box, showing a preview of data file
Preliminary Run 001.dat
Figure 124. Open Data File dialog box, showing a description of data file
Preliminary Run.dat
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Adding Integration Events to the Method
Adding Integration
Events to the Method
By default, the Integration Events table for each wavelength channel
contains a Width integration event, set to a value of 0.2 minutes, and a
Threshold integration event, set to a value of 50. See Figure 125.
Figure 125. Default Integration Events table for the 230 nm wavelength channel
You can optimize the integration of your chromatograms by adding
integration events to their respective Integration Events tables or by
changing the default values for the Width and Threshold integration events.
In this tutorial, you will be adding a Threshold integration event to the
Integration Events tables for the 230 nm and the 260 nm wavelength
channels of your method Test Mix.met.
Note You can use one of the stored data files provided with
ChromQuest to perform this tutorial. These data files are located in the
Drive:ChromQuest\Data folder. The data files named Multi Level
Calibration 1.dat to Multi Level Calibration 6.dat contain a single
chromatogram. The data files named LabTest2001.dat to
LabTest2007.dat contain a chromatogram for one discrete wavelength,
as well as scan data from 220 nm to 360 nm.
This section contains the following topics:
• Adding Integration Events to the 230 nm Chromatogram
• Adding Integration Events to the 260 nm Chromatogram
Adding Integration Events
to the 230 nm
Chromatogram
To add an integration event to the 230 nm chromatogram
1. If it is not already open, open the Preliminary Run 001.dat data file as
described in the previous section: Opening a Stored Data File.
2. If your system has a PDA detector and you collected a scan along with
the two discrete wavelength channels, close the Mixed View window.
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3. Display the chromatogram for 230 nm. See Figure 126.
Figure 126. Chromatogram for the 230 nm wavelength channel
4. Add a Threshold event to the Integration Event table for the 230 nm
wavelength channel:
a. From the Select Channel list in the Instrument window toolbar,
select the 230 nm wavelength. See Figure 127.
Select Channel List
Figure 127. Instrument window toolbar, showing the 230 nm channel selected
b. Click the chromatogram to activate the integration toolbar at the
bottom of the Instrument window. See Figure 128.
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Suggest Sampling Frequency
Define Groups
Define Peaks
Adjust Group Range
Define Single Peak
Adjust Retention Time Window
Reset Baseline At Valley
Reset Baseline
Move Baseline Stop
Move Baseline Start
Force Peak Stop
Force Peak Start
Split Peak
Manual PeaK
Manual Baseline
Reassign Peak
Disable Peak End
Turn On Negative Peak
Minimum Area
Front Tangent Skimming
Tangent Skinning
Lowest Point Horizontal Baseline
Backward Horizontal Baseline
Horizontal Baseline
Valley To Valley
Integration Off
Shoulder Sensitivity
Threshold
Width
Adding Integration Events Graphically
Adding Integration Events to the Method
Figure 128. Integration Events toolbar
c. In the Integration Events toolbar, click the Threshold integration
event button.
The message in the Status bar, which is located at the bottom of the
Instrument window just below the Integration Events toolbar,
instructs you to “Select Start of Baseline Segment”.
d. Click the baseline at approximately the 0.0 minute time point.
The Status bar message instructs you to “Select End of Baseline
Segment.”
e. Click the baseline just in front of the toluene peak, but not
including the toluene peak, at approximately the 1.9 minute time
point.
The Threshold dialog box appears. See Figure 129. By default, the
Insert Into Integration Events Table option is selected for the
Threshold integration event.
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Adding Integration Events to the Method
Figure 129. threshold dialog box
f. In the Threshold dialog box, click Analyze Now (see Figure 129).
This adds the integration event to the integration events table,
analyzes the chromatogram, and redraws the baseline. See
Figure 130.
Figure 130. Chromatogram for 230 nm
5. In the Command toolbar, click the Integration Events button to review
the Integration Events table for the 230 nm wavelength channel. See
Figure 131.
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Adding Integration Events to the Method
Figure 131. Integration Events table for the 230 nm wavelength channel
Adding Integration Events
to the 260 nm
Chromatogram
To add an integration event to the chromatogram for 260 nm
1. Display the chromatogram for 260 nm. See Figure 132.
Figure 132. Chromatogram for 260 nm with default integration
2. Add a Threshold event to the Integration Event table for the 260 nm
discrete channel:
a. From the Channel Selection list, select the 260 nm wavelength. See
Figure 133.
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Select Channel List
Figure 133. Instrument window toolbar, showing the 260 nm channel selected
b. Click the chromatogram to activate the integration toolbar at the
bottom of the Instrument window.
c. In the Integration Events toolbar, click the Threshold integration
event button.
d. Click the chromatogram at approximately the 0.0 min time point.
e. Click the chromatogram at approximately the 1.9 min time point.
The Threshold dialog box appears. See Figure 134.
Figure 134. Threshold dialog box
f. In the Threshold dialog box, click Analyze Now to add the event to
the integration events table and analyze the chromatogram using the
event. Figure 135 shows the results of the analysis.
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Adding Integration Events to the Method
Figure 135. Chromatogram window for the 230 nm wavelength channel
3. In the Command toolbar, click the Integration Events button to review
the Integration Events table for the 230 nm wavelength channel. See
Figure 136.
Figure 136. Integration events table for the 230 nm wavelength channel
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Adding a Manual Integration Fix to a Data File
Adding a Manual
Integration Fix to a
Data File
Occasionally one or more of the data files acquired with a method will
contain chromatographic anomalies that you might want to correct. In this
case, you can modify the integration of the affected chromatogram, not
the method.
By default, a Manual Integration Fixes table for a specific wavelength in a
data file does not contain any integration events. See Figure 137.
Figure 137. Default Manual Integration Fixes table for the 254 nm wavelength
channel for multi Calibration Level 6.dat
To add a manual integration fix integration event
1. Open the stored data file, multi Calibration Level 6.dat:
a. Choose File > Data > Open to open the Open Data File dialog box
(see Figure 138).
Figure 138. Open Data File dialog box
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b. Browse to the following directory: Drive:\ChromQuest\Data.
c. Select the Multi Calibration Level 6.dat data file from the list.
d. Under Options, select Original / Acquisition from the Method list.
e. Click Open to open the data file with its original method.
The chromatogram shown in Figure 139 appears. The active
method listed in the title bar will be named, Multi Level
Calibration.met.
Figure 139. Instrument window, showing the multi calibration level 6 data file integrated with its original method
2. From the Integration Events toolbar at the bottom of the Instrument
window, click the Move BL Start button.
Instructions appear in the Status Bar at the bottom of the Instrument
window. See Figure 140.
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Status Bar
Figure 140. Instrument window, showing instructions in the Status Bar
3. Follow the instructions in the Status Bar:
a. Click a baseline segment at approximately 8.2 minutes.
b. Adjust the baseline slightly upwards on the third peak in the
chromatogram. See Figure 141.
c. Press ESC to finish.
The Move BL Start dialog box appears. See Figure 142.
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Figure 141. Moving the baseline start
Figure 142. Move BL Start dialog box
4. Click Analyze Now to add the event to the table and to analyze the
chromatogram using the event.
The integration of your chromatogram changes, as shown in Figure 143.
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Figure 143. Result of moving the start of the baseline
5. In the Command toolbar, click the Manual Integration Fixes button.
The Manual Integration Fixes table appears (see Figure 144).
Figure 144. Manual Integration Fixes table for the 254 nm discrete wavelength
channel
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Chapter 8
Specifying the Calibration
Curve Parameters
In ChromQuest, the peak table contains the information required to
identify the peaks in the chromatograms and the information required to
build the calibration curves to quantitate your analytes. In this tutorial, you
learn how to add a peak table to your method.
There is one peak table for each detector in the instrument configuration.
For multi-wavelength analyses, you use the Analysis Channel column in the
peak table to specify the analysis wavelength(s) for the peaks. The
wavelength listed in the Title bar is the current selection in the Channel
Selection list.
Figure 145. Peak table, showing Analysis Channel list for Test Mix.met
With the UV/Vis detector, you can simultaneously collect data on two
wavelength channels. With the PDA detector, you can collect scan data
from 190 nm to 800 nm, and then quantitate each analyte at its optimal
analysis wavelength by adding multi-chromatogram channels to the
method.
This chapter contains the following sections:
• Adding a Peak Table Graphically
• Modifying the Properties of the Peak Table
• Performing Multi-Wavelength Analyses
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Adding a Peak Table Graphically
Adding a Peak Table
Graphically
You can graphically add a peak table to your method using either the Define
Peaks dialog box or the Define Single Peak dialog box. Shortcut buttons to
these dialog boxes are located in the Integration Events toolbar at the
bottom of the Instrument window.
With the Define Single Peak dialog box, you can add define the calibration
curve parameters for the peaks in your chromatograms one-by-one. With
the Define Peaks dialog box, you can define the calibration curve parameters
for a set of peaks.
This section contains the following topics:
• Using the Define Single Peak Dialog Box
• Using the Define Peaks Dialog Box
Using the Define Single
Peak Dialog Box
In ChromQuest, the peak table contains the information required to
identify and quantitate your analytes. In this procedure, you graphically
enter the information for a 3-point calibration curve using the data file that
you acquired by injecting a solution of toluene (or an analyte of your
choice).
To create a peak table for your method
1. If it is not already open, open your stored data file, Preliminary Run.dat:
a. From the Instrument window menu bar, choose File > Open >
Data.
b. In the Open Data File dialog box (see Figure 123), browse to the
directory where the data file is stored:
Drive:\ChromQuest\Projects\Tutorial\Data
c. Select the data file.
d. Under Options, select Results in the Method list.
e. Click Open.
2. Click anywhere in the Chromatogram window to activate the
Integration Events toolbar, and then click the Define Single Peak
button.
The Define Single Peak dialog box appears. See Figure 146.
3. Click the toluene peak in your chromatogram.
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The time listed in the Define Single Peak dialog box should match the
retention time of the peak you just selected.
Retention Time of
Currently Selected
Peak
Figure 146. Define Single Peak dialog box
4. Type Toluene in the Peak Name box.
In Chapter 10, “Creating a Sequence Table,” you create a sequence table
that contains a set of calibration standards and a set of unknowns. The
calibration levels that you enter in the peak table in steps 5, 6, and 7 are
used to create a calibration curve for the quantitation of the unknowns.
In Chapter 11, “Running and Reprocessing a Sequence.” you inject 1,
2, and 3 μL of the toluene solution, instead of preparing a set of
calibration standards.
5. To enter the concentration for the first level of the 3-point calibration
curve:
a. Leave the value of 1 in the Conc. Level box to the left of the colon.
b. Type 1 (or the concentration of your lowest calibration standard) in
the box to the right of the colon.
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6. To enter the concentration for the second level of the 3-point calibration
curve:
a. Click the toluene peak a second time.
b. Type 2 (or the concentration of your lowest calibration standard) in
the Conc. Level box to the left of the colon
c. Type 2 in the box to the right of the colon.
7. To enter the concentration for the third level of a 3-point calibration
curve:
a. Click the toluene peak a third time.
b. Type 3 (or the concentration of your lowest calibration standard) in
the Conc. Level box to the left of the colon.
c. Type 3 in the box to the right of the colon.
Note The concentration values in the Levels columns are
unitless. The entry in the Unit column of the peak table is only a
label.
8. Leave the other settings in the Define Single Peak dialog box at their
defaults.
9. Click Done.
10. Click the Peak/Group Tables button to review the information in the
Peak table.
Figure 147. Peak table for the PDA Plus detector, showing the entries for the toluene peak
11. Scroll to the right in the peak table until you find the Fit Type column.
Change the fit type to linear by clicking the down arrow in the Fit Type
column and selecting Linear from the list.
Note By default, the fit type is point-to-point.
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12. Scroll further to the right to review the calibration levels. See
Figure 148.
Figure 148. Peak table for the PDA Plus detector, showing the calibration levels
Leave the peak table open and proceed to “Modifying the Properties of the
Peak Table” on page 191.
Using the Define Peaks
Dialog Box
If you do not have a stored data file of your own, perform this tutorial to
familiarize yourself with the peak table feature. Use the following data file
that is supplied with ChromQuest: Multi Calibration Level 6.dat. This data
file contains one chromatogram.
To create a peak table using the Define Peaks dialog box
1. Open the data file named Multi Calibration Level 6.dat:
a. From the Instrument window menu bar, choose File > Open >
Data.
b. In the Open Data File dialog box, browse to the appropriate
directory.
Drive:\ChromQuest\Data\Projects\Default
c. Select the data file Multi Calibration Level 6.dat.
d. Under Options, select Original / Acquisition in the Method list.
e. Click Open.
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Figure 149. Open Data File dialog box, showing the selection of the
multi-calibration level 6 data file
2. Click anywhere in the Chromatogram window to activate the
Integration Events toolbar, and then click the Define Peaks button.
Instructions appear in the Status bar, which is located below the
Integration Events toolbar. The first message instructs you to click the
beginning of the range of the peaks you want to define (see Figure 150).
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Instructions in Status Bar
Figure 150. Instrument window, showing instructions in the status bar
3. Click the chromatogram at approximately the 5 minute time point.
The message in the Status bar instructs you to click the end of the range
of the peaks you want to define.
4. Click the chromatogram at approximately the 10 minute time point.
The Define Peaks dialog box appears (see Figure 151). The Start time
box shows the beginning of the selected peak range and the Stop time
box shows the end of the selected peak range.
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Figure 151. Define Peaks dialog box
5. Display the list of integrated peaks by clicking the down arrow for the
Minimum Peak Area list (see Figure 152).
Use the listed areas to determine the minimum peak area for your
analysis. The peak areas in the list originate from the areas of the
integrated peaks in the data file. The last four areas in the table represent
the four analyte peaks. The remaining peak areas are due to noise or
contaminants.
Figure 152. List of integrated peaks in the selected chromatogram
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6. Keep all parameters in the Define Peaks dialog box set to the default
settings except those that are listed in the following table and are shown
in Figure 153.
By default, the retention time window is set to ± 2.5% of the retention
time of a named peak and peaks are quantitated based on their areas.
By selecting the Replace existing peaks in table option, you clear any
existing entries in the peak table.
Parameter
Minimum Peak Area
Replace Existing Peaks In
Table
Setting
6500
*
Result
Specifies that peaks with areas below
6500 area counts are not named in the
peak table
Specifies that the existing peak table will
be replaced with the current settings
* Note. To enter this setting, type 6500 in the Minimum Peak Area text box.
Figure 153. Define Peaks dialog box, showing entries
7. Click OK to close the dialog box and create the peak table.
8. Click the Peak/Group Tables button in the toolbar to review the
information in the Peak table. See Figure 154.
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Peak Table for Surveyor PDA Plus Detector
Figure 154. New Peak table for Multi Level Calibration.met
9. Scroll to the right in the peak table. Notice that by default the fit type of
the calibration curve is set to point-to-point. See Figure 155.
Note The Multi Calibration Level data files contain only one
chromatogram. For multi-wavelength analyses, select the analysis
channel in the Analysis Channel list. Unlike the Integration Events
tables, the wavelength listed in the title bar of the Peak/ Group Tables
window has no real meaning.
