s1 Agilent Capillary Electrophoresis System User’s Guide

s1 Agilent Capillary Electrophoresis System User’s Guide
Agilent Capillary
Electrophoresis System
User’s Guide
s1
 Copyright Agilent
Technologies 2000
All rights reserved.
Reproduction, adaption,
or translation without
prior written permission
is prohibited, except as
allowed under the
copyright laws.
Part No. G1600-90009
Edition 04/00
Printed in Germany
Warranty
WARNING
The information
contained in this
document is subject to
change without notice.
For details of safety,
see Safety Information
on page 224.
Agilent Technologies
makes no warranty of
any kind with regard to
this material,
including, but not
limited to, the implied
warranties or
merchantability and
fitness for a particular
purpose.
Agilent Technologies
shall not be liable for
errors contained herein
or for incidental or
consequential damages
in connection with the
furnishing, performance,
or use of this material.
Warning Symbols Used
In This Book
!
The apparatus is marked
with this symbol when
the user should refer to
the instruction manual
in order to protect the
apparatus against
damage.
Eye damage may result
from directly viewing
the light produced by the
deuterium lamp used in
this product. Always
turn off the deuterium
lamp before opening the
metal lamp door on the
side of the instrument.
Agilent Technologies Deutschland GmbH
Hewlett-Packard-Strasse 8
76337 Waldbronn
Germany
Agilent Capillary Electrophoresis System
User’s Guide
Introduction
This handbook gives an overview on your Agilent capillary electrophoresis
system. It is designed to get you started working with the system. We strongly
advise you to make extensive use of the help system when working with the
system. There you find detailed reference and task information which
complements the overview given in this guide. Refer to Getting Help later in
this section for information on using the online help system. Refer to Where
to Find Information for help in finding information for certain tasks.
Getting Help
You can get help by choosing the Help item from the top menu. This puts you
into the help index. From there you can browse through the information by
selecting the appropriate jumps (underlined words). To access specific
information in help:
When you are in the Agilent ChemStation software:
•
select a context-sensitive element and press F1.
When you are in the online help.
•
search for a specific keyword using the search command,
•
select a jump – a jump is an underlined word you can select to go to a topic
related to the word, or
•
select a word or phrase with a dotted underline to see its definition.
The help system also comprises online information on how to use help.
Table 1
Where to Find Information
Task
Refer to
Installation of the Agilent CE system, installing Chapter 1 “The Core Agilent CE Instrument”
the external water bath, installing the high
sensitivity cell
Setting up the Agilent CE system for CE
analysis, fraction collection, CE+p
4
Chapter 2 “Capillary Electrophoresis”
Introduction
Table 1
Where to Find Information, continued
Task
Refer to
How to use the Agilent CE system for capillary
electrochromatography
Chapter 3 “Capillary Electrochromatography”
How to connect and operate the Agilent CE
system for CE-MS and what else can be done
with CE-MS adapter kit
Chapter 4 “Online CE-ESI-MS with the Agilent CE System”
How to maintain the Agilent CE system
Chapter 5 “Maintaining Your Agilent CE Instrument”
Setting up a sequence
Help system
Data analysis
Help system
Setting up a report
Help system
Spectral library search
ChemStation Applications handbook and help system
Setting up customized reports
ChemStation Applications handbook
Safety information
“Safety Information” on page 224
Learning ChemStation concepts
Understanding Your ChemStation handbook
Learning the theory of the technique
High Performance Capillary Electrophoresis: An Introduction
The CE Partner CD-ROM: The comprehensive, interactive tool for beginners and
advanced users of CE
What Learning Products are Available?
The Agilent CE system is supplied with:
•
•
User’s Guide
High Performance Capillary Electrophoresis: An Introduction
The Agilent ChemStation is supplied with:
•
•
•
•
Installing Your Agilent ChemStation
Installing and Understanding Your Spectra Module
Understanding Your Agilent ChemStation
Help system
5
In This Book
•
Chapter 1 “The Core Agilent CE Instrument” describes the installation of
the Agilent CE system, external water bath for tray cooling, detector filter
assembly, changing fuses and the core CE diagram screen, which is used
for instrument control.
•
Chapter 2 “Capillary Electrophoresis” with user interface explanation
describes the tasks that need to be performed on the Agilent CE system
when preparing an analysis. In addition some new functions are described
such as fraction collection and capillary electrophoresis plus high
pressure.
•
Chapter 3 “Capillary Electrochromatography”, a new separation
technique, lists parameters that make up a CEC method and describes
their meaning.
•
Chapter 4 “Online CE-ESI-MS with the Agilent CE System” describes the
online CE coupling to the Agilent 1100 Series LC/MSD.
•
Chapter 5 “Maintaining Your Agilent CE Instrument” describes standard
operating procedures which should be used to maintain the functionality
of your system.
6
Contents
1 The Core Agilent CE Instrument
How to install the Agilent CE instrument
11
Installing the Agilent CE Instrument 13
Setting the Line Power Switch 20
Changing the Fuses for the Power Supplies 25
Supplement 30
External Water Bath for Tray Cooling (Optional) 31
Installing Drainage Tubing 35
Installing the Detector Filter Assembly G1600-62700
(Optional) 38
Installing the High Sensitivity Cell (Optional) 47
What is Needed? 48
Preparing Capillaries and Fittings for Coupling to High Sensitivity Detection Cell 49
Coupling the Capillaries to the High Sensitivity Detection
Cell 51
Realigning the Capillaries to the High Sensitivity Detection
Cell 52
Storing the High Sensitivity Detection Cell 54
Troubleshooting 55
Controlling the Agilent CE Instrument 61
Working with the CE Diagram 63
2 Capillary Electrophoresis
How to use your Agilent CE instrument for capillary electrophoresis separations 65
7
Contents
Agilent CE Method Parameters 70
Home Values 71
Conditioning 73
CE Injection 75
CE Electric 77
Timetable 79
Detector Parameters 81
How to Prepare your Agilent CE Instrument for Analysis 84
Switching the Lamp On 85
Preparing the Replenishment System if needed 86
Preparing Vials for Buffer and Sample 89
Installing Capillary in the Alignment Interface 96
Installing Capillary in an Empty Capillary Cassette 99
Inserting the Cassette 103
Problem Solving 106
Fraction Collection 112
The Fraction Collection Screen 114
Summary 119
Capillary Electrophoresis plus High Pressure: CE+p
Requirements 123
Preparing the Agilent CE Instrument for High-Pressure
Use 124
Operation and Function in CE+p Mode 126
3 Capillary Electrochromatography
How to use your Agilent CE instrument for capillary
electrochromatography 129
8
122
Contents
Requirements 131
Preparing the Agilent CE Instrument for High-Pressure
Use 132
CEC-Specific GUI Explanation 134
CEC Control through the GUI 138
Running the Analysis 139
4 Online CE-ESI-MS with the Agilent CE System
Requirements 143
Setting up the Agilent CE Instrument 144
Installing the Capillary and the CE-MS Cassette 146
Installing the G1607A Sprayer Kit 150
Preparation of Buffers and Test Sample 152
Preparing the Agilent 1100 Series Pump and Vacuum
Degasser 154
Preparing the Agilent Mass Selective Detector 157
Installing the CE-ESI-MS Sprayer 160
Method to Analyze the Test Sample 162
Storing the CE-MS Capillary, the Sheath Flow Splitter and the
CE-MSD Sprayer Needle After Usage 168
Maintenance 169
Troubleshooting Matrix 177
What else can you do with the G1603A CE-MS Adapter kit? 178
5 Maintaining Your Agilent CE Instrument
Standard operating procedures for maintenance of your Agilent CE instrument 181
9
Contents
Quick overview about maintenance 183
Cleaning the Electrodes, Prepunchers and Insulation Plate
Cleaning the Electrodes 196
Cleaning the Detection Window and the Alignment
Interface 200
Preparing/Cleaning the Replenishment System 204
Maintaining the Functionality 212
Changing the Air Inlet Filter 213
Changing the Lamp 214
Practical Considerations 218
Legal, Safety and Warranty Information
Warranty Statements 222
Safety Information 224
Sound Emission 268
Agilent Technologies on Internet
10
269
185
1
1
The Core Agilent CE
Instrument
How to install the Agilent CE instrument
The Core Agilent CE
Instrument
This chapter describes how to install the Agilent CE instrument.
For details of how to install the computer and software, refer to the
Installing Your Agilent ChemStation handbook.
12
The Core Agilent CE Instrument
Installing the Agilent CE Instrument
Installing the Agilent CE Instrument
What You Will Do
• choose a suitable place,
• unpack the Agilent CE instrument, and
• set up the Agilent CE instrument.
Choosing a Suitable Place
Check carefully before you begin to install, that the place you have chosen
meets the requirements below.
Dimensions
425 × 575 × 520 mm
(16.8 × 22.7 × 20.5 inches)
(width × height × depth)
Leave adequate space, at least 80 mm (3 inches), at rear
for access to connectors.
Add space required for PC and printer.
Weight
56 kg (115 lbs)
Environment
Temperature 5–40 °C
Humidity: 15–80 % < 31 °C (non condensing)
Line Power
100–120 V AC ± 10 % or 220–240 V AC ± 10 %
Line Frequency 48–55 Hz or 57–66 Hz
Power
Consumption
650 VA
BTU Rating
2216.5 Btu/h
CEC/CE+p
External pressure supply (15 bar max) of oil-free air or
nitrogen
13
The Core Agilent CE Instrument
Installing the Agilent CE Instrument
Unpacking the Agilent CE Instrument
1 The Agilent CE instrument is heavy—it should be carried by two people. Use
the recessed handles on both sides to carry the Agilent CE instrument.
2 Open the box in which the Agilent CE instrument was shipped.
3 Pull the Agilent CE instrument out of the box and position it in a suitable
place.
Figure 1
Unpacking the Agilent CE Instrument
Recessed handle
Accessories
The accessories shipped with the Agilent CE instrument are in the drawer on
top of the instrument box. Check that you have the complete set using
Table 1.
14
The Core Agilent CE Instrument
Installing the Agilent CE Instrument
Table 1
Accessories
Description
Part Number
Agilent CE accessory kit, comprising:
G1600-68701
Tools
Fuses
Fittings for external water bath tubing
Fitting and tube for external pressure (CE+p and CEC mode needed)
Spare filters
Spare screws
Caps
Vials
Agilent CE start-up kit, comprising:
Straight capillary, 40-cm length, 50-µm id
Straight capillary, 56-cm length, 50-µm id
Extended light path capillary, 56-cm length, 50-µm id
Alignment interface for straight capillary, 50-µm id
Alignment interface for extended light path capillary,
50-µm id
Capillary insertion tools
G1600-68706
15
The Core Agilent CE Instrument
Installing the Agilent CE Instrument
Setting up Your Agilent CE Instrument
WAR NI N G
Ensure that line-power cord is disconnected before setting up your
Agilent CE instrument. For translations, see page 227.
After you have unpacked the Agilent CE instrument and positioned it in a
suitable place on your bench you need to
1 Connect cables at the rear of the Agilent CE instrument.
2 Install the bottles of the replenishment system.
3 Check the line power setting and the fuses.
Connectors
The Agilent CE instrument has the following connectors at the rear.
16
The Core Agilent CE Instrument
Installing the Agilent CE Instrument
Figure 2
Connections at Rear of Instrument
RS-232 (not used)
External contact 24V (not used)
Remote control
External contact closure (not use
GPIB cable
RS-232 (not used)
External high-pressure gas inlet
Power connectors (internal)
Remote control
GPIB cable
Line power
Ground cable
(only required for CE-MS)
Connecting Cables
Connect the following cables as shown in Figure 2.
1 Two GPIB cables—the first GPIB cable is used to transfer signals from the
core instrument to the detector, the second GPIB Cable connects the Agilent
ChemStation to the Agilent CE instrument
2 One start/stop-cable that is used to connect the core of the instrument to the
DAD.
3 One power cable (two power cables are already installed).
4 One ground cable (required for CE-MS).
17
The Core Agilent CE Instrument
Installing the Agilent CE Instrument
Installing the Replenishment Bottles
Figure 3
Installing the Replenishment Bottles
Cap of electrolyte bottle
WAR NI N G
Use only bottles with part number 9300-1748 with this instrument.
There is pressure and vacuum applied to the bottles. The bottles
specified above have a protective coating. For translations, see page
228.
1 Inspect the bottles supplied with the Agilent CE instrument. Make sure you
only use clean glassware.
2 Install the bottles in the Agilent CE instrument.
3 Tighten the caps. Make sure the caps are tightened firmly.
4 Before using the replenishment system for the first time it needs to be cleaned.
Refer to “Preparing/Cleaning the Replenishment System” on page 204.
18
The Core Agilent CE Instrument
Installing the Agilent CE Instrument
Checking the Line Power Switch and the Fuses
The following procedure describes how to set the line power select switch
located in the rear of the instrument. The line power select switch is only
changed if the Agilent CE instrument is moved from one power setting to
another, for example from 120 V AC to 240 V AC.
WAR NI N G
Make sure that the line power setting of the instrument complies with
the specification of your country before connecting the line power
cord. The line power setting can be checked through the slit of the fuse
holder on the rear of the instrument, see Figure 2. See instructions on
how to set the line power setting. For translations, see page 229.
WAR NI N G
Make sure that the fuses installed in the instrument comply with the
specifications before connecting the line power cord. See instructions
on how to access the fuses. For translations, see page 229.
WAR NI N G
The Agilent CE instrument panels and cabinet must be grounded
during operation. The Agilent CE instrument, and any other
instruments connected to it, must be operated from a three-contact
power source, with the ground contacts securely connected to ground.
Get an electrician to check and verify ground at the source. For
translations, see page 229.
19
The Core Agilent CE Instrument
Setting the Line Power Switch
Setting the Line Power Switch
What You Will Do
• access and remove the fuse holder,
• remove and reposition the line select plug, and
• change the line fuses and the power supply fuses.
WAR NI N G
When changing the setting of the line power select switch the line
fuses and the fuses of the power supplies need to be replaced with
fuses complying with the new setting before the instrument is
switched on again. For translations, see page 232.
WAR NI N G
Make sure line-power cord is disconnected before changing
line-voltage setting. For translations, see page 232.
Accessing and Removing the Line Fuse Holder
Material Needed
Small flat screwdriver
1 The fuse holder must be removed to access the line select plug.
2 The fuse holder is attached to the instrument by a latch. Put a flat screwdriver
M3 into the slit to release the latch.
20
The Core Agilent CE Instrument
Setting the Line Power Switch
Figure 4
Location of Line Fuse Holder
Figure 5
Accessing the Line Fuse Holder
3 Remove the fuse holder from the instrument.
21
The Core Agilent CE Instrument
Setting the Line Power Switch
Figure 6
Removing the Line Fuse Holder
Fuses
Line select viewer
4 Note the line select viewer in the line fuse holder.
Removing and Repositioning the Line Select Plug
1 Grasp the line select plug and pull it out. The line select plug is held tightly in
place. Use a pair of needle nose pliers to pull it out.
Figure 7
Removing the Line Select Plug
Line select plug
2 Position the line select plug so that the desired line voltage will be seen in the
line select viewer. If the power setting of your network is 230 V set the line
power select switch to 220 V.
22
The Core Agilent CE Instrument
Setting the Line Power Switch
Figure 8
Positioning the Line Select Plug
3 Push the line select plug back into its socket.
Continue with changing the fuses as described in “Changing the Line Fuses of
the Instrument” on page 23.
Changing the Line Fuses of the Instrument
The following procedure describes how to change the line fuses located in
the rear of the instrument.
The fuses should be changed when they have blown or after the line power
setting has been changed. When a fuse has blown you should first try to find
out which problem caused this before replacing the fuse.
What You Will Do
• access the fuse holder,
• replace the fuses, and
• reinstall the fuse holder.
23
The Core Agilent CE Instrument
Setting the Line Power Switch
Material Needed
Description
Part Number
Two line fuses T3A/250 V (line voltage 220–240 V), or
Two line fuses T6.25A/250V (line voltage 100–120 V)
2110-0029
2110-0422
Small flat screwdriver
For 110 V a 6-A time delay fuse is also acceptable.
Replacing the Line Fuses
1 Remove the fuses from the fuse holder by gently pulling them out.
2 Replace the burnt fuses with the new ones specified above.
WAR NI N G
Make sure line-power cord is disconnected before installing or
replacing a fuse. For continued protection against risk of fire replace
only with fuses of specified type and current ratings. For translations,
see page 234.
Reinstalling the Fuse Holder
1 Reinstall the fuse holder by pushing it back into its original position.
2 You will hear the latch being closed again with a click.
Make sure the desired line voltage appears in the line select viewer.
24
The Core Agilent CE Instrument
Changing the Fuses for the Power Supplies
Changing the Fuses for the Power Supplies
The following procedures describe the steps necessary to change the fuses
for the power supplies located in the rear of the instrument.
The fuses should only be changed when the line power setting has been
changed. If the fuses have blown there is a severe problem in the instrument,
call Agilent Technologies.
What You Will Do
• access the fuse holder,
• replace the fuse, and
• reinstall the fuse holder.
Material Needed
Description
Part Number
Four fuses 220/240 V, F2 A/250 V, or
Four fuses 100/120 V, 3 A/250 V
2110-0002
2110-0003
Small flat screwdriver
WAR NI N G
Make sure line-power cord is disconnected before installing or
replacing a fuse. For translations, see page 235.
25
The Core Agilent CE Instrument
Changing the Fuses for the Power Supplies
Accessing the Fuse Holder for the Power Supply Fuses
1 Put a small flat screwdriver into the slit of one of the fuse holders and turn it
to the left to release the fuse holder.
Figure 9
Location of the Fuse Holders for the Power Supply Fuses
Power supply fuses
26
The Core Agilent CE Instrument
Changing the Fuses for the Power Supplies
Figure 10
Accessing the Power Supply Fuses
2 Remove the fuse holder from the power supply.
Replacing the Fuse
1 Remove the fuse from the fuse holder by gently pulling it out.
2 Replace the fuse with a new one specified above.
Reinstalling the Fuse Holder
1 Reinstall the fuse holder by pushing it back into its original position.
2 Put a flat screw driver into the slit of the fuse holder and turn to the right to
fasten the fuse holder.
Repeat the above steps for all four fuses of the two power supplies.
27
The Core Agilent CE Instrument
Changing the Fuses for the Power Supplies
Table 2
Specifications of Fuses
Customer
Replaceable
Description
Part Number
Two line fuses
(line voltage 220–240 V) T3 A/250 V
2110-0029
Yes see “Changing the
Line Fuses of the
Instrument” on
page 23
Or two line fuses
(line voltage 100–120 V) T6.25 A/250 V
2110-0422
Yes see “Changing the
Line Fuses of the
Instrument” on
page 23
Four fuses for power supplies
(line voltage 220–240 V) F 2 A/250 V
2110-0002
Only when changing
power setting
Or four fuses for power supplies
(line voltage 100–120 V) F 3 A/250 V
2110-0003
Only when changing
power setting
Air-pump fuse T 6.25 A/250 V
2110-0422
Yes
ELC-PCB F 8 A/250 V
2110-0342
No
TDR-PCB F 8 A/250 V
2110-0342
No
Four fuses for motors (tray and lifts)
T 1.5 A/250 V (PDV)
2110-0304
No
Two fuses on acquisition board (AQB)
F 250 mA/250 V
2110-0004
No
Eight fuses for valves F 500 mA/125 V (PDV)
2110-0716
No
28
The Core Agilent CE Instrument
Changing the Fuses for the Power Supplies
29
Supplement
This section describes:
• installing the external water bath for tray cooling, and
• installing the DNA filter kit.
• installing the high sensitivity cell
30
The Core Agilent CE Instrument
External Water Bath for Tray Cooling (Optional)
External Water Bath for Tray Cooling
(Optional)
The two connections at the side panel (see Figure 11) of the instrument can
be used to install an external water bath for the tray of the Agilent CE
instrument.
The temperature setting for the cooling liquid is done at the external water
bath. The temperature display in the CE Diagram displays the actual
temperature of the tray controlled by the cooling liquid. The external water
bath can’t be controlled by the Agilent CE instrument.
Good Practices
To avoid problems when using the external water bath consider the following
points.
• The tubes used should have a thermal insulation to avoid condensation.
• Use a displacer to minimize the amount of liquid that is released in case of
leaking cooling lines.
• The device should be equipped with a liquid level sensor to switch off
automatically when liquid is lost.
• Do not operate the devices at temperatures below +1°C to avoid formation
of ice on internal surfaces.
External Water Bath Requirements
• Minimum cooling power 100 W, at bath temperatures of 5 °C.
• Temperature precision better than ± 0.2 °C.
• Safety requirements must meet national standards and also:
be suitable for unattended operation,
be suitable for continuous operation, and
have controllable high temperature protection.
• Requirements for external liquid circulation:
suitable for external circulating of liquid,
31
The Core Agilent CE Instrument
External Water Bath for Tray Cooling (Optional)
suitable for connection of 8-mm id tubes,
minimum circulating liquid flow rate 8-l/min,
minimum differential pressure of 150 mbar, and
maximum pressure of 500 mbar.
• Requirements for pressure pump:
A pump with the capacity to generate a differential pressure of at least
0.15 bar. For a single action pump, a high pressure capability of 0.15 bar is
required. The high pressure must not exceed 0.5 bar and the low pressure
must not exceed -0.5 bar.
• Cooling liquid:
distilled water plus maximum 3 % anti-freeze.
• Bath temperature:
minimum allowed temperature is 1 °C, and
maximum allowed temperature is 50 °C.
Connecting an External Water Bath
WAR NI N G
Make sure both the water bath and the Agilent CE instrument are
switched off when installing the external water bath. For translations,
see page 236.
32
The Core Agilent CE Instrument
External Water Bath for Tray Cooling (Optional)
1 Remove the plastic protectors on the side panel of the instrument using a flat
screwdriver.
Figure 11
Connections for External Tray Cooling
Connector
Fitting
2 The startup kit supplied with the Agilent CE instrument includes two fittings
for flexible tubing with 8mm internal diameter. Use those fittings for the
flexible tubing used with your external water bath.
3 Connect the fittings to the connectors on the instrument (see Figure 12). Use
the upper connector for incoming liquid, the lower connector for outgoing
liquid.
33
The Core Agilent CE Instrument
External Water Bath for Tray Cooling (Optional)
Figure 12
Connecting the Tubing for an External Tray Cooling
34
The Core Agilent CE Instrument
Installing Drainage Tubing
Installing Drainage Tubing
The tubing drains condensation out of the instrument which may appear if
you connect an external water bath to cool your samples in the sample tray.
1 Behind the lower of the two plastic grommets is 10 cm of corrugated tubing.
Place a beaker of at least 200-ml capacity under this tubing to collect the
drained condensation, see Figure 13.
Figure 13
Condensation Drain
2 If you want to use a larger reservoir under the lab bench, remove the 10 cm of
corrugated tubing and connect the 100-cm length of tubing supplied in the
startup kit, see Figure 14.
35
The Core Agilent CE Instrument
Installing Drainage Tubing
Figure 14
Changing the Drain Tubing
Disconnecting the External Water Bath
To disconnect the external water bath.
1 Switch off the external water bath and the Agilent CE instrument.
2 Push the shutter (see Figure 15) on the connector to release the self shutting
connector.
Figure 15
Releasing the Fitting
3 Secure the two connections with the plastic caps.
36
The Core Agilent CE Instrument
Installing Drainage Tubing
Disconnect external water baths and empty the tubing inside the Agilent CE
instrument before shipping it. Temperatures below 0° C can cause traces of
liquid in the tubing to freeze.
To empty the tubing inside the Agilent CE instrument, connect the two self
shutting connectors and pump the liquid out.
If you operate the external water bath at very low temperatures and at a high
ambient humidity level there is a possibility that water can condense inside
the Agilent CE instrument. This water can drip onto the base plate inside the
instrument. If the amount of condensing water is very high, the leak sensor of
the Agilent CE instrument is activated and the instrument reaches an error
state. In this case shut off the Agilent CE instrument and the external water
bath immediately. Use a tissue to dry the leak sensor. Avoid condensation by:
• raising the temperature at the water bath, and/or
• lowering the ambient humidity level.
37
The Core Agilent CE Instrument
Installing the Detector Filter Assembly G1600-62700 (Optional)
Installing the Detector Filter Assembly
G1600-62700 (Optional)
The detector filter transmits light of a defined wavelength range. Figure 16
shows a typical transmission spectrum. Maximum transmission is between
256–266 nm.
Figure 16
Transmission Spectrum
Lamp Intensity
The filter kit (part number G1600-62700) for gel-filled capillaries contains:
• filter assembly (part number G1600-62701), and
• filter tool (part number G1600-03801).
Do not touch the surface of the filter or any other optical parts. Store the
filter or other optical parts in the shipping container. Fingerprints may lead to
baseline drifts and may also increase baseline noise of detector response.
Parts required:
• 12 mm hexagonal socket screwdriver (included in startup kit),
• Pozidriv screwdriver (included in startup kit), and
• a pair of tweezers.
Installing the Filter
Before you install the filter, do the following:
38
The Core Agilent CE Instrument
Installing the Detector Filter Assembly G1600-62700 (Optional)
1 Select the detector icon in the CE diagram screen.
2 Select Lamp Off from the menu to switch the lamp off.
3 Select Maintenance from the instrument menu to lower all lifts.
4 Select Exit from the File menu to exit the Agilent ChemStation. Exit both the
online and offline copy (if offline is also activated).
5 Turn off line power of the Agilent CE instrument.
6 Disconnect the line power cord from the Agilent CE instrument.
Removing the Detector Cover
1 Open the top cover.
2 Remove the capillary cassette.
WAR NI N G
Make sure you have disconnected the line power cord of the Agilent
CE instrument before you proceed. For translations, see page 237.
3 Take off the detector cover by opening the two push turn locks. To open them
press them down and turn them counterclockwise, see Figure 17.
Figure 17
Before Removing the Detector Cover
Detector cover
Push turn lock
Push turn lock
39
The Core Agilent CE Instrument
Installing the Detector Filter Assembly G1600-62700 (Optional)
Figure 18
After Removing the Detector Cover
WAR NI N G
If the Agilent CE instrument has been in use, the lamp housing can be
very hot. Avoid touching the lamp housing. For translations, see page
238.
40
The Core Agilent CE Instrument
Installing the Detector Filter Assembly G1600-62700 (Optional)
Removing the Insulation Plate
1 Use a Pozidriv screwdriver to unscrew the 2 polypropylene screws which
secure insulation plate, see Figure 19.
Figure 19
Screws Securing the Insulation Plate
Polypropylene screws
Insulation plate
2 Use a Pozidriv screwdriver to remove the screw that secures the blue ground
cable, see Figure 20.
3 Use the 12 mm hexagonal socket screwdriver to unscrew the inlet electrode,
see Figure 20.
Figure 20
Removing the Inlet Electrode
12 mm hexagonal
socket screwdriver
Red high voltage cable
Blue ground cable
4 Carefully lift the red high voltage wire. The inlet electrode will come out.
41
The Core Agilent CE Instrument
Installing the Detector Filter Assembly G1600-62700 (Optional)
5 Slowly slide the insulation plate to the right (1), lift it up (2 and 3), and remove
it, ensuring that the outlet electrode does not hit the foam of the tray cooling
or the lift station, see Figure 21.
Figure 21
Removing the Insulation Plate
42
The Core Agilent CE Instrument
Installing the Detector Filter Assembly G1600-62700 (Optional)
Removing the Standard Lens Holder Assembly
1 Mark the position of the lens holder assembly with a colored pen. If you
subsequently have to reinstall the lens holder assembly, ensure that it is
rotated to the original position. This is to avoid loss in light throughput.
Figure 22
Lens Holder Assembly
2 Remove the screw which secures the lens holder from the detector housing
using the Pozidriv screwdriver.
3 Note the orientation of the spring which holds the lens assembly and pull it out
from the detector housing. It may be necessary to use a pair of tweezers.
4 Remove the lens assembly and store it in the filter shipping container.
5 Take the filter tool and slide it into the grooves of the filter assembly, see
Figure 23.
43
The Core Agilent CE Instrument
Installing the Detector Filter Assembly G1600-62700 (Optional)
Figure 23
Filter Tool
6 Insert the filter assembly with the help of the tool. Take care that the filter is
seated correctly.
7 Reinsert the spring, taking care that the spring is fitted in the correct way.
8 Secure the spring with the screw.
9 Reassemble the instrument.
Checking the Proper Function of the Filter
Restart the instrument and check the proper function of the filter as follows.
1 Install the red alignment interface (G1600-60230) without the capillary into the
cassette.
2 The detector lamp should be switched on for one hour, to allow stabilization,
before proceeding with the DAD test. Select More DAD followed by DAD Test
from the Instrument menu.
3 Start the DAD test with Measure. If the filter is correctly installed the intensity
curve and the holmium spectra curve must look like the typical curve in
Figure 24.
44
The Core Agilent CE Instrument
Installing the Detector Filter Assembly G1600-62700 (Optional)
Figure 24
DAD Test with Filter Installed
Tips and Hints
For operation set the wavelength in a range from 256 nm to 266 nm, for
example:
• sample wavelength 262 nm and bandwidth 2 nm,
• sample wavelength 262 nm and bandwidth 4 nm, or
• sample wavelength 262 nm and bandwidth 6 nm.
A reference wavelength is not recommended. If you have to use it, choose
370 nm with bandwidth 40 nm.
Useful parts, see Figure 22.
• Screw (part number 0515-1508)
• Spring clip (part number G1600-21200)
45
The Core Agilent CE Instrument
Installing the Detector Filter Assembly G1600-62700 (Optional)
What Can Happen if you Work in the Low UV Range with
the Filter Installed
With the filter installed, only a limited wavelength range can be used. A signal
recorded outside this range will be extremely noisy and even; no peaks are
shown.
Figure 25
Signal at 200 nm with Filter Installed (Attenuation 3)
46
The Core Agilent CE Instrument
Installing the High Sensitivity Cell (Optional)
Installing the High Sensitivity Cell
(Optional)
General Information
The high sensitivity detection cell increases sensitivity (signal to noise) up to
10-fold over standard 75 µm id capillaries. It comes prealigned in a special
optical alignment interface (cell holder) that matches the Agilent CE system.
Part numbers and accessories for the high sensitivity detection cell:
G1600-68713 High Sensitivity Detection Cell Kit
• G1600-60027 High Sensitivity Detection Cell
• G1600-63200 Replacement Fittings
• G1600-60002 Capillary Cassette
• G1600-68715 High Sensitivity Detection Cell Capillary Kit-72
Replacement Capillary Kits
• G1600-68716 High Sensitivity Detection Cell Capillary Kit-56:
56 cm inlet capillary (75 µm id), and
8.5 cm outlet capillary (75 µm id)
• G1600-68715 High Sensitivity Detection Cell Capillary Kit-72:
72 cm inlet capillary (75 µm id), and
8.5 cm outlet capillary (75 µm id)
• G1600-68714 High Sensitivity Detection Cell Capillary Kit-88:
88 cm inlet capillary (75 µm id), and
8.5 cm outlet capillary (75 µm id)
47
The Core Agilent CE Instrument
What is Needed?
What is Needed?
Older capillary cassettes do not match the new interface design of the high
sensitivity detection cell. The new cassettes are modified with a cut-out for
fitting, see Figure 26. Please make sure that you have a modified cassette
before you continue.
• All buffers, samples and solvents should be filtered through a 0.2 µm filter.
• All vials should be cleaned inside.
• The electrodes, prepunchers and replenishment system should be clean.
Figure 26
Cut-out on New Capillary Cassette for Fitting High Sensitivity Detection Cell
CA UT IO N
Make sure that you prepare a clean bench for the following procedure. If dust
particles enter the capillary or the high sensitivity detection cell, this may lead
to poor performance of the cell. Compressed air is very useful for cleaning the
parts before they are assembled. For translations, see page 239.
48
The Core Agilent CE Instrument
Preparing Capillaries and Fittings for Coupling to High Sensitivity Detection Cell
Preparing Capillaries and Fittings for
Coupling to High Sensitivity Detection
Cell
The capillaries have one end prepared for coupling to the high sensitivity
detection cell. The polyimide coating is removed from this end and the edges
are bevelled. This end is protected by a cover sleeve upon delivery.
Figure 27
Prepared End of Capillary (Polyimide Removed)
1 Take the fittings and fitting cap out of the bag.
Loosely screw the fitting cap onto the fitting
screw until you feel it stop. Don’t tighten the
screw. Purge with compressed air to remove
any particles.
Never slide the capillary through the
fitting screw without the fitting cap
screwed on. The seal inside the fitting
screw could come out, and is difficult to
replace.
Fitting screw
(grey)
Seal
Fitting cap
(black)
49
The Core Agilent CE Instrument
Preparing Capillaries and Fittings for Coupling to High Sensitivity Detection Cell
2 Start with the shorter capillary (outlet
capillary). Pull the protective cap off the
bevelled capillary tip. Only the capillary end
with bevelled edges and without polyimide
fits correctly to the cell. Do not touch the
capillary tip. Push the capillary through the
fitting screw until the capillary tip is just
visible through the end of the fitting cap.
3 Remove the fitting cap from the fitting screw.
Check the capillary tip for particles and
remove them if necessary with compressed
air. Always store the fitting cap in a bag to
protect it from dust.
Capillary
4 Pull the capillary back until it is
approximately 1 mm proud of the seal. Don’t
slide it forward, as this could cause the seal to
come out of the fitting.
Approximately 1 mm
50
5 Repeat steps 1 to 4 with the inlet capillary.
The capillaries and fittings are now ready for
coupling to the cell.
The Core Agilent CE Instrument
Coupling the Capillaries to the High Sensitivity Detection Cell
Coupling the Capillaries to the High
Sensitivity Detection Cell
Make sure that you have a clean bench for
the following procedure. If dust particles
enter the capillary or the cell, this may
lead to poor performance of the cell. Also
check the ends of both capillaries and
fittings for dust particles.
Inlet capillary
(long)
1 Hold the capillary straight and avoid
touching the sides of the cell with the
capillary tip. Lightly screw the outlet capillary
to the cell holder until you feel the stop. Don’t
tighten the screw yet.
Outlet capillary
(short)
2 Screw the inlet capillary to the cell holder.
Hold both capillaries just behind the screw
fitting and gently push the capillaries towards
each other. Tighten fittings.
3 The cell is now ready for installing into the
cassette. Avoid pulling on the capillaries
during installation of the cassette in the
instrument.
Flat
51
The Core Agilent CE Instrument
Realigning the Capillaries to the High Sensitivity Detection Cell
Realigning the Capillaries to the High
Sensitivity Detection Cell
Realigning the capillaries is necessary if
one of the problems described in
“Troubleshooting” on page 55 occurs.
1 Open the outlet fitting screw by turning it
approximately 360° counterclockwise.
Outlet capillary (short)
Make sure that you have a clean bench for
the following procedure. If dust particles
enter the capillary or the cell, this may
lead to poor performance of the cell.
Inlet capillary (long)
2 Push the outlet capillary gently towards the
cell holder. Lightly screw the outlet capillary
back to the cell holder. Be sure not to pull the
capillary. Don’t tighten the screw yet.
3 Open the inlet fitting screw by turning it
approximately 360° counterclockwise.
4 Push the inlet capillary gently towards the
cell holder.
5 Screw the inlet capillary to the cell holder.
Tighten the fittings.
52
The Core Agilent CE Instrument
Realigning the Capillaries to the High Sensitivity Detection Cell
The high sensitivity detection cell is now ready for installing into the cassette.
NOTE: Avoid pulling on the capillaries during installation into the cassette and
instrument.
53
The Core Agilent CE Instrument
Storing the High Sensitivity Detection Cell
Storing the High Sensitivity Detection Cell
Short term
• Leave the capillaries connected to the high sensitivity detection cell.
• Put a buffer vial on the inlet and the outlet capillary.
Long term
• For standard and coated capillaries leave them connected to the high
sensitivity detection cell.
• Place a vial filled with water at the inlet and flush for 15 minutes.
• Place an empty vial at the inlet and flush for 15 minutes. That will dry the
capillary and the high sensitivity detection cell with air.
• For CEC capillaries remove the CEC capillary first and store according the
care and use sheet.
• Flush the high sensitivity detection cell with water to remove the buffer.
• Dry the high sensitivity detection cell with air.
The high sensitivity detection cell should be stored in a clean environment
(e.g. the plastic container in which the cell is shipped).
54
The Core Agilent CE Instrument
Troubleshooting
Troubleshooting
If, after installation of the high sensitivity detection cell you have problems
like those shown in Figure 28 and Figure 29, there are several procedures you
can try to get the system running properly.
Figure 28
Signal Trace Showing Abnormal Baseline—Indicates Air
DAD1 B, Sig=235,30 Ref=off
Absorbance
[mAU]
2000
1500
1000
500
0
4
8
6
10
12
14
16
20
18
Time [min]
Figure 29
Current Trace Showing Current Drops
Current
[µA]
80
HPCE1 C, Current
60
40
20
4
6
8
10
12
14
16
18
20
Time [min]
If you encounter one or several of the following problems:
• baseline jumps in the UV-signal due to coupling problems,
• increased baseline noise due to insufficiently filtered buffers, samples or
dirty vials,
• current breakdown when high voltage is applied,
• no current,
• leak current (indicated by yellow bar (current) in graphical user interface),
• current trace showing current drops due to air bubbles in the cell,
• buffer leaks due to improperly coupled capillaries.
55
The Core Agilent CE Instrument
Troubleshooting
Then one of the following may have occurred:
• the capillaries are not properly coupled to the high sensitivity detection
cell,
• air bubbles are in the cell,
• particles (from a dirty workbench or vials or from a broken capillary tip)
are in the cell,
• the cell is not properly cleaned.
If particles enter the high sensitivity detection cell, this may lead to air
bubbles and/or increased baseline noise. As the air bubbles grow, you will
observe large baseline drifts or jumps and current drops (see Figure 28 and
Figure 29).
CA UT IO N
Make sure you have a clean bench when you couple/decouple the high
sensitivity detection cell assembly. Store the capillaries with the protection
cap attached and the cell in the dust-protected box. For translations, see page
240.
Loose fittings can lead to dead volumes at the capillary/cell interface and
thus generate band broadening. Also air bubbles may enter the high
sensitivity detection cell. Tighten the fittings to the cell holder.
Steps to solve these problems:
1 Perform the realignment.
2 Use degassed buffer. Look for air bubbles in buffer and sample vials.
3 Flush the high sensitivity detection cell and capillary for a longer period of
time with running buffer (approximately 10 minutes), then apply 50 mbar
pressure for 5 minutes.
4 Filter buffers and samples with a 2 µm pore-size filter or use buffer prepared
under cleanroom conditions.
5 If steps 1 through 4 do not improve the situation, flush with 1 N NaOH at
elevated temperature (40–60 °C) for at least 60 minutes followed by a flush
with water for 3 minutes. This is also recommended if you encounter high
baseline noise after repeatedly running samples which stick to the cell
windows. The capillary has to be conditioned again with your running buffer
after this procedure.
56
The Core Agilent CE Instrument
Troubleshooting
CA UT IO N
Do not use step 5 with coated capillaries, packed columns or any other
capillary where NaOH is problematic. For translations, see page 241.
6 Use the “Cleaning Procedure for the High Sensitivity Detection Cell” on
page 58.
CA UT IO N
The high sensitivity detection cell and fittings are made out of different
materials: fused Silica, PEEK (polyetheretherketone) and FVMQ
(fluorosilicone rubber). Do not use solvents that can degrade these materials.
For translations, see page 242.
57
The Core Agilent CE Instrument
Troubleshooting
Cleaning Procedure for the High Sensitivity Detection Cell
As in micro-scale technique the operational parts must be protected from
contamination with µ-particles. These particles are frequently smaller than
can be seen although their effects can be extremely detrimental to the
separations. This procedure should ensure that the effects of blockage or
contamination with µ-particles can be reversed even in extreme cases.
4.5 ml of HELLMANEX II is included as part of the kit. For replacement,
please order 1.3 kg bottle part number 5062-8529.
1 Prepare a 2 % solution (v/v) of HELLMANEX II in capillary electrophoresis
grade water (double distilled de-ionized—part number 5062-8578) by adding 1
ml of HELLMANEX II to approximately 50 ml water in a clean glass beaker.
2 Place the cell housing in the beaker and make sure that it is completely
immersed in the solution.
3 Flush this diluted solution through the cell via the fitting holes using a 1 ml
pipette to ensure maximal wetting of the cell assembly. This procedure will
also remove air bubbles from inside the cell. See Figure 30.
Figure 30
Flushing the High Sensitivity Detection Cell
4 Place the beaker in an ultra-sonic bath and sonicate for at least 15 minutes.
The high sensitivity detection cell housing may change color slightly.
5 Remove the high sensitivity detection cell from the beaker and flush with CE
grade water.
6 Place the high sensitivity detection cell in a fresh clean beaker containing
CE-grade water and sonicate again for approximately 10 minutes.
7 Remove the high sensitivity detection cell from the beaker and take special
care to dry the window area thoroughly. Do not allow water to evaporate from
this area as this may deposit a film over the window.
58
The Core Agilent CE Instrument
Troubleshooting
8 Using a microscope check that the windows are clean before recoupling the
capillaries to the high sensitivity detection cell.
59
The Core Agilent CE Instrument
Troubleshooting
Troubleshooting Matrix
Table 3
Troubleshooting Matrix
What Can Be Seen?
Step 1
Step 2
Current drop, current leaks
Realign
Change fittings, then
capillary, use the “Cleaning
Procedure for the High
Sensitivity Detection Cell” on
page 58
Excess baseline noise
Flush with buffer
Use the “Cleaning Procedure
for the High Sensitivity
Detection Cell” on page 58
Tailing peaks
Realign
Change inlet capillary
Baseline jumps
Flush with buffer
for 10 minutes
Realign then use the
“Cleaning Procedure for the
High Sensitivity Detection
Cell” on page 58
60
The Core Agilent CE Instrument
Controlling the Agilent CE Instrument
Controlling the Agilent CE Instrument
After your Agilent CE system has been properly installed (see “Installing the
Agilent CE Instrument” on page 13) you can begin exploring the system. Your
Agilent CE system comprises the Agilent CE instrument and the Agilent
ChemStation. The Agilent CE instrument doesn’t have a keyboard itself. It is
completely controlled by the Agilent ChemStation through the CE diagram.
Each component of the instrument is reflected by an icon in the CE diagram.
Before You Start the Agilent CE Instrument
Use the following as a checklist before you start the Agilent CE instrument.
Make sure that:
• the replenishment bottles are installed and properly tightened,
• the cassette (with a capillary installed) is inserted into the system,
• you do not need to put the vials into the tray before you start the Agilent
CE instrument, and
• the cassette door and the tray door are closed.
Starting Up the Agilent CE Instrument
The power switch of the instrument is located in the front left corner of the
instrument. Press this switch to turn on line power.
After the instrument is started, the fans and the pump start working. The
instrument starts to build-up internal pressure (~ 1bar) first followed by
vacuum.
The LED’s colors on the front cover have the following meaning:
Color
Meaning
Green
Analysis running, do not use instrument with the
exception of manual operation of the tray. Some entry
fields are shaded gray.
Yellow
Not-ready condition. The run cannot be started yet. When
you start the LED will come on after a certain time. Click
on the Agilent CE system status bar to see what is not
ready (e.g. temperature).
61
The Core Agilent CE Instrument
Controlling the Agilent CE Instrument
Red
Error condition. There is a problem with the instrument or
communication to the PC, which requires your action.
Check the logbook. To get more information double click
Starting Your Computer and the Agilent ChemStation
Software
Starting Your Computer and the Agilent ChemStation
Software
When you have switched on your computer and it has booted up start the
Agilent ChemStation by double click on the Instrument Online Icon from the
Agilent ChemStation menu.
If you have problems starting the Agilent ChemStation, check that the
software has been properly installed.
When you have double clicked the Instrument Online icon the software is
loaded with the last method that was used. The software boots in exactly the
status you had left it. If in the graphical instrument control screen, the CE
diagram is not active after startup, choose Instrument actuals from the
Window menu to reactivate it, or type Diagram in the command line and
press ENTER.
After startup of the instrument the CE State shows Not initialized in the
Status window. Choose System INIT from the instrument menu, to initialize
the system.
During initialization the instrument tests different functions and adjustments.
The tray as well as all three lift stations are moved to a defined position, and
the lamp is switched on. Do not use the instrument or the graphical user
interface during initialization. Wait until the system has reached the ready
state. If there is a vial in tray position 49 or 50 (reserved positions) or in any
other position the lift is moving then the system asks you to remove that vial
before you proceed.
If there are no bottles (electrolyte or waste) in the replenishment system or
they are not properly tightened, the Error LED (red) will come on because no
pressure can be built up. Check the logbook for more information.
62
The Core Agilent CE Instrument
Working with the CE Diagram
Working with the CE Diagram
When you start the Agilent ChemStation the CE diagram appears by default
as the main screen. The CE diagram displays the Agilent CE system showing
the actual status of the system as a whole. Each icon represents one
component of the system.
Common SW Toolbar
To change values for a component you choose the appropriate icon. This will
bring up the menu. The menu may comprise sets of items; for help, for
changing parameters in your method and for control of the instrument. For
more assistance click HELP in the menu bar.
Figure 31
SW Toolbar
3
4
5
7 8 9 10 11
6
1
2
15
14
13
12
16
1 Work with sequences
2 Work with single vial
3 Load method
4 Store method
5 Load sequence
6 Store sequence
7 Change the layout to show the sampling area and the diagram
8 Change the layout to show the sampling area and two online samples
9 Change the layout to show the sampling area and online signal display
10 Display the current logbook
11 Display the current sequence logbook
12 View the current sequence
13 View the current method
14 View the run time
15 Status display
16 Displays sample tray when working with sequences otherwise shows single vial information
63
The Core Agilent CE Instrument
Working with the CE Diagram
Where to Find More Info:
• online help system,
• Installing your ChemStation manual,
• Understanding your ChemStation manual,
• Installing and Understanding your Spectra Module manual, and
• Macro Programming information (see help system).
64
2
2
Capillary
Electrophoresis
How to use your Agilent CE instrument for
capillary electrophoresis separations
Capillary Electrophoresis
Graphical User Interface Explanation
Figure 32
Components of the Agilent CE Instrument in the CE Diagram
1
2
3
4 5
6
7
8
17
9
16
15
14
1 CE status
2 Tray
3 Injection
4 Diode array detector
5 Timing
6 Analyses modes
66
13 12
11
10
7 Doors
8 On line windows
9 Cassette temperature
10 Inlet/outlet lift
11 Electrical values
12 Waste bottle
13 Internal pressure
14 Electrolyte bottle
15 Status bar
16 Energy values
17 Detector wavelengths/spectra
Capillary Electrophoresis
Figure 33
Corresponding Hardware Components
Capillary cassette
Diode-array detector
Status display (LED)
Electrodes
Inlet lift Electrodes
Outlet lift
Replenishment lift
Tray
Replenishment bottles
Menus in the CE Diagram
To change values for a component, select the appropriate icon. A menu will
appear. This menu comprises three sets of items:
• help menu,
• method parameters, and
• instrument control.
67
Capillary Electrophoresis
Help Menu
Each menu associated with an icon has a help option. Click on help for more
information on the subject you are interested.
Method Parameters
Items that refer to method parameters are marked with a book icon. When
you change settings using these items you change your current method. The
changes are reflected in the CE diagram when the method is executed. You
can access the same items from the instrument menu of the CE diagram, in
which case the book icon will not be present.
Instrument Control
Items without the book icon are used for direct instrument control. The
actions are performed immediately and the changes are reflected in the CE
diagram. Such actions are, for example:
• switching the lamp on,
• controlling the sample tray,
• lowering the lift stations to remove the capillary cassette, or
• releasing pressure to change the replenishment bottles.
Working with these items does not change the method currently loaded.
Status Display and Simulation
The CE diagram can be used to monitor the progress of your analysis and the
current status of the system. This is done in the CE diagram itself as well as
in the status display on the bottom of the diagram. You can try this status
monitoring using the Simulation function of the instrument menu. This will
simulate the active method in the CE diagram and in the status display.
Simulation can also be used to check your active method for inconsistencies.
The online help system provides information on this.
System Vial Table
The vial table is a graphical tool which facilitates the management of vials in
the tray. It can be used as an intelligent notepad, which exchanges its
information with methods, sequences and the graphical user interface.
Some features:
• If a vial is defined in the vial table this definition (name of vial) will be used
68
Capillary Electrophoresis
whenever the vial is referenced (Method editing, Method print).
• The vial information part used by the method is stored together with the
method and will be reloaded if the method is loaded again.
• Consistency checks can be done in order to verify if there are vial conflicts
between method and sequence usage of vials.
• Documentation of vial positions, contents.
69
Agilent CE Method Parameters
This section describes the parameters of an Agilent CE Method for data
acquisition and their meaning. Example sets of parameters for a method to
analyze the test sample are in the procedures part of the online help system.
After you have completely set up the Agilent CE Method and supplied the
sample information you can run your method. Choose Run Method from the
Runcontrol Menu.
Overview
A convenient way to access the parameters of an Agilent CE method is the
item Setup CE Method from the Instrument menu. This displays the CE
Method screen. On this screen the parameters are grouped as follows:
• Home Values
• Conditioning
• Injection Parameters
• Electric Parameters
• Timetable
• Detector Parameters
Each of these groups is located on a separate screen which can be accessed
via a button from the CE Method screen.
You can change parts of the method either using the dialog boxes from the
menu or menus in the CE Diagram marked with a book icon. If you do not
save the method to the hard disk this changed method is executed when
choosing Run Method. When you refer to a method in a sequence the method
is loaded from disk. So if you did not save your changes using Save Method
the original method from the hard disk is used.
70
Capillary Electrophoresis
Home Values
Home Values
You can program the following settings in the CE Home Values screen of the
Agilent ChemStation software.
• Lift offsets
• Cassette Temperature
• External pressure (only in CEC mode shown)
• Inlet Home Vial
• Outlet Home Vial
The Home Values are activated at five points in your analysis:
• When a method is loaded
• When you activate Run Method or Run Sequence from the RunControl
Menu
• After Injection is completed
• After the Stoptime has elapsed
• After the whole analysis (including Postconditioning) is completed
Lift Offsets
The lift offset is the distance between the bottom of the vial and the end of
the capillary. You can use the lift offset to adjust the depth to which the
capillary reaches into the vial. This lift offset setting is used for both the inlet
and outlet lift. It does not affect replenishment. The lift offset also affects the
level of liquid needed in the vials.
Cassette Temperature
The temperature set in the home value section is the default temperature of
the capillary cassette.
71
Capillary Electrophoresis
Home Values
External Pressure
In CEC mode the external pressure can be set which keeps the inlet AND the
outlet of the capillary pressurized. This maybe necessary to supress bubble
formation inside the capillary. Any value between 2 and 12 bar can be set.
Inlet Home Vial and Outlet Home Vial
The Inlet Home vial and Outlet Home vial define the default buffer vials that
will be used for the separation. Inlet Home refers to the vial at the capillary
inlet, where the voltage is applied. Outlet Home (GND) refers to the outlet
vial, at the detector end of the capillary. Via the items InHomeVial and
OutHomeVial you can refer to these settings in other screens; for example the
Conditioning screen.
72
Capillary Electrophoresis
Conditioning
Conditioning
Replenishment
During multiple analyses the composition of the buffer can change. One way
to refresh the buffer is to access different buffer vials in the tray. This is more
useful for example in method development.
Another way to change the buffer is replenishment.
The replenishment system automatically empties and refills the buffer vials
in the tray with new buffer from the electrolyte bottle. Typically
replenishment is done after every 3 or 4 runs. Depending on the stability of
the running buffer it is sometimes necessary to do it before each run.
Replenishment can be programmed via a table in the Replenish section of the
Conditioning Screen. If you want to use the replenishment system prepare it
as described in “ Preparing the Repelnishment System”.
You have a choice to perform the replenishment in serial or parallel mode.
Using parallel replenishment, you can decrease the total time needed for
your analysis as replenishment and preconditioning are done at the same
time. When using this feature you have to make sure that vials used in
conditioning are not replenished at the same time otherwise you will get a
vail conflict. Use Simulation to check your method for this potential problem.
Also note that the time for a replenishment step can vary from vial to vial.
When using detergent containing buffers (e.g. SDS) the replenishment may
not fulfill your needs. The limit for SDS concentration is 70 mM. Do not
exceed this concentration. Add some organic liquid to the waste bottle so
that bubbles do not form.
Conditioning of the Capillary
In order to have stable conditions for your analysis conditioning of the
capillary is important.
Depending on the separation mode used it can comprise:
• conditioning the capillary with conditioning agents, and
• equilibrating the capillary with buffer.
73
Capillary Electrophoresis
Conditioning
These conditioning steps can be programmed using a table in the
Preconditioning or Postconditioning section of the Conditioning screen
depending on whether they should be done before or after injection and run.
74
Capillary Electrophoresis
CE Injection
CE Injection
You have several choices for CE Injection:
• hydrodynamic injection (by pressure or vacuum),
• electrokinetic injection (applying a voltage, current or power), or
• using an injection program.
These modes and the corresponding parameters (time and unit
corresponding to mode) can be set using the CE Injection screen of the
Agilent ChemStation software. There you also have the choice to disable
injection by choosing No Injection.
Hydrodynamic Injection
In hydrodynamic injection the inlet buffer reservoir is replaced with the
sample vial. A pressure is applied for a certain time to introduce the sample
in the capillary. The system constantly controls the pressure and corrects for
the rise time effects of valves and the compressibility of air. When pressure is
applied, the pressure to the sample vial is increased gradually to its setpoint
after which the pressure decreases gradually to approximately a fifth. Then a
correction time is inserted after which the pressure decreases gradually to
atmospheric pressure. This results in accurate and reproducible injection as
well as exeptional injection linearity.
Injection by pressure is the most frequently used injection technique. There
are no differences in injection concentration for molecules with different
mobilities as in electrokinetic injection.
Electrokinetic Injection
In electrokinetic injection the inlet buffer vial is replaced with the sample
vial. A voltage, current or power is applied for a certain time that causes the
sample to migrate into the capillary.
This injection technique is used for capillaries filled with fixed or
cross-linked gels or other material of high viscosity, where injection by
pressure is not applicable.
75
Capillary Electrophoresis
CE Injection
Difference between Hydrodynamic and Electrokinetic Injection
To do electrokinetic injection the electrode must touch the sample in the
sample vial. Whereas in hydrodynamic injection the sample only needs to
touch the capillary tips.
Using an Injection Program
The injection table is used for advanced injection tasks such as:
• injection from different vials, e.g. ITP,
• multiple injection modes, e.g. spiking, and
• injecting a buffer plug after the sample to prevent bsample loss after
applying voltage.
The online help system provides more information on this.
76
Capillary Electrophoresis
CE Electric
CE Electric
The following parameters of the power supply can be controlled using the CE
Electric screen in the Agilent ChemStation software:
• Polarity
• Voltage
• Current
• Power
In addition you can set a Lower Alarm Limit for Current. Polarity
The standard polarity setting is positive polarity, that is the positive electrode
is at the inlet vial.
You can reverse the polarity to negative. This means that the inlet vial
becomes the negative electrode where a negative voltage is applied.