Figure 155. View of the peak table after scrolling to the right
10. Save the method by choosing File > Method > Save.
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Table
Specifying the Calibration Curve Parameters
Modifying the Properties of the Peak Table
By default the peak table contains all the columns shown in Figure 156. You
can reduce the number of columns in the table to those that you actually use
by clearing the check boxes next to the columns that you do not use.
Because the column selections in the peak table are saved on a
per-instrument and per-user basis, you can set the peak table parameters for
each instrument controlled from the ChromQuest data system.
Figure 156. List of default columns in the peak table
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Specifying the Calibration Curve Parameters
Modifying the Properties of the Peak Table
To modify the properties of the peak table
1. Right-click in the Peak Tables window and choose Properties from the
shortcut menu.
2. To avoid excessive scrolling, clear the check boxes next to the columns
that will not be used in this tutorial. Keep the columns listed in the
following table.
Columns
#
Selection check box
Name
ID
Ret Time
Window
Detection
Analysis Channel
Quantitate
Fit Type
Calib Flag
Levels
3. Change the number of calibration levels displayed in the peak table:
a. In the Properties dialog box, double-click Levels to open the Max #
of levels box.
b. Highlight the current value of 10.
c. Type 3 in the box, and then press ENTER.
d. Verify that the Levels check box is still selected. See Figure 157.
e. Click OK.
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Modifying the Properties of the Peak Table
Figure 157. Properties dialog box, showing the number of calibration levels
displayed in the peak table
4. Save the method by choosing File > Method > Save.
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Performing Multi-Wavelength Analyses
Performing
Multi-Wavelength
Analyses
The ability to identify and quantitate a peak on more than one analysis
channel is a useful feature, especially if your chromatographic peaks are
impure or if the optimal analysis wavelengths of the analytes in your sample
differ significantly.
This section contains the following topics:
• Modifying the Analysis Channels in the Peak Table
• Modifying the Annotations Listed on the Screen
Modifying the Analysis
Channels in the Peak
Table
To identify and quantitate the toluene peak on two Analysis Channels
1. Verify that the Analysis Channel in the first row is the 230 nm
wavelength
2. Copy the information in row 1 to row 2:
a. In row 1 of the peak table, click the # column to select the
entire row.
b. Press CTRL+C to copy the information in row 1.
c. In row 2 of the peak table, click the # column to select the
entire row.
d. Press CTRL+V to paste the information in row 1.
3. In the Analysis Channel column, select the 260 nm wavelength from the
list of available wavelengths.
4. Right-click in the peak table and choose Renumber Peak IDs from the
shortcut menu.
5. Change the name of the peak on the first row of the peak table to
Toluene_230nm.
6. Change the name of the peak on the second row of the peak table to
Toluene_260nm.
Give the two toluene peaks unique names to help you identify their
respective calibration curves when you review the calibration
information. See Figure 158.
7. Save the method by choosing File > Method > Save.
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Peak Names
Specifying the Calibration Curve Parameters
Performing Multi-Wavelength Analyses
Analysis Channels
Figure 158. Peak table for Test Mix.met
Modifying the Annotations
Listed on the Screen
To view the names of the peaks on your view screen, add the Name
annotation.
To add the Name annotation to the chromatograms
1. Click a chromatogram on your view screen to open the Chromatogram
shortcut menu.
2. In the shortcut menu, choose Annotations to open the Trace
Annotation Properties dialog box. See Figure 121 on page 162.
3. In the Available Annotations box, double-click the Name annotation to
add it to the Show The Following Annotations box.
4. Click Apply To All.
5. Click OK to exit the Trace Annotation Properties dialog box.
6. Save the method by choosing File > Method > Save.
7. In the Command toolbar, click the Analyze button.
The peak in the chromatogram for the 230 nm wavelength channel is
named Toluene_230nm.
The peak in the chromatogram for the 260 nm wavelength channel is
named Toluene_260nm.
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Chapter 9
Adding a Custom Report to
the Method
In this tutorial, you learn how to add one of the standard method custom
report templates to your method and how to create your own method
custom report template that can be stored in a template folder and then
added later to a method.
To print an individual report for each data file, you must add a method
custom report to your method. You can add one of the six standard custom
templates, or you can create your own method custom report template.
After you create and save a method custom report template, you can add it
to other methods, as well as the current method.
This chapter contains the following sections:
• Adding a Standard Custom Report to the Method
• Creating a Custom Report Template
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Adding a Standard Custom Report to the Method
Adding a Standard
Custom Report to the
Method
By default, the Custom Report section of the method is blank until you add
a report template.
To add a Custom Report to your method
1. Open the Offline Instrument window for your instrument.
2. Ensure that the method you created while performing the tutorial in
Chapter 4, “Creating Methods,” is open.
The name of the active method is listed in the title bar of the Instrument
window.
3. Click the Edit Custom Report button.
If this is a new method, a blank page appears.
4. From the Instrument window menu bar, choose
File > Report Template > Open to open the Open Report Template
File dialog box and access one of the six standard custom report
templates:
• External Standard-horiz.rep
• External Standard-Vertical.rep
• External Standard.srp
• Internal Standard.srp
• Area%.srp
• Normalization.srp
5. Select the External Standard.srp custom report template. See
Figure 159.
Templates with the file extension .srp can be automatically viewed by
choosing Reports > View, and then making a selection from the menu
shown in Figure 160.
The External Standard.srp custom report template appears on your
screen. The contents of the active data file, Preliminary Run 001.dat are
displayed. Your custom report page will look similar to the one shown in
Figure 161.
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6. Choose File > Method > Save to save the method.
Figure 159. Open Report Template dialog box
Figure 160. Selecting the report that you want to view on the view screen
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Adding a Standard Custom Report to the Method
Header
Data Graph
Run Report
Footer
Figure 161. Method Custom Report created using the External Standard.srp
template
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Adding a Custom Report to the Method
Creating a Custom Report Template
To suit your reporting requirements, you can modify a custom report
template or you can create a completely new custom report template.
If it is not already open, click the Edit Custom Report button to open the
Custom Report page of the method. Ensure that the report page contains
the ESTD Standard.rep custom report template that you added while
performing the procedure “Adding a Standard Custom Report to the
Method” on page 198.
Create your own custom report template by modifying the ESTD
Standard.rep custom report template by performing the following
procedures in this section:
• Adding Text to a Custom Report Template
• Modifying Data Graph Annotations
• Changing the Appearance of the Data Graph
• Modifying the Run Report
Adding Text to a Custom
Report Template
To add the name of your company to the top of the report
1. Place the cursor between the report header and the data graph of the
chromatogram (see Figure 161 on page 9-200). If the header is not
visible, right-click the report and choose Header / Footer from the
shortcut menu.
2. Type the name of your company, for example, Quality Performance
Laboratories.
3. Using the Custom Report toolbar (see Figure 162), change the font type
for the company name to arial, 14 point, bold, and then center justify
the heading:
a. Highlight the company name.
b. Select Arial from the Font Name combo box.
c. Select 14 from the Font Size combo box.
d. Click the Bold (B) button.
e. Click the Center Justify button to horizontally center the company
name at the top of the report page.
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Font Name
Font Size
Bold
Center Justify
Figure 162. Custom Report toolbar
Modifying Data Graph
Annotations
By default, the template contains one data graph. The retention time of the
peaks to 3 decimal places is its only annotation.
To modify the annotations on the data graph
1. Double-click the data graph to open the Data Graph Properties
dialog box.
The Data Graph Properties dialog box opens with the Trace Setup page
displayed (see Figure 163).
Figure 163. Data Graph Properties dialog box – Trace Setup page
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2. In the Trace Setup page, click Annotations to open the Trace
Annotation Properties dialog box shown in Figure 164.
Figure 164. Trace Annotation Properties dialog box
3. In the Trace Annotation Properties dialog box, do the following:
a. Add the Name annotation by double-clicking Name in the Available
Annotations list.
b. Change the number of decimal places for Retention Time to 2:
i. Click the Retention Time annotation in the Show The
Following Annotations list.
ii. Highlight the number 3 in the Decimals box. Then, type 2.
c. Under Other, select the RT Window check box.
d. Click Apply To All.
e. Click OK to exit the Trace Annotation Properties dialog box.
Leave the Data Graph Properties dialog box open and go to the next topic,
Changing the Appearance of the Data Graph.
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Changing the Appearance
of the Data Graph
By default, the font size of the data graph legend is 6 point and its color is
gray. By default, annotations are listed at a 90° angle.
To change the appearance of the data graph
1. In the Data Graph Properties dialog box, click the Appearance tab to
open the Appearance page shown in Figure 165.
2. Change the appearance of the Legend, which is located in the top left
corner of the data graph:
a. From the Item list, select Graph.
b. From the SubItem list, select Legend.
c. From the Size list, select 10.
d. From the Color list, select the black square.
e. Click Apply.
Figure 165. Data Graph Properties dialog box – Appearance page
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3. Change the appearance of the trace Annotations:
a. From the Item list, select the trace. For example, the trace for the
Surveyor PDA will be named, 1:(Current Data)-PDA-230 nm. See
Figure 166.
b. From the Font list, select Arial.
c. From the Size list, select 10.
d. From the Angle list, select 45.
e. Click the Bold button.
f. Click Apply.
Figure 166. Appearance Properties dialog box
Modifying the Run Report
To modify the Run Report
1. Click the Run Report section of the Custom Report template. Then
right-click the Run Report section (see Figure 161 on page 200) to open
a shortcut menu. See Figure 167.
2. Verify that the menu item Show Data At Design Time is checked.
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Figure 167. Run Report shortcut menu
3. In the shortcut menu, choose Report Properties to open the Run
Report dialog box. See Figure 168.
Figure 168. Run Report dialog box
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Creating a Custom Report Template
4. In the Run Report dialog box, do the following:
a. In the Report the following parameters box, click Retention Time
to select it.
b. Change the number of decimal places to 2 by highlighting the
number 3, and then typing 2 in the Decimals box.
c. In the Report the following parameters box, click ESTD
Concentration to select it.
d. Change the number of decimal places to 2 by highlighting the
number 3, and then typing 2 in the Decimals box.
e. Click OK to exit the Run Report dialog box.
5. Save the new custom report template in the Templates folder of the
Tutorial project:
a. Choose File > Report Template > Save As from the menu bar to
display the Save Report Template As dialog box. See Figure 169.
Figure 169. Save Report Template As dialog box
b. Browse to the appropriate directory.
Drive:\ChromQuest\Projects\Tutorial\Template
c. In the Filename box, type in the name My Custom Report
Template for the Custom Report Template.
d. Click Save.
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Creating a Custom Report Template
ChromQuest adds the .rep extension to the file.
Note If you want the template to appear in the list that you access by
choosing Reports > View from the menu bar, add an [.srp] extension.
6. Save the method, Test Mix.met, by choosing File > Method > Save.
The custom report produced using your new template will look similar to
the one shown in Figure 170.
Heading
Data Graph
Run Report
Footer
Figure 170. Modified method custom report
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Chapter 10
Creating a Sequence Table
In this tutorial, you learn how to use the Sequence Wizard to create a basic
acquisition sequence table. In addition, you learn how to modify this basic
sequence table to suit your applications.
The sequence table contains the information that is required to inject a set
of samples or to reprocess a set of stored data files. ChromQuest contains a
Sequence Wizard that guides you through the creation of a sequence table.
This chapter contains the following sections:
• Using the Sequence Wizard to Create a Sequence Table
• Modifying the Sequence Table
• Creating a New Sequence Summary Template
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Creating a Sequence Table
Using the Sequence Wizard to Create a Sequence Table
Using the Sequence
Wizard to Create a
Sequence Table
In this tutorial you use the Sequence Wizard to create an acquisition
sequence that contains three calibration standards and three unknowns. For
convenience, you make all the injections from one vial.
To use the Sequence Wizard to create a sequence table
1. From the Instrument window, choose File > Sequence > Sequence
Wizard to start the Sequence Wizard.
The Sequence Wizard opens to the Method page.
2. In the Sequence Wizard – Method page, do the following:
a. Except for the method, leave the settings in the Method page at their
defaults, as shown in Figure 171. If Test Mix.met is not listed in the
Method list, browse to Test Mix.met or an appropriate alternative
method.
b. Click Next to open the Unknowns page of the Sequence Wizard.
Note When you open the Sequence Wizard, the Method list
displays the active method listed in the title bar of the
Instrument window.
Figure 171. Sequence Wizard – Method page
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3. In the Unknowns page, leave the settings at their defaults except for the
following changes, as shown in Figure 172:
a. Type Toluene in the Sample ID box.
b. Verify that the appropriate Data path is displayed.
Note If you are using the Tutorial project to organize your data,
verify that the following data path is displayed:
Drive:\ChromQuest\Projects\Tutorial\Data.
c. Give unique names to the data files in this sequence table:
i. Type Test Mix in the Data file box. Then add a space.
ii. Click the blue arrow next to the Data file box.
iii. From the pop-up menu, choose Line Number.
d. Type 3 in the Number of unknown runs in sequence box.
e. Click Next to open the Autosampler page of the Sequence Wizard.
Typing
Space
Line Number
Figure 172. Sequence Wizard – Unknowns page
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4. In the Autosampler page, make the entries described below and shown
in Figure 173:
a. In the Unknown vials of sequence area, type A;1 (A semicolon 1)in
the First Vial box.
Note You must type a semicolon between the tray type and the
tray location.
b. In the Calibration vials of sequence area, type A;1 in the First Vial
box.
All the injections for this sequence will be made from the
standard 1.8 mL vial in the A1 position of the standard tray.
c. In the Autosampler injection volume box, enter an injection volume
in μL:
• Type 1 if you are using a 5 cm LightPipe flow cell.
• Type 5 if you are using a standard 1 cm flow cell.
d. Verify that the Pretreatment program file box is empty.
e. Click Next to open the Calibration page of the Sequence Wizard.
Figure 173. Sequence Wizard – Autosampler page
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5. In the Calibration page, leave the settings at their defaults except for the
following entries and selections, as shown in Figure 174:
a. In the Number of calibration levels box, type 3.
The peak table that you created while performing the tutorial
contained in Chapter 8 has three calibration levels. To simulate
three concentration levels, you will inject variable amounts of
sample as described in the topic “Changing the Injection Volume”
on page 221.
b. Select the Clear all calibration at start of sequence check box.
This allows ChromQuest to clear the absorbance values in the
calibration table of the method at the beginning of a sequence run
or a sequence reprocessing.
c. Click Next to open the Reports page of the Sequence Wizard.
Figure 174. Sequence Wizard – Calibration page
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6. In the Reports page, leave the settings at their defaults except for the
following changes in the Summary area, as shown in Figure 175:
• Select the Include unknown runs in summary report check box.
• Select the Include calibration runs in summary report check box.
7. Click Finish.
The unedited sequence table appears. See Figure 176.
Figure 175. Sequence Wizard – Reports page
Figure 176. Unedited sequence table
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8. Save the sequence table with the name, Test Mix Sequence.seq:
a. Choose File > Sequence > Save As to display the Save Sequence File
As dialog box, shown in Figure 177.
b. Type Test Mix Sequence in the File Name box.
c. Click Save.
ChromQuest appends the file extension .seq to the sequence name.