The outlet electrode is always grounded to ensure that the potential at the
point of detection is close to ground. Even with negative polarity the inlet
electrode is the point where the power is applied and the outlet electrode is
close to ground.
Although the outlet electrode is always at the ground potential you should
make sure that the electrode is not short-circuited to ground. The electrode is
connected with the power supply to measure the current flowing through the
capillary. If the ground electrode is short-circuited to ground all current
flowing through the capillary is signaled as leakage current.
Voltage, Current and Power
Voltage, current and power are related to each other by the resistance of your
capillary/buffer system. You can set values for each of these three
parameters. The individual values are treated as limits. The limit that is
reached first applies. For current and voltage you can only enter absolute
values. The sign is determined by the polarity setting.
If you only want to control voltage and not current and power, you can set
those two to syslimit and the system will allow the maximum value.
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Capillary Electrophoresis
CE Electric
Lower Alarm Limit for Current
When using constant voltage mode, the current is normally the indicator of
the stability of the system. A rapid current decrease or drop can indicate an
instability of the system. By setting a lower alarm limit you can define when
you want to be warned of such instable conditions during the run.
Instable conditions can be caused by:
• no buffer in the capillary,
• air bubble in the capillary,
• clogged capillary, or
• broken capillary.
Current values are dependent on the buffer concentration, the type of buffer
used, the inner diameter and length of the capillary, and the temperature.
When running the Example Method for the Test Sample normal current
values are below 50 µA. If the lower alarm limit is not exceeded 30 seconds
after start of the run, the run is stopped. When the lower Alarm Limit is
reached during the Run part of the analysis (when the HV is applied) your
current run is stopped. During a sequence the system continues with the next
analysis.
Do not use the Lower Alarm Limit or time program it when changing the inlet
or outlet vial during the run (for example for fraction collection). Otherwise
the system will stop when changing the vial because the lower alarm limit is
reached.
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Capillary Electrophoresis
Timetable
Timetable
In the CE Timetable you can choose to:
• specify the raw data that can be monitored and stored in the data file,
• define stoptime and posttime of your analysis, or
• time-program certain events.
Specifying the CE-specific Raw Data
In the Agilent CE system you can choose to monitor and store the following
CE-specific raw data. If selected, they are stored in addition to the detector
signals defined:
• voltage,
• current,
• power,
• pressure, and
• temperature.
These CE-specific raw data are acquired with an acquisition rate of one data
point per 600 ms. The signal you want to store in the data file has a little
camera attached.
Posttime and Stoptime
The stoptime defines the time for your separation, also referred to as Run
part of the analysis. It does not include replenishment, preconditioning,
injection and postconditioning. After the stoptime has elapsed, the system
changes from the Run state to Postconditioning or Postrun (if defined) and
then to the Ready state.
The posttime is normally used for equilibration. It defines the time after the
analysis, that is after the stoptime has elapsed and data analysis is completed.
During the posttime the instrument is in the Postrun state. After both
stoptime and posttime have elapsed the instrument completes the
PostConditioning and is then ready for the next analysis.
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Capillary Electrophoresis
Timetable
Time Programming Certain Events
The Timetable panel of the CE: Timetable can be used to set values for the
following parameters at a certain time during the run.
• voltage,
• current,
• power,
• pressure,
• inlet,
• outlet,
• temperature,
• polarity, and
• lower alarm limit for current.
• Highpressure (only in CEC mode)
Time programming of these parameters can be useful for example :
• define gradients (for voltage or temperature),
• change the buffer during the analysis (inlet and outlet),
• apply pressure during the analysis,
• change the outlet vial during analysis,
• change the alarm limit for current for example when changing the buffer
system during the analysis.
Please note that the time table is not execible when fraction collection has
been activated.
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Capillary Electrophoresis
Detector Parameters
Detector Parameters
The parameters for the detector are set in the DAD Signals screen of the
Agilent ChemStation. You can define the following settings:
• stored signals, detection and reference wavelengths, bandwidths,
• spectra,
• peak width,
• autobalance,
• detector stoptime and posttime, and
• detector timetable.
The Detector Parameters are accessed via the Detector button from the CE
Method screen.
Signals, Wavelengths and Bandwidths
Detector parameters allow you to define the detector signals you want to
acquire during the analysis and store in the register file. For each signal the
wavelengths and bandwidths of sample and reference wavelengths are
defined.
It is recommended to do runs without using reference wavelengths to
minimize baseline noise. Reference wavelengths can help to compensate for
signal drifts. As reference wavelengths are measured at higher wavelengths,
the baseline noise is increased when using reference wavelengths. Therefore
it is recommended to record the two signals at the same wavelength; one
using the reference wavelength and one without.
The signal you want to store in the data file has a little camera attached.
Spectra
You can define at which points on a signal, spectra will be taken and saved.
You can choose to acquire them:
• at the upslope, apex and downslope of the peak,
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Capillary Electrophoresis
Detector Parameters
• all in a peak,
• all spectra during analysis, or
• none.
You can combine this setting with a wavelength range. By restricting the
wavelength range to the range of your interest you can save some disk space;
the size of your data files will be smaller because the full spectrum is not
stored.
A spectra icon is attached to the detector window in the graphical user
interface.
Peakwidth
The peakwidth sets the optimum response time of your detector. If you have
similar peak widths in your electropherogram, set it to the width of one of the
peaks in your electropherogram. If the peak widths are very different you can
use the detector timetable to adjust the peakwidth setting during the analysis.
If your peakwidth setting is too narrow, no spectra for the wider peaks are
acquired.
Autobalance
When selecting autobalance the baseline of the detector is set to zero after
each analysis.
Detector Stoptime and Posttime
You can define a special stoptime and posttime for your detector. If you want
to use the times set for the Agilent CE instrument set them to as CE.
Detector Timetable
Using the timetable for the detector you can time program the following
parameters during your analysis:
• measuring wavelength for signal A to E,
• measuring bandwidth for signal A to E,
• reference wavelength for signal A to E,
• reference bandwidth for signal A to E,
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Capillary Electrophoresis
Detector Parameters
• store spectrum,
• peakwidth, and
• threshold.
By time programming these parameters you can optimize the required disk
space for your spectra by switching the spectra acquisition on and off. Thus
you can acquire spectra for parts of the electropherogram only.
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Capillary Electrophoresis
How to Prepare your Agilent CE Instrument for Analysis
How to Prepare your Agilent CE Instrument for
Analysis
What You Will Do
To prepare the Agilent CE instrument for the analysis you need to do the
following tasks:
• start the Agilent CE instrument,
• start the computer,
• start the Agilent ChemStation software
• allow the lamp time to warm up (approximately 1 hour),
• prepare vials for sample and buffer,
• insert a capillary into the alignment interface,
• insert the capillary into the capillary cassette,
• insert the capillary cassette into the system, and
• prepare the Replenishment system (optional).
After you have completed all these tasks you can create a Agilent CE method.
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Switching the Lamp On
Switching the Lamp On
As part of the initialization the lamp is automatically switched on.
To switch the lamp on manually.
1 Click the DAD Lamp Off icon.
2 Select Lamp On from the menu. When you have switched on the lamp you can
see the light beam from the lamp to the detector in the Graphical User
Interface.
3 Allow the Lamp to warm up for about 1 hour.
If the warm up time of the lamp is too short, a baseline drift in the
electropherogram can occur.
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Capillary Electrophoresis
Preparing the Replenishment System if needed
Preparing the Replenishment System if needed
During multiple analyses the composition of the buffer can change. One way
to refresh the buffer is replenishment.
The replenishment system automatically empties and refills the buffer vials
in the tray with new buffer from the electrolyte bottle. Typically
replenishment is done before every or after every 3 to 4 runs depending on
the stability and volume of the running buffer.
If you do NOT want to use the replenishment system you still have to put
empty bottles (electrolyte and waste) into the replenishment system at the
bottom of the instrument. The bottles are also used as pressure and vacuum
reservoir. Missing bottles lead to pressure and/or vacuum error messages.
WAR NI N G
Use only bottles with Part Number 9300-1748 with this instrument.
There is pressure applied to the bottles. The bottles specified above
have a protective coating. For translations, see page 243.
If you want to use the replenishment system, you have to fill the electrolyte
bottle with buffer. Use about 100 ml to make sure the frit is submerged in
buffer. Do not fill more than 400 ml buffer into the bottles to ensure the
system can work properly. When using detergent containing buffers (e.g.
SDS) the waste bottle should contain some liquid (e.g. a few ml of isopropyl
alcohole) to destroy the detergent bubbles. Detailed information on buffers is
given in the primer High Performance Capillary Electrophoresis or the
CE-Partner CD-ROM, delivered together with the instrument.
Make sure the waste bottle is empty so it can hold the same amount of liquid
as in the electrolyte bottle.
WAR NI N G
Do not use sulfuric acid in the Agilent CE system. It may damage the
instrument. For translations, see page 243.
Before Filling the Replenishment System
The replenishment system (bottles and tubing) should be cleaned when:
• using it for the first time,
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Capillary Electrophoresis
Preparing the Replenishment System if needed
• changing the replenishment buffer, or
• if the replenishment system will be idle for some time.
Filling the Replenishment System
To prepare the replenishment system.
1 Click on one of the replenishment bottles in the CE diagram.
2 Choose Change Bottles to release the pressure before you open the bottles.
3 Open the transparent door at the bottom of the front panel.
WAR NI N G
The waste can contain organic solvents and residue of your sample.
Wear rubber gloves and safety spectacles when handling electrolyte
and deposit the waste in a safe waste container. For translations, see
page 245.
4 Take out the electrolyte bottle and unscrew the cap. The electrolyte bottle has
a frit and tubing inside. Avoid the frit becoming contaminated (see Figure 34).
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Capillary Electrophoresis
Preparing the Replenishment System if needed
Figure 34
Filling the Replenishment Bottles
Cap of electrolyte bottle
Filling the Replenishment Bottles
1 Make sure the buffer is filtered (use a 0.25 µm filter. For buffers of high
viscosity a bigger pore size, for example, 0.45 µm is also applicable.)
2 Fill the electrolyte bottle with buffer. Use a minimum of 100 ml to make sure
the frit is submerged in buffer. Maximum 400 ml.
3 Tighten the cap. Make sure the cap is tightened firmly, as pressure will be
applied to the bottle.
4 Choose Done in the Change Bottles dialog box. Wait until the pressure has
built up again, i.e. the instrument reaches the ready state.
The right bottle is the waste bottle. It is used to store buffer emptied from
vials of the tray. Empty the bottle whenever you refill the electrolyte bottle,
following the same procedure as described above. Empty the bottle
frequently. The error message Waste Bottle Full may be the result of a
full waste bottle.
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Capillary Electrophoresis
Preparing Vials for Buffer and Sample
Preparing Vials for Buffer and Sample
Vial Handling
• Use only recommended vials (see Table 4) when working with the Agilent
CE instrument.
• It is recommended to clean the vials prior to use them (flush them with
water and some alcohol, use compressed air to dry them) otherwise it can
happen that particles enter the capillary (current problems or blocked
capillary).
• All vials must be capped. Use recommended caps only, see Table 4.
WAR NI N G
Do not use crimp caps or any other metal caps when working with the
Agilent CE instrument. For translations, see page 246.
• Make sure there are no air bubbles in the vials, especially when using
microvials for sample. Current problems may occure or in worst case the
capillary can break (heat generation or no conductance to the outlet
buffer).
• Ensure an adequate liquid level for sample, buffer and waste vials. See
Figure 35 on page 91.
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Capillary Electrophoresis
Preparing Vials for Buffer and Sample
Table 4
Recommended Vials and Caps for Agilent CE Instrument
Item
Description
Part Number
Vial clear glass 2 ml
pack of 100
pack of 500
5182-9697
5183-4623
Vial amber glass 2 ml
pack of 100
pack of 500
5183-4619
5183-4622
Sample and buffer
Vial polypropylene 1 ml
pack of 100
5182-0567
Sample and buffer
Vial polypropylene glass lined 100 µl
pack of 100
9301-0977
Sample only
Vial polypropylene 100 µl
pack of 1000
9301-0978
Sample only
Snapcap polyethylene olefin septum
for chemical resistance (‘white’ color)
pack of 100
pack of 500
PUR polyurethane septum. Prefered
standard caps (‘clear’ color)
pack of 100
90
To Be Used For:
5181-1507
5181-1513
5181-1512
Sample and buffer,
but not for multiple
use
Sample and buffer
Capillary Electrophoresis
Preparing Vials for Buffer and Sample
The recommended maximum liquid level in the vials is 1.8 cm measured from
the bottom of the vial (see Figure 35). The minimum level depends on the
type of liquid the vial is used for (sample, buffer or waste).
Figure 35
Liquid Level and Lift Offset
5 mm
Liquid level
4 mm
NOTE
The liquid level also depends on the lift offset and the type of vials used.
To touch the electrode the liquid level must follow the condition:
Liquid Level = L = Lift offset + 5 mm up to electrode + 1 mm guard band.
See Figure 35.
To touch the capillary the liquid level must follow the condition:
Liquid Level = L = Lift offset + 1 mm guard band
All values given below only hold when using the default lift offset of 4mm and
the standard vials.
Sample Vials
The level of sample in the vials depends on the injection technique used.
Hydrodynamic
injection
When using injection by pressure the sample level only
needs to touch the capillary. The minimum liquid level in
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Capillary Electrophoresis
Preparing Vials for Buffer and Sample
the sample vials for this technique is 5 mm.
Electrokinetic
injection
When using injection by voltage or current the sample
level needs to touch the electrodes. Therefore the
minimum liquid level in the sample vial is 1 cm.
Buffer Vials
When the run is started the liquid level in the vial should touch the electrodes
to enable a current to flow. Therefore the minimum liquid level in the buffer
vials is 1 cm. A buffer level of 1.4 cm is recommended. You can use the
replenishment system to fill the buffer vials.
Outlet Vials
Outlet vials (for example, waste vials) should also be filled with enough
liquid to touch the capillaries. Thus the liquid flow through the capillary does
not produce droplets at the capillary end, which can cause pressure. This can
influence the injection amount. If the waste vial is filled with liquid, outgoing
droplets cannot be drawn up the electrode by capillary effects but are flushed
completely out of the outlet electrode. Therefore they cannot contaminate
the lift head or the next outlet vial. Outlet vials that are used during analysis
(for example, for fraction collection) need to be filled to a higher level
(1 cm). The liquid must touch the electrode to enable a current to flow.
Vial in position 49 (replenishment lift)
Using the replenishment it is a good practice to have a vial filled with water in
position 49 (= home position of the replenishment lift). The replenishment
needle stays in the water and is prevented from buffer precipitation and the
resulting blockage of the needle.
Inserting the Vials
The tray of the Agilent CE instrument is used for both buffer and samples.
The CE diagram of the Agilent ChemStation controls the tray.
To operate the sample tray, choose the tray icon in the CE diagram. Select
Tray control from the menu that displays the Direct Tray Control screen.
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Capillary Electrophoresis
Preparing Vials for Buffer and Sample
To access vials individually:
1 Close the tray door.
2 Enter the vial number.
3 Click Get Vial.
4 Wait until the tray has stopped motion.
5 Open the tray door.
6 Access the vial which is in front. The number is engraved in the sample tray.
NOTE
For safety reasons the tray can only be moved by the Agilent ChemStation
when the tray door is closed. The tray door icon of the CE diagram displays
the status of the tray door.
Click Done after you have accessed all the vial positions you needed.
Manual Operation
If you want to operate the tray manually or access segments of the tray, it is
recommended to remove the vials from the lifts first. This avoids filling the
positions of those vials in the tray again. Use the Unload button of the Direct
Tray Control screen to remove the vials from the lifts.
During the run you can access the vial tray any time the software is not trying
to access it. When accessing the tray during the run you cannot remove the
vials from the inlet and outlet lifts. Therefore you have to be careful not to fill
the positions of those vials in the tray again. Check in the CE diagram which
vials are currently located in the lifts.
Positioning Vials for Sample and Buffer
1 Fill the sample into a sample vial and put the vial in the tray, for example into
position 7.
2 Note the position. When you want to run the Agilent CE method you must
enter the position of the sample vial in the Sample Information screen or the
Sequence Table screen later. The Vial Table also helps.
3 Fill two vials with the buffer you want to use for your analysis.
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Capillary Electrophoresis
Preparing Vials for Buffer and Sample
4 Position those vials in the sample tray. For example, use positions 5 and 6.
Write down the positions. When creating the Agilent CE method you must
enter the positions of the buffer vials into the CE Home Values screen for inlet
home and outlet home vial. After the sample has been injected those two vials
are positioned at the two electrodes.
5 When using the replenishment system you can start off with empty buffer vials
and use the replenishment system to fill them automatically with buffer.
Removing the Capillary Cassette from the Agilent CE
Instrument
Your Agilent CE instrument comes with an empty capillary cassette installed.
WAR NI N G
Never apply pressure (start a run) when there is no capillary installed.
For translations, see page 247.
To remove the capillary cassette from the Agilent CE instrument you have to
do the following steps:
1 Choose the Cassette icon in the CE Diagram and select Change Cassette from
the menu. This removes the buffer vials from the ends of the capillary. This
will take approximately 5 seconds. If this is not done, buffer which may be
transferred via the capillary into the electrode inserts, precipitates and may
block the electrodes or damage the capillary. Refer to the instructions on how
to clean blocked electrodes.
2 Open the top cover to access the cassette. Press the button in the middle of
the lid front to release the latch. Figure 36 shows the position of an installed
cassette.
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Capillary Electrophoresis
Preparing Vials for Buffer and Sample
Figure 36
Position of Installed Cassette
Cassette
3 Move the cassette guide to the front.
4 Carefully pull the cassette out of the cassette guide. See Figure 37.
Figure 37
Removing the Cassette
Cassette guide
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Capillary Electrophoresis
Installing Capillary in the Alignment Interface
Installing Capillary in the Alignment Interface
The following procedure describes how to install a capillary into an
alignment interface. A capillary must have an alignment interface installed
before it can be installed into the capillary cassette.
Material Needed to Install Capillary in Alignment
Interface
• capillary
• corresponding alignment interface
• capillary insertion tool (separate item) or part incorporated in cassette
• safety glasses
Color Coding of Capillaries and Alignment Interfaces
The alignment interface must correspond to the type of capillary used. There
are special alignment interfaces for straight capillaries and for extended light
path capillaries. The alignment interface must also correspond to the inner
diameter of the capillary. To facilitate matching of capillary and
corresponding alignment interface we use the following color coding on both
the sleeve of the alignment interface and the plastic stop on the capillary.
Table 5
Alignment Interfaces
Sleeve Color
Part Number
Used For*
Black
G1600-60150
25 µm id capillaries with extended light path
Green
G1600-60210
50 µm id straight capillaries
Red
G1600-60230
50 µm id capillaries with extended light path
Blue*
G1600-60310
75 µm id straight capillaries
Yellow
G1600-60330
75 µm id capillaries with extended light path
*
For 100 or 150 µm od capillaries use the blue alignment interface
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Capillary Electrophoresis
Installing Capillary in the Alignment Interface
1 Make sure the color of the sleeve in the alignment interface matches the color
of the plastic stop on the capillary.
2 Remove the protective plastic cap which covers the detection window.
CA UT IO N
Do not touch the capillary at the detection window or the capillary tips, where
the polyimide is burnt off. The capillary is very fragile at that point and it is
important to keep the detection window clean. Clean the detection window
and the capillary tips as described. Put the insertion tool on a flat surface for
example, your lab bench. For translations, see page 248.
Figure 38
Alignment Interface and Insertion Tool
Colored sleeve
Alignment interface
Capillary insertion tool
3 Put the alignment interface on the insertion tool and press down firmly. Press
only on the outer ring to release the spring that secures the capillary in the
alignment interface.
Figure 39
Installing Capillary in Alignment Interface
Colored plastic stop
You can also use the insertion tool integrated into the capillary cassette. See
Figure 43.
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Capillary Electrophoresis
Installing Capillary in the Alignment Interface
4 Slide the capillary through the opening in the direction of the arrow printed on
the interface.
5 The capillary has a plastic stop fitted. Pull the capillary through the alignment
interface until the stop is reached.
6 While still slightly pulling the capillary stop against the alignment interface,
release the alignment interface from the installation tool to secure the
capillary in the alignment interface.
The detection window or the extended light path of the capillary must be
visible through the detection interface. If aligned correctly, you can see the
slit of the interface behind the detection window. See Figure 40.
Figure 40
Extended Light Path Aligned Correctly
Figure 41
Extended Light Path Aligned Incorrectly
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Capillary Electrophoresis
Installing Capillary in an Empty Capillary Cassette
Installing Capillary in an Empty Capillary
Cassette
This procedure describes how to install a capillary into an empty capillary
cassette. If the cassette has already been installed into the Agilent CE
instrument remove it as described.
What You Will Do
• open the capillary cassette,
• install the capillary into the empty cassette, and
• close the capillary cassette.
Material Needed for Installation of a Capillary
Table 6
Material Needed for Installation of a Capillary
Description
Part Number
Capillary cassette
G1600-60002
Capillary with alignment interface installed
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Capillary Electrophoresis
Installing Capillary in an Empty Capillary Cassette
Opening the Capillary Cassette
1 Use your finger to push down the cassette lock in the center of the cassette
cover.
Figure 42
Opening the Cassette Lock
Cassette lock
Capillary lock
2 The cassette cover is hinged to the cassette body.
3 Lift the cover at the side opposite to the detection window and open the hinge.
4 Remove the cassette cover from the cassette body.
Installing the Capillary
1 Hold the capillary at the alignment interface.
2 Insert the alignment interface into the alignment interface holder of the
cassette. Make sure the flat side of the alignment interface is at the outer edge
of the cassette, see Figure 43.
3 Make sure the capillary slides into the capillary guiding slit.
4 Wind the capillary around the reel in the center of the capillary cassette. The
pins surrounding the reel have grooves to secure the capillary.
5 Start with the inside of the upper pin to avoid the capillary being squeezed
when the cassette is closed.
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Capillary Electrophoresis
Installing Capillary in an Empty Capillary Cassette
Figure 43
Installing the Capillary
Alignment interface
Hook
Capillary insertion tool
Capillary lock
6 Alternate the sides of the pins when inserting the capillary. See Figure 44.
Figure 44
Installing the Capillary Alternating the Pins
NOTE
Make sure the windings of the capillary do not come into contact with each
other. As the heat cannot be led away at contact points they get hot and may
cause separation and detection problems.
7 Insert the other end of the capillary into the guiding hook at the right.
8 Press the white capillary lock towards the center of the cassette and insert the
capillary into the capillary lock. Release the capillary lock to secure the
capillary.
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Capillary Electrophoresis
Installing Capillary in an Empty Capillary Cassette
9 Make sure the two capillary ends outside the cassette are both the same length
as the cassette guiding pins (see Figure 44) and are parallel to the pins as
shown in Figure 45. Use a sheet of paper or another soft item with a straight
line to check this. Use the capillary lock to make adjustments at the inlet side.
Closing the Capillary Cassette
1 Connect the hinge at the side of the cassette body, which has the detection
interface installed, to the one of the cassette cover.
Figure 45
Closing the Capillary Cassette
Hook
2 Make sure the hinge is closed firmly at that side.
3 Connect the other sides and latch them together.
4 Make sure the cassette cover and body form one unit.
5 Make sure the two capillary ends outside the cassette are both the same length
as the cassette guiding pins and are parallel to them. Use the capillary lock to
make adjustments.
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Capillary Electrophoresis
Inserting the Cassette
Inserting the Cassette
NOTE
Use Change Cassette to access an already installed cassette.
To insert the capillary cassette into the system you have to perform the
following steps.
1 Open the top cover to access the capillary cassette. Press the button in the
middle of the lid front to release the latch.
Figure 46
Insertion of Cassette
Cassette guide
2 Move the cassette guide to the front.
3 Insert the cassette into the guiding slit of the cassette guide. Make sure the
ends of the capillary are not bent.
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Capillary Electrophoresis
Inserting the Cassette
Figure 47
Insertion of Cassette into Electrodes
Electrodes
4 Slowly lower the cassette until it reaches the insulation plate. While lowering
the cassette, insert the capillary ends into the capillary seats of the electrodes.
WAR NI N G
Make sure the ends of the capillary are not bent when lowering the
capillary cassette. They should slide directly into the capillary seats.
Otherwise they might break or be damaged. If you have problems
inserting the capillary into the electrodes, check if they are blocked
with salt crystals. For translations, see page 249.
5 Move the cassette guide back again while holding down the cassette. The
alignment interface aligns the capillary automatically to the detector.
WAR NI N G
Do not close the top cover, if the cassette is not properly installed.
This may damage the cassette. For translations, see page 249.
6 Close the top cover and secure it by latching it onto the body of the case. Make
sure the latch is tightened. For safety reasons no power is applied if the top
cover is open. Check in upper right corner of the CE diagram whether the top
cover is closed.
Because the cassette was changed, the Agilent ChemStation automatically
displays the Wavelength Calibration screen. It tests the wavelength
calibration of your detector and advises you to do adjustments if necessary.
The cassette is recognized during the wavelength calibration means there is
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Capillary Electrophoresis
Inserting the Cassette
no switch etc. If the lamp is off or the cassette is not inserted properly the
wavelength can not be adjusted correctly. This may result in following safety
functions:
• no voltage can be applied
• no pressure can be applied
• cassette temperature does not work
Following messages can be displayed:
Cassette not recognized or
Cassete fan switched off
Choose the Help button for more information on this feature.
After you have finished Wavelength Calibration, you can close the Change
Cassette dialog box by choosing Done and proceed with setting up a method.
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Capillary Electrophoresis
Problem Solving
Problem Solving
The problem solving section in the instrument online help system assists you
in solving the most common failures. Nevertheless a short list of problems
are featured here.
Leaks
The leak sensor of the Agilent CE instrument is located in the lower right part
of the instrument, see Figure 48.
Figure 48
Position of Leak Sensor
When the leak sensor is activated:
• the error LED on the front of the instrument is illuminated,
• the CE Status displays an error,
• a logbook entry is created,
• the run is stopped, and
• pressure is released.
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Capillary Electrophoresis
Problem Solving
If the leak sensor is activated or you think there is a leak in the instrument:
1 Switch off the Agilent CE instrument (and the external water bath, if
connected) immediately.
2 Disconnect the line power cord.
3 Remove the detector cover as described.
4 Remove the front cover as describedto diagnose the cause for the leakage.
5 Dry the leak sensor using tissue.
Possible Reasons for Leaks and Appropriate Action
Spilled Liquid From Other Sources
If you spilled some liquid into the Agilent CE instrument it is sufficient to
thoroughly clean and dry the Agilent CE instrument and the leak sensor.
Leaking Water Bath Tubing Inside the Agilent CE Instrument
Call Agilent Technologies. Do not use the Agilent CE instrument until Agilent
Technologies has repaired it.
Leaks of Replenishment Tubing or Valves
Call Agilent Technologies.
Reason Unknown
If you can not trace the reason for the leak, call Agilent Technologies. There
may be an instrument problem.
Blocked Replenishment Needle
If you are using the replenishment function of the Agilent CE instrument,
buffer may precipitate inside the replenishment needle. To avoid this, put a
vial filled with water (to a height of 1.8 cm) in vial position 49. Vial position 49
is the home position of the replenishment lift. The vial will be picked up by
the lift and brought to the replenishment needle. The needle will dip into the
water and this prevents the buffer from precipitating.
Leakage Current
Leakage current is indicated by a yellow bar in the current part of the energy
status display in the CE diagram. Leakage current means that the current at
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Capillary Electrophoresis
Problem Solving
the inlet electrode is different from the one at the outlet electrode and thus a
current flows outside the capillary.
It can be caused by:
• broken capillary,
• empty inject vial,
• air bubble in injection vial/or run buffer vials,
• air bubble in capillary,
• contaminated insulation plate,
• high humidity level, or
• conductive capillary surface.
In case you found a broken capillary in the alignment interface use an
ultrasonic bath to clean the interface to remove quarz residues.
Broken Capillary
To check for a broken capillary, remove the capillary cassette and open it as
described in the User’s Guide. The capillary is especially sensitive to
breaking at the detection window and the capillary ends, therefore check
these parts of the capillary. If the capillary is broken at the detection window,
a leakage current of nearly the same value as the expected current can occur.
Small instabilities in the current curves as shown in Figure 49 can also be a
sign of a broken capillary.
Figure 49
Current Instabilities
If the capillary is broken at the ends, it may no longer reach into the liquid in
the vial. In that case the analysis is stopped if lower alarm limit for current
has been reached (must be set first). Sometimes the capillary has very small
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Capillary Electrophoresis
Problem Solving
cracks which are hardly recognized under a microscope. Exchange the
broken capillary.
Contaminated Insulation Plate
Salt crystals or other contamination of the insulation plate can cause leakage
current. Clean the insulation plate as described.
Empty Injection Vial
Access the vial position specified in Sample info or Sequence table using the
Get vial function in the Tray control screen. Check if the vial is filled.
Dirt in the vials
Clean all vials prior to use them. First flush them with water followed by e.q.
some isopropyl alcohol. Use compressed air to dry the vials inside.
Air Bubble in Injection Vial/or Run Buffer Vials
When filling sample or buffers into the vial, an air bubble is sometimes
formed. This happens very frequently when using microvials. Air bubbles can
cause air to be injected instead of the sample. To avoid air bubbles, carefully
fill vials starting from the bottom. If you can’t avoid the formation of air
bubbles, adjust the lift offset in the home values dialog box so that the
capillary doesn’t reach into the bubbles.
Air Bubble in Capillary
Small air bubbles in the capillary can cause spikes in your electropherogram.
Larger air bubbles expand so much during analysis that the current drops
below the lower alarm limit. Flush your capillary with buffer to remove air
bubbles.
Problems with the Replenishment System and SDS
Containing Buffers
When a vial is replenished the SDS foam may stick to the side of the vial. The
empty function draws air around the foam into the replenishment needle.
When filling this vial a set of new foam bubbles is introduced into the vial
before the fresh buffer. The surface sensing system recognizes the first foam
bubble as liquid. So the final liquid level is not at the height it should be. It
may also happen that the vial contains no buffer liquid but only foam. The
result could be seen as low current or even no current supplied.
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Capillary Electrophoresis
Problem Solving
As a workaround we recommend programming a replenishment program in
two steps.
1 Replenishment to a level about 0.2 cm higher than required.
2 Fill to the desired level.
If the fill finds a level higher than the desired, the vial is emptied to the level
without using the sensing function and the foam has no influence.
As an example: the desired height is 1.4 cm
1 Replenish vial XX to 1.6 cm (0.2 cm more)
2 Fill vial XX to 1.4 cm
Alternative
No replenishment necessary.
Fill several vials with buffer. Program methods which use these vials as the
inhome and outhome vials (e.g. vials 4, 5, 6, 7).
Method 1:
inhome = 4, outhome = 5
Method 2:
inhome = 6, outhome = 7
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Capillary Electrophoresis
Problem Solving
111
Fraction Collection
In the bioscience and pharmaceutical areas the capability of fraction
collection is frequently required in order to characterize separated
components by off-line analysis techniques such as MALDI/TOF-MS or
protein sequencing. Figure 50 shows general principle of how fraction
collection in CE works.
Figure 50
Fraction Collection
Apply pressure
or voltage
Peak detection
Buffer
Buffer
Collection vial
Fraction collection uses information from the built-in diode array detector to
find the exact time when a peak appears at the outlet electrode. A peak is
detected if the peak signal exceeds the threshold set in the diode array set up
screen. Therefore some diode array settings have to be adjusted such as
selecting spectra storing. When a new peak is detected, the time is calculated
in which the peak will leave the capillary. The length of the capillary between
the detection window and the outlet is fixed to 8.5 cm.
In addition some settings have to be entered, such as the inner diameter of
the capillary, the correct total and effective capillary length in the Capillary
Information screen, e.g. inner diameter is 100 µm, total length is 96.5 cm and
effective length 88 cm as shown in Figure 51:
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Capillary Electrophoresis
Fraction Collection
Figure 51
Capillary Information Screen
Peaks may be collected either by using pressure or electrokinetic elution.
Since peak volumes are generally in the size of nl volumes, peaks are
collected into microvials containing about 10–15 µl of an appropriate solution
(for correct selection, see Table 7). The lift offset should be set to 3 mm in the
CE Home Values menu. Applications include fraction collection from CZE,
MECC, from CGE analyses using fixed gels and even from CIEF separations.
The instrument logbook shows the steps done during fraction collection e.q.
when a peak has been detected, the vial in which the peak has been collected
etc.
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Capillary Electrophoresis
The Fraction Collection Screen
The Fraction Collection Screen
In order to use fraction collection you must have entered values for the
effective capillary length in the Capillary Information screen.
Collection Mode
OFF:
No fraction collection set up.
Pressure:
Moves the peak into the collection vial by applying 50
mbar pressure.
Voltage:
Moves the peak into the collection vial by applying the
voltage. The collection vial should be filled with run buffer
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Capillary Electrophoresis
The Fraction Collection Screen
or other electrolyte to ensure conductance.
IEF:
50 mbar pressure is used to move the peak into the
collection vial. In the IEF mode the peaks have no
electrophoretic mobility so the algorithm to calculate the
time when the peaks reach the outlet is different than in
collection with pressure only.
Collect
Depending on the collection mode selected the items in the Collect panel
change:
Select All Peaks if you want to have all detected peaks above a certain
threshold collected. Select Selected Peaks and provide migration times into
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Capillary Electrophoresis
The Fraction Collection Screen
the fields to collect up to 4 selected peaks expected within each time window
specified by the migration time.
In First Vial the start vial where the first fraction is collected can be entered.
The second fraction will be collected in the next vial.
An error message will be logged in the logbook if the actual vial number
exceeds 48. No further peaks will be collected.
Collection Width
The collection width defines the peak collection time window around the
peak apex. Depending on the collection mode, it is specified in percentage of
the time the peak appears or an absolute time. This is the migration time plus
the time the peak needs from the detector to the outlet vial. This is because
slower peaks (will become broader and therefore) need a wider migration
time window.
Example
If a collection width of 5% is used for a peak which has an expected migration
time of 1 minutes (to capillary outlet-end) then the collected peak width will
be 0.6 minutes. Therefore peak collection will begin at 11.7 minutes and
continue until 12.3 minutes.
If peaks are being collected from an IEF separation, the peak width is not
dependent upon migration time so the peak collection time can be defined
and remains the same for all collected peaks.
Migr. Time with 50 mbar, Migr. Time With Pressure
If you use collection by Pressure or IEF the instrument needs to know at
which velocity the peaks are moving. In other words, how long it would take
for an injected sample plug to move from the capillary inlet to the detector if
moved by pressure only. To find this value, create the following method:
1 switch electronic off (means no voltage applied)
2 in the timetable enter: At time 0.01 minutes 50 mbar and a stoptime of ‘no
limit’
3 inject your sample or any absorbing liquid (e.q. NaOH, isopropyl alcohol)
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Capillary Electrophoresis
The Fraction Collection Screen
4 set a wavelength where your sample or the liquid absorbes (e.q. 200 nm for
NaOH)
5 in Conditioning flush the capillary with your run buffer for 5 minutes
6 start the method
7 the time when your sample or NaOH appears in the detector window
should be entered as ‘Migr. Time with pressure’.
Voltage Ramp for Separation
Sometimes a method starts with a voltage ramp to prevent rapid heating of an
injected sample. The conditions are set in the Set up CE Timetable screen.
NOTE
When using fraction collection all your timetable entries which use voltage,
outlet vial or pressure settings will be deleted from the timetable. Therefore
set the voltage you want to use in the electric screen and the time for the
voltage ramp in the fraction collection screen. The voltage ramp must be
completed before the first peak to be collected is expected.
Activating the Peak Detector of the Diode Array Detector
In order to run fraction collection you have to activate the peak detector in
the DAD Signals screen
Figure 52
DAD Signals Screen
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Capillary Electrophoresis
The Fraction Collection Screen
Go to the Spectrum section and activate store Apex+Slopes+Baselines. Set
the range to 200–350 nm and set the threshold to a value below the expected
mAU of the compound you want to collect.
Because threshold always refers to Signal A, in ‘Store Signals’ set the desired
wavelength for Signal A (e.g. 200/20 nm). The setting for peak width has to be
a value which is similar to the real peak width. The value can be found in the
integration results report of a standard analysis.
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Capillary Electrophoresis
Summary
Summary
Steps for Fraction Collection
• Fill microvials with 10–15 µl of appropriate solution (see Table 7).
• place microvials in sample tray (random choice)
• enter the correct capillary information in the ‘capillary information screen’
• set lift offset to 3 mm in the CE Home Values menu
• find the migr. time with pressure values
• set threshold accordingly, store Apex+Slope+ Baseline spectra and the
signal wavelength A in the DAD settings
• set peak width similar to the real peak width
• run fraction collection method. Look into the logbook to see the fraction
collection steps
• mix the collected fraction thoroughly when you want to re-analyse the
collected fractions. Be aware that the result depends on many factors like
sample concentration, injection time and pressure, capillary length and
internal diameter
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Capillary Electrophoresis
Summary
Recommended Collection Modes
Table 7 gives an overview of the recommended collection modes using the
various CE separation techniques and proper fraction collection solutions.
Table 7
NOTE
Collection Modes
Technique
Collection Mode
Pressure
Collection Mode
Voltage
Collection Liquid
CZE
Preferred
Yes
2-% acetic acid
ITP
Preferred
Yes
2-% acetic acid
CGE (using gels)
Do not use!
Yes
Electrolyte
MECC
Yes
Yes
Electrolyte
IEF
Yes
No
Carrier ampholyte
In order to use fraction collection you must have specified values for the
effective capillary length in the Capillary Information screen. An error
message will be logged in the logbook if the actual vial number exceeds 48. No
further peaks will be collected. When using fraction collection all timetable
entries which require voltage, outlet vial or pressure settings will be removed
from the timetable. The voltage ramp must be completed before the first peak
to be collected is expected.
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Capillary Electrophoresis
Summary
121
Capillary Electrophoresis plus High
Pressure: CE+p
The Agilent CE instrument is capable of accepting an external gas pressure in
addition to the internal pressure. The external high pressure source can be
up to 15 bar, and is regulated within the instrument via GUI control between
2–12 bar. This pressure is applied to one or both vials at the capillary ends.
The high pressure option may be required if using highly viscous replaceable
gels in CGE.
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Capillary Electrophoresis
Requirements
Requirements
WAR NI N G
Use the new design of capillary cassette when your application or
method requires organic solvents. The new design of capillary cassette
conforms to the higher flammability class (CV01). For translations,
see page 251.
• External pressure supply (< 15 bar) of oil-free air or nitrogen.
• Push-fit connector and gas line (supplied in startup kit).
The inlet for the external gas high pressure is situated at the back of the
instrument (Figure 53).
Figure 53
External High Pressure Gas Inlet
External high pressure
gas inlet
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Capillary Electrophoresis
Preparing the Agilent CE Instrument for High-Pressure Use
Preparing the Agilent CE Instrument for
High-Pressure Use
1 Ensure gas source is switched off prior to connection.
2 Connect push-fit connector and gas line to the receptor at the rear of the
Agilent CE instrument.
3 Connect the gas line to the source.
4 Switch on gas supply.
5 Select CE Mode from Instrument in the Method and Run Control screen.
6 Select CE+p from the CE mode selection menu (Figure 54).
Figure 54
Select CE Mode
7 CE+p Mode can also be selected by clicking on the CE Mode icon on the GUI.
8 A new icon will appear on the GUI and the Mode field will indicate CE+p
(Figure 55).
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Capillary Electrophoresis
Preparing the Agilent CE Instrument for High-Pressure Use
Figure 55
GUI
125
Capillary Electrophoresis
Operation and Function in CE+p Mode
Operation and Function in CE+p Mode
By selecting the CE+p mode some new functionality is available for control
via the GUI which will assist in the operation of CGE with highly viscous
buffers. This includes the ability to apply high pressure during
preconditioning and postconditioning and the ability to use high pressure to
introduce samples.
Preconditioning
When the instrument is operating in the CE+p mode the user can chose to
apply HIFLUSH during preconditioning (Figure 56). This means that high
pressure will be applied to the capillary inlet vial which may be:
• a set value (2–12 bar),
• raw external pressure (up to 15 bar),
• or it is still possible to use ‘Flush’ (= internal pressure up to 1 bar).
Figure 56
CE Preconditioning Menu
Other parameters which can be set in the HIFLUSH field include:
• applied voltage (kV),
• duration (min),
• inlet vial, and
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Capillary Electrophoresis
Operation and Function in CE+p Mode
• outlet vial.
Injection Options with CE+p
High pressure may also be used for injection purposes using the timetable
option in the Injection field (Figure 57).
Figure 57
CE Injection Table
From this menu you may select the HIFLUSH option during injection with the
same parameter choices as are available during Preconditioning.
This may be used to apply a pressure injection while using a replaceable gel
filled capillary.
Please note that the external pressure is not as precise as the 50 mbar
injection pressure!
CE+p Control Using the GUI
High pressure may be applied independently of the method for capillary
conditioning or cleaning by clicking on the External pressure icon on the GUI
(Figure 54). This will cause a parameter setting field to appear (Figure 58).
From this field you may chose to apply pressure to the inlet, outlet or both
vials, and also whether to apply a simultaneous voltage of -30 to 30 kV.
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Capillary Electrophoresis
Operation and Function in CE+p Mode
Figure 58
CE+p Control via GUI
128
3
3
Capillary
Electrochromatography
How to use your Agilent CE instrument for
capillary electrochromatography
Capillary
Electrochromatography
Capillary electrochromatography (CEC) is a fusion of capillary
electrophoresis and capillary chromatography. In CEC, solvent is mobilized
through a packed capillary column by electroosmotic flow instead of the
hydraulic flow used in LC. Higher efficiencies are achievable because of the
uniform flow velocity profile and the ability to use smaller particle sizes in
longer columns than is possible with LC. Operation of CEC requires the equal
application of high pressure to the inlet and outlet vials in order to suppress
bubble formation while avoiding hydrodynamic flow due to a pressure
differential.
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Capillary Electrochromatography
Requirements
Requirements
WAR NI N G
Use only the new design of capillary cassette. Never use the old design
of cassette when organic solvents are required. This applies especially
when performing CEC applications. For translations, see page 252.
• External pressure supply (<= 15 bar) of oil-free air or nitrogen to get
between 2 and 12 bar regulated.
• Push-fit connector and gas line (supplied in startup kit).
The inlet for the external high-pressure gas is situated at the back of the
instrument (Figure 59).
Figure 59
External High-Pressure Gas Inlet
External
high-pressure gas
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Capillary Electrochromatography
Preparing the Agilent CE Instrument for High-Pressure Use
Preparing the Agilent CE Instrument for
High-Pressure Use
1 Ensure gas source is switched off prior to connection.
2 Connect push-fit connector and gas line to the receptor at the rear of the
Agilent CE instrument.
3 Connect the gas line to the source.
4 Switch on gas supply.
5 Select CEC from the CE mode selection menu or by clicking on the option
button next to CEC (Figure 60).
Figure 60
CE Mode Selection Menu
6 A new icon will appear on the GUI and the Mode field will indicate CEC
(Figure 61).
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Capillary Electrochromatography
Preparing the Agilent CE Instrument for High-Pressure Use
Figure 61
GUI
133
Capillary Electrochromatography
CEC-Specific GUI Explanation
CEC-Specific GUI Explanation
When the CEC mode is selected a new icon in the form of a green gas bottle
appears on the lower right side of the GUI (Figure 61). There are also a
number of new parameters which may be set either from the Edit entire
method option or from the Individual parameter settings within the
Instrument menu selected from the Method & Run Control toolbar. The new
parameter choices appear in the following menu selections.
Home Values
When the CEC Mode is selected a new parameter setting field appears in the
Home Values menu from the method edit menu (Figure 62). In the Pressure
field there is the option to select:
• pressure off,
• external, and
• set pressure from 2–12 bar.
Figure 62
Home Values Menu
Pressure off is self-explanatory as is the default setting. Selecting external
applies the raw external pressure with no control through the internal valve
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Capillary Electrochromatography
CEC-Specific GUI Explanation
block. The pressure may be set to a value from 2–12 bar at increments of
0.1 bar.
Injection Options with CEC
High pressure may also be used for injection purposes using the Time Table
option in the Injection field (Figure 63).
Figure 63
CE Injection Table Menu
From this menu the user may select the HIFLUSH option during injection
with the same parameter choices as are available during preconditioning.
This may be used to apply a pressure injection while using a packed capillary.
The internal pressure of up to 1 bar is generally insufficient to introduce
sample onto a packed capillary.
The external pressure is not as precisely delivered as the 50 mbar injection
pressure. However electrokinetic injection can be readily used in CEC mode.
Preconditioning
In order to precondition a packed capillary an applied pressure of
approximately 12 bar alone is usually insufficient to move mobile phase
rapidly through the column. Therefore the applied high pressure may be
assisted by the application of an electric field. This serves to augment the
high pressure flow with an electroosmotic flow. In the Preconditioning menu
HIFLUSH can be selected for this purpose (Figure 64). HIFLUSH applies
pressure with or without voltage to the INLET vial only.
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Capillary Electrochromatography
CEC-Specific GUI Explanation
Figure 64
CE Preconditioning Menu
After selecting HIFLUSH, options appear for applying pressure without
voltage (Figure 64). The applied pressure may be selected from:
• (enter value) which will allow you to apply pressure from 2–12 bar to the
inlet vial,
• external applies raw external pressure (up to 15 bar) to the inlet vial,
• internal applies internal pressure (up to 1 bar) to the inlet vial, or
• simultaneously applied voltage can be set from -30 to 30 kV.
NOTE
When applying voltage simultaneously the OUTLET vial must contain
electrolyte in order to maintain electrical contact.
Postconditioning
The available fields and setting parameters are identical to those in
Preconditioning.
Time Table
In the Time Table field the option for applying high pressure is available from
the command selection menu (Figure 65).
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Capillary Electrochromatography
CEC-Specific GUI Explanation
Figure 65
CE Timetable Menu
In this case by selecting HIGHPRESS, the pressure value which was set in
HOMEVALUES is used. A further option is to apply this pressure to inlet,
outlet or both vials.
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Capillary Electrochromatography
CEC Control through the GUI
CEC Control through the GUI
High pressure and voltage may be applied independently of the method for
capillary conditioning or cleaning by clicking on the external pressure icon
on the GUI (Figure 61). This will cause a parameter setting field to appear
(Figure 66). From this field you may chose to apply pressure to the inlet,
outlet or both vials, and a simultaneous voltage of -30 to 30 kV.
Figure 66
External Pressure Field
138
Capillary Electrochromatography
Running the Analysis
Running the Analysis
After setting up the operational parameters you can initiate the run from the
Method & Run Control menu by clicking on Run Method or by clicking on the
Start button on the GUI. Some other operational aspects should be noted
when running CEC separations.
Capillaries
Packed capillaries are very fragile and must be handled
with great care. Offline purging of the capillary using an
LC pump may be necessary if air bubbles form within the
capillary.
Buffers
CEC buffers are generally of lower concentrations
(4–20 mM) than conventional CE buffers. These may also
contain a larger proportion of organic modifier than is
found in CZE or MECC buffers. The currents generated are
generally in the order of 2–10 µA for an applied field of
approximately 600 V/cm.
Injection
Because of the resistance of the packed bed the usual
applied pressure of up to 50 mbar is insufficient to
introduce sample onto the capillary. In this case high
pressure and/or electrokinetic loading may be used,
although the limitations of this technique should be noted.
Problem solving
Offline purging If air bubbles occur during an analysis the current will
become unstable or very low (< 0.3 µA). If this occurs,
remove the packed capillary and connect it to an LC pump
capable of delivering a flow rate of 40 µL/min to the
capillary.
NOTE
Great care should be taken when removing and handling the capillary and
when connecting and disconnecting the capillary to the pump. Fused silica
packed capillaries are extremely fragile.
Instrument
purging
If the capillary is filled with solvent then this may be
replaced by inserting the capillary into the instrument and
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Capillary Electrochromatography
Running the Analysis
applying a HIFLUSH through the external gas supply icon
on the GUI. Generally 10–60 minutes are sufficient to
replace the storage solvent with the operating mobile
phase, depending on the velocity of EOF.
140
4
4
Online CE-ESI-MS with
the Agilent CE System
How to use your Agilent CE instrument for online
CE-ESI-MS
Online CE-ESI-MS with the
Agilent CE System
This chapter describes how to connect a Agilent CE capillary electrophoresis
system to an G1946A or G1946B Agilent 1100 Series LC/MSD. Typical method
parameters are given to analyze a simple test sample. Detailed information on
how to set up the MSD can be found in the System Installation Manual for
the Agilent 1100 LC/MSD. Information on how to configure the capillary
electrophoresis with the Agilent G1946A/B can be found in the Installing
your ChemStation Manual.
NOTE
All Accessories and documentation needed to interface the capillary
electrophesis system to a non-Agilent mass spectometer are the responsibility
of the respective vendor and will not be provided by Agilent. See information
attached to this chapter.
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Online CE-ESI-MS with the Agilent CE System
Requirements
Requirements
Equipment
❏ Agilent CE system with serial number greater than 3534G00644 or
upgraded to CE-MS capability.
❏ Agilent G1603A CE-MS Adapter kit.
❏ Agilent G1946A or G1946B Agilent 1100 Series LC/MSD G1948A
API-Electrospray source.
❏ Agilent 1100 Series pump with an optional Agilent 1100 Series vacuum
degasser to add the sheath liquid or syringe pump which can deliver a
precise flow rate of 1–5 µl/min.
❏ Agilent G1607A CE-ESI-MS sprayer kit
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Online CE-ESI-MS with the Agilent CE System
Setting up the Agilent CE Instrument
Setting up the Agilent CE Instrument
If your G1603A CE-MS adapter kit and the G1607A Sprayer kit is going to be
installed by a Hewlett-Packard engineer, go to ‘Method to analyse the test
sample’.
WAR NI N G
Make sure you have disconnected the line power cord of the Agilent
CE instrument before you proceed. For translations, see page 264.
Leveling the CE-MSD
When setting-up the CE-MSD, the two instruments should be positioned close
together to minimize the total capillary length. Placing the Agilent CE on a
cart simplifies positional adjustment or changeover to a HPLC-system.
The capillary inlet should be at a similar height to that of the the capillary
outlet to avoid siphoning. Placing the Agilent CE and the Agilent 1100 Series
LC/MSD on the same bench results in the outlet being at nearly the same
height as the inlet.
Installing the MS Cover
The new top cover insertion piece (MS-cover) must be installed to enable the
use of the CE-MS capillary cassette. To install do the following:
1 Open the instrument lid.
2 Remove the black detector cover (quick lock mechanism).
3 unscrew the two screws behind the cassette which hold the sheet metal cover
and exchange this for the new part and replace the screws.
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Online CE-ESI-MS with the Agilent CE System
Setting up the Agilent CE Instrument
Figure 67
Screws
Installing the MS Cover
MS cover
145
Online CE-ESI-MS with the Agilent CE System
Installing the Capillary and the CE-MS Cassette
Installing the Capillary and the CE-MS
Cassette
Preparing the Capillary
To have a short capillary connection to the Agilent mass selective detector,
approximately 45 cm can be cut off from the long end of the CE-MS capillary.
The remaining length is now about 80 cm.
The precision of the spray depends on the quality of the cut. Ordinary scoring
devices gather and rip the coating of the capillary. Jagged edges are left that
prevent a perfect spray and can act as adsorptive sites for sample
components. A CE column cutter (5183-4620) can be used to reduce the
capillary length. If not available, please follow the procedure described
below.
Figure 68
Cutting the Capillary
1 Place the capillary over a large radius surface under slight tension.
2 Hold the scribe (delivered with the capillary) at an angle of approximately 30°
to the capillary.
3 Draw the edge of the scribe across the capillary penetrating the polyimide.
4 Pull the capillary horizontal until it breaks. If the capillary will not break, the
polyimide has not been cut. Repeat above steps.
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Online CE-ESI-MS with the Agilent CE System
Installing the Capillary and the CE-MS Cassette
Installing the Capillary and the CE-MS cassette
WAR NI N G
Wear safety glasses when opening the cassette and handling the
capillary to protect your eyes. For translations, see page 257.
NOTE
Cover the exposed capillary section with the supplied external PTFE tube.
The maximum stored energy in the CE power-supply is 200 mJ. Due to this and
the fixed current limit of 50 µA the high voltage applied is not defined as
hazardous voltage. However, to avoid electrical shock always switch off the
CE high voltage during capillary handling.
Should you set a current limit higher than 50 µA in the Electric field of method
set up, a warning will appear on the ChemStation screen. The non-metal
alignment interface should be used only when operating in CE-MS mode.
Figure 69
Capillary Position in Cassette
1 Procedures for inserting the capillary into the alignment interface, installing
this in a cassette and inserting the cassette into the CE are described in
Chapter 2. Figure 69 shows the position of the capillary in the CE-MS cassette.
Note the following points:
• In contrast to the regular CE-mode, the non-metal alignment interface is
nearer to the inlet than to the outlet (inlet to detector = 21.6 cm.)
• The long end of the capillary must be sufficient to reach the MS. For best
results the shortest total length of capillary should be used (80 cm for use
with the Agilent 1100 Series LC/MSD including UV detection).
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Online CE-ESI-MS with the Agilent CE System
Installing the Capillary and the CE-MS Cassette
2 Cut the PTFE tube to an appropriate length to cover the exposed capillary
between the end of the CE-MS cassette and the inlet of the MS. Place the PTFE
tube over the capillary and screw it on tightly at the CE-MS cassette.
3 Insert the CE-MS cassette in the same position as for the regular CE mode.
Make sure that the long part of the capillary stays outside the instrument after
closing the top cover.
Figure 70
Inserting the CE-MS Cassette
Configuring the Graphical User Interface for CE-MS Mode
1 Select Instrument from the Method & Run Control menu of the Agilent
ChemStation.
2 From the Instrument menu choose Select CE mode.
3 Open the CE mode box and select CE-MS. The screen shown in the next figure
will appear. The Select CE mode dialog box can also be opened by clicking on
Mode in the Method & Run Control menu.
148
Online CE-ESI-MS with the Agilent CE System
Installing the Capillary and the CE-MS Cassette
Figure 71
Select CE-MS Mode
Figure 72
GUI for CE-MS Mode
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Online CE-ESI-MS with the Agilent CE System
Installing the G1607A Sprayer Kit
Installing the G1607A Sprayer Kit
Table 8
Contents of Agilent G1607A CE-ESI-MS Sprayer Kit
Description
Quantity
Part Number
ES needle assembly
1
G1607-60041
CE-MS Neb. cover
1
G1607-60034
Hex key set II
1
8710-2164
DIN3115 screwdriver
1
5022-2142
CE-ESI sprayer
1
G1607-60001
Splitter assembly
1
G1607-60000
Ferrule 360 µm
1
5022-2141
Nut fingertight
2
0100-1543
Flex loc element
2
1520-0401
Screw M4 x 0.7
2
0515-0982
Gasket
2
G1607-20030
Fitting
2
0100-1544
Instructions
1
G1600-90106
Ion kit (ammonium acetate)
1
8500-4410
CE-MS test sample
1
5063-6590
Chemicals and Parts Needed
❏ 1 N sodium hydroxide (not supplied, for example, part number 5062-8576).