Figure 177. Save Sequence File As dialog box
9. Verify the file folders for the sequence:
a. From the Instrument window, choose Sequence > Properties.
The Sequence Properties dialog box appears. See Figure 178.
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Figure 178. Sequence Properties dialog box, showing the file paths of
the method files and the data files
b. Verify that the method and the data files are in the appropriate
project folder. Then, close the Sequence Properties dialog box.
Leave the sequence table open on the view screen and proceed to the next
topic, “Modifying the Sequence Table” on page 217.
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Sequence Table
Creating a Sequence Table
Modifying the Sequence Table
You can create a basic sequence table using the Sequence Wizard. If you
want to turn off the solvent flow from the pump or turn off the lamps at the
end of a sequence run, you must modify the sequence table.
If your sequence is not already open, click the Edit Sequence button to
open it.
This section contains the following procedures for common modifications
made to sequence tables:
• Deleting Unused Columns from the Sequence Table
• Changing the Vial Locations
• Changing the Injection Volume
• Adding a Shutdown Line
• Adding an Action
Deleting Unused Columns
from the Sequence Table
By default, the Sequence table contains 22 columns. To make it easier to
work with the table, delete some of the columns from view.
To delete unused columns from the sequence table
1. Right-click in the sequence table to open its shortcut menu. See
Figure 179.
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Figure 179. Sequence shortcut menu
2. From the shortcut menu, choose Properties to open the Properties
dialog box. See Figure 180.
Figure 180. Properties dialog box
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3. Clear the check boxes for all the columns except for those listed below.
Run #
Status
Run Type
Level
Reps
Vial
Volume
Sample ID
Method
Filename
Action
4. Click OK to exit the Properties dialog box.
Changing the Vial
Locations
In the next chapter, you make a sequence of injections from the same vial.
Therefore, you need to modify the vial locations listed in the current
sequence table, Text Mix Sequence.seq.
To change the vial locations and make the injections from the same vial
1. Select the Vial column by clicking its header.
2. Right-click in the sequence table to open the shortcut menu, as shown
in Figure 181.
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Figure 181. Sequence shortcut menu
3. From the shortcut menu (see Figure 181), choose Fill Down to open
the Fill Down dialog box.
4. Except for the following change in the Increment Vials area, leave the
settings in the Fill Down dialog box at their defaults, as shown in
Figure 182:
a. In the Increment by box, highlight the number 1, and then type 0.
b. Click OK.
A;1 is now the only vial listed in the Vial column.
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Figure 182. Fill Down dialog box
5. To save the sequence, click the Save button in the Command toolbar,
and then choose Save Sequence.
Changing the Injection
Volume
In this tutorial, you use one vial for all your injections. Instead of injecting a
set of calibration standards to create your calibration curve, you inject a
variable amount of sample solution.
To change the injection volumes in your sequence table
1. Click the first cell in the Volume column. Then enter an appropriate
injection volume for the first calibration level:
• If you are using a 5 cm LightPipe flow cell, type 0.5.
• If you are using a standard 1 cm flow cell, type 2.5.
2. Click the second cell in the Volume column. Then, enter an appropriate
injection volume for the second calibration level:
• If you are using a 5 cm LightPipe flow cell, type 1.
• If you are using a standard 1 cm flow cell, type 5.0.
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3. Click the third cell in the Volume column. Then, enter an appropriate
injection volume for the third calibration level:
• If you are using a 5 cm LightPipe flow cell, type 1.5.
• If you are using a standard 1 cm flow cell, type 7.5.
As the calibration data files in the sequence are acquired, the injection
volumes that you entered will create a calibration curve proportional to the
values entered in the peak table. See page 183, which describes the
concentration entries in the peak table.
Adding a Shutdown Line
At the end of a sequence run, you might want to turn off the pump flow or
the lamps, or both. Turning off the pump flow conserves solvent. Turning
off the lamps extends their lifespan. To turn off the pump flow or the lamps,
you must create a shutdown method and add a shutdown line to the end of
your sequence table. You created a shutdown method named Shutdown.met
while performing the tutorial “Creating a Shutdown Method” on page 110.
To add a shutdown line at the end of your sequence table
1. In the seventh row of the sequence table (see Figure 181 on page 220),
click the blue arrow in the Run Type column.
The Sample Run Types dialog box appears (see Figure 183).
2. In the Sample Run Type(s) dialog box, select the Shutdown check box.
3. Click OK to exit the Sample Run Types dialog box.
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Figure 183. Sample Run Type dialog box
4. In the Method column of the sequence table (see Figure 181 on
page 220), click the green arrow.
The Open Method File dialog box appears (see Figure 106 on
page 138).
5. Select the Shutdown.met shutdown method, and then click Open.
Row seven of your sequence table now contains a Shutdown run type
and lists the Shutdown.met. The entire row is highlighted in red.
6. Save the sequence table by choosing File > Sequence > Save.
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Adding an Action
The Action feature automates a variety of operations, such as setting off an
alarm is a baseline check fails or performing a shutdown if a hardware status
error occurs.
To shut down the LC in the event of a hardware error
1. In row 1 of the sequence table (see Figure 181 on page 220), click the
blue arrow in the Action column.
The Action dialog box appears (see Figure 184).
2. Make the following selections in the Action dialog box:
a. In the Test column, select Hardware Status.
b. In the Result column, select Fail.
c. In the Action column, select Run Shutdown.
3. Click OK to close the dialog box.
4. Save the sequence by choosing File > Sequence > Save.
Figure 184. Action dialog box, showing a Hardware Status test
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Creating a New
Sequence Summary
Template
By default, the basic sequence created with the Sequence Wizard uses the
sequence summary template Summary.tpl, which reports the concentrations
of the named peaks for the first wavelength listed in the Channel Selection
list. ChromQuest contains several other sequence summary templates from
which to choose, but none of these templates reports the concentrations of
your analytes for two or more wavelengths. Therefore, to print a summary
of the results for both of your wavelength channels, you must create your
own sequence summary template and then add it to your sequence table.
To create your own customized sequence summary report and add it to your
sequence table, perform the following procedures:
1. Opening the Sequence Editor
2. Using the Table Wizard
3. Selecting a Sequence Summary Template
Opening the Sequence
Editor
Create your own sequence summary template by using the Sequence Editor.
to modify one of the standard sequence summary templates. The sequence
summary template Summary.tpl reports the concentrations of named peaks
at one wavelength. This summary report can be easily modified to report the
concentrations of your named peak at two wavelengths.
To open the sequence summary template for editing
1. From the Instrument window, choose File > Advanced Reports >
Open.
The Open Advanced Report File dialog box appears (see Figure 185).
2. Select the sequence summary report template Summary.tpl.
3. Click Open.
The Summary template opens in the Template Editor window, as shown
in Figure 186.
Tip Double-click the formula cells (EX.R...) to view the formulas.
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Figure 185. Open Advance Report File dialog box
Figure 186. Template Editor, showing the sequence summary template, Summary.tpl
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Using the Table Wizard
Now that you have the sequence summary template open for editing, add an
additional table to report the results of the 260 nm analysis channel.
To add an additional table to the sequence summary template
1. Ensure that your method Test Mix.met is the active method, so that the
traces are available.
2. Place your cursor in cell A15 of the sequence summary template.
3. Click the Table Wizard button.
The Table Wizard appears. See Figure 187.
Figure 187. Table Wizard – Table Type page
4. Click Next.
The Parameters page appears.
5. In the Parameters page, make the following selections, as shown in
Figure 188:
a. Click the Trace Index button to display the available traces.
b. Select the 260 nm wavelength (trace index 2).
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Creating a Sequence Table
Creating a New Sequence Summary Template
c. Click Select Trace.
d. Double-click ESTD Concentration to add it to the list of
parameters on the right.
Figure 188. Table Wizard – Parameters page, showing appropriate settings
e. Click Next.
The Types page appears.
6. Leave the parameters in the Types page at their default settings, as
shown in Figure 189, and then click Next.
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Creating a New Sequence Summary Template
Figure 189. Table Wizard – Types page, showing default settings
The Run Parameters page appears.
7. In the Run Parameters page, double-click Data Filename to add it to
the box on the right, as shown in Figure 190, and then click Next.
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Creating a New Sequence Summary Template
Figure 190. Table Wizard – Run Parameters page, showing Data Filename
selected
The Run Direction page appears (see Figure 191).
8. In the Run Direction page, leave the Run Direction at its default setting
of Down, and then click Next.
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10 Creating a Sequence Table
Creating a New Sequence Summary Template
Figure 191. Table Wizard – Run Direction page, showing default settings
The Statistics page appears (see Figure 192).
9. In the Statistics page, leave the selection at its default setting of Yes, as
shown in Figure 192, and then click Finish.
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Figure 192. Table Wizard – Statistics page, showing default settings
Your new template appears. See Figure 193.
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Creating a New Sequence Summary Template
Figure 193. New template, showing formula for second trace
10. Save the new template with the name Summary_ESTD_Dual
Wavelength.tpl:
a. Choose File > Advanced Reports > Save As.
The Save dialog box appears (see Figure 194).
b. Type Summary_ESTD_Dual Wavelength in the File Name
text box.
c. Click Save.
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Creating a Sequence Table
Creating a New Sequence Summary Template
Figure 194. Save As dialog box, showing the new file name
Selecting a Sequence
Summary Template
To select the sequence summary template
1. Double-click in the Run Type cell that contains the Begin Summary
(SMB) run type in row 1. See Figure 195.
Figure 195. Test Mix Sequence table, showing Run Type column
The Sample Run Types dialog box appears. See Figure 196.
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10 Creating a Sequence Table
Creating a New Sequence Summary Template
Figure 196. Sample Run Types dialog box, displaying the current report template
2. In the Sample Run Types dialog box, select the Begin Summary
check box.
The Report Template box shows the current report template.
3. Click the file folder next to the Report Template box to open the Select
Sequence Report Template dialog box. See Figure 197.
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Creating a Sequence Table
Creating a New Sequence Summary Template
Figure 197. Select Sequence Report Template dialog box
4. Select the Summary_ESTD_Dual Wavelength.tpl template that you
created from the list of templates.
5. Save the sequence table by choosing File > Sequence > Save.
Your modified sequence table is shown in Figure 198.
Figure 198. Final sequence table
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Chapter 11
Running and Reprocessing a
Sequence
In this tutorial, you learn how to start a sequence run. In addition, you learn
how to reprocess the data files acquired by running the sequence and review
the calibration table contained in your method.
To start a sequence run, you must be working in the Online Instrument
window. To reprocess a sequence file, you can be working in either the
Online or Offline Instrument window.
This chapter contains the following sections:
• Starting a Sequence Run
• Reviewing the Peak Calibration
• Reprocessing a Sequence Run
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Running and Reprocessing a Sequence
Starting a Sequence Run
Starting a Sequence
Run
To start a sequence run, you must be working in the Online Instrument
window. If you have not already created a sequence table listing your sample
set, see Chapter 10, “Creating a Sequence Table,” for information on
creating a sequence table.
To start a sequence run
1. Ensure that your standard tray contains a vial in the A1 vial location.
2. From the Online Instrument window toolbar, click the Sequence Run
button.
The Sequence Run dialog box appears.
3. Set the parameters to the settings that are shown in the following table
and in Figure 199.
Parameter
Setting
Result
Drive:\ChromQuest\
Projects\Tutorial\
Sequence\
Test Mix Sequence.seq
Lists the sequence that will
be run
Sequence Information
Sequence Name
Your directory might be
different. Browse to the
appropriate directory.
Printing
Printing
Print Method Reports
Specifies that the method
custom reports will be
printed after the data file is
acquired
Printing
Print Sequence Reports
Specifies that your
sequence summary report
will be printed at the end of
the sequence run
4. Click Start.
A warning message appears because your sequence table contains the
same vial locations for your calibration standards as it does for your
unknowns. See Figure 200.
5. In the Overwriting Sample Type dialog box, click OK To All.
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Starting a Sequence Run
The sequence run commences. As each data file is acquired, the method
custom report that you created while performing the tutorial contained
in Chapter 9, “Adding a Custom Report to the Method,” is printed.
Figure 199. Run Sequence dialog box
Figure 200. Overwriting Sample Type warning box
6. To see which sequences or single runs are currently being processed,
click the Run Queue button.
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Running and Reprocessing a Sequence
Starting a Sequence Run
The Run Queue window appears. See Figure 201.
Figure 201. Run Queue window, showing the sequence in progress
After ChromQuest acquires the data file Test Mix 003.dat, it prints the
the sequence summary report that you created while performing the
tutorial contained in Chapter 10, “Creating a Sequence Table.” See
Figure 202.
Thermo Fisher Scientific
ChromQuest 4.2
Sequence Summary Report
Sequence name:
Analyst
Test Mix Sequence
B. A. Cook
PDA-230 nm
Toluene_230nm
Data Filename
ESTD
Cal_Test Mix 001.dat
Cal_Test Mix 002.dat
Cal_Test Mix 003.dat
1.00
Test Mix 001.dat
Test Mix 002.dat
Test Mix 003.dat
2.03
2.04
1.99
2.00
3.00
Min:
Max:
Mean:
Std Dev:
%RSD:
1.00
3.00
2.01
0.58
28.75
PDA-260 nm
Toluene_260nm
Data Filename
ESTD
Cal_Test Mix 001.dat
Cal_Test Mix 002.dat
Cal_Test Mix 003.dat
1.00
2.00
3.00
Test Mix 001.dat
Test Mix 002.dat
Test Mix 003.dat
2.04
2.04
2.00
Min:
Max:
Mean:
Std Dev:
%RSD:
1.00
3.00
2.01
0.63
31.43
Figure 202. Sequence Summary Report, showing data for two wavelengths
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Starting a Sequence Run
7. After the sequence run has ended (approximately 30 min), review the
information in the title bar.
The current method is the shutdown method named Shutdown.met.
8. Open the Instrument Status window by choosing Control >
Instrument Status, and verify that the lamps are off and that the flow
rate is set to zero.
9. Open the method Test Mix.met by choosing File > Method > Open.
Select Test Mix.met from the list, and then click Open.
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Reviewing the Peak Calibration
Reviewing the Peak
Calibration
After you have completed running all of the calibration standards for your
method, you can review the calibration curve and associated data.
To review the peak calibration information
1. Click the Review Peak Calibration button to review the calibration
information contained in the method Test Mix.met for the two
wavelengths: 230 nm and 260 nm.
2. Click the Toluene_230nm peak in the Peak list to view its calibration
information (see Figure 203).
3. Click the Toluene_260nm peak in the Peak list to view its calibration
information (see Figure 204).
The calibration curve for the selected peak appears in the lower left
corner of the window. The box in the lower right corner of the window
displays information about the calibration data.
Figure 203. Review Peak Calibration window, showing the calibration data for the peak named Toluene_230 nm
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Reviewing the Peak Calibration
Figure 204. Review Peak Calibration window, showing the calibration data for the peak named Toluene_260 nm
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Reprocessing a Sequence Run
Reprocessing a
Sequence Run
To review the results of your sequence run
1. Choose Sequence > Process or click the Sequence Process button.
The Process Sequence dialog box appears.
2. Set the parameters in the dialog box to the settings that are shown in the
following table and in Figure 205.