❏ CE grade water (not supplied, for example, part number 5062-8578).
❏ HPLC grade methanol (not supplied).
❏ 100 mM ammonium acetate (5 ml ampules containing 1.9 g ammonium
acetate in 20/80 (v/v) methanol/water. part number 8500-4410,
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Installing the G1607A Sprayer Kit
Thermospray Ionization Reagent. Included in the Agilent G1607A kit.
❏ Test sample quinine sulfate dihydrate (part number 5063-6590). Included
in the Agilent G1607A kit.
❏ CE-MS capillary o.d. 360 µm, i.d. 50 µm (part number G1600-67311, one set
is included in the Agilent G1603A CE-MS adapter kit.
❏ CE-MS alignment interface (part number G1600-60400, one interface is
included in the Agilent G1603A CE-MS adapter kit).
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Preparation of Buffers and Test Sample
Preparation of Buffers and Test Sample
The following solutions have to be prepared:
❏ 1 N sodium hydroxide (initial conditioning of the capillary).
❏ 1 ml water (initial conditioning of the capillary).
❏ 10 mM ammonium acetate pH 6.9 (running buffer).
❏ 5 mM ammonium acetate in 50% methanol (sheath liquid).
❏ 1 mg/ml quinine sulfate dihydrate in water (test sample).
NOTE
Solvents used for CE should be filtered through a 0.2 µm filter prior to use.
Test Sample
1 Weigh 1 mg of the test sample in a 2 ml Eppendorf cup.
2 Add 1 ml water.
3 Stir until the compound is completely dissolved (place the vial for 30 min in a
mixer or for 15 min in an ultrasonic bath).
4 Prepare a CE vial with the test solution (1 ml in a glass vial or 500 µl in a
polypropylene, PP, vial).
Solvents Used for Initial Conditioning
1 Prepare 1 vial with 1 N sodium hydroxide (300 µl in a PP vial).
2 Prepare 1 vial with water (1 ml in glass vial or 500 µl in PP vial).
Running Buffer
1 Dilute the content of one 5 ml ampule (containing 1.9 g ammonium acetate in
20/80 (v/v) methanol/water) in 245 ml water. This gives a stock solution of
100 mM ammonium acetate pH 6.9, 0.4% (v/v) methanol.
2 Dilute a certain amount of the stock solution to 1:10.
3 Prepare two vials (1 ml in glass vials or 500 µl in PP vials) with running buffer.
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Preparation of Buffers and Test Sample
Sheath Liquid
1 Mix 225 ml of CE grade water with 250 ml of methanol.
2 Add 25 ml of the 100 mM ammonium acetate stock solution.
3 Stir thoroughly.
4 Fill the sheath liquid in solvent bottle A of the Agilent 1100 Series pumping
system. In case a syringe pump is used to deliver the sheath liquid, fill a
syringe.
NOTE
Prepare fresh sheath liquid at least once a week.
Prepare the test sample fresh prior to use.
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Preparing the Agilent 1100 Series Pump and Vacuum Degasser
Preparing the Agilent 1100 Series Pump
and Vacuum Degasser
The following steps have to be done if the pump and degasser are used.
❏ Install the pump and the degasser (optional) as described in their
installation documentation. Otherwise an infusion pump has to be
installed and connected.
❏ The sheath flow splitter (included in the Agilent G1607A CE-MS sprayer
kit) has to be connected to the pump outlet. The following section
describes the setup.
Figure 73
The Sheath Flow Splitter
Recycle or waste
1:100 Splitter
From
pump
RECYCLE
OUT
G1607-60000
Out to CE-ESI-MS interface
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Preparing the Agilent 1100 Series Pump and Vacuum Degasser
1 Install the flow splitter in the isocratic pump.
Put the 2 rubber plugs from the
Agilent G1607A kit into the sheet metal as
shown below and use the two screws also
included to mount the splitter.
2 Connect the tubing labeled pump to the
pump outlet. The waste tubing may be drawn
back into the sheath liquid bottle to reuse the
solvent).
3 Still leave the tubing labeled Out disconnected from the sprayer but place its end in a beaker. It
will be connected later.
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Preparing the Agilent 1100 Series Pump and Vacuum Degasser
5 Fill the sheath liquid in solvent bottle A of the 1100 pumping system. Connect
the tubing of bottle A to the degasser (optional). Open the purge valve and
flush the pump at 2 ml/min (100% A) for 10 min. Reduce the flow rate to 0.4
ml/min and close the purge valve. The splitter splits the sheath liquid in the
ratio 1:100, so the sheath liquid flow rate will be 4 µl/min.
If no degasser is in use the sheath liquid MUST be vacuum degassed prior use,
and the flush time may be shorter.
6 The backpressure at 0.4 ml/min is approximately 40 bar.
Setup Pump Parameters
• Flow: 0.400 ml/min (1:100 flow splitter = 0.004 ml/min)
• %B: 0
• Active channel: A
• Max pressure/flow: 400 bar
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Preparing the Agilent Mass Selective Detector
Preparing the Agilent Mass Selective
Detector
The API-Electrospray source must be installed. Please refer to appropriate
documentation. It is assumed that the MSD has been successfully tuned for
single charged molecules (atunes.tun).
Inserting and Adjusting the CE Capillary in the
CE-ESI-MS Sprayer
Figure 74
The CE-ESI-MS Sprayer
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Preparing the Agilent Mass Selective Detector
1 Remove the protection plastic tube from the
sprayer tip. Turn the adjustment screw
counterclockwise (+ direction) until its
mechanical stop. Then turn two complete
turns clockwise (- direction).
158
2 Open the fitting for the CE and insert the
capillary. Fix the capillary so that it still can
be moved up or down. The capillary should
be aligned flat with the sprayer tip. You can
use your finger nail. Tighten the fitting screw
tight enough to keep the capillary in place.
Online CE-ESI-MS with the Agilent CE System
Preparing the Agilent Mass Selective Detector
3 Turn the adjustment screw 1/4 turn counter clockwise (2 marks in + direction). Finally the
capillary should protrude approximately 0.1 mm out of the sprayer tip
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Installing the CE-ESI-MS Sprayer
Installing the CE-ESI-MS Sprayer
1 Connect the nebulizing gas and the sheath
liquid to the CE sprayer.
160
2 Remove the cover of the ion source.
Carefully insert the CE sprayer into the
electrospray chamber of the MSD. Do not
touch the electrospray chamber with the
sprayer tip, because it can damage the
sprayer tip easily. Do not hold the sprayer at
the adjustment screw while inserting it into
the ion source. This can misalign the sprayer.
Online CE-ESI-MS with the Agilent CE System
Installing the CE-ESI-MS Sprayer
3 Mount the ion-source cover included in the Agilent G1607A sprayer kit.
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Method to Analyze the Test Sample
Method to Analyze the Test Sample
The parameters given in this section are typical for the analyzed test sample.
However, they might have to be adapted when other samples are analyzed.
Typical settings for the spray chamber parameters are:
Drying gas: 6–10 l/min
Drying gas temperature: 100–300 °C
Nebulizing gas: 10–20 psi
HV (positive mode): 3.5–4 kV
HV (negative mode): 3–3.5 kV
Prior to first use, a new capillary should be properly conditioned. A
procedure including a 5 min 1N NaOH flush, followed by a 10 min flush with
water and a 20 min flush with run buffer is appropriate for the analysis of the
test sample. There is no need to remove the capillary from the system prior to
this procedure provided the sheath liquid is running.
Vial table:
1 = 1 N NaOH,
2 = water
5 = run buffer (10 mM ammonium acetate, pH 6.9, 0.04% methanol),
6 = flush buffer (same as run buffer)
7 = 1 test sample (1 mg/ml quinine sulfate dihydrate).
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Method to Analyze the Test Sample
The method described here is also stored in the Agilent ChemStation for
CE-MS under C:\HPCHEM\1\methods\cems\quinine.m
HIGH PERFORMANCE CAPILLARY ELECTROPHORESIS
CE mode:
CE-MS
Home values:
Lift Offset
4
Cassette Temperature 20 °C
Inlet Home Vial
5: buffer
Outlet Home Vial
none
Replenishment and Preconditioning:
serial processing
Replenishment Entries:
No Replenishment used
Preconditioning Entries:
1 FLUSH
5.00 min, I:6, O:don’t care
Postcondition Entries:
No Postcondition used
Electric:
Electric
Polarity
Voltage
Current
On
Positive
0.00 kV
50.00 µA
Power
System Limit
Low Current Limit 0.00 µA
Injection Table Entries:
1 PRESSURE 50.0 mbar, 2.0 sec, I:InjectVial, O:don’t care
2 PRESSURE 50.0 mbar, 2.0 sec, I:InHomeVial, O:don’t care
Store Data:
Collect current:
Yes
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Method to Analyze the Test Sample
Time entries:
Stoptime
10.00 min
Posttime
Off
Timetable:
Time [min]
0.3
Function
VOLTAGE
Parameter
27.00 kV
DIODE ARRAY DETECTOR
Settings:
Stop Time
no Limit
Post Time
Off
Response Time
0.2 sec
Peakwidth
>0.01 min
Prerun Autobalance
On
Postrun Autobalance
Off
Spectrum:
Store
From
To
Treshold
All in peak
190 nm
450 nm
2.00 mAU
Signals:
Store Signal,Bw Reference,Bw [nm]
A: Yes
254,16
450,80
Agilent 1100 ISOCRATIC PUMP 1
Control:
Flow
Stoptime
Posttime
164
0.400 ml/min
No Limit
Off
Online CE-ESI-MS with the Agilent CE System
Method to Analyze the Test Sample
Solvents:
Solvent A
100.0% (5 mM Ammac pH 6.9 in 50% MeOH)
Pressure Limits:
Minimum Pressure
0 bar
Maximum Pressure
400 bar
Auxiliary:
Maximal Flow Ramp
100.00 ml/min^2
Compressibility
75*10^-6/bar
Minimal Stroke
100 µl
MASS SPECTROMETER DETECTOR Agilent G1946B
Use MSD : Enabled
Ionization Mode : API-ES
Tune File: atunes.tun
StopTime: asPump
Time Filter: Enabled
Data Storage: Condensed
Peakwidth: 0.12 min
Scan Speed Override: Disabled
Signals:
Signal 1
Polarity: Positive
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Method to Analyze the Test Sample
Fragmentor Ramp: Disabled
Scan Parameters:
Time |
(min) |
Mass Range
Low
|
High
|Frag- | Gain|Thres-| Step|mentor| EMV | hold | size
-------|----------|---------|------|-----|------|------0.00
150.00
350.00
65
3.0
50
0.10
Spray Chamber:
MSZones:
Gas Temp
: 130 C
DryingGas
: 10.0 l/min
Neb Pres
VCap
: 10 psig
maximum 350 C
maximum 13.0 l/min
maximum 60 psig
: 4000 V
Establish and run a sequence with 5 consequent injections from the vial with
the test sample.
Due to the equilibration of the system the first run of the sequence may not
be successful and should be discarded. The following runs should result in
UV and MS traces similar to those shown in Figure 75.
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Method to Analyze the Test Sample
Figure 75
UV and MS Traces
MS spectra taken from the peak in the MS trace should be similar to the one
shown in Figure 76. The current should be approximately 4 uA.
Figure 76
MS Spectra
MW: 325, 326
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Online CE-ESI-MS with the Agilent CE System
Storing the CE-MS Capillary, the Sheath Flow Splitter and the CE-MSD Sprayer
Needle After Usage
Storing the CE-MS Capillary, the Sheath
Flow Splitter and the CE-MSD Sprayer
Needle After Usage
To avoid plugging of the capillary, the sheath flow splitter and the CE-MSD
sprayer needle need to be cleaned if not in use.
Cleaning of the Sheath Flow Splitter and the CE-MSD
Sprayer Needle
1 Replace the sheath liquid bottle with a bottle filled with water.
2 Leave the CE-MS capillary, the sheath flow splitter and the nebulizing gas
connected.
3 Prime the pump and flush for 10 minutes pump water through the sheath flow
splitter and CE-MSD sprayer needle.
4 Replace the water in the sheath liquid bottle with iso-propanol.
5 Prime the pump and flush for 10 minutes pump iso-propanol through the
sheath flow splitter and CE-MSD sprayer needle.
Storing the CE-MS capillary
1 Flush the capillary with water for 10 minutes.
2 Insert an empty vial with a cap into the tray and flush the capillary with air for
10 minutes.
3 The capillary can now be removed and stored.
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Maintenance
Maintenance
In general the CE-MSD sprayer needs little maintenance. But it may happen
that from time-to-time or in case of a problem some parts need to be
replaced. The parts shown in Table 9 can be exchanged:
Table 9
Parts for Maintenance or Repair (see Figure 77)
Item Number
Description
Part number
1
Sprayer body
No part number
2
Sprayer head
No part number
3a, 3b
Gasket
G1607-20030
4
0-ring
0905-1022
5
Spray needle
G1607-60041
6
Spring
5022-2140
7
Screw body
G1607-20029
8a, 8b
Seal holding screw
G1607-20022
9
Protection tube for the sprayer
0890-0581
Not shown
PEEK screw capillary and nebulizing gas
0100-1543
Not shown
PEEK ferrule capillary
5022-2141
Not shown
PEEK ferrule nebulizing gas
0100-1544
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Maintenance
Figure 77
The G1607A CE-MSD Sprayer
7
6
1
2
3a
8a
8b 8
4
5
9
3b
The Agilent G1607A CE-ESI-MS Sprayer needs in general little maintenance.
Following parts can be exchanged if necessary.
Replacing the Sprayer Gasket
(item 3a, 3b, part number G1607-20030)
A leaky gasket can generate an unstable MSD signal. The gasket can be
damaged if the flow rate of the sheath liquid exceeds than 200 µl/min.
A set of gaskets is provided with the Agilent G1607A CE-ESI-MS sprayer kit.
Parts Needed
❏ Hex key 2 mm
❏ Hex key 1.3 mm
❏ Gasket (part number G1607-20030)
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Maintenance
Steps to Exchange the Gasket
1 Disconnect the nebulizing gas, the sheath liquid tube and the CE-MS capillary
from the sprayer.
2 Remove the CE-MS sprayer from the MSD.
3Open the two body screws (7). Be careful not to
lose the attached springs (6).
4 Unscrew the sprayer head (2) from the
sprayer body (1).
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Maintenance
5 Using the special screw driver open the seal holding screw (8a) in the bottom of the sprayer
head. Replace the gasket (3a)
3 Carefully reassemble the G1607A CE-ESI-MS Sprayer.
4 Make the connections and reinstall it into the MSD.
5 Use the test sample to verify the proper function.
The Sprayer Needle (item 5, part number G1607-60041)
Periodically check the sprayer tip for erosion under a microscope (use the
nebulizer adjustment fixture (part number G1946-20215) and the
corresponding pocket microscope (part number G1946-80049) which are part
of the accessories kit of the G1946A/B MSD).
You also can examine the sprayer tip by applying a sheath flow of 50 µl/min.
At this flow it is possible to see the spray cone. The cone must be
symmetrical.
If the tip shows some erosion or the cone isn’t symmetrical the sprayer
needle (5) needs to be exchanged.
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Maintenance
Steps to Exchange the Sprayer Capillary
1 Disconnect the nebulizing gas, the sheath liquid tube and the CE-MS capillary
from the sprayer.
2 Remove the CE-MS sprayer from the MSD.
4Open the two body screws (7). Be careful not to
lose the attached springs (6).
5 Unscrew the sprayer head (2) from the
sprayer body (1).
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Maintenance
6 Open the seal holding screw (8b) of the
sprayer body.
7 Remove the defective sprayer needle (5).
Add a new gasket (3b) and carefully insert
the new sprayer needle.
8b
5
3b
3 Carefully reassemble the Agilent G1607A CE-ESI-MS Sprayer.
4 Check, if the sprayer needle juts out about 0.1–0.2 mm (ca. 1/3 of its diameter)
from the sprayer tip. Use the nebulizer adjustment fixture (part number
G1946-20215) and the corresponding pocket microscope (part number
G1946-80049) which are part of the accessories kit of the Agilent G1946A
MSD. If necessary readjust the sprayer needle.
5 Make the connections and reinstall it into the MSD.
6 Use the test sample to verify the proper function.
NOTE
A new Agilent G1607A CE-ESI-MS Sprayer is already preadjusted. Therefore
do not open the hex-key screws on the body part.
Check, if the sprayer needle protrudes about 0.1–0.2mm (~ 1/3 × d) from the
sprayer tip. Use the nebulizer adjustment fixture (part number G1946-20215)
and the corresponding pocket microscope (part number G1946-80049) which
are part of the accessories kit of the Agilent G1946A MSD. If necessary
readjust the sprayer needle.
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Maintenance
Readjusting the Sprayer Needle
A new Agilent G1607A CE-ESI-MS Sprayer is already preadjusted. The
sprayer needle should protrude from the sprayer tip about 0.1–0.2mm. This
setting gives highest sensitivity and baseline stability. During normal
operation there is no need to readjust the sprayer needle. Only readjust the
CE-ESI-MS Sprayer if the sprayer needle position differs from the settings
above.
Steps to Readjust the Sprayer Needle
1 Turn the adjustment screw counterclockwise (+ direction) until its
mechanical stop.
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Online CE-ESI-MS with the Agilent CE System
Maintenance
2 Open the 3 hex-key screws on the lower adjustment ring by 1/2 turn.
3 During the following adjustment procedure press sprayer head and body part
together to remove backlash (see picture above)
4 Turn the lower adjustment ring counterclockwise until the sprayer needle is
flat with the sprayer tip.
5 Now turn the lower adjustment screw by 1/4 turn clockwise.
6 Tighten the 3 hex-key screws on the lower adjustment ring again.
Check that the sprayer needle now protrudes about 0.1–0.2mm (~ 1/3 × d)
from the sprayer tip. This can be done by using the nebulizer adjustment
fixture (part number G1946-20215) and the corresponding pocket
microscope (part number G1946-80049) which are part of the accessories kit
of the Agilent G1946A/B MSD.
If necessary readjust the sprayer needle.
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Troubleshooting Matrix
Troubleshooting Matrix
Table 10
Troubleshooting Matrix
What Can Be Seen?
Cause
Test sample delayed or
not shown in the UV
signal
Pressure difference
Adjust the level of the inlet and outlet of the CE-MS capillary (e.g.
between inlet and outlet height adjustable table). Check for a slight overpressure due to
of the CE-MS capillary the parameters of the vent system (e.g. drying gas).If possible
remove the overpressure situation otherwise apply 50 mbar
during run (Timetable of the CE).
MSD signal not stable
Capillary not well cut
Solution
Cut the capillary again. Make sure the cut is flat and the capillary
is not shattered at the end.
Polyimide left at the end Remove the polyimide by burning it off. Clean that area with
iso-propanol.
Capillary not well
adjusted
The capillary should be 0.1 mm (2 marks) protrude of the sprayer
capillary, if that is not the case readjust the CE-capillary. See
“Inserting and Adjusting the CE Capillary in the CE-ESI-MS
Sprayer” on page 157.
Sprayer tip eroded
Exchange the sprayer tip. See “Steps to Exchange the Sprayer
Capillary” on page 173.
Sprayer capillary gasket Replace the sprayer capillary gasket. See “Steps to Exchange the
is leaking
Gasket” on page 171.
Sheath flow too low
The sheath flow splitter might be plugged. But be aware that
4 µl/min (about1droplet/8 minutes) leaving the restriction
capillary is not much!.
Backflush the different capillaries of the sheath flow splitter or
exchange the flow splitter.
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What else can you do with the G1603A CE-MS Adapter kit?
What else can you do with the G1603A
CE-MS Adapter kit?
Connecting the Agilent CE system to a non Agilent MS
All accessories and documentation needed to interface the Agilent capillary
electrophesis system to a non-Agilent mass spectometer are the
responsibility of the respective vendor and will not be provided by Agilent
Technologies.
The ground cable provided in the G1603A CE-MS Adapter kit should be
connected between the two instruments to ensure that they both refer to the
same ground.
For connection of the Agilent CE to any other MS where the electrospray
needle is on high voltage, an additional resistor sink, which is the
responsibility of the MS vendor, is required. This is mandatory to prevent the
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Online CE-ESI-MS with the Agilent CE System
What else can you do with the G1603A CE-MS Adapter kit?
power supply of the MS from being destroyed by the incomming CE current.
In this case the maximum CE current should not exceed 50 uA.
Agilent parts
Agilent CE
G1600A
MS Vendor
G1603A Adapter kit
•Cassette
•Capillary
MS interface must be
ordered by the MS
vendor
MS
Agilent CE
ChemStation
•Ground Cable
HPIB
HPIB
HPIB Cable
• ships with
G1600A/G1602A
Sink resistor is
requested on some MS
PC for MS
Connecting the Agilent capillary electrophesis system to
an external detector
The G1603A CE-MS Adapter kit makes it possible to get the capillary out of
the instrument. This maybe helpfull to connect an external detector e.q.
fluorescence detector or a laser induced fluorescence detector to the
capillary electrophoresis system.
NOTE
Check with the vendor of the external detector to verify what the detector
ships with. All accessories and documentation needed to interface the Agilent
capillary electrophesis system to an external detector are the responsibility of
the respective vendor and will not be provided by Agilent.
To connect to an external detector you typically need:
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What else can you do with the G1603A CE-MS Adapter kit?
• G1603A CE-MS Adapter kit
• A/D Converter 35900E#011 (GPIB)
• Signal Cable 35900-60750
• Remote Cable G1530-60930
Figure 78
180
5
5
Maintaining Your
Agilent CE Instrument
Standard operating procedures for maintenance
of your Agilent CE instrument
Maintaining Your Agilent CE
Instrument
This chapter describes standard operating procedures (SOPs) which should
be used to do maintenance on your instrument at regular intervals. Do only
maintenance on the Agilent CE instrument specified in this chapter. Other
maintenance or repairs must be done by Agilent Technologies trained
personnel. Unauthorized maintenance can be dangerous and damages are not
covered by warranty.
182
Maintaining Your Agilent CE Instrument
Quick overview about maintenance
Quick overview about maintenance
Injection System
• Electrodes
do not open top cover or remove cartridge without lowering lifts ->
electrodes filled with buffer -> carry over
clean frequently (e.q. weekly)
inspect for salt deposits, o-ring integrity, bending
• Prepunchers
clean frequently (e.q. weekly)
inspect for salt deposits, bent tip, deposits in top funnel
• Replace if either electrodes or prepunchers are bent or cannot be
cleanned.
Replenishment System
• System cleaning
should be performed when new buffer is added to the electrolyte reservoir.
Clean with isopropanol/water
filter all solutions prior to use (0.2 or 0.45 um)
• Replenishment Needle
keep water-filled vial in carousel position 49
inspect for bending if clogged, remove and syringe flush with
water/isopropanol or use ultrasonic device
• O-ring in buffer reservoir cap
inspect for damage
remove salt deposits
• Inlet frit
inspect and clean (especially if abosorbing buffers have been used)
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Maintaining Your Agilent CE Instrument
Quick overview about maintenance
replace when dirty
• Vial caps
do not overuse them (especially the PEO-caps)
inspect for damage
• Do not use buffer reservoir for long-term storage (prevents oxygen
saturation and bacteria growing)
Detection System
• Optical alignment interface
inspect for occlusion under microscope or magnifier (dust or shards of
polyimide)
if capillary is broken while in the interface, inspect and remove fragments
(ultrasonic device and air burst)
• Lamp
perform DAD test regularly (e.q. weekly) and monitor lamp counts using
the red labled alignment interface without capillary
replace lamp when lamp counts are low and signal to noise increases
• Capillary window
gently clean with isopropanol and lint-free wipe
184
Maintaining Your Agilent CE Instrument
Cleaning the Electrodes, Prepunchers and Insulation Plate
Cleaning the Electrodes, Prepunchers and
Insulation Plate
Objective
Buffer precipitates, dust or other solid material in the electrodes and
prepunchers or under the insulation plate can cause arcing or current
leakage, buffer contamination or carryover. Cleaning these parts will prevent
these from occurring.
Frequency
The electrodes, prepunchers and the insulation plate must be cleaned
regularly, about once a week, or if peaks show an unusual tailing or unusual
peak shoulders or if arcing or current leakage can be observed or if creeping
buffers such as urea are used.
What You Will Do
• prepare the Agilent CE instrument for this task,
• remove the detector cover,
• access the electrodes,
• remove the front cover,
• access the prepunchers,
• clean the electrodes,
• clean the prepunchers,
• clean the insulation plate,
• reinstall the prepunchers, and
• reinstall the electrodes.
Instrumentation
• This SOP is applicable to the Agilent CE instrument with firmware revision
1.4 or higher, built-in diode array detector with firmware revision 1.0 or
higher.
185
Maintaining Your Agilent CE Instrument
Cleaning the Electrodes, Prepunchers and Insulation Plate
• Agilent ChemStation for control and data evaluation.
• Check revision numbers: Under INSTRUMENT menu click on Revision &
Serial Numbers.
Parts Required
❏ 12 mm hexagonal socket screwdriver (included in the startup kit).
❏ Pozidriv screwdriver (included in the startup kit).
❏ Wash bottle with isopropanol.
❏ Wash bottle with water.
❏ Compressed inert gas, oil-free.
WAR NI N G
Follow the common safety regulations for laboratories when doing
this task. Wear safety spectacles and rubber gloves. For translations,
see page 255.
Preparing the Agilent CE Instrument
Prior to maintenance do the following:
1 Select the Detector icon in the CE Diagram screen.
2 Select Lamp Off from the menu to switch the lamp off.
3 Select Maintenance from the Instrument menu to lower all lifts.
4 Select Exit from the File menu to exit the Agilent ChemStation. Exit both the
online and offline copy.
5 Turn off line power to the Agilent CE instrument.
6 Disconnect the line power cord from the Agilent CE instrument.
Removing the Detector Cover
1 Open the top cover.
2 Remove the capillary cassette.
WAR NI N G
Make sure you have disconnected the line power cord of the Agilent
CE instrument before you proceed. For translations, see page 255.
186
Maintaining Your Agilent CE Instrument
Cleaning the Electrodes, Prepunchers and Insulation Plate
3 Take off the detector cover by opening the two push turn locks. To open press
down and turn them counterclockwise.
Figure 79
Push Turn Locks Securing the Detector Cover
Detector cover
Push turn locks
WAR NI N G
If you were using the Agilent CE instrument shortly before, the lamp
may be very hot. Avoid touching the lamp or wear protective gloves.
For translations, see page 257.
187
Maintaining Your Agilent CE Instrument
Cleaning the Electrodes, Prepunchers and Insulation Plate
Figure 80
After Removing the Detector Cover
188
Maintaining Your Agilent CE Instrument
Cleaning the Electrodes, Prepunchers and Insulation Plate
Accessing the Electrodes
1 Use a Pozidriv screwdriver to unscrew the two polypropylene screws which
secure the insulation plate.
Figure 81
Screws Securing the Insulation Plate
Polypropylene screws
Insulation plate
2 Use a Pozidriv screwdriver to remove the screw that secures the blue ground
cable.
Figure 82
Removing the Inlet Electrode
12 mm hexagonal
socket screw driver
’Red’ high voltage cable
Ground cable screw
’Blue’ ground cable
189
Maintaining Your Agilent CE Instrument
Cleaning the Electrodes, Prepunchers and Insulation Plate
3 Use the 12 mm hexagonal socket screwdriver to unscrew the inlet electrode.
4 Carefully lift the red high voltage wire. The inlet electrode will come out.
5 Slowly slide the insulation plate to the right (1), tilt it up (2 and 3) and take it
out. Make sure that the outlet electrode is not touching the foam of the tray
cooling or the lift station.
Figure 83
Removing the Insulation Plate
6 Remove the outlet electrode from the insulation plate using the 12 mm
hexagonal socket screwdriver.
190
Maintaining Your Agilent CE Instrument
Cleaning the Electrodes, Prepunchers and Insulation Plate
Removing the Front Cover
1 Loosen the two screws that secure the front cover. It is not necessary to
remove the screws completely.
Figure 84
Removing the Front Cover
Front cover screws
Front cover
2 Carefully pull the front cover away from the mainframe. Pull steadily at both
sides. You need slightly more force to remove the right side due to a connector.
191
Maintaining Your Agilent CE Instrument
Cleaning the Electrodes, Prepunchers and Insulation Plate
Figure 85
Connector
Connector
192
Maintaining Your Agilent CE Instrument
Cleaning the Electrodes, Prepunchers and Insulation Plate
Accessing and removing the Prepunchers
1 Inlet and outlet lifts are located behind the sample tray. The replenishment lift
is located at the right side under the replenishment needle.
Figure 86
Outlet lift
Inlet lift
Replenishment needle
Replenishment lift
CA UT IO N
Do not touch or bend the replenishment needle. For translations, see page 258.
193
Maintaining Your Agilent CE Instrument
Cleaning the Electrodes, Prepunchers and Insulation Plate
WAR NI N G
The prepunchers have very sharp tips. Be careful when touching them.
For translations, see page 259.
2 Remove the plastic screw which secures the inlet prepuncher using a Pozidriv
screwdriver.
Figure 87
Teflon tubing
Inlet prepuncher
Hole
Screw
3 Unscrew the teflon inlet prepuncher tubing which is labeled 7.
4 The prepuncher has a hole which can be used to gently lever it out using a
paper clip. Carefully pull the prepuncher out of the lift.
CA UT IO N
Do not damage the funnel surface of the prepuncher or the teflon air pressure
tube connection. For translations, see page 260.
5 Unscrew the teflon outlet prepuncher tubing labelled 8. Carefully pull the
prepuncher out of the lift.
NOTE
For cleaning it is NOT necessary to remove the teflon tube from the
prepunchers.
6 Remove the prepuncher from replenishment lift.
194
Maintaining Your Agilent CE Instrument
Cleaning the Electrodes, Prepunchers and Insulation Plate
Cleaning the prepuncher
Check for damaged tips before cleaning the prepunchers. If the tips are
damaged replace the prepunchers.
1 Flush the prepunchers with water. Watch for salt crystals. All salt crystals
must be removed. Take particular care with the teflon tube connector.
Figure 88
2 Flush the prepunchers with isopropanol.
3 Use compressed air to dry the prepunchers and remove all traces of liquid. Do
not forget to dry the connected tubes as well.
If dirt is still accumulated in the prepuncher funnel, put the prepuncher with
connecting tubing in a beaker with water and place it in an ultrasonic bath for
five minutes followed by steps 2 and 3 of this section.
CA UT IO N
To avoid contamination the prepunchers and connected tubes must be
completely dry before reinstallation. For translations, see page 261.
195
Maintaining Your Agilent CE Instrument
Cleaning the Electrodes
Cleaning the Electrodes
1 Flush the electrodes with water. Watch for salt crystals. All salt crystals must
be removed.
Figure 89
2 Flush the electrodes with isopropanol.
3 Use compressed air to dry the electrodes and remove all traces of liquid.
CA UT IO N
To avoid contamination, the inside of the electrodes must be completely dry
before reinstallation. For translations, see page 262.
If there is still an accumulation of dirt in the upper funnel start the cleaning
procedure again or place it in a beaker of iso-propanol and place this in an
ultrasonic bath for five minutes followed by steps 2 and 3 of this section. If
this fails then replace the electrode.
Cleaning the Insulation Plate
1 Moisten a tissue with water and use it to clean the insulation plate.
2 Moisten a tissue with isopropanol and use it to clean the insulation plate.
3 Dry the insulation plate with tissue and compressed air.
The insulation plate can also be cleaned in a dishwasher at a maximum
temperature of 60 °C.
196
Maintaining Your Agilent CE Instrument
Cleaning the Electrodes
To avoid arcing the insulation plate must be completely dry before
reinstallation.
Reinstalling the Prepunchers
1 Carefully insert the prepuncher labeled 8 into the outlet lift. Connect the tube
to its connector (labeled 8).
Figure 90
Prepuncher
The fitting of the outlet prepuncher must be tight. Otherwise problems during
CEC mode may occur.
2 Carefully insert and tighten the plastic screw to secure the prepuncher. The
prepuncher will assume its correct position as the screw is tightened.
3 Carefully connect the inlet prepuncher with the air pressure tube (labeled 7)
connected into the inlet lift. Connect the tube to its connector (labeled 7).
The fitting of the inlet prepuncher must be tight. Otherwise pressure or
injection problems may result.
4 Carefully insert and tighten the plastic screw to secure the prepuncher. The
prepuncher will assume its correct position as the screw is tightened.
197
Maintaining Your Agilent CE Instrument
Cleaning the Electrodes
Reinstalling the Electrodes and the Insulation Plate
1 Reinstall one electrode into the outlet electrode position of the insulation .
plate.
2 Thread the electrode into place by hand . first.
3 Tighten the electrode with the 12 mm hexagonal socket screw driver. Do not
overtighten. Do not bend the . electrodes.
4 Holding the insulation plate by the cassette holder, slide the base plate under
the detector (1) then tilt it up into place (2) and push it completely to the left
(3).
Figure 91
Installation of the Base Plate
5 The insulation base plate should be laying flat in position. The outlet electrode
should be positioned correctly under the detector.
198
Maintaining Your Agilent CE Instrument
Cleaning the Electrodes
Figure 92
Alignment to the Detector
6 Secure the base plate by reinstalling the two plastic screws.
7 Reconnect the blue ground wire and fasten the screw.
8 Insert the inlet electrode into the ring connector of the red high voltage wire.
Thread the inlet electrode (and the ring connector) into the base plate by hand
then tighten the inlet electrode with the 12 mm hexagonal socket screwdriver.
Do not overtighten.
9 Reinstall the front cover and fasten the two screws.
10 Reinstall the detector cover and fasten the two push turn locks by pushing
down and turning them clockwise.
11 Reinstall the capillary cassette.
12 Close the top cover.
13 Connect the power cord and turn on line power to the Agilent CE instrument.
14 Start your computer and Agilent ChemStation.
199
Maintaining Your Agilent CE Instrument
Cleaning the Detection Window and the Alignment Interface
Cleaning the Detection Window and the
Alignment Interface
The following procedures describes how to clean the detection window of
the capillary and the slit of the alignment interface. An occluded interface
may result in increased noise or instrument error.
Frequency
As necessary, especially if the signal-to-noise ratio isn’t within specification
or if the slit of the alignment interface is contaminated (e.g. a capillary was
broken or polyimide fragments or dust deposits).
Instrumentation
• This SOP is applicable for the Agilent CE instrument with firmware
revision 1.4 or higher, built-in diode array detector with firmware revision
1.0 or higher.
• Agilent ChemStation for control and data evaluation.
• Check revision numbers: Click on Serial Numbers in Instrument menu.
Parts/Material Required
❏ Cotton swab or a soft tissue.
❏ Isopropanol.
❏ Capillary insertion tool.
❏ Compressed inert gas, oil-free.
❏ Ultrasonic bath.
❏ 100 ml beaker.
❏ Safety glasses.
What You Will Do
• remove the capillary cassette,
200
Maintaining Your Agilent CE Instrument
Cleaning the Detection Window and the Alignment Interface
• remove the capillary from the cassette,
• remove the alignment interface from the capillary,
• clean the detection window, and
• clean the alignment interface.
Removing the Capillary Cassette
1 Select Change Cassette from the CE Diagram.
2 Open the top cover.
3 Remove the cassette from the Agilent CE instrument.
Removing the Capillary from the Cassette
WAR NI N G
Wear safety glasses when opening the cassette and handling the
capillary to protect your eyes. For translations, see page 263.
1 Open the capillary cassette by pushing the cassette lock.
Figure 93
Removing the Capillary from the Cassette
Cassette lock
Capillary lock
2 Carefully remove the capillary by pushing the white capillary lock.
3 Remove the alignment interface.
201
Maintaining Your Agilent CE Instrument
Cleaning the Detection Window and the Alignment Interface
Removing the Alignment Interface from the Capillary
1 Put the insertion tool on a flat surface. You can also use the tool integrated into
the capillary cassette.
2 Put the alignment interface on the insertion tool and press down firmly. Press
only on the outer ring to release the black plastic spring.
3 Hold the capillary at the colored stopper. Pull the capillary out of the
alignment interface.
Figure 94
Removing the Alignment Interface
Alignment interface
Capillary insertion tool
Do not release the alignment interface while the capillary is moving. The
capillary may break.
Cleaning the Detection Window
1 Dip a cotton swab or a soft tissue into isopropanol and wipe the detection
window of the capillary carefully.
Cleaning the Alignment Interface
1 Put the alignment interface into the beaker filled with isopropanol. Place the
beaker in an ultrasonic bath for 5 minutes.
2 Use compressed inert gas to dry the alignment interface.
The gas should have a maximum pressure of two bar. Make sure it is free of
oil residues.
3 View slit interface under a microscope or magnifier to verify cleanliness.
202
Maintaining Your Agilent CE Instrument
Cleaning the Detection Window and the Alignment Interface
Figure 95
Detection Interface and Alignment of Extended Light Path
Reinstalling the Capillary
Install the capillary as described in “ Installing the Capillary in the Alignment
Interface”.
203
Maintaining Your Agilent CE Instrument
Preparing/Cleaning the Replenishment System
Preparing/Cleaning the Replenishment
System
Objective
The Replenishment System is used to automatically empty and refill vials
with buffer. This is used to prevent changes in the composition of the buffer
during multiple analyses. Preventive maintenance is required to keep the
system functioning properly.
Frequency
The replenishment system (bottles and tubing) should be cleaned when:
• using it for the first time,
• changing the buffer, or
• the replenishment system will be idle for some time.
Instrumentation
• This SOP is applicable for the Agilent CE instrument with firmware
revision 1.4 or higher, built-in diode array detector with firmware revision
1.0 or higher.
• Agilent ChemStation for control and data evaluation.
• Check revision numbers: Click on Serial Numbers in the Instrument menu.
Parts required
❏ Vials and caps:
Plastic vials, part number 5182-0567
Vial caps, part number 5181-1507
Vials are part of the Agilent CE test kit G1600-68703.
❏ Replenishment bottles.
❏ Water and/or buffer and isopropanol (depending on the protocol selected).
204
Maintaining Your Agilent CE Instrument
Preparing/Cleaning the Replenishment System
Preparing the Replenishment System for Initial Use
1 Click on one of the replenishment bottles in the CE diagram.
2 Choose Change Bottles to release the pressure before you open the bottles.
Figure 96
CE-Diagram—Change Bottles
3 Open the transparent door at the bottom of the front panel.
4 Take out the electrolyte bottle and unscrew the cap. The electrolyte bottle has
a frit and tubing inside. The frit should not get dirty.
5 Fill 200 ml of the water in the electrolyte bottle. Make sure the water is filtered
by using a 0.45 µm filter.
6 Make sure the waste bottle is empty.
7 Cap the bottles tightly.
8 Choose Done in the Change Bottles Dialog box. Wait until the pressure has
built up again, i.e. the instrument reaches the Ready state.
9 Put a capped empty vial into tray position number 46.
10 Click on the vial between the electrolyte and waste bottle in the CE diagram.
11 Choose Clean Tubes to flush the tubing which is involved in the replenishment
function.
205
Maintaining Your Agilent CE Instrument
Preparing/Cleaning the Replenishment System
Figure 97
CE Diagram—Clean Tubes
12 Enter the vial number of the empty vial when prompted and set a flushing time
of 2 minutes.
13 Start the program.
206
Maintaining Your Agilent CE Instrument
Preparing/Cleaning the Replenishment System
Changing the Buffer Composition
1 Click on one of the replenishment bottles in the CE diagram.
2 Choose Change Bottles to release the pressure before you open the bottles.
Figure 98
CE Diagram—Change Bottles
3 Open the transparent door at the bottom of the front panel.
4 Take out the electrolyte bottle and unscrew the cap. The electrolyte bottle has
a frit and tubing inside. The frit should not get dirty.
5 Fill minimum 200 ml of buffer in the electrolyte bottle. Make sure the buffer is
filtered by using a 0.45 µm filter.
6 Make sure the waste bottle is empty.
7 Cap the bottles tightly.
8 Choose Done in the Change Bottles Dialog box. Wait until the pressure has
built up again, i.e. the instrument reaches the Ready state.
9 Put a capped empty vial into a tray position number 46.
10 Click on the vial between the electrolyte and waste bottle in the CE diagram.
11 Choose Clean Tubes to flush the tubing which is involved in the replenishment
function.
207
Maintaining Your Agilent CE Instrument
Preparing/Cleaning the Replenishment System
Figure 99
CE Diagram—Clean Tubes
12 Enter the vial number of the empty vial when prompted and set a flushing time
of 0.5 minutes.
13 Start the program.
The involved volume of the replenishment system is about 5 ml. A 10-time
flush is sufficient. The flush time depends strongly on the viscosity of your
buffer. Therefore choose a short time and repeat Clean Tubes if necessary,
otherwise it may happen that the buffer is flushed completely into the waste
bottle.
208
Maintaining Your Agilent CE Instrument
Preparing/Cleaning the Replenishment System
The Replenishment System will not be used some time
The replenishment system should be flushed first with water and then with
isopropanol.
1 Click on one of the replenishment bottles in the CE diagram.
2 Choose Change Bottles to release the pressure before you open the bottles.
Figure 100
CE Diagram—Change Bottles
3 Open the transparent door at the bottom of the front panel.
4 Take out the electrolyte bottle and unscrew the cap. The electrolyte bottle has
a frit and tubing inside. The frit should not get dirty.
5 Fill 200 ml of the water in the electrolyte bottle. Make sure the water is filtered
by using a 0.45 µm filter.
6 Make sure the waste bottle is empty.
7 Cap the bottles tightly.
8 Choose Done in the Change Bottles Dialog box. Wait until the pressure has
built up again, i.e. the instrument reaches the Ready state.
9 Put a capped empty vial into a tray position number 46.
10 Click on the vial between the electrolyte and waste bottle in the CE diagram.
11 Choose Clean Tubes to flush the tubing which is involved in the replenishment
function.
209
Maintaining Your Agilent CE Instrument
Preparing/Cleaning the Replenishment System
Figure 101
CE Diagram—Clean Tubes
12 Enter the vial number of the empty vial when prompted and insert a flushing
time of 2 minutes.
13 Start the program.
14 Repeat the action by using isopropanol instead of water. Empty the waste
bottle whenever you fill or refill the electrolyte bottle.
Acceptance
• The air pressure and vacuum must come up without showing an Error
message.
• The vial in position 46 must appear in the replenishment lift.
• The pressure inside the electrolyte bottle must be reduced to
approximately 400 mbars.
• Liquid inside the electrolyte bottle must be flushed to the waste bottle
(volume depends on the procedure you choose).
• Vial 46 must be filled with the liquid of the electrolyte bottle and emptied
afterwards without any Error message.
210
Maintaining Your Agilent CE Instrument
Preparing/Cleaning the Replenishment System
Further Action
If the instrument can’t build-up pressure or vacuum, check the following:
❏ proper seal of the electrolyte bottle, and
❏ proper seal of the waste bottle
❏ tight fittings
If none of these reasons apply, call Agilent Technologies.
211
Maintaining Your Agilent CE Instrument
Maintaining the Functionality
Maintaining the Functionality
This section describes how to exchange parts which may alter during the
operation, such as the air inlet filter and the detector lamp.
212
Maintaining Your Agilent CE Instrument
Changing the Air Inlet Filter
Changing the Air Inlet Filter
Objective
The following procedure is used to replace the air inlet filter. The installed air
pump uses air from the environment (through this filter) to create pressure
inside the electrolyte bottle. The compressed air is needed to flush a capillary
and to inject the sample.
Frequency
Air filter replacement should be considered if the environment is very dusty
or if the system can’t generate enough pressure (a typical Error message is:
ER 5207: Airpump failed to generate pressure). We suggest you should
change the air filter at least every three months.
Material Needed
❏ Pozidriv screwdriver (part of the accessory kit)
❏ air filter (a spare filter is supplied with the standard accessory kit)
Accessing the Air Filter
The air filter is located inside the electrolyte and waste bottle compartment.
Therefore it is very easy to replace the air filter frequently.
If the instrument can't build-up either pressure or vacuum, check the
following:
❏ proper seal of the pressure bottle,
❏ proper seal of the vacuum bottle, and
❏ loose fittings on the bottles
If none of these reasons apply, call Agilent Technologies.
213
Maintaining Your Agilent CE Instrument
Changing the Lamp
Changing the Lamp
Objective
The following procedure is used to replace the lamp. Loss of light intensity
over time increases baseline noise.
Frequency
Exchange of the lamp should be considered if a drastic increase of the noise
level is encountered which is not caused by any other reason (for example a
contaminated slit, something absorbing inside the capillary, etc.) or the lamp
refuses to ignite. Generally the light intensity drops 50 % (measured at
230 nm) of its original value after approximately 700 hours of operation. We
suggest you check the lamp intensity before you change the lamp to be sure
the noise increase is not due to other reasons (air bubbles inside the
capillary, alignment interface or detection window contaminated).
It may happen that a hot lamp refuses to ignite at the first attempt. Please
wait until the lamp has cooled down.
Material Needed
❏ Pozidriv screwdriver (part of the accessory kit).
❏ Deuterium lamp for Agilent CE instrument.
Removing the Existing Lamp
1 Select Exit from the File menu to exit the Agilent ChemStation. Exit both
online and offline copy.
2 Turn off the line power to the Agilent CE instrument.
WAR NI N G
If the lamp is still on after you removed the detector cover the UV
radiation can be harmful to your eyes. For translations, see page 264.
3 Open the top cover.
4 Open the push turn locks fastening the detector cover by pushing them down
and turning them counterclockwise.
214
Maintaining Your Agilent CE Instrument
Changing the Lamp
5 Remove the detector cover.
6 Disconnect the lamp cable connector. Pull at the connector, not at the cable.
WAR NI N G
If you were using the Agilent CE instrument shortly before, the lamp
may be very hot. For translations, see page 265.
7 Loosen the two lamp screws using the #1 Pozidriv screwdriver.
Figure 102
Position of Cable Connector and Lamp Screws
Lamp screws
Cable connector
8 Remove the used lamp.
215
Maintaining Your Agilent CE Instrument
Changing the Lamp
Inserting the New Lamp
1 Hold the new lamp at the metal ring to avoid touching the glass surface.
Fingerprints absorb light and can reduce light intensity.
2 Insert the lamp into the lamp compartment. Position the lamp such that the
notch in the lamp ring matches with the pin on the detector housing.
Figure 103
Inserting the Lamp
Positioning pin
Lamp
Positioning slit
3 Tighten the two lamp screws.
4 Connect the lamp cable connector.
5 Reinstall the detector cover and close the push turn locks. Close the top cover.
6 Turn line power on.
7 Boot the Agilent ChemStation.
8 Do an Initialization in the Instrument menu. The lamp will be automatically
switched on. Allow the lamp about 1 hour to warm up.
9 Select More DAD from the Instrument menu and reset the lamp life counter.
Acceptance
Check sensitivity, noise and drift of the detector.
216
Maintaining Your Agilent CE Instrument
Changing the Lamp
Further Action
If the instrument fails this test, check the following:
❏ correct seating of the lamp,
❏ correct seating of the cassette, and
❏ if the red alignment interface without capillary is used, check for
contamination of the interface.
If none of these reasons apply, call Agilent Technologies.
217
Maintaining Your Agilent CE Instrument
Practical Considerations
Practical Considerations
• Capillary
one capillary per method
re-cut end if broken or replace it
keep detection window clean as well as the alignment interface
storage: flush with water and air (if possible)
when in doubt, throw it out
• Sample
remove particles (filter, centrifuge)
thermostate carousel if temperature sensitive or small volume
let the temperature stabalize when taking the sample from the
refrigerator -> reproducibility problems
use clean and dust-free vials at all time
• Buffers
do not overuse (e.q. use replenishment) -> migration time drift or
spurious peaks
try to use buffers not just electrolytes -> migration time drift
use CE quality water ( 18 Mohm and organics removed)
use highest quality buffer components available
remove particles with 0.2 um or 0.45 um filter
use clean and dust-free vials at all time
use good, calibrated pH meter or purchase pre-made buffers
prepare fresh buffer often -> phospahte and organic buffers susceptible to
microbial growth
let the temperature stabalize when taking the buffer from the refrigerator
-> reproducibility problems
218
Index
A
accessories, 14
accessory kit, 14
acquisition
current, 79
detector signals, 81
power, 79
pressure, 79
spectra, 81
temperature, 79
voltage, 79
alignment interface
cleaning, 200
color coding, 96
installation of capillary, 96
removing, 202
arcing, 185
autobalance, 81
B
buffer, 243
change, 73
change during run, 80
buffer vials
liquid level, 92
preparation, 89
C
capillary
conditioning, 73
corresponding alignment interface,
96
detection window, 98
extended light path, 96
installation into alignment interface,
96
installation into cassette, 99, 100
removing from cassette, 201
capillary cassette
opening, 100
removing, 201
temperature, 71
capillary detection window
cleaning, 200
capillary electrophoresis start-up kit,
14
capillary electrophoresis test kit, 14
capillary insertion tool, 97
capillary lock, 100
carousel, 92
cassette
closing, 102
installation, 103
installation of capillary, 99
opening, 100
removing, 201
removing from instrument, 94
temperature, 71
cassette lock, 100
CE diagram, 61, 63
CE Timetable, 79
change cassette function, 103
changing
line fuses, 23
power supply fuses, 25
ChemStation
starting, 62
cleaning
alignment interface, 200
capillary detection window, 200
electrodes, 185
insulation plate, 185
prepunchers, 185
condensing water, 37
conditioning, 73
connecting
GPIB cables, 17
start/stop cables, 17
connectors, 16
current, 77
D
DAD test, 214
detector
icon, 85
lamp, 85, 214
parameters, 81
posttime, 82
raw data, 81
signals, 81
stoptime, 82
timetable, 81, 82
detector cover
removing, 39, 186, 256
diagram, 63
diode array detector, 81
drift, 81
E
electric
parameters, 77
electrodes
accessing, 41, 189
cleaning, 185
installation, 198
electrolyte bottle, 87
environment conditions, 13
events
timeprogramming, 80
extended light path capillaries, 96
external contacts, 16
F
fraction collection, 80
front cover
removing, 191
fuses
changing, 23, 25
line, 23
power supply, 25
replacing, 24
G
gradients, 80
H
home values, 71
humidity
environment, 13
I
injection
by current, 75
by power, 75
by pressure, 75
by voltage, 75
electrokinetic, 75
hydrodynamic, 75
parameters, 75
table, 76
inlet default vial, 72
installation, 12
instrument
initialization, 62
219
Index
starting, 61
unpacking, 14
instrument control, 61, 63
insulation plate
alignment to detector, 198
cleaning, 185, 196
installation, 198
removing, 41, 189
L
lamp
changing, 214
installation, 216
switching on, 85
lamp intensity, 214
leak, 37
lift offset, 71
line frequency, 13
line fuse holder, 20
line power, 13
line power switch
setting, 20
line select plug, 22
liquid level
buffer vials, 92
outlet vials, 92
sample vials, 91
waste vials, 92
lower alarm limit, 78
liquid level, 92
P
peakwidth, 81
polarity, 77
switching, 80
posttime, 79
power, 77
power supply
changing of fuses, 25
prepuncher
installation, 197
prepunchers
cleaning, 185
pressure
programming, 80
R
raw data
CE-specific, 79
current, 79
detector, 81
detector signals, 81
power, 79
pressure, 79
spectra, 81
voltage, 79
reference wavelength, 81
removing
cassette, 201
replenishment parameters, 73
replenishment system, 73
installation of bottles, 18
preparation, 86
RS-232 connection, 16
M
maintenance
alignment interface, 200
detector lamp, 214
electrodes, 185
insulation plate, 185
prepunchers, 185
method
parameters, 70, 141
setup, 70
multiple analysis, 73
S
safety, 224
sample vials
liquid level, 91
preparation, 89
setting
line power switch, 20
setup CE method, 70
signals, 81
slit
cleaning, 200
spectra, 81
N
noise level, 214
O
outlet default vial, 72
outlet vials
220
starting
instrument, 61
stoptime, 79
straight capillaries, 96
T
temperature
environment, 13
temperature control, 31
timeprogramming, 80
timetable
detector, 81
top cover, 250
tray, 92, 93
cooling, 31
manual operation, 93
tray cooling
requirements, 31
V
vials
changing during run, 80
handling, 89
liquid level, 90
positioning in tray, 93
recommended, 89
vitamin test sample, 14
voltage, 77
programming, 80
W
warranty, 222
waste bottle, 88
waste vials
liquid level, 92
water bath, 31
connecting, 32
disconnecting, 36
requirements, 31
shipping the instrument, 36
wavelength
switching during run, 82
wavelength calibration, 104
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All Analytical Products
Agilent Technologies (Agilent) warrants its chemical analysis products
against defects in materials and workmanship. For details of the warranty
period in your country, call Agilent. During the warranty period, Agilent will,
at its option, repair or replace products which prove to be defective.
Products that are installed by Agilent are warranted from the installation
date, all others from the ship date.
If Buyer schedules or delays installation more than 30 days after delivery,
then warranty period starts on 31st day from date of shipment (60 and 61
days, respectively for products shipped internationally).
Agilent warrants that its software and firmware designed by Agilent for use
with a CPU will execute its programming instructions when properly
installed on that CPU. Agilent does not warrant that the operation of the
CPU, or software, or firmware will be uninterrupted or error-free.
Limitation of Warranty
On-site warranty services are provided at the initial installation point.
Installation and on-site warranty services are available only in Agilent service
travel areas, and only in the country of initial purchase unless Buyer pays
Agilent international prices for the product and services. Warranties requiring
return to Agilent are not limited to the country of purchase.
For installation and warranty services outside of Agilent’s service travel area,
Agilent will provide a quotation for the applicable additional services.
If products eligible for installation and on-site warranty services are moved
from the initial installation point, the warranty will remain in effect only if the
customer purchases additional inspection or installation services, at the new
site.
The foregoing warranty shall not apply to defects resulting from:
1 Improper or inadequate maintenance, adjustment, calibration or operation
by Buyer;
2 Buyer-supplied software, hardware, interfacing or consumables;
3 Unauthorized modification or misuse;
222
Warranty Statements
4 Operation outside of the environmental and electrical specifications for
the product;
5 Improper site preparation and maintenance; or
6 Customer induced contamination or leaks.
THE WARANTY SET FORTH IS EXCLUSIVE AND NO OTHER WARRANTY,
WHETHER WRITTEN OR ORAL, IS EXPRESSED OR IMPLIED. AGILENT
SPECIFICALLY DISCLAIMS THE IMPLIED WARRANTIES OF
MERCHANTABILITY AND FITNESS FOR A PARTICULAR PURPOSE.
Limitation of Remedies and Liability
THE REMEDIES PROVIDED HEREIN ARE BUYER’S SOLE AND
EXCLUSIVE REMEDIES. IN NO EVENT SHALL AGILENT BE LIABLE FOR
DIRECT, INDIRECT, SPECIAL, INCIDENTAL, OR CONSEQUENTIAL
DAMAGES (INCLUDING LOSS OF PROFITS) WHETHER BASED ON
CONTRACT, TORT OR ANY OTHER LEGAL THEORY.