Instead of selecting a run range of All as you did when you originally ran
your sequence, select a range consisting of rows 1 through 6. You
exclude row 7 from the selection because it contains a shutdown run
without a specified file name.
Parameter
Setting
Result
Drive:\ChromQuest\
Projects\Tutorial\
Sequence\
Test Mix Sequence. seq
Lists the sequence that will
be processed
Sequence Information
Sequence Name
Run Range
Run Range
Run Range
Range
1-6
Specifies the reprocessing
of rows 1 through 6
Reintegrate
Specifies that the data files
will be reintegrated
Mode
Processing Mode
Review
Review
Review
Review
Results Review
(Pause After Each Run)
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Turns on Review
Pauses after each run. To
review the next data file,
you must click the green
down arrow at the bottom
of the Instrument window
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Running and Reprocessing a Sequence
Reprocessing a Sequence Run
Figure 205. Process Sequence dialog box, showing entries for reviewing your sequence
3. Click Start.
The review process commences.
4. Click the green up and down arrows in the bottom-right of the
Instrument window to move through the rows of the sequence. See
Figure 206.
5. After you review the last data file, click the green down arrow one more
time to exit the sequence reprocessing mode.
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Reprocessing a Sequence Run
Figure 206. Instrument window, showing the Chromatogram windows for both the 230 nm and the 260 nm
analysis channels
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Diluting Samples with a
Pretreatment Method
Chapter 12
In this tutorial, you learn how to create and run a pretreatment method that
dilutes samples ten-fold.
Pretreatment methods allow you to perform automated sample preparation
operations. Pretreatment methods are created separately from acquisition
methods. To perform a pretreatment method you must open it in the Single
Run dialog box or add it to the sequence table. The Surveyor Autosampler
performs the specified pretreatment method before it injects the sample. If
you create a special type of “prep only” acquisition method, the autosampler
does not make an injection.
This chapter contains the following sections:
• Creating a Pretreatment Method
• Creating a Prep Only Method
• Using a Pretreatment Method
• Pretreatment Rules
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Creating a Pretreatment Method
Creating a
Pretreatment Method
In ChromQuest, pretreatment routines are created as separate methods. The
Pretreatment dialog box allows you to create a multi-function pretreatment
method in a table format, which can contain up to 512 rows. This section
describes how to create a pretreatment method that dilutes samples ten-fold.
Create a pretreatment method that dilutes samples ten-fold by performing
the following procedures:
1. Opening the Pretreatment Window
2. Adding Functions to the Pretreatment Table
3. Saving the Pretreatment Method
Opening the Pretreatment
Window
To open the Pretreatment window from the Instrument window
1. Choose File > Pretreatment > New.
2. Choose Pretreatment > Edit to open the Pretreatment window
containing an empty pretreatment table. See Figure 207.
Figure 207. Pretreatment window, showing empty Pretreatment table
Adding Functions to the
Pretreatment Table
248
The Pretreatment window contains a table. Each line of the table contains
an individual function that performs a particular task, such as transferring a
specified volume of liquid from one vial location to another vial location.
When you click the down-arrow in the Function column, a list of functions
appears. Each function contains a set of parameters, such as transfer volume
source vial location, and destination vial location. After you enter the
parameters for a function, the Description column of the table lists the
parameters that you selected.
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Creating a Pretreatment Method
To create a pretreatment table that dilutes samples ten-fold, perform the
following procedures:
1. Adding the Transfer From Sample to Sample Function
2. Adding the Transfer From Reservoir To Sample Function
3. Adding the Wash Needle Function
4. Adding the Mix At Sample Function
Adding the Transfer From Sample
to Sample Function
Your pretreatment method will transfer sample from the current vial
location in the sequence table to the current + 1 vial location. For example,
if the current vial location is A1, sample solution will be drawn from vial
location A1 and transferred to vial location A2.
To add the Transfer From Sample To Sample function
1. Click the first cell of the Function column to make the down-arrow
appear.
2. Click the down-arrow in the Function column to open the list of
pretreatment functions. See Figure 208.
Figure 208. Function list
3. Select the Transfer From Sample To Sample function.
ChromQuest opens a dialog box for the Transfer From Sample To
Sample function.
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4. Keep all parameters in the Transfer From Sample To Sample Setup
dialog box set to the default settings except those that are listed in the
following table and are shown in Figure 209.
5. Click OK.
Parameter
Setting
Result
Volume
50
Specifies that 50 μL of sample will be
withdrawn
Source Sample
Location
Source Sample Location
Relative List
Relative
Current
Turns on the Relative vial location option
Specifies that the sample will be
withdrawn from the current vial location in
the sequence table
Destination Sample
Location
Destination Sample
Location option
Relative List Item
Relative
Current + 1
Turns on the Relative vial location option
Specifies that the sample will be
transferred to the current + 1 vial location
in the sequence table
Figure 209. Transfer From Sample To Sample function dialog box
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Adding the Transfer From
Reservoir To Sample Function
Diluting Samples with a Pretreatment Method
Creating a Pretreatment Method
To transfer more than 265 μL of liquid, you have to use the outer plunger of
the syringe, which is less precise than the inner plunger. Therefore, to keep
the operation in the inner plunger, your pretreatment method will transfer
225 μL of solvent from reservoir bottle 1 to the current + 1 vial location in
the sequence table, twice, for a total of 450 μL of diluent.
To add the Transfer From Reservoir To Sample Function
1. Click the second cell of the Function column, and then click the
down-arrow to open the list of pretreatment functions.
2. Select the Transfer From Reservoir To Sample function.
ChromQuest opens a dialog box for the Transfer From Reservoir To
Sample function.
3. Keep all parameters in the Transfer From Reservoir To Sample Setup
dialog box set to the default settings except those that are listed in the
following table and are shown in Figure 210.
4. Click OK.
5. Click the third cell of the Function column, and then repeat step 2
through step 4 to add a second Transfer From Reservoir To Sample step.
Parameter
Setting
Result
Volume
225
Specifies that 225 μL of solution will be
withdrawn
Source RV
RV1
Specifies that the solution will be drawn
from reservoir vial 1
Sample Location
Sample Location option
Relative List Item
Thermo Scientific
Relative
Current +1
Turns on the Relative vial location option
Specifies that the diluent drawn from the
reservoir vial will be delivered to vial
location current +1
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Figure 210. Transfer From Reservoir To Sample Setup dialog box
Adding the Wash Needle
Function
After performing the Transfer From Sample To Sample function and the
Transfer From Reservoir To Sample function, both of which used the inner
plunger of the concentric syringe, the autosampler must home the plungers
of the concentric syringe before it can perform the Mix At Sample function,
which uses the outer plunger of the concentric syringe.
There are two functions that home the syringe: Wash Needle and Flush To
Waste. For your pretreatment method, you will use the Wash Needle
function to home the plungers of the concentric syringe.
To add the Wash Needle function to the pretreatment table
1. Click the third cell of the Function column, and then click the
down-arrow to open the list of pretreatment functions.
2. Select the Wash Needle function.
The Wash Needle Setup dialog box appears.
3. Keep all parameters in the Wash Needle Setup dialog box set to the
default settings, as shown in Figure 211.
4. Click OK.
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Figure 211. Wash Needle Setup dialog box
Adding the Mix At Sample
Function
At this point, the vial in the current + 1 vial location contains 50 μL of the
original 0.5% toluene in methanol sample solution and an additional
450 μL of methanol. Before you inject this solution, you want to mix it. The
mixing function in ChromQuest aspirates liquid into the needle tubing, and
then expunges it.
To add the Mix At Sample function to your pretreatment method
1. Click the fourth cell of the Function column, and then click the
down-arrow to open the list of pretreatment functions.
2. Select the Mix At Sample function.
The Mix At Sample Setup dialog box appears.
3. Keep all parameters in the Mix At Sample Setup dialog box set to the
default settings except those that are listed in the following table and are
shown in Figure 212.
4. Click OK.
Parameter
Setting
Result
Sample Location
Relative
Sample Location Mode
Thermo Scientific
Specifies that 50 μL of sample will be
withdrawn
Relative List Item
Current + 1
Specifies that the sample will be
transferred to the current + 1 vial location
in the sequence table
Volume
250 μL
Specifies that 250 μL of sample will be
aspirated into the needle tubing, and then
expunged back into the vial
Cycles
10
Specifies that the sample will be aspirated
and expunged 10 times
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Figure 212. Mix At Sample Setup dialog box
You have entered the functions required to perform a ten-fold sample
dilution. Verify that your pretreatment table matches the one shown in
Figure 213. Then save your pretreatment method as described in the next
topic Saving the Pretreatment Method.
Figure 213. Pretreatment table, displaying a Pretreatment method that performs a ten-fold dilution
Saving the Pretreatment
Method
To save the Pretreatment method
1. Choose File > Pretreatment > Save As to open the Save Pretreatment
File As dialog box. See Figure 214.
2. Browse to the Pretreatment folder.
3. Type the name Ten Fold Dilution in the filename box.
4. Click Save.
Pretreatment methods are saved with the file extension .ape.
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Creating a Pretreatment Method
Figure 214. Save Pretreatment File As dialog box
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Creating a Prep Only Method
Creating a Prep Only
Method
To create a method that can run without triggering an injection or data
acquisition, you must select the Prep Only Method check box for every
Surveyor module of your instrument.
In a “prep only method”, the mobile phase conditions are maintained at the
initial settings in the pump program. No injection and no data acquisition
occurs. If the instrument setup page for the autosampler specifies timed
events they are not performed.
To create a prep only method
1. From the Instrument window, choose File > Method > Open.
2. From the Open Method File dialog box, select the method that you
created while performing “Creating an Acquisition Method” on page 91
or a method that contains the pump program that you want to use.
3. From the Instrument window, choose Method > Instrument Setup.
4. Click the Surveyor LC Pump tab, and then select the Prep Only
Method check box. See Figure 215.
Figure 215. Prep only method for the Surveyor LC Pump
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Creating a Prep Only Method
5. Click the tab for the detector, and then, select the Prep Only Method
check box. See Figure 216.
Figure 216. Prep only method for the Surveyor PDA Detector
6. Click the Surveyor AS tab, and then select the Prep Only Method
check box. See Figure 217.
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Creating a Prep Only Method
Figure 217. Prep only method for the Surveyor Autosampler
7. Choose File > Method > Save As to display the Save Method File As
dialog box.
8. Type PrepOnly in the File Name box, and then click Save.
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Using a Pretreatment
Method
Diluting Samples with a Pretreatment Method
Using a Pretreatment Method
Pretreatment methods are separate from data acquisition methods. If you
run a pretreatment method with a “prep only” method, the autosampler
does not make an injection.
To dilute the 0.5% toluene solution with methanol
1. Transfer the contents of an ampule of Autosampler Test Mix to a
standard 1.8 mL vial.
2. Place the vial into vial location A1 of a standard tray.
3. Place an empty vial into vial location A2 of a standard tray.
4. Open the method named PrepOnly.met.
5. Click the Single Run button to open the Single Run Acquisition
dialog box.
6. Keep all parameters in the Single Run Acquisition dialog box set to the
default settings except those that are listed in the following table and
shown in Figure 218.
The active method PrepOnly.met is listed in the Method box.
Parameter
Setting
Result
PrepOnly.met
Downloads this method
Run Information
Method
Autosampler
Use Program
Enables the selection of a pretreatment method
Program File
Ten Fold
Dilution.ape
Specifies that the autosampler will perform the
Ten Fold Dilution.ape pretreatment method
Vial
A;1
Specifies the sample location
Injection Volume
1
No injection occurs. However this field requires a
minimum value of 0.1 μL.
7. Click Run.
The run starts. The autosampler performs the pretreatment method, but
does not make an injection.
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Using a Pretreatment Method
Figure 218. Single Run Acquisition dialog box, showing the parameters for creating a diluted sample
Table 10. Steps that occur during pretreatment method
260
Step
Action
Transfer From Sample To
Sample
The autosampler draws 50 μL of sample from vial location A1
(current), and then deposits the sample in vial location A2
(current + 1).
Transfer From Reservoir To
Sample
The autosampler draws 225 μL of diluent from reservoir vial
1, and then deposits this solvent in vial location A2
(current + 1).
Transfer From Reservoir To
Sample
The autosampler draws 225 μL of diluent from reservoir vial
1, and then deposits this solvent in vial location A2
(current + 1).
Wash Needle
The needle moves to the wash station where it expels 400 μL
of solvent from the wash bottle. The autosampler initializes
the concentric syringe.
Mix At Sample
The needle moves to vial location A2 where it aspirates and
expels 250 μL of sample solution 10 times.
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Diluting Samples with a Pretreatment Method
Using a Pretreatment Method
To inject the diluted sample
1. Click the Single Run button to open the Single Run Acquisition
dialog box.
2. Keep all parameters in the Single Run Acquisition dialog box set to the
default settings except those that are listed in the following table and
shown in Figure 219.
Parameter
Setting
Result
Sample ID
0.05% Toluene in Methanol
Gives the data file a sample ID,
which can be used in the search
feature within ChromQuest
Method
Test Mix.met
Downloads this method
Data Path
Drive:\ChromQuest\
Projects\Tutorial\Data
Stores the data file in this folder.
Data File
Diluted Sample.dat
The data file will be named Diluted
Sample.dat
Run Information
Print Method
Report
Specifies that the custom report will
be printed after the data file is
acquired.
Autosampler
Use Program
Specifies that the autosampler will
not perform a pretreatment method
Vial
A;2
Specifies that the autosampler
withdraws sample from
vial location A2.
Injection Volume
If you are using a 5 cm
LightPipe flow cell, enter a
value of 1.
Specifies that the autosampler will
inject 1 μL or 5 μL, respectively, of
sample into the mobile phase
stream.
If you are using a standard
1 cm flow cell, enter a value
of 5.
3. Click Run.
The run starts. The autosampler does not perform a pretreatment
method because you have removed the method from the Program File
box. Compare the custom reports for data files Preliminary Run.dat and
Diluted Sample.dat. The area of the toluene peak in the diluted sample
will be approximately one-tenth the area for the toluene in the original
sample.
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Using a Pretreatment Method
Figure 219. Single Run Acquisition dialog box, showing parameters for injecting the diluted sample
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Pretreatment Rules
Diluting Samples with a Pretreatment Method
Pretreatment Rules
The maximum volume that can be deposited, drawn, or transferred during a
pretreatment step depends on the syringe type, the function requested, and
the previous step in the pretreatment method.
For the concentric syringes (see Figure 220), the manner in which the
various pretreatment functions can be linked together is affected by whether
the function takes place in only the large bore of the syringe, only the small
bore of the syringe, or in either bore of the syringe. The pretreatment
functions can be divided into three groups depending on whether they use
only the small bore of the syringe, only the large bore of the syringe, or
either bore of the syringe. See Table 11.
Table 11. Functions Grouped According to Bore Usage
Small Bore Only
Large Bore Only
Small or Large Bore
Draw from Sample
Mix at Sample
Draw from Reservoir
Transfer from Sample to Sample
Mix at Reservoir
Deposit Liquid in Sample
Transfer from Sample to Reservoir
Wash Needle
Deposit Sample in Reservoir
Flush to Waste
Transfer from Reservoir to Sample
Transfer from Reservoir to
Reservoir
Inner Plunger
Outer Plunger
Figure 220. Concentric syringe, showing inner and outer plungers
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Diluting Samples with a Pretreatment Method
Pretreatment Rules
If you are using a concentric syringe, the arrangement of the functions in the
pretreatment table is restricted by the following four rules:
Note You cannot save a method that does not conform to these rules.
1. For functions that are performed with the small bore of the concentric
syringe, the maximum volume (sample + air bubble) that you can draw,
deposit, or transfer is limited to the nominal size of the syringe.
2. Functions performed with the large bore of the concentric syringe, the
maximum volume (sample + air bubble) that you can draw, deposit, or
transfer is limited to 500 μL. The volume range for the Wash Needle
and Flush to Waste functions is 100 to 6000 μL. For flush or wash
volumes greater than 539 μL, multiple draws are performed.
3. If a function can be performed from either the small or the large bore:
•
The concentric syringe uses the small bore if the requested volume
(liquid + air bubble) plus any volume left in the needle tubing from
a previous step is less than the nominal syringe size.
•
The syringe uses the large bore if the requested volume (liquid + air
bubble) is greater than the nominal syringe size.
•
If the previous step started in the small bore, and the requested
volume plus any volume left in the needle tubing from the previous
step is greater than the nominal syringe size, the step will not be
allowed. Crossover between bores is not allowed.
4. You cannot add a function that requires the use of the large syringe bore
immediately following a function that requires the use of the small
syringe bore. To switch from the small bore to the large bore of the
syringe, you must insert a Flush to Waste step or a Wash Needle step.
These functions initialize the plungers of the syringe.
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Appendix A
Calibration Procedures
The modules of your Surveyor Plus instrument are factory calibrated. The
PDA detector is tested for linearity, noise, and drift. The LC pump is
calibrated to produce an accurate flow rate while pumping water at
1 mL/min. Its ability to accurately proportion binary mobile phases is also
tested at a flow rate of 1 mL/min. The column oven and the tray
temperature zones of the autosampler are calibrated at 30 °C. The
positioning of its XYZ arm mechanism is calibrated for the carrier trays
supplied by Thermo Fisher Scientific.
Because of the sensitivity of its optical bench, it is best to recalibrate the
Surveyor PDA Plus Detector after you install it, each time you move it,
change its flow cell, or replace either of the lamps. In addition, you might
need increase or decrease the amount of incident light reaching the diode
array by adjusting the attenuators. Over time, the performance of the lamps
deteriorates. Using the built-in holmium oxide filter, you can monitor the
performance of the lamps and establish an appropriate lamp replacement
schedule.
Perform the validation procedure for the Surveyor RI Plus Detector after
you install it and periodically thereafter. The validation procedure for the RI
detector verifies the full-scale range of the detector based on the detector’s
response to a 0.35% by weight sucrose / water solution. In addition, the
software contains procedures for verifying the analog output signals so that
you can use a chart recorder or an integrator to collect data.
The Surveyor Autosampler Plus does not require calibration upon arrival at
its shipping destination. However, if you plan to use custom vials or custom
microplates, you must perform the “Bottom Distance Calibration” on
page 295 to determine their depth. If problems occur with the column
oven, the tray temperature control, or the arm positioning of autosampler,
contact a Thermo Fisher Scientific service representative. Procedures for
calibrating the temperature of the column oven and tray compartments are
included in this appendix for those users who prefer to maintain their own
instruments.
Like the autosampler, the Surveyor LC Pump Plus does not require
calibration upon arrival at its shipping destination. However, over time, the
pressure readings from its built-in pressure transducer can drift, thereby
requiring re-zeroing.
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This appendix contains the following sections:
• Verifying the Performance of the PDA Detector
• Calibrating the Autosampler
• Calibrating the LC Pump
• Calibrating the RI Detector
Verifying the
Performance of the
PDA Detector
This section contains the following procedures:
• Adjusting the Light Intensity
• Performing a Wavelength Calibration
• Performing an Array Calibration
• Checking the Status of the Lamps
Adjusting the Light
Intensity
The Surveyor PDA Plus Detector has two attenuators that control the light
output from the lamps. During the lifetime of the Surveyor FL Plus
Detector it might be necessary to adjust the attenuators to increase or
decrease the amount of light falling onto the array.
Decreasing light output increases baseline noise. Increasing light output can
cause saturation of the diode array. If the array is saturated the response from
the Surveyor FL Plus Detector will be a flat baseline.
The attenuators require adjustment when either lamp is replaced or when
the flowcell is replaced. When any of these items are replaced, check the
light intensity by following the Operational Verification procedure and
adjust the attenuators to provide light intensities in the specified operating
ranges.
Note Before you adjust the attenuators, replace the column with a flow
restrictor, and set the pump to deliver HPLC-grade water at a flow rate
of 1 mL/min through the flowcell.
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To adjust the light output from the lamps, follow these procedures:
1. Turning on the Lamps
2. Setting the Parameters for the Spectral Display
3. Determining the Diode of Maximum Intensity for the UV Range
4. Determining the Diode of Maximum Intensity for the Visible Range
5. Setting the Discrete Channel Displays
6. Adjusting the Attenuators
Turning on the Lamps
To turn on the lamps
1. From the Windows XP taskbar, choose Start > All Programs >
Chromatography > ChromQuest to open ChromQuest.
2. In the ChromQuest Main Menu, double-click the icon for your
instrument to open the online Instrument window.
3. From the menu bar, choose Control > Instrument Status to open the
Instrument Status window.
4. Click the Surveyor PDA Plus tab to open the Surveyor PDA Plus
Instrument Status page.
5. Note the usage hours for each lamp.
6. Verify that both lamps are On. If they are not On, click On for both
lamps.
Setting the Parameters for the
Spectral Display
To set the parameters for the spectral display of the lamp intensities
1. From the Instrument Status page for the Surveyor PDA Plus, click
Diagnostics.
2. Click the Control tab.
3. Under Mode, select the Intensity option.
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4. Click Default, and then verify that the following parameters are
specified:
Start = 2
End = 511
Step = 1
Bandwidth = 1
Scan rate = 1
5. Click Load To Detector.
A dialog box containing the following message “Method Has Been
Downloaded” appears.
6. Click OK.
Determining the Diode of
Maximum Intensity for the UV
Range
To determine the diode of maximum intensity for the deuterium lamp (UV
range)
1. Click the Display tab to open the Display page.
2. Click Start located in the upper right corner of the Display page.
3. Right-click in the top pane containing the intensity spectrum and
choose Axis Setup.
The Axis Properties dialog box, shown in Figure 221, appears.
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Figure 221. Axis Properties dialog box
4. In the Axis Properties dialog box, do the following:
a. Select X-axis from the list.
b. Select the Use this range option.
c. Type a Min value of 30 and a Max value of 50 for the diode range.
d. Click OK to close the dialog box and update the scaling of the
Spectrum view.
5. From the spectrum displayed, determine and record the pixel of
maximum intensity within the 30 to 50 diode range. This is the diode of
maximum output for the deuterium lamp.
Determining the Diode of
Maximum Intensity for the
Visible Range
To determine the diode of maximum intensity for the visible range
1. Open the Axis Setup dialog box.
2. Type a a Min value of 400 and a Max value of 500 for the diode range.
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3. Click OK to close the dialog box and update the scaling of the Spectrum
view.
4. In the spectrum displayed, determine and record the pixel of maximum
intensity within the 400 to 500 diode range. This is the diode of
maximum output for the tungsten lamp.
5. Turn off the data stream by clicking Off for Status and Stop for Data.
Setting the Discrete Channel
Displays
To set the discrete channel displays
1. Click the Control tab to open the Control page.
2. Type the value for the diode of maximum intensity for the deuterium
lamp in the Channel A box.
3. Type the value for the diode of maximum intensity for the tungsten
lamp in the Channel C box.
4. Click Load To Detector.
A dialog box containing the following message “Method Has Been
Downloaded” appears.
5. Click OK.
Adjusting the Attenuators
To adjust the attenuators
1. Open the front doors of the detector.
2. Unscrew the captive screw, and then remove the flowcell cover.
The attenuators are located on the right side of the front panel. There
are two black tabs attached to the attenuators for manual adjustments.
See Figure 222.
The left tab is the deuterium lamp attenuator and the right tab is the
tungsten lamp attenuator. Pushing the attenuator tab up increases light
output and pulling the tab down decreases light output.
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Deuterium
Attenuator
Tungsten
Attenuator
Figure 222. Front view of the detector, showing the attenuators
3. Click the Display tab to open the Display page (see Figure 223)
4. Adjust the attenuators:
a. Adjust the attenuator with the left tab on the PDA (UV
attenuation) to achieve a Channel A value of between 750000 and
775,000 intensity counts.
b. Adjust the attenuator with the right tab (Visible attenuation) to
achieve a Channel C value of between 750000 and 775000
intensity counts.
5. After you finish adjusting the attenuators, replace the flowcell access
cover and close the front doors of the detector.
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Figure 223. Diagnostics dialog box - Display page, showing adjustment of Discrete Channels A and B
Performing a Wavelength
Calibration
The alignment of the spectrum on the diode array is dependent upon the
physical alignment of various components of the optical bench. The
alignment can become offset if the detector is sharply jolted, for example, in
shipping. ChromQuest allows you to determine the wavelength accuracy of
the optical bench and to mathematically correct for any alignment changes,
if necessary. We recommend that you perform a wavelength calibration
whenever you move the detector or download new firmware.
To perform a wavelength calibration
1. Prepare the detector for a wavelength calibration:
a. Replace the column with a flow restrictor.
b. Pump HPLC- grade water or HPLC-grade methanol through the
flow cell for at least one hour.
c. Turn on both lamps by clicking the On buttons for the lamps in the
Surveyor PDA Plus page of the Instrument Status window.
d. Wait 1 hour for the D2 lamp to equilibrate.
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Lamp On/Off
Buttons
Figure 224. ChromQuest Instrument Status dialog box, showing the Surveyor PDA Plus page
2. In the Surveyor PDA Plus Instrument Status page, click Diagnostics to
open the Surveyor PDA Plus Diagnostics dialog box.
3. Click the Calibration tab to open the Calibration page. See Figure 225.
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Figure 225. PDA Plus Diagnostics dialog box - Calibration page
4. Open a Wavelength Calibration file:
a. Under Wavelength File, click Open to open the Select Wavelength
Calibration dialog box.
b. In the Select Wavelength Calibration dialog box, shown in
Figure 226, select an appropriate wavelength calibration file from
the list. An appropriate wavelength file should include the range of
wavelengths that you use under normal operation conditions.
c. Click Open.
Note ChromQuest has four calibration files to choose from.
The HolmiumUV file contains five wavelengths in the UV
region while the other files, such as Holmium12, use sets of
wavelengths from both the UV and Visible wavelength regions.
The holmium oxide absorbance maxima are selected from a
spectrum published in “Holmium Oxide Solution Wavelength
Standard from 240 to 640 nm - SRM 2034 (NIST Special
Publication 260-54)”.
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The holmium oxide bands of the selected file are displayed in the
Wavelength File area, as shown in Figure 227.
Figure 226. Select Wavelength Calibration dialog box
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Figure 227. Diagnostics dialog box - Calibration page, showing the Holmium12
Wavelength Calibration File selected
5. In the Wavelength area, click Execute.
A message box opens to remind you of all of the required preconditions.
See Figure 228.
Figure 228. Calibration Preconditions message box
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6. If you have met the preconditions, click OK in the message box.
ChromQuest collects a background spectrum, which is used to remove
the absorbance contribution of the mobile phase. When the background
collection is complete, the message box, shown in Figure 229, appears.
Figure 229. Calibration message box
7. Move the filter wheel to position 2 as directed, and then click OK in the
message box.
The detector takes a holmium oxide scan, performs iterative calculations
while applying the rise time and bandwidth filters, and then displays a
new message box. See Figure 230.
Figure 230. Calibration Complete message box
8. Move the filter wheel back to position 1, and then click OK to close the
message box and view the results.
9. Check the delta values in the Wavelength File area (see Figure 231).
a. If the delta values are not within the range of ±1 nm, repeat the
wavelength calibration procedure for verification.
b. If, after applying a new calibration, the delta values are still not
within the range of ±1 nm, call your Thermo Fisher Scientific
service representative for assistance.
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10. If your data system PC is connected to a printer, click Print to print a
hardcopy report. To store the results, click Export.
The date and time of calibration are displayed and are stored in memory.
Tip You can click Cancel in any of the Calibration dialog boxes at any time
to abort the calibration process.
Figure 231. Calibration dialog box, showing acceptable Delta values
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Performing an Array
Calibration
The function of the array calibration is to measure and correct for the dark
current produced by the diodes of the photodiode array. The dark current is
the small amount of background signal that is produced by the diodes of the
array even when both lamps are turned off. Typical dark current values range
from 1500 to 3000 counts.
Because the environmental conditions of your laboratory can cause the dark
current of the diode array to increase over time, we recommend that you
perform an array calibration (dark current) after 100 hours of use or
monthly, whichever comes first; whenever a significant temperature change
occurs; after you move the detector; after you replace the lamps; and after
you download a new firmware file.
Because the dark current produced by the diodes rises as the temperature
within the detector rises, it is important to warm up the lamps for 1 hour
before you perform a dark current calibration. Warming up the lamps for
1 hour allows your detector to equilibrate to its normal operating
temperature.
ChromQuest briefly turns the lamps off as it performs the dark current
calibration routine. After it completes the dark current calibration routine,
ChromQuest turns the lamps back on.
Note The dark current calibration program will not run when data
collection is enabled on the Display page.
To perform a dark current calibration of the diode array
1. Pump HPLC grade water or methanol at 1 mL/min through the flow
cell for at least one hour.
2. Place the filter wheel of the Surveyor FL Plus Detector in position 1
(Open).
3. Turn on both lamps and wait 1 hour for the D2 lamp to equilibrate:
a. From the online Instrument window, choose Control > Instrument
Status to display the Instrument Status window.
b. Click the Surveyor PDA Plus tab to display the Surveyor PDA Plus
page. See Figure 224.
c. Click the D2 (deuterium) Lamp On button and the W (tungsten)
Lamp On button.
This allows the detector to reach its normal operating temperature.
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4. Click Diagnostics to open the Surveyor PDA Plus Diagnostics dialog
box. Then click the Calibration tab to display the Calibration page. See
Figure 227 on page 276.
5. In the Array area, click Execute.
A message box opens to remind you of the necessary preconditions. See
Figure 232.
Figure 232. Calibration Preconditions message box
6. If you have met the preconditions, click OK.
The status of the calibration procedure is displayed by the Status
readback area on the Calibration page. During the dark current
calibration, intensity scans are collected with both lamps Off. After the
last event is completed, the lamps are turned back On.
7. Click OK to finish the calibration.
The date and time of calibration are displayed and are stored in
memory.
Checking the Status of the
Lamps
The performance of the deuterium lamp degrades over time, whereas the
tungsten lamp tends to fail abruptly. As the performance of the deuterium
lamp degrades, you might notice a decrease in the signal to noise ratio for
the UV range. Replace the deuterium lamp after 1000 h of usage or as
needed. Replace the tungsten lamp after 2500 h of usage or as needed.