Responsibilities of the Customer
The customer shall provide:
1 Access to the products during the specified periods of coverage to perform
maintenance.
2 Adequate working space around the products for servicing by Agilent
Technologies personnel.
3 Access to and use of all information and facilities determined necessary by
Agilent Technologies to service and/or maintain the products. (Insofar as
these items may contain proprietary or classified information, the
customer shall assume full responsibility for safeguarding and protection
from wrongful use.)
4 Routine operator maintenance and cleaning as specified in the Agilent
Technologies operating and service manuals.
5 Consumables such as paper, disks, magnetic tapes, ribbons, inks, pens,
gases, solvents, columns, syringes, lamps, septa, needles, filters, frits,
fuses, seals, detector flow cell windows, etc.
223
Safety Information
Safety Information
The following general safety precautions must be observed during all phases
of operation, service, and repair of this instrument. Failure to comply with
these precautions or with specific warnings elsewhere in this manual violates
safety standards of design, manufacture, and intended use of the instrument.
Agilent Technologies Company assumes no liability for the customer’s failure
to comply with these requirements.
General
This is a Safety Class I instrument (provided with terminal for protective
earthing) and has been manufactured and tested according to international
safety standards.
Operation
Before applying power, comply with the installation section. Additionally the
following must be observed:
Do not remove instrument covers when operating. Before the instrument is
switched on, all protective earth terminals, extension cords,
auto-transformers, and devices connected to it must be connected to a
protective earth via a ground socket. Any interruption of the protective earth
grounding will cause a potential shock hazard that could result in serious
personal injury. Whenever it is likely that the protection has been impaired,
the instrument must be made inoperative and be secured against any
intended operation.
Make sure that only fuses with the required rated current and of the specified
type (normal blow, time delay, etc.) are used for replacement. The use of
repaired fuses and the short-circuiting of fuse holders must be avoided.
Some adjustments described in the manual, are made with power supplied to
the instrument, and protective covers removed. Energy available at many
points may, if contacted, result in personal injury.
Do not operate the instrument in the presence of flammable gases or fumes.
Operation of any electrical instrument in such an environment constitutes a
definite safety hazard.
224
Safety Information
Do not install substitute parts or make any unauthorized modification to the
instrument.
Capacitors inside the instrument may still be charged, even though the
instrument has been disconnected from its source of supply. Dangerous
voltages, capable of causing serious personal injury, are present in this
instrument. Use extreme caution when handling, testing and adjusting.
Safety Symbols
Table 11 shows safety symbols that are used on the instrument and
throughout the documentation.
Table 11
Safety Symbols
Symbol
!
Description
The apparatus is marked with this symbol when the user should refer to the
instruction manual in order to protect the apparatus against damage.
Indicates dangerous voltages.
Indicates a protected ground terminal.
225
Safety Information
Warnings and Cautions
WAR NI N G
A warning alerts you to situations that could cause physical injury or
damage to the equipment. Do not proceed beyond a warning until you
have fully understood and met the indicated conditions. For
translations, see page 266.
CA UT IO N
A caution alerts you to situations that could cause a possible loss of data. Do
not proceed beyond a caution until you have fully understood and met the
indicated conditions. For translations, see page 267.
226
Safety Information
Translations of the WARNING on page 16.
WAR NU N G
Während der Installation Ihres Agilent Capillary Electrophoresis Systems
darf das Netzkabel nicht angeschlossen sein.
CU IDA DO
Asegúrese que el cable de red está desconectado antes de instalar su Agilent
Capillary Electrophoresis System.
AT T E N T IO N
Assurez vous que le cable secteur n’est pas connecté avant d’installer votre
Agilent Capillary Electrophoresis System.
AT T E N Z IO NE
Assicuratevi che il cavo di alimentazione sia scollegato prima di installare il
Agilent Capillary Electrophoresis System.
WAAR SC HUW ING
Zorg dat het netsnoer is losgekoppeld, voordat de Agilent Capillary
Electrophoresis System wordt geïnstalleerd.
227
Safety Information
Translations of the WARNING on page 18.
WAR NU N G
Mit diesem Instrument sind ausschließlich Flaschen mit der Teilenummer
9300-1748 zu benutzen. Die Flaschen stehen unter Druck. Die oben
genannten Flaschen weisen eine Schutzbeschichtung auf.
CU IDA DO
Con este instrumento utilizar sólo botellas con el Número de Pieza 9300-1748.
Se ha aplicado presión a las botellas. Las botellas arriba especificadas tienen
un revestimiento de protección.
AT T E N T IO N
Avec cet instrument, utilisez exclusivement les bouteilles portant le numéro
de pièce 9300-1748. Ces bouteilles sont soumises à une pression. Les
bouteilles mentionnées ci-dessus portent un revêtement protecteur.
AT T E N Z IO NE
Con questo strumento usare esclusivamente bombole Num.Art. 9300-1748.
Nelle bombole è contenuta pressione. Le bombole sopra citate sono dotate di
rivestimento di protezione.
WAAR SC HUW ING
Gebruik voor dit instrument alleen flesjes met het onderdeelnummer
9300-1748. De voornoemde flesjes zijn voorzien van een beschermende laag.
228
Safety Information
Translations of the WARNING on page 19.
WAR NU N G
Vergewissern Sie sich, daß die Spannungseinstellung Ihres Instruments mit
der Netzspannung Ihres Landes übereinstimmt, bevor Sie das Netzkabel in
die Steckdose stecken. Die Spannungseinstellung kann durch den Schlitz im
Sicherungschalter hinten im Instrument überprüft werden. Siehe Figure 2.
Anweisungen zum Einstellen der Spannung siehe “Setting the Line Power
Switch” on page 20.
CU IDA DO
Asegurarse de que el reglaje de la línea de corriente del instrumento se ajusta
a las especificaciones del pais, antes de conectar el cable de la línea de
corriente. El reglaje de la línea de corriente puede ser comprobado a través de
la rendija del portador de fusibles, en la parte posterior del instrumento.
Véase Figure 2. Véase “Setting the Line Power Switch” on page 20 para las
instrucciones sobre cómo fijar el regalje de la línea de corriente.
AT T E N T IO N
Vérifiez que le réglage de l’alimentation secteur de l’instrument est conforme
aux spécifications de votre pays avant de brancher le fil d’alimentation
secteur. Vous pouvez contrôler le réglage de l’alimentation secteur à travers
la fente ménagée dans le porte-fusible à l’arrière de l’instrument. Voir
Figure 2. Pour les instructions sur la façon de régler l’alimentation secteur,
voir “Setting the Line Power Switch” on page 20.
AT T E N Z IO NE
Assicurarsi che l’impostazione del voltaggio corrente corrisponda alle
caratteristiche nazionali prima di connettere il cavo di alimentazione. La
giusta impostazione del voltaggio corrente puó essere controllata attraverso
la fessura del portafusibile sulla parte posteriore dello strumento. Si veda
Figure 2 e “Setting the Line Power Switch” on page 20 riguardo alle istruzioni
sulla selezione della corrente di rete.
WAAR SC HUW ING
Wees ervan overtuigd dat de netspanningsafstelling van het instrument
voldoet aan de specificaties van uw land voordat het netsnoer wordt
aangesloten. De netspanningsafstelling kan worden gecontroleerd via de gleuf
van de zekeringhouder aan de achterkant van het instrument. Zie Figure 2. Zie
“Setting the Line Power Switch” on page 20 voor instructies betreffende het
afstellen van de netspanningsafstelling.
229
Safety Information
Translations of the WARNING on page 19.
WAR NU N G
Vergewissern Sie sich, daß die im Gerät eingesetzten Sicherungen den
Vorschriften entsprechen, bevor das Netzkabel in die Steckdose gesteckt
wird. Anweisungen zum Auswechseln der Sicherungen siehe “Changing the
Line Fuses of the Instrument” on page 23 und “Changing the Fuses for the
Power Supplies” on page 25.
CU IDA DO
Asegurarse de que los fusibles instalados en el instrumento se ajustan con las
especificaciones, antes de conectar el cable de la línea de corriente. Véanse las
“Changing the Line Fuses of the Instrument” on page 23 y “Changing the
Fuses for the Power Supplies” on page 25 para las instrucciones sobre cómo
introducir los fusibles.
AT T E N T IO N
Avant de brancher le fil d’alimentation secteur, assurez-vous que les fusibles
installés dans l’instrument respectent les spécifications. Pour les instructions
sur la façon d’accéder aux fusibles, voir “Changing the Line Fuses of the
Instrument” on page 23 et “Changing the Fuses for the Power Supplies” on
page 25.
AT T E N Z IO NE
Assicurarsi che i fusibili installati sullo strumento rispondano alle sopra
citate caratteristiche prima di connettere il cavo di alimentazione. Si veda
“Changing the Line Fuses of the Instrument” on page 23 e “Changing the
Fuses for the Power Supplies” on page 25 sul modo di accedere ai fusibili.
WAAR SC HUW ING
Wees ervan overtuigd dat de geïnstalleerde zekeringen in het instrument
voldoen aan de specificaties, voordat het netsnoer wordt aangesloten. Zie
“Changing the Line Fuses of the Instrument” on page 23 en “Changing the
Fuses for the Power Supplies” on page 25 voor instructies betreffende de
toegang tot de zekeringen.
230
Safety Information
Translations of the WARNING on page 19.
WAR NU N G
Um den Benutzer zu schützen, muß das Agilent Capillary Electrophoresis
System geerdet sein. Ihr Agilent Capillary Electrophoresis System ist mit
einem 3-adrigen Netzkabel und einem 3-poligen Stecker ausgestattet, der Ihr
Agilent Capillary Electrophoresis System mit der Schutzerde verbindet,
sobald der Stecker in eine passende Steckdose gesteckt wird.
CU IDA DO
El Agilent Capillary Electrophoresis System debe ser conectada a una toma de
tierra para protegerle durante su operación. El Agilent Capillary
Electrophoresis System y cualquier otro instrumento conectado a ella deben
operar desde una fuente de energía de tres contactos, con el contacto de tierra
conectado con certeza a tierra. Pídale a un electricista que compruebe la
tierra de la fuente.
AT T E N T IO N
Le Agilent Capillary Electrophoresis System doivent avoir leurs panneaux et
chassis mis à la terre pour votre protection pendant l’utilisation. Le Agilent
Capillary Electrophoresis System et tous les instruments qui lui sont
connectés doivent être alimentés à l’aide d’un cable à trois fils avec les
contacts terre fermement reliés à la terre. Demandez à un électricien de
verifier la mise à la terre.
AT T E N Z IO NE
Per proteggere l’operatore i pannelli dello strumento e la struttura esterna
devono essere collegati a terra. Il sistema il rivelatore ed ogni alto strumento
connesso ad essa deve lavorare con un cavo di alimentazione a tre conduttori,
con il filo di terra adeguatamente collegato a terra. Verificate la vostra linea
di terra prima di installare lo strumento.
WAAR SC HUW ING
De panelen en de behuizing van de Agilent Capillary Electrophoresis System
moeten gedurende de werking geaard zijn. De Agilent Capillary
Electrophoresis System en alle andere instrumenten die hierop zijn
aangesloten, moeten worden gevoed door een drie-aderige voeding, waarvan
de aardielding op de juiste manier is aangesloten. Schakel een elekticien in om
te controleren of de voeding op de juiste wijze geaard is.
231
Safety Information
Translations of the WARNING on page 20.
WAR NU N G
Bei einer anderen Netzspannung muß der Wahlschalter entsprechend
eingestellt werden. Die Sicherungen am Hauptnetzteil und den internen
Netzteilen müssen gegen Sicherungen ausgetauscht werden, die der neuen
Netzspannung entsprechen, bevor das Instrument wieder eingeschaltet
werden darf. Anweisungen siehe “Changing the Line Fuses of the
Instrument” on page 23 und “Changing the Fuses for the Power Supplies” on
page 25.
CU IDA DO
Al cambiar el reglaje del conmutador selector de la línea de corriente, los
fusibles de línea y los fusibles del suministro de corriente tienen que ser
reemplazados por fusibles ajustados al nuevo reglaje, antes de conectar de
nuevo el instrumento. Se remite a las “Changing the Line Fuses of the
Instrument” on page 23 y “Changing the Fuses for the Power Supplies” on
page 25 para instrucciones.
AT T E N T IO N
Quand vous modifiez le réglage du sélecteur d’alimentation de secteur, vous
devez, avant de remettre l’instrument en circuit, remplacer les fusibles de
secteur et les fusibles des blocs d’alimentation par des fusibles correspondant
au nouveau réglage. Voir les instructions dans “Changing the Line Fuses of the
Instrument” on page 23 et “Changing the Fuses for the Power Supplies” on
page 25.
AT T E N Z IO NE
Nel variare l’impostazione dell’interruttore di selezione voltaggio corrente
bisogna sostituire i fusibili di linea e i fusibili di alimentazione con fusibili
corrispondenti alla nuova impostazione prima che lo strumento sia messo di
nuovo in funzione. Fare riferimento a “Changing the Line Fuses of the
Instrument” on page 23 e “Changing the Fuses for the Power Supplies” on
page 25 per quanto riguarda le istruzioni.
WAAR SC HUW ING
Wanneer de afstelling van de netspanningskeuzeschakelaar wordt veranderd,
dan moeten de netzekeringen en de zekeringen van de voedingen worden
vervangen door zekeringen die overeenkomen met de nieuwe afstelling
voordat het instrument opnieuw wordt ingeschakeld.
232
Safety Information
Translations of the WARNING on page 20.
WAR NU N G
Wenn der Netzspannungswahlschalter betätigt wird, darf das Netzkabel nicht
angeschlossen sein.
CU IDA DO
Asegúrese que el cable de red está desconectado antes de cambiar el selector
de voltaje.
AT T E N T IO N
Assurez-vous que le câble secteur n’est pas connecté avant de changer la
tension d’alimentation.
AT T E N Z IO NE
Assicuratevi che il cavo di alimentazione sia scollegato prima di spostare il
selettore di voltaggio.
WAAR SC HUW ING
Zorg dat de voedingskabel losgekoppeld is, voordat de voedingsspanning
wordt veranderd.
233
Safety Information
Translations of the WARNING on page 24.
WAR NU N G
Sicherungen dürfen nur bei nicht angeschlossenem Netzkabel installiert oder
gewechselt werden. Zum Schutz gegen Feuergefahr dürfen nur Sicherungen
von angegebenem Typ und Stärke verwendet werden.
CU IDA DO
Asegúrese que el cable de red está desconectado antes de instalar o cambiar
un fusible. Para una protección continuada frente a riesgo de fuego, reponer
sólo fusibles de tipo y régimen de corriente especificados.
AT T E N T IO N
Assurez-vous que le câble secteur n’est pas connecté avant de changer un
fusible. Pour assurer une protection permanente contre les risques
d’incendie, n’utilisez que des fusibles du type et de l’intensité nominale
spécifiés.
AT T E N Z IO NE
Assicuratevi che il cavo di alimentazione sia scollegato prima di installare o
sostituire un fusibile. Per garantire efficace protezione contro rischi di
incendio usare solo fusibili di ricambio del tipo specificato e della potenza
prestabilita.
WAAR SC HUW ING
Zorg dat de voedingskabel losgekoppeld is, voordat een zekering wordt
geplaatst of vervangen. Voor een permanente bescherming tegen brand
adviseren wij bij vervanging alleen zekeringen van het gespecificeerde type en
de juiste stroomsterkte te gebruiken.
234
Safety Information
Translations of the WARNING on page 25.
WAR NU N G
Sicherungen dürfen nur bei nicht angeschlossenem Netzkabel installiert oder
gewechselt werden.
CU IDA DO
Asegúrese que el cable de red está desconectado antes de instalar o cambiar
un fusible.
AT T E N T IO N
Assurez-vous que le câble secteur n’est pas connecté avant de changer un
fusible.
AT T E N Z IO NE
Assicuratevi che il cavo di alimentazione sia scollegato prima di installare o
sostituire un fusibile.
WAAR SC HUW ING
Zorg dat de voedingskabel losgekoppeld is, voordat een zekering wordt
geplaatst of vervangen.
235
Safety Information
Translations of the WARNING on page 32.
WAR NU N G
Stellen Sie sicher, daß Wasserbad und Agilent CE Instrument abgeschaltet
sind, bevor Sie das Wasserbad installieren.
CU IDA DO
Asegúrese de que el baño y el Agilent CE Instrument están desenchufados
cuando instale el baño de agua externo.
AT T E N T IO N
Assurez-vous que vous avez bien éteint le thermostat à circulation ET le
Agilent CE Instrument avant de procéder à l’installation du thermostat à
circulation.
AT T E N Z IO NE
Fate attenzione che, sia il bagno refrigerante che le Agilent CE Instrument
siano spenti quando installate il bagno refrigerante.
WAAR SC HUW ING
Schakel het Agilent CE Instrument en het waterbad uit wanneer u het
waterbad installeerd.
236
Safety Information
Translations of the WARNING on page 39.
WAR NU N G
Vergewissern Sie sich, daß das elektrische Netzanschlußkabel des Agilent CE
Instruments aus der Steckdose gezogen ist, bevor Sie mit Ihrer Arbeit
fortfahren.
CU IDA DO
Asegurarse de que se ha desconectado el cable de la línea de corriente del
instrumento Agilent CE, antes de seguir adelante.
AT T E N T IO N
Assurez-vous que vous avez débranché le fil d’alimentation secteur de
l’instrument Agilent CE avant de poursuivre les opérations.
AT T E N Z IO NE
Prima di procedere assicurarsi di avere staccato il cavo di alimentazione dello
strumento Agilent CE.
WAAR SC HUW ING
Wees ervan overtuigd dat het netsnoer van het Agilent CE instrument uit het
stopcontact is getrokken voordat u begint.
237
Safety Information
Translations of the WARNING on page 40.
WAR NU N G
Das Lampengehäuse des Agilent CE kann bei Gebrauch sehr heiß werden.
Bitte nicht berühren.
CU IDA DO
La lámpara del detector puede estar muy caliente si hace poco que el
instrumento ha sido utilizado. Evite tocar la lámpara.
AT T E N T IO N
Si vous avez utilisé votre Agilent CE juste avant de suivre la procédure, la
lampe peut etre brulante, ne pas la toucher.
AT T E N Z IO NE
Se si esegue questa operazione dopo un utilizzo recente dello strumento la
lampada potrebbe essere ad una temperatura elevata. Evitare il contatto
diretto con la lampada.
WAAR SC HUW ING
Als u kort voor deze procedure het Agilent CE instrument gebruikt heeft, kan
de lamp nog zeer heet zijn. Vermijd het de lamp aan te raken.
238
Safety Information
Translations of the CAUTION on page 48.
ACHTUNG
Reinigen Sie die Arbeitsfläche gründlich, bevor Sie mit der folgenden Prozedur
beginnen. Wenn Staub- oder Schmutzpartikel in die Kapillare oder die
Hochempfindlichkeits-Detektorzelle geraten, kann dies zu erheblichen
Empfindlichkeitseinbußen der Zelle führen. Druckluft eignet sich gut zum Reinigen der
Teile, bevor sie zusammengesetzt werden.
PR E C AU C IO N
Preparar un banco limpio para la realización del siguiente procedimiento. Si entrasen
partículas de polvo al capilar o a la celda de detección de alta sensibilidad, podría
producirse un bajo rendimiento de la celda. El aire comprimido resulta de gran utilidad
para la limpieza de las piezas antes de su ensamblaje.
AT T E N T IO N
Pour exécuter la procédure suivante, veillez à ce que votre espace de travail sur la
paillasse soit bien propre. Si des particules de poussière devaient pénétrer dans le
capillaire ou dans la cellule de détection haute sensibilité, ceci aurait pour
conséquence de détériorer les performances de la cellule. L’air comprimé s’avère très
utile pour nettoyer les pièces avant leur assemblage.
PR E C A U Z I ON E
Assicurarsi di predisporre una zona pulita sul banco di lavoro per effettuare la
procedura seguente. Se nel capillare o nella cella di rivelazione ad elevata sensibilità
dovessero entrare particelle di polvere, questo potrebbe ridurre le prestazioni della
cella. L’aria compressa si rivela molto utile per la pulizia delle parti prima del loro
riassemblaggio.
VO OR ZI C HT IG
Voor de volgende procedure moet er op een schone ondergrond worden gewerkt.
Wanneer er stofdeeltjes in de capillair of in de zeer gevoelige detectiecel komen kan dit
leiden tot slechte resultaten. Perslucht is goed bruikbaar voor het schoonmaken van de
onderdelen voordat ze in elkaar worden gezet.
239
Safety Information
Translations of the CAUTION on page 56.
ACHTUNG
Beim Zusammensetzten/Auseinanderbauen der HochempfindlichkeitsDetektorzellen-Einheit muß der Arbeitstisch sehr sauber sein. Lagern Sie die Zelle und
die Kapillaren mit aufgesetzter Schutzkappe im Staubschutzbehälter.
PR E C AUC ION
Asegurarse de que el banco de trabajo esté limpio cuando se vaya a acoplar/desacoplar
el conjunto de la celda de detección de elevada sensibilidad. Guardar los capilares con
la protección adecuada y la celda en una caja protegida contra el polvo.
AT T E N T IO N
Assurez-vous de la propreté de votre paillasse avant d’assembler/de désassembler
l’ensemble cellule de détection haute sensibilité. Rangez les capillaires munis de leur
capuchon de protection ainsi que les cellules dans la boîte hermétique étanche à la
poussière.
PR E C A U Z I ON E
Quando si accoppia/disaccoppia il gruppo della la cella di rivelazione ad elevata
sensibilità, assicurarsi di operare in una zona pulita del banco di lavoro. Riporre nel
contenitore antipolvere i capillari, chiusi con i tappi di protezione, e la cella.
VO O RZ I C H T IG
Zorg voor een schone ondergrond als u de onderdelen van de zeer gevoelige detectiecel
uit elkaar haalt of in elkaar zet. Bewaar de capillairen met bijgevoegde
beschermingskap en de cel in het stofbeschermende doosje.
240
Safety Information
Translations of the CAUTION on page 57.
ACHTUNG
Führen Sie Schritt 5 nicht durch, wenn Sie beschichtete Kapillaren, gepackte
Kapillaren oder andere Kapillaren verwenden, bei denen die Verwendung von NaOH
problematisch ist.
PR E C AU C IO N
No utlilizar el paso 5 con capilares recubiertos, columnas empaquetadas o con
cualquier otro capilar donde el hidróxido sódico, NaOH, resulte problemático.
AT T E N T IO N
Ne procédez pas à l’étape 5 si vous utilisez des capillaires revêtus, des colonnes
remplies ou tout autre capillaire avec lequel NaOH peut être source de problème.
PR E C A U Z I ON E
Non effettuare il passaggio 5 con i capillari ricoperti, le colonne impaccate o con
qualsiasi altro capillare per il quale l’uso di NaOH potrebbe risultare dannoso.
VO OR ZI C HT IG
Stap 5 is NIET van toepassing bij: gecoate capillairen, gepakte kolommen of andere
capillairen die problemen kunnen geven met NaOH.
241
Safety Information
Translations of the CAUTION on page 57.
ACHTUNG
Die Hochempfindlichkeits-Detektorzelle und die Anschlüsse bestehen aus
verschiedenen Materialien: Fused Silika, PEEK (Polyetheretherketon) und FVMQ
(Fluorsilikonkautschuk). Verwenden Sie keine Lösungsmittel, die diesen Materialien
schaden.
PR E C AUC ION
La celda de detección de alta sensibilidad y las conexiones están hechas de diferentes
materiales: silice fundida, PEEK (polieteretercetona) y FVMQ (goma de
fluorosilicona). No utilizar disolventes que puedan degradar estos materiales.
AT T E N T IO N
La cellule de détection haute sensibilité et les raccords sont fabriqués à partir de
matériaux divers: silice fondue, PEEK (polyétheréthercétone), et FVMQ (silicone
fluoré). N’utilisez pas de solvants susceptibles de les détériorer.
PR E C A U Z I ON E
La cella di rivelazione ad elevata sensibilità ed i connettori sono costituiti da materiali
diversi: silice fusa, PEEK (polietere chetonetere) e FVMQ (gomma fluorosiliconica).
Non utilizzare solventi che possano degradare tali materiali.
VO O RZ I C H T IG
De zeer gevoelige detectiecel en toebehoren bestaan uit verschillende materialen:
fused silica, PEEK (polyetheretherketone) en FVMQ (fluorosilicone rubber). Gebruik
geen oplosmiddelen die deze materialen kunnen afbreken.
242
Safety Information
Translations of the WARNING on page 86.
WAR NU N G
Mit diesem Instrument sind ausschließlich Flaschen mit der Teilenummer
9300-1748 zu benutzen. Die Flaschen stehen unter Druck. Die oben
genannten Flaschen weisen eine Schutzbeschichtung auf.
CU IDA DO
Con este instrumento utilizar sólo botellas con el Número de Pieza 9300-1748.
Se ha aplicado presión a las botellas. Las botellas arriba especificadas tienen
un revestimiento de protección.
AT T E N T IO N
Avec cet instrument, utilisez exclusivement les bouteilles portant le numéro
de pièce 9300-1748. Ces bouteilles sont soumises à une pression. Les
bouteilles mentionnées ci-dessus portent un revêtement protecteur.
AT T E N Z IO NE
Con questo strumento usare esclusivamente bombole Num. Art. 9300-1748.
Nelle bombole è contenuta pressione. Le bombole sopra citate sono dotate di
rivestimento di protezione.
WAAR SC HUW ING
Gebruik voor dit instrument alleen flesjes met het onderdeelnummer
9300-1748. De voornoemde flesjes zijn voorzien van een beschermende laag.
243
Safety Information
Translations of the WARNING on page 86.
WAR NU N G
Sie sollten keine Schwefelsäure mit dem Agilent CE System verwenden. Das
Gerät könnte dadurch beschädigt werden.
CU IDA DO
No usar ácido sulfúrico en el Sistema Agilent CE. Esto puede dañar el
instrumento.
AT T E N T IO N
N’utilisez pas d’acide sulfurique dans le système Agilent CE. Cela pourrait
endommager l’instrument.
AT T E N Z IO NE
Nel sistema Agilent CE non è ammesso l’uso di acido solforico. Ció potrebbe
danneggiare lo strumento.
WAAR SC HUW ING
Gebruik geen zwavelzuur in het Agilent CE systeem. Hierdoor kan het
instrument worden beschadigd.
244
Safety Information
Translations of the WARNING on page 87.
WAR NU N G
Im Abfall können organische Lösungsmittel und Rückstände Ihrer Proben
enthalten sein. Beim Umgang mit Elektrolyt unbedingt Gummihandschuhe
und Schutzbrille tragen, Rückstände in einen dafür vorgesehenen
Abfallbehälter verbringen.
CU IDA DO
El desechado puede contener disolventes orgánicos y residuos de la muestra.
Utilizar guantes de goma y gafas de seguridad al manejar el electrolito y
depositar el desechado en un recipiente de residuos seguro.
AT T E N T IO N
Le résidu peut comporter des solvants organiques et des restes de votre
échantillon. Lors de la manipulation de l’électrolyte, portez des gants en
caoutchouc et des lunettes de sécurité; déposez le résidu dans un conteneur
de résidu assurant une bonne sécurité.
AT T E N Z IO NE
Le bombole vuote possono contenere solventi organici o residui del provino in
esame. Nel fare uso di elettroliti munirsi di guanti di gomma e di occhiali
protettivi. Depositare i vuoti in un contenitore di sicurezza.
WAAR SC HUW ING
Het afval kan organische oplosmiddelen en restanten van uw monster
bevatten. Draag rubber handschoenen en een veiligheidsbril wanneer met het
elektrolyt wordt gewerkt en deponeer het afval in een speciale, beveiligde
container.
245
Safety Information
Translations of the WARNING on page 89.