You can monitor the status of the lamps from the Diagnostics dialog box –
Lamps page.
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This topic contains the following procedures:
• Viewing an Intensity Scan of Both Lamps Together
• Viewing an Intensity Scan of the Deuterium Lamp
• Viewing an Intensity Scan of the Tungsten Lamp
• Recording the Performance of the Lamps
Viewing an Intensity Scan of
Both Lamps Together
To view an intensity scan of the deuterium (D2) and tungsten (W) lamps
1. If they are not already On, turn on both lamps and wait 1 h for the D2
lamp to equilibrate:
a. From the online Instrument window, choose Control > Instrument
Status to display the Instrument Status window.
b. Click the Surveyor PDA Plus tab to display the Surveyor PDA Plus
page. See Figure 224.
c. Click the D2 (deuterium) Lamp On button and the W (tungsten)
Lamp On button.
d. Note the status and usage for each lamp.
2. Click Diagnostics to open the Surveyor PDA Diagnostics dialog box.
3. Open the Control page by clicking the Control tab. Then, set the
parameters for viewing an intensity scan for the both lamps:
a. Select the Intensity option in the Mode area to set the units to
Intensity.
b. Click Default.
c. In the Discrete Channels area, set Channels A, B, and C to monitor
diodes 94, 177, and 260, respectively.
d. Click Load To Detector.
Note These diodes roughly correspond to the wavelengths of
300 nm, 400 nm, and 500 nm, respectively. The Discrete
Channels area on the Display page contains a digital readout for
the selected diodes. See Figure 233. Record these readout values
to track the performance of the deuterium lamp. You can also
select other diode values to track the performance of the lamps .
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4. Open the Display page, and click Start to refresh the display. See
Figure 233.
5. Save a printout or an electronic copy of the spectrum. Date the printout
and add it to your maintenance records. See “Recording the
Performance of the Lamps” on page 286.
Figure 233. Diagnostics dialog box - Display page, showing an intensity scan with both lamps
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Viewing an Intensity Scan of the
Deuterium Lamp
To view an intensity scan of the deuterium (D2) lamp
1. If it is not already On, turn on the deuterium lamp and wait 1 hour for
it to equilibrate:
a. From the online Instrument window, choose Control > Instrument
Status to display the Instrument Status window.
b. Click the Surveyor PDA Plus tab to display the Surveyor PDA Plus
page. See Figure 224.
c. Click the D2 (deuterium) Lamp On button.
d. Note the status and usage for the deuterium lamp.
2. Ensure that the W (tungsten) lamp is Off.
3. Click Diagnostics to open the Surveyor PDA Plus Diagnostics
dialog box.
4. Open the Control page by clicking the Control tab. Then, set the
parameters for viewing an intensity scan for the deuterium lamp:
a. Select the Intensity option in the Mode area to set the units to
Intensity.
b. Click Default.
c. In the Discrete Channels area, set Channels A, B, and C to monitor
diodes 35, 52, and 77 respectively. See Figure 234.
Note These diodes roughly correspond to the wavelengths of
230 nm, 250 nm, and 280 nm, respectively. The Discrete
Channels area on the Display page contains a digital readout for
the selected diodes. See Figure 234. Record these readout values
to track the performance of the deuterium lamp. You can also
select other diode values to track the performance of the
deuterium lamp.
d. Click Load To Detector.
This will allow you to view the emission intensity spectrum of the
deuterium lamp.
5. Open the Display page and click Start in the Data area to refresh the
display. See Figure 234.
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6. Save a printout or an electronic copy of the spectrum. Date the printout
and add it to your maintenance records. Compare this scan with similar
scans that you obtain in the future to monitor any degradation in light
intensity. See “Recording the Performance of the Lamps” on page 286.
Figure 234. Diagnostics dialog box - Display page, showing an intensity scan with the deuterium lamp
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Viewing an Intensity Scan of the
Tungsten Lamp
To view an intensity scan of the tungsten (W) lamp
1. If it is not already On, turn on the Tungsten lamp:
a. From the online Instrument window, choose Control > Instrument
Status to display the Instrument Status window.
b. Click the Surveyor PDA Plus tab to display the Surveyor PDA Plus
page. See Figure 224.
c. Click the W (Tungsten) Lamp On button.
d. Note the status and usage for the Tungsten lamp.
2. Ensure that the D2 (deuterium) lamp is Off.
3. Click Diagnostics to open the Surveyor PDA Plus Diagnostics
dialog box.
4. Open the Control page and set the parameters for viewing an intensity
scan for the tungsten lamp:
a. Select the Intensity option in the Mode area to set the units to
Intensity.
b. Click Default.
c. In the Discrete Channels area, set Channels A, B, and C to monitor
diodes 219, 302, and 427, respectively.
d. Click Load To Detector.
Note These diodes roughly correspond to the wavelengths of
450 nm, 550 nm, and 699 nm, respectively. The Discrete
Channels area on the Display page contains a digital readout for
the selected diodes. See Figure 235. Record these readout values
to track the performance of the deuterium lamp. You can also
select other diode values to track the performance of the tungsten
lamp.
e. Open the Display page and click Start to refresh the display.
5. Save a copy of the scan for your maintenance records. See Figure 235.
6. Turn on the deuterium lamp again and allow sufficient warm-up time
before you begin acquiring data.
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Figure 235. Diagnostics dialog box - Display page, showing an intensity scan for the tungsten lamp
Recording the Performance of
the Lamps
There are several methods available to you to record the spectrum data on
the Display page in ChromQuest.
To store the spectral data by using the print screen key
1. As you are collecting the data stream in the Display page, press
SHIFT+PRT SCR.
2. Open Microsoft Paint and save the screen capture as a bitmap or open
Microsoft Word and paste the screen capture into a Word document.
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ChromQuest contains a print utility that allows you to print a copy of the
Spectrum window.
To use the print utility
1. In the Display page, place the cursor in the Spectrum window.
2. Right-click to open the shortcut menu.
3. Choose Utilities > Print.
The snapshot option allows you to create a Microsoft Excel Comma
Separated Values file that contains information about the spectrum in the
Display page. This file, which is overwritten each time you click the
Snapshot button, is stored in the ChromQuest directory and is named
WaveData.csv. The file contains three columns: diode number, wavelength,
and intensity value.
To take a snapshot
1. In the Display page, stop the data stream by clicking Stop in the Data
area.
2. Click Snapshot.
3. Using Explorer, browse to the ChromQuest directory.
4. Click the WaveData.csv file. Microsoft Excel opens.
5. In Microsoft Excel, save the file with an appropriate name.
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Calibrating the Autosampler
Calibrating the
Autosampler
There are four calibration options for the Surveyor Autosampler Plus:
column oven calibration, tray calibration, arm calibration, and bottom
distance calibration.
If you are using custom vials or custom microplates, you must perform the
the bottom distance calibration. The temperature calibration procedures for
the column oven and tray compartments are typically performed by a
Thermo Fisher Scientific service representative. If you want to perform these
temperature calibrations, you must order the field service calibration tool kit
which contains a customized temperature probe for the Surveyor
Autosampler Plus. The XYZ arm calibration is best performed at the factory
and is therefore not described in this section.
This section contains the following topics, which describe your calibration
options:
• Column Oven Calibration
• Tray Calibration
• Bottom Distance Calibration
Column Oven Calibration
The column oven calibration wizard is used calibrate the column oven
temperature by using an external temperature sensor.
To perform this calibration, you must install the Oven Sensor Test Fixture
that is contained in the field service calibration tool kit, and then open the
Column Oven Calibration wizard in ChromQuest.
Note The calibration of the temperature sensor for the column oven is
performed by a Thermo Fisher Scientific service representative. To
perform this calibration yourself, you need to order the Field Service
Calibration Kit (P/N 60053-62001).
This topic contains the following procedures:
• Installing the Oven Sensor Test Fixture
• Performing a Column Oven Calibration
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Installing the Oven Sensor Test
Fixture
To install the Oven Sensor Test Fixture
1. Open the oven door, and then loosen the top thumbscrew that holds the
column clamp.
2. With the sensor facing down, slide the metal cable protector under the
right side of the clamp.
3. Verify that the sensor is between the upper and lower column clamps
and that it is not touching any metal.
4. Tighten the thumbscrew on the sensor, route the cable of the sensor so
as not to interfere with the door, and then close the column oven door.
Performing a Column Oven
Calibration
Before you open the Column Oven Calibration Wizard, install the Oven
Sensor Test Fixture as described above in “Installing the Oven Sensor Test
Fixture” on page 289.
To calibrate your column oven
1. Open the Column Oven Calibration Wizard:
a. From the Instrument window, choose Control > Instrument
Status.
b. Click the Surveyor AS tab to display the Surveyor AS page. See
Figure 236.
Figure 236. Instrument Status window – Surveyor AS page
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c. Click Diagnostics to open the Diagnostics dialog box.
d. Click the Calibration tab, and then select the Column Oven
Calibration option to open the first page of the Column Oven
Calibration wizard, shown in Figure 237.
Figure 237. Column Oven calibration option - page 1
2. Size the Diagnostics dialog box so that you can see the Temp actual
readout in the Column Oven area of the Instrument Status window –
Surveyor Autosampler page (see Figure 236).
3. In the Oven area of the Column Oven Calibration wizard, type 30 in
the box, and then click Set Temp.
The second page of the Column Oven Calibration wizard appears. See
Figure 238.
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Figure 238. Column Oven Calibration Wizard – page 2
4. In the Instrument Status window – Surveyor AS page – Column Oven
area, verify that the Temp actual readout is moving towards the set point
of 30.0 °C (Figure 236).
5. When the Temp actual readout reaches exactly 30.0 °C, type the reading
from the 869C thermometer in the Oven Calibration box of the
Column Oven Calibration wizard (Figure 238), and then click
Calibrate.
Repeat steps 4 and 5 until the readout on the 869C thermometer and
the Temp actual readout in the Column Oven area of the Instrument
Status window - Surveyor AS are in agreement (± 0.2 °C)
6. After the Temp actual readout and the 869C thermometer reach
agreement, click Done to exit the Column Oven Calibration wizard.
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Tray Calibration
The Tray Calibration wizard is used to calibrate the temperature of the tray
compartment by using an external temperature sensor. The Field Service
Calibration kit (P/N 60053-62001) that you need to perform this
calibration includes the following items:
• Calibrated Omega 869C RTD thermometer
• Surveyor A/S Vial Tray Sensor
Note The calibration of the tray temperature compartment is
performed by a Thermo Fisher Scientific service representative. To
perform this calibration, you need to order the Field Service
Calibration Kit (P/N 60053-62001).
This topic contains the following procedures:
• Installing the Tray Temperature Sensor
• Performing a Tray Calibration
Installing the Tray Temperature
Sensor
To install the tray temperature sensor
1. Open the door to the tray compartment.
2. Install the tray temperature sensor, which is a standard tray with a
temperature sensor potted in a middle vial location of the tray, into
location E of the tray compartment.
3. Route the cable of the sensor through the notch at the top on the tray
compartment so that it does not interfere with the door closure.
4. Close the door to the tray compartment.
Performing a Tray Calibration
Before you open the Tray Calibration Wizard, install the Tray Temperature
Test Fixture as described above in “Installing the Tray Temperature Sensor”
on page 292.
To perform a tray calibration
1. Open the Tray Calibration wizard:
a. From the Instrument Setup window, choose Control > Instrument
Status to open the Instrument Status window.
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b. Click the Surveyor AS tab to open the status page for the Surveyor
Autosampler.
c. Click Diagnostics at the bottom of the Instrument Status –
Surveyor AS page to open the Diagnostics dialog box for the
Surveyor Autosampler.
d. Click the Calibration tab to open the Calibration page.
e. Select the Tray Calibration option to open the first page of the Tray
Calibration wizard, shown in Figure 239.
Figure 239. Tray Calibration wizard – page one
2. Size the Diagnostics dialog box so that you can see the Temp actual
readout in the Instrument Status window – Surveyor AS page (see
Figure 236).
3. In the Tray Calibration wizard – Tray Calibration area, type a value of
30 in the box, and then click the Set Temp button.
The second page of the Tray Calibration wizard, shown in Figure 240
appears.
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Figure 240. Tray Calibration wizard – page two
4. In the Tray area of the Instrument Status window – Surveyor AS page,
verify that the Temp actual readout is moving towards the set point of
30.0 °C. See Figure 236.
5. When the Temp actual readout reaches exactly 30.0 °C, type the reading
from the 869C RTD thermometer in the Tray Calibration box, and
then click Calibrate.
6. Repeat step 4 and step 5 until the readout on the 869C RTD
thermometer and the current temperature readout in the Tray area are in
agreement (± 0.2 °C)
7. After the Temp actual readout in the Tray area of the Instrument Status
window – Surveyor AS page and the 869C RTD thermometer reach
agreement, click Done to exit the Tray Calibration wizard.
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Bottom Distance
Calibration
You use the Bottom Distance wizard to calibrate the distance that the needle
must travel to reach the bottom of a vial or well. The XYZ arm uses this
value when you select one of the custom tray configurations.
Note Because the autosampler stores only one value for the custom
well bottom distance, you must perform the bottom distance
calibration procedure each time you select a new custom tray type
configuration and each time you use a different type of custom vial or
custom microplate.
To perform a bottom distance calibration
1. Open the Bottom Distance Calibration wizard:
a. In the Surveyor AS – Diagnostics dialog box, click the Calibration
tab to open the Calibration page.
b. Select the Bottom Distance option to open the first page of the
Bottom Distance wizard. See Figure 241.
Note The Surveyor Autosampler stores only one bottom distance
value for custom tray configurations. The number at the top of
this dialog box is the current value for the distance.
Figure 241. Bottom Distance Calibration wizard – page one
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2. Do one of the following:
• To enter a previously determined value for the bottom distance,
continue at step 3.
• To perform an active calibration using the custom vial or well,
continue at step 4.
3. To enter a previously determined value for the bottom distance:
a. Select the Enter the Value Manually option (see Figure 241), and
then click Next.
The Bottom Distance Manual Entry area, shown in Figure 242,
appears.
b. Type in a value in microns in the Enter Value box.
The allowable values are 15000 to 46990 (15 mm to 46.99 mm)
c. Click Calibrate in the Bottom Distance Manual Entry area to
upload this value into the persistent memory of the autosampler.
The autosampler will use this bottom distance value for all custom
configurations.
d. Click Done to exit the Bottom Distance Calibration wizard.
Figure 242. Calibration page, showing the Bottom Distance Manual Entry pane
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4. To perform an active calibration:
a. Select the Calibrate to Find Value option to perform an active
calibration, and then click Next.
The Bottom Distance pane, shown in Figure 243, appears.
Caution Remove vial caps or microtitre plate lids before
performing an active bottom distance calibration. As the needle
pierces a vial cap or a microtitre plate lid, the spring in the needle
mechanism is compressed, which can cause premature activation
of the needle sensor.
b. Type a vial or well position in the Calibrate box, and then click
Calibrate.
The XYZ arm moves to the selected vial or well position. The needle
descends until it senses the bottom of the vial or well. After the
autosampler has determined the bottom distance of your selection,
the current distance value appears in the Bottom Distance pane.
c. Click Done to exit the Bottom Distance Calibration wizard.
Figure 243. Calibration page, showing Bottom Distance (active calibration) pane
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Calibrating the LC
Pump
There are two calibration options for the Surveyor LC Pump Plus that affect
its performance. These are the alpha setting, which affects the flow rate, and
the compressibility setting, which affects the pressure pulsation of the
system. The compressibility setting is set to that for water, and then the
alpha setting is factory calibrated to produce an accurate flow rate for water.
If you are pumping solvents other than water and your application is
sensitive to pressure pulsation, you might want to optimize the
compressibility setting. Changing the compressibility setting will affect the
accuracy of the flow rate. Therefore, after you optimize the compressibility
setting to minimize the pressure pulsation of your system, you must check
the accuracy of the flow rate. If adjusting the compressibility setting has
affected the accuracy of your flow rate, you must adjust the alpha setting.
In addition to the alpha setting and the compressibility setting, the pressure
transducer that is attached to the back of the purge manifold assembly is
also factory calibrated. If you replace the pressure transducer, you will need
to update the pressure sensor adjustment setting. Even if you never need to
replace the pressure sensor, its readout tends to drift under normal usage.
Therefore, you need to occasionally re-zero its output.
This topic contains the following subtopics:
• Calibration Options
• Calibration Procedures
Calibration Options
The Surveyor LC Pump Plus has the following calibration options:
• Compressibility
• Pressure Recorder Full Scale
• Flow Rate Adjustment (Alpha)
• Pressure Sensor Adjustment
• Pressure Transducer Zero
Compressibility
298
The compressibility of a liquid is a measure of its resistance to a decrease in
volume caused by an increase in pressure. Compared to gases, most liquids
are relatively incompressible. Water, a commonly used mobile phase solvent,
is even less compressible than most organic solvents because of its extensive
hydrogen bonding and cluster structure.
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Effect of the Compressibility Setting
Even though liquids are relatively incompressible, the compressibility of
your mobile phase can affect your chromatography if you are running the
LC pump at the upper limit of its pressure range. The compressibility
setting of the Surveyor LC Pump Plus allows you to compensate for the
compressibility of your mobile phase. Changing the compressibility setting
simultaneously affects the pressure pulsation of the pump and flow rate of
the mobile phase.
The compressibility effect is caused by the portion of the cam cycle in which
the pistons are compressing the mobile phase. The compressibility portion
of the cam cycle extends from the time at which the primary piston starts
discharging until the time at which the secondary piston reaches full
intake—approximately 80° of the cam cycle.
To minimize the pressure pulsation and maintain a constant flow rate, the
on-board CPU compensates for this compression by making fine
adjustments to the speed of the stepping motor. If the compressibility
setting is incorrect, the LC pump will not be able to effectively minimize the
pressure pulsation.
Figure 244 shows the effect of the compressibility setting on the pressure
pulsation. The pressure trace was recorded as water was pumped at a flow
rate of 3 mL/min. Three runs were recorded and the compressibility setting
was changed between the runs. The compressibility settings for the three
runs were 0.45 GPa-1, 1.25 GPa-1, and 3.0 GPa-1.
Figure 244. Overlaid LC pump pressure traces, showing the effect of the compressibility setting
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Table 12 shows a comparison of water pumped at three different
compressibility settings. The correct compressibility setting for water is
0.45 GPa-1. The pressure pulsation as well as the actual flow rate increased
as the compressibility setting increased.
Table 12. Measured flow rate and pressure pulsation vs. compressibility setting
Compressibility
Setting
%Pressure Pulsation
Actual Flow Rate
(mL/min)
% Rel Diff
0.45 GPa-1
± 0.2%
3.01
+ 0.3%
1.25 GPa-1
± 0.4%
3.05
+ 1.8%
± 1%
3.18
+ 5.9%
3.0 GPa
-1
Compressibility Ratios for Common Solvents
Most mobile phases are made up of a mixture of solvents. Therefore, you
need to determine the correct compressibility setting for a given mobile
phase empirically. Table 13 lists the compressibility ratios for a few common
solvents.
Table 13. Compressibility ratios for common solvents
Pressure Recorder Full Scale
Solvent
Compressibility Ratio (GPa-1)
Water
0.45
Acetonitrile
1.20
Methanol
1.25
Hexane
1.60
The pressure recorder full-scale calibration option allows you to set the
scaling factor for the pressure trace if you choose to monitor the pressure
with a chart recorder. The pressure recorder terminals are located on the
back of the Surveyor LC Pump Plus. See Figure 245. The allowable range is
integer values from 1 to 10.
Full scale = Set value × 4.9 MPa (50 kgf/cm2) (full scale voltage = 1 mV)
Examples:
When the set value = 1, the full scale is 4.9 MPa (50 kgf/cm2)
When the set value = 10, the full scale is 49.0 MPa (500 kgf/cm2)
4.9 MPa = 50 kgf/cm2 = 49 bar = 711 psi.
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Note This parameter is not backed up when the power is turned off.
Remove vial caps or microtitre plate lids before performing an active
bottom distance calibration. As the needle pierces a vial cap or a
microtitre plate lid, the spring in the needle mechanism is compressed,
which can cause premature activation of the needle sensor.
Pressure
Terminals
UNIT ID
ENET
NC
FW DOWNLOAD
PRESSURE PRESSURE+
NC
NC
+5V @ 15OmA
GND
PUMP ON
GND
INJECT HOLD
PUMP READY
PROG. START
NC
+5V @ 150mA
GND
Figure 245. Terminals located on the back of the Surveyor LC Pump Plus
Flow Rate Adjustment (Alpha)
The alpha value for your Surveyor LC Pump Plus is factory calibrated based
on a flow rate of 1.000 mL/min for distilled water. A sticker containing the
factory calibration value is located inside the pump on the motor casing.
You can optimize the alpha value for your application by updating the alpha
setting. The allowable range for alpha values is 1 to 9.9. If you are pumping
water, decreasing the alpha value by 1, decreases the flow rate by 1%.
Whereas, increasing the alpha value by 1, increases the flow rate by 1%.
Pressure Sensor Adjustment
Reset this parameter when the pressure sensor is replaced. Each pressure
sensor is labeled with a data sticker that contains a “0.XXXX” value for the
pressure sensor adjustment setting. Multiply this value by 10,000 and then
update the pressure sensor adjustment setting for your Surveyor LC Pump
Plus.
Pressure Transducer Zero
The pressure readout for the Surveyor LC Pump Plus is produced by a cell
type potentiometer. This type of device tends to drift by small increments.
Therefore, you will occasionally need to re-zero the readout. To update the
pressure transducer zero value, turn off the pump flow and open the drain
valve knob before clicking the pressure transducer zero button in the
software program that operates your pump.
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Calibration Procedures
The Calibration page contains the options for fine-tuning the performance
of the LC pump.
This topic contains the following procedures:
• Opening the Calibration Page
• Updating the Compressibility Setting
• Optimizing the Compressibility Setting
• Scaling the Output of the Pressure Recorder Terminals
• Updating the Flow Rate Adjustment Setting
• Updating the Pressure Sensor Adjustment Setting
• Re-zeroing the Pressure Transducer
Opening the Calibration Page
The LC pump calibration options are accessed through the Calibration page
of the Diagnostics dialog box.
Note To access the Calibration page in ChromQuest, you must have
Instrument administration privileges.
To open the Calibration page in ChromQuest
1. Turn on the power to the pump.
2. Double-click the ChromQuest icon on the Windows desktop to launch
ChromQuest. (The icon should be the same as that shown here.)
3. Double-click the Instrument button to open the online Instrument
window. (The button should be the same as that shown here.)
4. In the online Instrument Window, choose Control > Instrument
Status to open the Instrument Status window.
5. Click the Surveyor LC Pump tab to open the Instrument Status
window – Surveyor LC Pump page.
6. Click Diagnostics at the bottom of the Surveyor LC Pump page to
open the Diagnostics dialog box for the pump.
7. Click the Calibration tab to open the Calibration page, shown in
Figure 246.
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Figure 246. Surveyor LC Pump Diagnostics dialog box – Calibration page
Updating the Compressibility
Setting
If you have already determined the best compressibility setting for your
application, update the compressibility setting of the LC pump.
To update the compressibility setting in ChromQuest
1. In the Instrument window, open the Calibration page for the pump as
described in “Opening the Calibration Page” on page 302.
2. Type a new value in the Compressibility setting box, and then click
Update.
A dialog box containing a warning message appears.
3. Click OK in the dialog box to update the compressibility setting.
Optimizing the Compressibility
Setting
To optimize the compressibility setting of the LC pump
1. Add an auxiliary LC Pump pressure trace to your method:
a. Open your method by choosing File > Method > Open.
b. Click the Auxiliary trace tab in the Instrument Setup window.
c. Select the LC Pump Pressure acquire check box.
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d. Save the method by choosing File > Method > Save.
2. Download the method that contains the pressure trace by choosing
Control > Download Method.
3. Preview the run and print out the pressure trace:
a. Choose Control > Preview Run.
b. Choose Window > LC Pump Pressure.
c. Preview the run for a few minutes, and then print out the LC pump
pressure trace. To print the trace, right-click to open the
Chromatogram shortcut menu and choose Utilities > Print.
4. Stop the preview run by clicking the Stop button in the Instrument
toolbar. (The button should be the same as that shown here.)
5. Change the compressibility setting as described in “Updating the
Compressibility Setting” on page 303.
6. Repeat step 3, step 4, and step 5 until you are satisfied with the results.
7. After you determine the best compressibility setting for your
application, enter the value in the Compressibility setting box and click
Update.
Note Changing the compressibility setting will also affect the flow
rate. After you change the compressibility setting, measure the flow
rate.
Scaling the Output of the
Pressure Recorder Terminals
The pressure recorder terminals are located on the back panel of the
Surveyor LC Pump Plus.
To set the full-scale output of the pressure recorder terminals
1. Open the Calibration page for the pump as described in “Opening the
Calibration Page” on page 302.
2. Type a value from 1 to 10 in the Pressure Recorder Full-Scale box, and
then click Update.
A dialog box containing a warning message appears.
3. In the dialog box containing the warning message, click OK to update
to scaling output of the pressure recorder terminals.
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Updating the Flow Rate
Adjustment Setting
Change the flow rate adjustment setting if the flow rate is not accurate.
To update the flow rate adjustment setting in ChromQuest
1. Open the Calibration page for the pump as described in “Opening the
Calibration Page” on page 302.
2. Enter a new value in the Flow rate adjustment (alpha) box.
3. Adjust the setting:
• If the actual flow rate is below the set value, increase the alpha value.
If you are pumping water, increasing the alpha value by 1, increases
the flow rate by 1%. For example, if the pump is set to deliver a flow
rate of 1 mL/min and the actual flow rate is 0.96 mL/min, which is
4% below the set rate, raise the current setting by 4. If the current
setting is 5.0, enter a new setting of 9.0.
• If the actual flow rate is above the set value, decrease the alpha value.
If you are pumping water, decreasing the alpha value by 1, decreases
the flow rate by 1%. For example, if the pump is set to deliver a flow
rate of 1 mL/min and the actual flow rate is 1.04 mL/min, which is
4% above the set rate, lower the current setting by 4. If the current
setting is 5.0, enter a new setting of 1.0.
4. Click Update.
A dialog box containing a warning message appears.
5. In the dialog box containing the warning message, click OK to update
the alpha value setting.
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Updating the Pressure Sensor
Adjustment Setting
Reset this parameter when the pressure sensor is replaced. Each pressure
sensor is labeled with a data sticker that contains a “0.XXXX” value for the
pressure sensor adjustment setting. Multiply this value by 10,000 and enter
the result in the Pressure Sensor Adjustment setting box.
To update the pressure sensor adjustment setting
1. Open the Calibration page in ChromQuest as described in “Opening
the Calibration Page” on page 302.
2. Enter the value on the data sticker “0.XXXX” multiplied by 10,000 in
the Pressure Sensor Adjustment setting box.
3. Click Update.
A dialog box containing a warning message appears.
4. In the dialog box containing the warning message, click OK to update
the Pressure Sensor Adjustment setting.
Re-zeroing the Pressure
Transducer
The offset error of the pressure transducer is the value that is displayed when
under a particular set of conditions, such as the purge manifold knob being
set to the open position, it should be zero. You can correct for this offset
error by re-zeroing the pressure readout of the pressure transducer when the
purge manifold knob is open.
To re-zero the output of the pressure transducer from ChromQuest
1. Stop the pump flow:
a. Choose Control > Instrument Status > Pump tab.
b. Click Stop Pump.
2. Open the drain valve by gently turning it counterclockwise 180° to
ensure that the transducer is actually sensing zero system pressure. See
Figure 247.
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Figure 247. Drain valve in open position
3. Open the Calibration page for the pump as described in “Opening the
Calibration Page” on page 302.
4. Click Pressure Transducer Zero.
5. When you are finished zeroing the pressure transducer, close the drain
valve by gently turning it clockwise until you feel resistance.
Note Applying excessive force to the drain valve knob will wear out
its O-ring at a faster rate.
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Calibrating the RI
Detector
Validate the accuracy of the RI detector on a regularly scheduled basis by
using the built-in validation procedure accessed from the ChromQuest data
system.
To validate the accuracy of the detector, perform the following procedures:
1. Preparing to Perform the Validation Procedure
2. Performing the Validation Procedure
3. Restoring the RI Detector to Normal Operation
Preparing to Perform the
Validation Procedure
Before you can perform the validation procedure, you must set up your LC
system to pump HPLC grade water and you must prepare a sucrose
validation standard. To prepare for the validation procedure, perform the
following procedures:
• Preparing the Sucrose Validation Standard
• Setting Up the LC System to Validate the RI Detector
Preparing the Sucrose Validation
Standard
You inject a 0.35% by weight sucrose in water solution as part of the
validation procedure.
To prepare a fresh sucrose solution
1. Weigh out 350 mg of sucrose, and transfer quantitatively to a 100 mL
volumetric flask.
2. Add approximately 50 mL of HPLC grade water to the flask. Swirl the
flask to dissolve the sucrose, and then fill the flask to volume with water.
Setting Up the LC System to
Validate the RI Detector
Before you perform the validation procedure for the RI detector, the LC
system must be set up to pump HPLC grade water. In addition, it is a good
practice to flush the flow cell of the RI detector with HPLC grade water.
To set up the LC system for the validation procedure
1. Fill one of the solvent reservoir bottles with 100% HPLC grade water.
2. To purge the flow cell of the RI detector, follow the instructions in
“Purging the Flow Cell of the RI Detector” on page 134.
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3. Leave the program open to the RI Diagnostic page and proceed to the
next procedure.
Performing the Validation
Procedure
Use the validation procedure to validate the accuracy of the RI detector and
to ensure the optimal performance of the optical system.
To perform the validation procedure, you must have the following items
provided in the accessory kit for the RI detector:
• 10 mL syringe
• Syringe adapter
To validate the accuracy of the detector
1. Initiate the validation procedure:
a. If it is not already open, open the RI Diagnostic page.
b. In the Calibration list, select Validation Procedure. See Figure 248.
c. Click Go.
The popup message, shown in Figure 249, appears.