WAR NU N G
Bördelkappen oder sonstige Metallkappen dürfen beim Arbeiten mit dem
Agilent CE Instrument nicht benutzt werden.
CU IDA DO
No utilizar casquetes ondulados o casquetes de cualquier otro metal, cuando
se trabaja con el instrumento.
AT T E N T IO N
Lors que vous travaillez avec l’instrument, n’utilisez pas de capuchons sertis
ni d’autres capuchons métalliques.
AT T E N Z IO NE
Durante l’uso dello strumento Agilent CE non deve essere fatto uso di tappi a
pressione né di alcun altro tipo di tappi metallici.
WAAR SC HUW ING
Maak geen gebruik van krimpkappen of andere metalen kappen wanneer met
het Agilent CE Instrument wordt gewerkt.
246
Safety Information
Translations of the WARNING on page 94.
WAR NU N G
Auf keinen Fall Druck geben (Arbeitsgang anlaufen lassen), wenn keine
Kapillare eingesetzt ist.
CU IDA DO
Nunca aplicar presión (iniciar una serie), si no hay capilar instalado.
AT T E N T IO N
N’appliquez jamais la pression (Lancement d’un cycle) quand le capillaire
n’est pas installé.
AT T E N Z IO NE
Non azionare mai Pressione (Inizio corsa) se non è stato prima installato il
capillare.
WAAR SC HUW ING
Gebruik nooit druk (omloop opstarten) wanneer geen buisje is geïnstalleerd.
247
Safety Information
Translations of the CAUTION on page 97.
ACHTUNG
Die Kapillare am Detektionsfenster oder die Kapillarspitzen, wo das Polyimid
abgebrannt ist, nicht berühren. Die Kapillare ist an dieser Stelle sehr zerbrechlich und
das Detektionsfenster muß unbedingt sauber gehalten werden. Detektionsfenster und
Kapillarspitzen gemäß Beschreibung in “Cleaning the Detection Window and the
Alignment Interface” on page 200 reinigen, wenn Sie sie berührt haben.
PR E C AUC ION
No tocar el capilar en la ventana de detección o las puntas del capilar, donde se ha
quemado la poliimida. El capilar es muy fragil en este punto y es importante el
mantener limpia la ventana de detección. Limpiar la ventana de detección y las puntas
del capilar, tal como se describe en “Cleaning the Detection Window and the Alignment
Interface” on page 200, si estas han sido tocadas.
AT T E N T IO N
Ne touchez pas le capillaire au niveau de la fenêtre de détection ni les extrémités du
capillaire, là où le polyimide est brûlé . Le capillaire est très fragile à cet endroit et il est
important que la fenêtre de détection demeure propre. S’il vous arrive de toucher la
fenêtre de détection et les extrémités du capillaire, nettoyez-les selon la description
donnée dans “Cleaning the Detection Window and the Alignment Interface” on
page 200.
PR E C A U Z I ON E
Non toccare il capillare, nel punto in cui è situata la cellula di rivelazione, o le sue
estremitá, dove è bruciato il poliammide, perchè in questo punto esso è molto fragile;
è inoltre importante che la cellula di rivelazione sia mantenuta pulita. La cellula di
rivelazione e le estremitá del capillare devono essere ripulite, se sono state toccate,
seguendo la descrizione in “Cleaning the Detection Window and the Alignment
Interface” on page 200.
VO O RZ I C H T IG
Raak het buisje niet aan bij het kijkglas en bij de uiteinden waar het polyamide is
weggebrand. Het buisje is bij deze punten uiterst breekbaar en bovendien is het
belangrijk dat het kijkglas schoon blijft. Maak het kijkglas en de uiteinden van het
buisje schoon zoals omschreven in “Cleaning the Detection Window and the Alignment
Interface” on page 200 wanneer u deze toch heeft aangeraakt.
248
Safety Information
Translations of the WARNING on page 104.
WAR NU N G
Vergewissern Sie sich, daß die Kapillarenden nicht gebogen sind, wenn die
Kapillarkassette nach unten gefahren wird. Sie müssen direkt in die
Kapillarsitze gleiten. Sonst können sie brechen oder beschädigt werden.
Wenn es beim Einsetzen der Kapillare in die Elektroden zu Problemen
kommt, überprüfen, ob diese durch Salzkristalle verstopft sind. Anweisungen
zum Reinigen der Elektroden siehe “Cleaning the Electrodes, Prepunchers
and Insulation Plate” on page 185.
CU IDA DO
Asegurarse de que los extremos del capilar no están doblados, al bajar el porta
capilares. Estos deberán deslizarse directamente en los asientos para
capilares. De otra forma estos pueden romperse o ser deteriorados. Si se tiene
problemas al insertar el capilar dentro de los electrodos, comprobar si estos
están bloqueados por cristalitos salinos. Se remite a la “Cleaning the
Electrodes, Prepunchers and Insulation Plate” on page 185 para instrucciones
sobre la limpieza de electrodos.
AT T E N T IO N
Assurez-vous que les extrémités du capillaires ne sont pas courbées quand
vous abaissez la cassette du capillaire. Ces extrémités doivent aller se loger
directement dans les sièges du capillaire. Sinon, elles pourraient se rompre ou
être endommagées. Si vous avez du mal à insérer le capillaire dans les
électrodes, vérifiez si ces dernières ne sont pas obturés par des cristaux de
sel. Voir “Cleaning the Electrodes, Prepunchers and Insulation Plate” on
page 185 pour les instructions de nettoyage des électrodes.
AT T E N Z IO NE
Assicurarsi che le estremitá del capillare non siano inclinate quando la
cassetta si posiziona verso il basso: esse dovrebbero scivolare inserendosi
direttamente all’interno del capillare; in caso contrario potrebbero rompersi
o venire danneggiate. Se si presentassero dei problemi nell’inserimento del
capillare negli elettrodi, controllare che questi non siano otturati da cristalli
di sale. Fare riferimento a “Cleaning the Electrodes, Prepunchers and
Insulation Plate” on page 185 per quanto riguarda istruzioni su come pulire gli
elettrodi.
WAAR SC HUW ING
Wees er van overtuigd dat de uiteinden van het buisje niet krom zijn, wanneer
de buiscassette naar beneden wordt gebracht. Deze moeten rechtstreeks in de
buis zittingen glijden, anders kunnen ze breken of worden beschadigd. Indien
het inbrengen van de buisjes in de elektroden problemen oplevert, controleer
dan of deze worden gehinderd door zoutkristallen. Zie “Cleaning the
Electrodes, Prepunchers and Insulation Plate” on page 185 betreffende de
instructies voor het schoonmaken van de elektroden.
249
Safety Information
Translations of the WARNING on page 104.
WAR NU N G
Deckel nicht schließen, wenn die Kassette nicht ordnungsgemäß eingesetzt
ist. Die Kassette könnte beschädigt werden.
CU IDA DO
No cerrar la cubierta superior, si el porta capilares no está instalado
adecuadamente. Esto podría deteriorar el porta capilares.
AT T E N T IO N
Ne fermez pas le couvercle supérieur si la cassette n’est pas installée
correctement. Cela pourrait endommager la cassette.
AT T E N Z IO NE
Non richiudere il coperchio superiore se la cassetta non è stata correttamente
inserita. Ció potrebbe danneggiarla.
WAAR SC HUW ING
Indien de cassette onjuist is geïnstalleerd mag het deksel niet worden
gesloten. Hierdoor kan de cassette worden beschadigd.
250
Safety Information
Translations of the WARNING on page 123.
WAR NU N G
Bitte beachten Sie, dass Sie die neue Kapillarkassette nehmen müssen, wenn
Ihre Applikation oder Methode organische Lösungsmittel enthält. Der Grun
dafür ist, dass die neue Kaillarkassette einer höheren Flammschutzklasse
(CV01) entspricht. Sie sollten die alte Kassette auf keinen Fall benutzen,
wenn organische Lösungsmittel benutzt werden.
CU IDA DO
Utilice el nuevo cassette de capilares siempre que trabaje con un método o
aplicación que haga uso de disolventes orgánicos. El diseño de este cassette
cumple el estandar de inflamabilidad (CV01). No use nunca el cassette de
antiguo diseño cuando trabaje con disolventes orgánicos.
AT T E N T IO N
Veuillez noter que vous devez utiliser la nouvelle cassette si une application
ou une methode necessite des solvants organiques. Cette nouvelle cassette
repond a la plus haute norme d'inflamabilite (CV01). Vous ne devez pas
utiliser l'ancienne cassette des que des solvants organiques sont utilises.
AT T E N Z IO NE
É obbligatorio usare sempre il nuove tipo di cassette capillare qualora
l’applicazione o il metodo usati prevendano l’impiego di solventi organica.
Questo perché il nuovo tipo di cassetta capillare é in conformità alle norme di
alta infiammabilità (CV01). Lei non deve assolotamente usare la cassetta
capillare vecchio modello quando vengono utilizzati solventi organici.
WAAR SC HUW ING
Gelieve uitsluitend de nieuw ontworpen capillaire cassette te gebruiken
wanneer U een applikatie of methode met organische oplosmiddelen toepast.
Dit is omdat dit nieuw type capillaire cassette voldoet aan de eisen van de
hogere ontvlambaarheidsklasse (CV01). Het oude type capillaire cassette
dient U niet te gebruiken met organische oplosmiddelen.
251
Safety Information
Translations of the WARNING on page 131.
WAR NU N G
Bitte benutzen Sie nur die neue Kapillarkassette. Für weitere Hinweise lesen
Sie die Warnung auf der Seite 226. Bitte niemals die alte Kapillarkassette
benutzen, wenn Ihre Applikation organische Lösungsmittel enthält. Dies gilt
besonders für CEC Applikationen.
CU IDA DO
Utilice solamente el nuevo cassette de capilares. Ver anotación de seguridad
en página 226. No use nunca el antiguo cassette cuando utilice disolventes
orgánicos, especialmente si trabaja con un método de CEC.
AT T E N T IO N
Utilisez la nouvelle cassette seulement. Voir la note de securite sur page 226.
Ne jamais utiliser l’ancienne cassette quand des solvants organiques sont
utilises.Cela s’applique essentiellement lors des applications CEC.
AT T E N Z IO NE
Usare solo il nuovo tipo di casstta capillare. Leggere la nota di sicurezza a
pagina 226. Mai usare la cassetta capillare vecchio modello quando vengono
utilizzati solventi organici. E questo particolarmente quando si eseguono
applicazioni CEC.
WAAR SC HUW ING
Gelieve uitsluitend het nieuw type capillaire cassette te gebruiken. Zie de
waarschuwing met betrekking tot de veiligheid op bladzijde 226. Gebruik
nooit het oude type capillaire cassette wanneer organische oplosmiddelen
gebruikt worden. Dit is in het bijzonder van belang bij het uitvoeren van CEC
applikaties.
252
Safety Information
Translations of the WARNING on page 144.
WAR NU N G
Vergewissern Sie sich, daß das elektrische Netzanschlußkabel des Agilent CE
Instruments aus der Steckdose gezogen ist, bevor Sie mit Ihrer Arbeit
fortfahren.
CU IDA DO
Asegurarse de que se ha desconectado el cable de la línea de corriente del
instrumento Agilent CE, antes de seguir adelante.
AT T E N T IO N
Assurez-vous que vous avez débranché le fil d’alimentation secteur de
l’instrument Agilent CE avant de poursuivre les opérations.
AT T E N Z IO NE
Prima di procedere assicurarsi di avere staccato il cavo di alimentazione dello
strumento Agilent CE.
WAAR SC HUW ING
Wees ervan overtuigd dat het netsnoer van het Agilent CE instrument uit het
stopcontact is getrokken voordat u begint.
253
Safety Information
Translations of the WARNING on page 147.
WAR NU N G
Beim öffnen der Kassette und berühren des Kapillars eine Sicherheitsbrille
zum Schutz der Augen tragen.
CU IDA DO
Protéjase los ojos con gafas de seguridad cuando abra el cassette y manipule
el capilar.
AT T E N T IO N
Mettre des lunettes de securité lors de l’ouverture de la cassette et du
changement du capillaire, afin de protéger vos yeux.
AT T E N Z IO NE
Al fine di evitare rischi, proteggere gli occhi utilizzando occhiali di sicurezza
quando si apre la cassetta portacapillari o si manipolano i capillari stessi.
WAAR SC HUW ING
Draag een veiligheidsbril om uw ogen te beschermen als u de cassette opent
en het capillair hanteert.
254
Safety Information
Translations of the WARNING on page 186.
WAR NU N G
Bei der Durchführung dieser Aufgabe allgemeine Sicherheitsvorschriften für
Laboratorien beachten. Schutzbrille und Gummihandschuhe tragen.
CU IDA DO
Observar las regulaciones usuales de seguridad para los laboratorios, cuando
se realiza esta labor. Utilizar gafas de seguridad y guantes de goma.
AT T E N T IO N
Respectez les règles de sécurité usuelles applicables aux laboratoires lors de
la réalisation de cette opération. Portez des lunettes de sécurité et des gants
en caoutchouc.
AT T E N Z IO NE
Nel procedere a tale scopo osservare le disposizioni di sicurezza in uso
comune nei laboratori. Munirsi di guanti di gomma e di occhiali protettivi.
WAAR SC HUW ING
Neem de algemene veiligheidsvoorschriften voor laboratoria in acht wanneer
een opdracht wordt uitgevoerd. Draag een veiligheidsbril en rubber
handschoenen.
255
Safety Information
Translations of the WARNING on page 186.
WAR NU N G
Vergewissern Sie sich, daß das elektrische Netzanschlußkabel des Agilent CE
Instruments aus der Steckdose gezogen ist, bevor Sie mit Ihrer Arbeit
fortfahren.
CU IDA DO
Asegurarse de que se ha desconectado el cable de la línea de corriente del
instrumento Agilent CE, antes de seguir adelante.
AT T E N T IO N
Assurez-vous que vous avez débranché le fil d’alimentation secteur de
l’instrument Agilent CE avant de poursuivre les opérations.
AT T E N Z IO NE
Prima di procedere assicurarsi di avere staccato il cavo di alimentazione dello
strumento Agilent CE.
WAAR SC HUW ING
Wees ervan overtuigd dat het netsnoer van het Agilent CE instrument uit het
stopcontact is getrokken voordat u begint.
256
Safety Information
Translations of the WARNING on page 187.
WAR NU N G
Wenn Sie das Agilent CE Instrument erst vor kurzem benutzt haben, ist die
Lampe möglicherweise noch sehr heiß. Lampe nicht berühren bzw.
Schutzhandschuhe tragen.
CU IDA DO
Si se ha utilizado el instrumento, inmediatamente después la lámpara puede
estar muy caliente. Evitar el tocar la lámpara, o usar guantes de protección.
AT T E N T IO N
Si vous avez utilisé l’instrument peu de temps auparavant, il est possible que
la lampe soit très chaude. Evitez de toucher la lampe ou bien portez des gants
protecteurs.
AT T E N Z IO NE
Se si è fatto uso dello strumento poco prima la lampada può avere raggiunto
un’alta temperatura. Evitarne il contatto oppure munirsi di guanti di
protezione.
WAAR SC HUW ING
Indien het instrument kort tevoren nog werd gebruikt, dan kan de lamp nog
zeer heet zijn. Raak de lamp niet aan of gebruik hiervoor speciale
handschoenen.
257
Safety Information
Translations of the CAUTION on page 193.
ACHTUNG
Nachfüllnadel nicht berühren oder verbiegen.
PR E C AUC ION
No tocar o doblar la aguja de reaprovisionamiento.
AT T E N T IO N
Il ne faut ni toucher ni courber l’aiguille de remplissage.
PR E C A U Z I ON E
Non toccare o piegare l’ago di riempimento.
VO O RZ I C H T IG
De vulnaald mag niet worden aangeraakt of gebogen.
258
Safety Information
Translations of the WARNING on page 194.
WAR NU N G
Der Vorlocher hat eine sehr scharfe Spitze. Vorsicht beim Berühren des
Vorlochers, Schutzhandschuhe tragen, um Verletzungen zu vermeiden.
CU IDA DO
La preperforadora tiene una punta muy aguda. Tener precaución al tocar la
preperforadora y usar guantes de protección, para evitar lesiones.
AT T E N T IO N
L’outil de pré-poinçonnage possède une extrémité très acérée. Soyez prudent
lorsque vous le touchez et portez des gants protecteurs pour éviter les
accidents.
AT T E N Z IO NE
Il punzone è dotato di estremitá estremamente appuntita. Prestare
particolare attenzione nel maneggiarlo e munirsi di guanti di protezione per
evitare di ferirsi.
WAAR SC HUW ING
De voorperforator heeft een zeer scherpe punt. Wees voorzichtig bij het
aanraken van de voorperforator en draag speciale handschoenen om
verwondingen te voorkomen.
259
Safety Information
Translations of the CAUTION on page 194.
ACHTUNG
Trichterfläche des Vorlochers nicht beschädigen.
PR E C AUC ION
No deteriorar la superficie del embudo de la preperforadora.
AT T E N T IO N
N’endommagez par la surface en forme d’entonnoir de l’outil de pré-poinçonnage.
PR E C A U Z I ON E
Non danneggiare la superficie a imbuto del punzone.
VO O RZ I C H T IG
Voorkom beschadiging van het trechteroppervlak van de voorperforator.
260
Safety Information
Translations of the CAUTION on page 195.
ACHTUNG
Die Vorlocher müssen vor dem Wiedereinbau völlig trocken sein, um Kontamination zu
vermeiden.
PR E C AU C IO N
Para evitar contaminación, las preperforadoras tienen que estar completamente secas,
antes de su reinserción.
AT T E N T IO N
Pour éviter la contamination, les outils de pré-poinçonnage doivent être complètement
secs avant le remontage.
PR E C A U Z I ON E
Allo scopo di evitare contaminazione i punzoni devono essere completamente asciutti
prima della reinstallazione.
VO OR ZI C HT IG
Om vervuiling van de voorperforators te voorkomen moeten deze absoluut droog zijn
voordat deze opnieuw worden geïnstalleerd.
261
Safety Information
Translations of the CAUTION on page 196.
ACHTUNG
Das Innere der Elektroden muß vor dem Wiedereinbau völlig trocken sein, um
Kontamination zu vermeiden.
PR E C AUC ION
Para evitar contaminación, el interior de los electrodos tiene que estar completamente
seco, antes de su reinserción.
AT T E N T IO N
Pour éviter la contamination, l’intérieur des électrodes doit être complètement sec
avant le remontage.
PR E C A U Z I ON E
Allo scopo di evitare contaminazione la parte interna degli elettrodi deve essere
completamente asciutta prima della reinstallazione.
VO O RZ I C H T IG
Om vervuiling te voorkomen moet de binnenzijde van de elektrodes absoluut droog
zijn voordat deze opnieuw worden geïnstalleerd.
262
Safety Information
Translations of the WARNING on page 201.
WAR NU N G
Beim öffnen der Kassette und berühren des Kapillars eine Sicherheitsbrille
zum Schutz der Augen tragen.
CU IDA DO
Protéjase los ojos con gafas de seguridad cuando abra el cassette y manipule
el capilar.
AT T E N T IO N
Mettre des lunettes de securité lors de l’ouverture de la cassette et du
changement du capillaire, afin de protéger vos yeux.
AT T E N Z IO NE
Al fine di evitare rischi, proteggere gli occhi utilizzando occhiali di sicurezza
quando si apre la cassetta portacapillari o si manipolano i capillari stessi.
WAAR SC HUW ING
Draag een veiligheidsbril om uw ogen te beschermen als u de cassette opent
en het capillair hanteert.
263
Safety Information
Translations of the WARNING on page 214.
WAR NU N G
Vergewissern Sie sich, daß das Netzanschlußkabel aus der Steckdose gezogen
ist, bevor der Detektordeckel abgenommen wird. Wenn die Lampe noch an ist,
wenn der Deckel abgenommen wird, kann die UV-Strahlung sehr schädlich für
die Augen sein.
CU IDA DO
Asegurarse de que el cable de la línea de corriente está desconectado, antes
de retirar la cubierta detectora. Si la lámpara está todavía encendida, cuando
se retira la cubierta, la radiación UV puede ser nociva para los ojos.
AT T E N T IO N
Assurez-vous que le fil électrique est débranché avant de retirer le couvercle
du détecteur. Si la lampe demeure allumée après que vous avez ôté le
couvercle, le rayonnement UV peut être dangereux pour vos yeux.
AT T E N Z IO NE
Assicurarsi che il cavo di alimentazione sia stato disinserito prima di
rimuovere il coperchio del rivelatore. Se la lampada fosse ancora accesa dopo
la rimozione del coperchio le radiazioni ai raggi ultravioletti potrebbero
essere dannose agli occhi.
WAAR SC HUW ING
Wees ervan overtuigd dat het netsnoer uit het stopcontact is getrokken
voordat de het deksel wordt verwijderd. Wanneer de lamp nog steeds brandt
terwijl u het deksel verwijderd, dan kan de UV-straling schadelijk voor uw
ogen zijn.
264
Safety Information
Translations of the WARNING on page 215.
WAR NU N G
Wenn Sie das Agilent CE Instrument erst vor kurzem benutzt haben, ist die
Lampe möglicherweise noch sehr heiß. Lampe nicht berühren bzw.
Schutzhandschuhe tragen.
CU IDA DO
Si se ha utilizado el instrumento, inmediatamente después la lámpara puede
estar muy caliente. Evitar el tocar la lámpara, o usar guantes de protección.
AT T E N T IO N
Si vous avez utilisé l’instrument peu de temps auparavant, il est possible que
la lampe soit très chaude. Evitez de toucher la lampe ou bien portez des gants
protecteurs.
AT T E N Z IO NE
Se si è fatto uso dello strumento poco prima la lampada può avere raggiunto
un’alta temperatura. Evitarne il contatto oppure munirsi di guanti di
protezione.
WAAR SC HUW ING
Indien het instrument kort tevoren nog werd gebruikt, dan kan de lamp nog
zeer heet zijn. Raak de lamp niet aan of gebruik hiervoor speciale
handschoenen.
265
Safety Information
Translations of the WARNING on page 226.
WAR NU N G
Das WARNUNG Zeichen weist auf eine Gefahr für den Menschen hin. Wenn
die Anweisungen und Verfahrensweisen oder ähnliches nicht befolgt und
korrekt ausgeführt werden, könnte das eine erhebliche Verletzungsgefahr
oder Lebensgefahr zur Folge haben. Solange die neben dem Warnungzeichen
angegebenen Anweisungen nicht vollständig verstanden und ausgeführt sind,
sollten keine weiteren Schritte unternommen werden.
CU IDA DO
El signo de CUIDADO denota un riesgo. Remite a un procedimiento o práctica
que de no ser llevada a cabo correctamente, podría resultar en un daño o
pérdida de vida. No continue cuando exista un signo de advertencia hasta que
las condiciones indicadas hayan sido completamente entendidas y satisfechas.
AT T E N T IO N
Le signe ATTENTION dénote un risque. Il appelle une précaution quant à une
procédure ou une pratique qui, si elle n’est pas correctement suivie, peut
résulter en une blessure ou même en un risque pour la vie. S’assurer d’avoir
rempli toutes les conditions indiquées avant de continuer.
AT T E N Z IO NE
La scritta ATTENZIONE indica un pericolo. Essa richiama l’attenzione su una
situazione che può portare a danni anche gravi per le persone. Non proseguire
oltre tale indicazione senza aver ben compreso il rischio ed aver seguito le
istruzioni per evitarlo.
WAAR SC HUW ING
Het teken WAARSCHUWING wijst op gevaar. Indien de aanwijzingen,
procedures etc. niet opgevolgd en korrekt uitgevoerd worden, kan dat een
aanzienlijk risiko en zelfs levensgevaar met zich mee brengen. Zolang de naast
het waarschuwingsteken aangegeven aanwijzingen niet volledig begrepen en
opgevolgd zijn, dienen geen verdere stappen ondernomen te worden.
266
Safety Information
Translations of the CAUTION on page 226.
ACHTUNG
Das ACHTUNG-Zeichen weist auf eine mögliche Beschädigung der Geräte hin. Wenn
Anweisungen und Verfahrensweisen oder ähnliches nicht befolgt und korrekt
ausgeführt werden, könnte das eine Beschädigung oder Zerstörung eines Teils oder des
gesamten Gerätes zur Folge haben. Solange die neben dem ACHTUNG-Zeichen
angegebenen Anweisungen nicht vollständig verstanden und ausgeführt sind, sollten
keine weiteren Schritte unternommen werden.
PR E C AU C IO N
El signo de PRECAUCION denota un riesgo. Remite a un procedimiento o práctica que
de no ser llevada a cabo correctamente podría resultar en un daño o destruccion
parcial o total del equipo. No continue cuando exista un signo de precaución hasta que
las condiciones indicadas hayan sido completamente entendidas y satisfechas.
AT T E N T IO N
Le signe AVERTISSEMENT dénote un risque. Il appelle une précaution quant à une
procédure ou une pratique qui, si elle n’est pas correctement suivie, peut résulter en un
dommage ou même une destruction du matériel. S’assurer d’avoir rempli toutes les
conditions indiquées avant de continuer.
PR E C A U Z I ON E
La scritta PRECAUZIONE indica un pericolo. Essa richiama l’attenzione su una
situazione che può portare a danni anche permanenti allo strumento. Non proseguire
oltre tale indicazione senza aver ben compreso il rischio ed aver seguito le istruzioni
per evitarlo.
VO OR ZI C HT IG
Het teken VOORZICHTIG wijst op gevaar voor een mogelijke beschadiging van de
instrumentatie. Het vraagt de aandacht voor een praktijk, werkwijze etc. welke, indien
niet opgevolgd en korrekt uitgevoerd, kan leiden tot beschadiging of vernieling van de
apparatuur of een deel ervan. Zolang de naast het teken voorzichtig aangegeven
aanwijzingen niet volledig begrepen en opgevolgd zijn, dienen geen verdere stappen
ondernomen te worden.
267
Sound Emission
Sound Emission
Manufacturer’s Declaration
This statement is provided to comply with the requirements of the German
Sound Emission Directive, from 18 January 1991.
This product has a sound pressure emission (at the operator position) < 70
dB.
• Sound Pressure Lp < 70 dB (A).
• At Operator Position.
• Normal Operation.
• According to ISO 7779:1988/EN 27779/1991 (Type Test).
Herstellerbescheinigung
Diese Information steht im Zusammenhang mit dem Anforderungen der
Maschinenlärminformationsverordung vom 18 Januar 1991.
• Schalldruckpegel Lp < 70 dB (A).
• Am Arbeitsplatz.
• Normaler Betrieb.
• Nach ISO 7779:1988/EN 27779/1991 (Typprüfung).
268
Agilent Technologies on Internet
Agilent Technologies on Internet
For the latest information on products and services visit our worldwide web
site on the Internet at:
http://www.agilent.com/chem
269
Agilent Technologies on Internet
270
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In This Book
Chapter 1 “The Core Agilent CE
Instrument” describes the installation
of the Agilent CE system, external
water bath for tray cooling, detector
filter assembly, changing fuses and the
core CE diagram screen, which is used
for instrument control.
Chapter 2 “Capillary Electrophoresis”
with user interface explanation
describes the tasks that need to be
performed on the Agilent CE system
when preparing an analysis. In
addition some new functions are
described such as fraction collection
and capillary electrophoresis plus high
pressure.
Chapter 3 “Capillary
Electrochromatography”, a new
separation technique, lists parameters
that make up a CEC method and
describes their meaning.
Chapter 4 “Online CE-ESI-MS with the
Agilent CE System” describes the
online CE coupling to the Agilent 1100
Series LC/MSD.
Chapter 5 “Maintaining Your Agilent
CE Instrument” describes standard
operating procedures which should be
used to maintain the functionality of
your system.
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