Calibration List
Go Button
Figure 248. RI Diagnostic page, showing the selection of the Validation Procedure
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Figure 249. Popup message, warning you not to switch between tabs
d. Click OK in the popup message.
Two messages appear, as shown in Figure 250. The message in the
Message box informs you how long the validation procedure takes to
complete. The message below the message box instructs you to start
the pump, which should be set to deliver HPLC grade water at a
flow rate of 1 mL/min.
Figure 250. Messages that appear at the beginning of the validation procedure
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2. Do one of the following:
• If the pump flow is already on, proceed to step 4.
• If the pump flow is off, proceed to step 3:
3. To turn on the pump flow:
a. Click the Surveyor LC Pump tab.
b. Click Start Pump.
c. Check that the flow rate is set to 1 mL/min and that the status of the
pump is READY, as shown in Figure 251.
Status
Flow Rate
Start Pump
and Stop
Pump
Buttons
Figure 251. Instrument Status window – Surveyor LC Pump page
d. Return to the RI Diagnostic page by clicking the Surveyor RI Plus
tab and the RI Diagnostic tab.
4. Click OK in the RI Diagnostic page.
ChromQuest performs the following tasks in the order listed:
• Sends a signal to the detector to open the purge valve.
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• Measures the baseline stability as water is pumped through both the
reference and sample compartments of the flow cell.
• Sends a signal to the detector to close the purge valve.
After the purge valve closes, the message, shown in Figure 252, appears
below the Message box. The message instructs you to stop the pump
flow.
Message
Figure 252. Message instructing you to turn off the pump flow
5. Click the Surveyor LC Pump tab, and then click Stop Pump.
6. Return to the RI Diagnostic page by clicking the Surveyor RI Plus tab
and the RI Diagnostic tab.
7. Click OK below the stop pump flow message.
ChromQuest performs a second baseline stability check, and then zeroes
the signal from the detector. See Figure 253.
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Figure 253. Ri Diagnostic page, showing the progress of the Validation Procedure
After ChromQuest zeroes the signal from the RI detector, the popup
message, shown in Figure 254, appears.
Figure 254. Popup message instructing you to inject the sucrose solution
8. To inject the sucrose solution, do the following:
a. Disconnect the LC system from the In port of the RI detector, and
then connect an adapter to the In port of the RI detector, as shown
in Figure 255.
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In
Out
Adapter
Syringe
Figure 255. Injecting the sucrose solution
CAUTION The flowcell cannot withstand a backpressure greater
than 520 kPa (75 psi). Therefore, you can easily break the
flowcell by forcing fluid through it.
b. Fill a syringe with the freshly prepared sucrose solution, and then
gently inject approximately 2 mL or less of the solution into the IN
port of the detector.
c. In the popup message instructing you to inject the sugar solution
(see Figure 254), click OK.
In the Progress area, the following messages appear: Calibrator
injected and Span value confirmed. The detector measures the
refractive index of the sucrose solution, and ChromQuest compares
the measured value to the expected value of μ512 RIU.
9. Depending on whether the span confirmation passes or an error message
appears, do the following:
• If the span confirmation passes, as shown in Figure 256, click OK in
the popup message that appears, and then proceed to the next
procedure, “Restoring the RI Detector to Normal Operation” on
page 316.
• If an error message appears, proceed to step 10.
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Figure 256. Validation procedure complete, showing the popup message
10. To recover from a span confirmation error:
a. Click OK in each popup message that appears. Figure 257 shows
the messages that appear.
Figure 257. Span confirmation failure messages
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b. Turn off the power to the RI detector.
c. Exit the Instrument window.
d. Turn the power to the RI detector back on.
e. Wait until the instrument becomes available in the Main Menu
window of ChromQuest, and then double-click the button for the
instrument to open the online Instrument window
f. Restart the validation procedure at step 1 on page 309.
Restoring the RI Detector
to Normal Operation
To restore detector to normal operation
1. Remove the syringe from the IN port of the detector.
2. Re-attach the tubing to the IN port of the detector.
3. Click the Surveyor LC Pump tab, and then click Start Pump.
4. Return to the RI Diagnostic page by clicking the Surveyor RI Plus tab
and the RI Diagnostic tab.
5. Under Purge Control, click On to rinse the sample and reference
compartments of the flow cell with HPLC grade water.
6. After a period of approximately 5 to 10 minutes, click Off in the Purge
Control area to close the purge valve and to return to normal operation.
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Thermo Scientific
Index
A
C
accuracy validation for RI detector 308
acquisition methods 88
actions, added to sequences 224
Advanced Method Options window, opening 106
air, removing from solvent lines 126
analog icon 80
analysis channels 194
Analyze button 152
annotations 195, 202
appearance 204
asymmetry of peak, calculating 108
autosampler
configuring 78
Diagnostics dialog box - Direct Controls page (figure) 132
Instrument Setup window - Surveyor AS page (figure) 97
auxiliary traces 90
averaging
bandwidth 2
standard replicates 90
cables
Ethernet 31
system interconnect 28
USB 31
calibration
analysis channels 194
bottom distance 295
dark current 279
identifying peaks 181
levels 192
Review Peak Calibration button 242
reviewing 242
setting up 213
tables 90
capacity factor, calculating 108
Channel Selector list 36
channels 98
chromatograms
adding annotations 161
overlaying 153
tiling 153
viewing 140, 153
ChromQuest
dark current calibration 279
Instrument window 36
launching 120
Main Menu window 36, 70
navigating 36
column
Column/Performance page (figure) 108
connecting 116
connections (figure) 117
entering parameters for 106
length 107
performance 90
void volume, entering in method 107
column oven
calibrating 289
control 24
column oven test fixture, installing 289
Comm LED 26
compressibility
defined 298
updating value 303
B
backpressure 9, 10, 78
bandwidth 98, 98
baseline
previewing 140
stability 140
zeroing 5
baseline check
configuration option 85
dialog box (figure) 142
performing 141
viewing 143
baseline noise
effect of bandwidth 3
effect of rise time 3
Bottom Distance
calibrating 295
caution 297, 301
note 295
buffered mobile phases
effect on pump seals 9
rinsing pump heads 9
Thermo Scientific
Surveyor Plus Getting Started with the ChromQuest 4.2 Chromatography Data System
317
Index: D
concentric syringe
described 18
figure 263
Configuration Options dialog box (figure) 85
configuring
autosampler 78
baseline check option 85
FL detector 82
LC pump 77
PDA detector 80
RI detector 84
UV/Vis detector 81
custom reports
figure 208
header/footer, viewing 201
run report 205
templates, viewing (note) 208
toolbar 201
custom tray, calibration of bottom distance 295
D
dark current calibration 279
data files
adding the file extension (note) 149
creating unique names 211
file path in sequences 216
opening 166
Data Graph Properties dialog box
Trace Setup page (figure) 202
data graphs, appearance 204
dead volume 78
Define Single Peak dialog box (figure) 183
Degas LED 27
degassing chambers, volume 8
Description button 166
Detect button 79
detectors
peak table 181
deuterium lamp 138
Diagnostics dialog box
Operation page (figure) 128
Diagnostics Program
performing a dark current calibration 279
performing a wavelength calibration 272
Wavelength Calibration 272
diode voltage leakage 279
discrete channels 2
drain valve knob 10, 126
drift test 142
318
E
Edit Custom Report button 198
Edit Sequence button 217
E-mail 61
Enterprise
adding instruments 42, 70
defined 41
errors, resetting for pump 128, 139
Ethernet cables 31
exporting data 90
Extend Run
command 151
dialog box (figure) 151
external trigger 105
F
file extensions
custom report templates 198
data files 149
methods 109
pretreatment methods 254
sequence tables 215
Fill Down dialog box (figure) 221
filter frit in purge manifold assembly 10
fixed loop injection 23
FL detector
configuring 82
method parameters 101
flow rate adjustment, setting the alpha value 305
flow rates
gradient 8
isocratic 8
Flush Syringe direct command 132
full loop injection, description 23
functions
adding to a pretreatment method 249
Flush to Waste 264
Mix at Sample 253
pretreatment 248
Transfer from Reservoir to Sample 251
Transfer from Sample to Sample 249
Wash Needle 252, 264
G
gradient delay volume
column oven tubing 24, 34
flow path 35
pulse dampener 8, 35
Surveyor Plus Getting Started with the ChromQuest 4.2 Chromatography Data System
Thermo Scientific
Index: H
H
M
high-pressure tubing 34
Main Menu window
accessing 70
figure 36
returning to 86
manual integration fixes
adding 175
default 165
manual trigger 105
maximum pressure 95
mercury (Hg) lamp, setting the usage limit 82
method custom report (figure) 208
Method Wizard
dialog box (figure) 94
invoking 93
methods
acquisition 88
audit trail 61, 90
downloading 137
prep only 256
pretreatment 247
properties 90
saving 109
shutdown 110
microplates 11
minimum pressure 95
mobile phase, proportioned 95
modes of operation
full loop injection 21
no waste injection 21
partial loop injection 21
multi-chromatograms, selecting 90
I
injection port 18
injection valve 20
Installing the Oven Sensor Test Fixture 289
Instrument Configuration dialog box (figure) 74
Instrument Status window
Surveyor AS page (figure) 131
Surveyor LC Pump page 127
Surveyor PDA page (figure) 124
Instrument window
figure 36
opening 36, 91
instruments
adding to Enterprise 70
configuring 74
control parameters 88
control parameters, entering 95
naming 72
status 124
integration events
button 171
table, default 168
toolbar (figure) 170
integration, effect of scan rate 2
K
keys, assigning privileges 66
N
L
lamps
deuterium 5, 125
LED 27
safety interlock 5
shutting down 222
tungsten 5
type 2
LEDs, status 25
library search parameters 90
LightPipe sensitivity 150
line filter body 10
linear range of no waste injections 21
loop size 78
low-pressure lines 34
Thermo Scientific
needle assembly, description 14
new instrument 43
no waste injection
description 21
effect of flush solvent 21
noise test 142
O
Offline Instrument window, opening 91
Online Instrument window, opening 120
Open Data File dialog box dialog box (figure) 167
Open Method File dialog box (figure) 138
Options dialog box - Enterprise page (figure) 48
Surveyor Plus Getting Started with the ChromQuest 4.2 Chromatography Data System
319
Index: P
P
partial loop injection, description 22
particle diameter 88, 107
PDA detector
configuring 80
method parameters 99
peak purity
calculating 158
parameters 90
performing manually 159
peak table
creating 182, 185
definition 181
modifying 192
properties 191
performance parameters, calculating 107
power
LED 25
switches (figure) 119
turning on 118
prep only method 256
pressure
maximum 95
minimum 95
recorder terminals 300
sensor adjustment 306
pressure recorder terminals, scaling the output 304
pressure sensor 9
pressure transducer
replacing 301
zeroing 301, 306
pressure units 77
Pretreatment dialog box 248
pretreatment methods
rules 263
saving 254
use 247
pretreatment program file 212
pretreatment table
adding functions 248
described 248
Preview button 166
Preview Run button 140
Process Sequence dialog box (figure) 245
project management, enabling 45
Project Wizard 41
projects, creating 51
Properties dialog box for peak table (figure) 193
pump
calibrating 61
components 8
320
Instrument Setup - Surveyor LC Pump page (figure) 96
purging 126
resetting errors 128, 139
shutting down 222
purge command 126
purge manifold assembly 9, 34
purging the flowcell of the RI detector 134
R
report properties 206
reservoir vials 11
Reset Error button 128, 139
resolution, calculating 108
response factor definition 90
Review Peak Calibration button 242
Review Peak Calibration window (figure) 242
RI detector
calibrating 308
configuring 84
method parameters 103
rinse tube assembly 9
rise time 3
rules, pretreatment 263
Run LED 26
Run Queue window (figure) 240
run report 205
Run Sequence dialog box (figure) 239
run time 100
runs
analyzing 152
extending 151
starting 149
stopping 151
unknown 211
S
safety interlock, lamps 5
sample trays, conventional 11
samples, loading 145
Save all analysis results feature 61
Save As dialog box for templates (figure) 207
Save Pretreatment File As dialog box (figure) 255
Save Sequence File As dialog box (figure) 215
saving
custom report templates 208
methods 108
pretreatment methods 254
sequences 215
Surveyor Plus Getting Started with the ChromQuest 4.2 Chromatography Data System
Thermo Scientific
Index: T
scan range, maximum 2
scan rates
effect on file size 2
effect on integration 2
scans
bandwidth setting 2
effect of bandwidth 2
effect of scan rate 2
viewing 154
security features, enabling 45
Sequence Run button 238
Sequence Summary Report (figure) 240
sequence tables 209
adding a shutdown line 222
adding actions 224
deleting columns 217
editor 225
fill down 220
modifying 217
Sequence Wizard
Autosampler page (figure) 212
Calibration page (figure) 213
creating a sequence table 210
Method page (figure) 210
Reports page (figure) 214
Unknowns page (figure) 211
sequences
reprocessing 244
running 238
Show Data At Design Time 206
shutdown methods 110
Single Run Acquisition dialog box (figure) 150
Single Run button 149
solvent bottles 34
solvent platform 34
span confirmation error, recovering from 315
Spectral Options window - Purity page (figure) 159
spectral views 154
spectrum calculations, enabling 158
Stack ID 69, 78, 81
status, instrument 124
step, wavelength 98
Stop Run button 141
Stop Run dialog box (figure) 151
sucrose solution
injecting 313
preparing 308
sucrose test solution 308
summary report templates, creating 227
Surveyor dialog box (figure) 75
Surveyor LC Pump Configuration dialog box (figure) 77
Thermo Scientific
syringe
concentric 18
flushing 61, 130
syringe drive assembly 15
syringe valve 16
System Administration Wizard locked 51
system interconnect cable 28
system suitability parameters 90
T
Table Wizard 227
temperature
column oven temperature range 24
LED 27
tray temperature range 24
templates 208
test solution, suggested 308
Trace Annotation Properties dialog box (figure) 162, 203
transfer tube 18
tray compartment 11
tray temperature
calibrating 292
control 11, 24
tray temperature sensor, installing 292
trays, conventional 11
trigger 90
tubings
behind column oven 24
high-pressure 34
inside degassing chambers 34
insulated, red, PEEK 116
transfer tube 18
wash bottle line 12
U
unit ID 69
units
pressure 77
Y-axis multiplier 80
unknown runs 211
unretained peak time 107, 137
USB cable 31
user lists
obtaining from a domain controller 49
obtaining from data system 47
user, assigning privileges 61
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321
Index: V
UV/Vis detector
configuring 81
method parameters 100
wavelength modes 5
V
vacuum degasser 8
Validation Procedure for the RI detector 308
variable loop injection 22
Verify door is closed option 78
vial tray, temperature calibration 292
vials
reservoir 11
standard 1.8 mL 11
viewing
calibration information 242
run queue 240
void volume of column 107
W
wash bottle
described 12
tubing 130
Wash Needle command 13
wash station 18
wash tube assembly 9
waste cup 18
X
XYZ arm, described 13
Y
Y-axis units 80
Z
zero on wavelength change 5
zeroing pressure transducer 306
322
Surveyor Plus Getting Started with the ChromQuest 4.2 Chromatography Data System
Thermo Scientific