Agilent Technologies All Troubleshooting guide

Agilent 1100/1200 HPLC
ChemStation Operation
Course Number H4033A
Laboratory Manual
Agilent 1100/1200 HPLC
ChemStation Operation
Course Number H4033A
Laboratory Manual
ChemStation B.03
Printed in April, 2007
Notice
The information contained in this document is subject to change without notice.
Agilent Technologies makes no warranty of any kind with regard to this material,
including but not limited to the implied warranties of 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.
No part of this document may be photocopied or reproduced, or translated to
another program language without the prior written consent of Agilent
Technologies, Inc.
Agilent Technologies, Inc
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 2007 by Agilent Technologies, Inc.
All rights reserved
Printed in the United States of America
ii
Table Of Contents
LAB EXERCISE: INTRODUCTION TO THE HPLC CHEMSTATION..... 1
IN THIS LABORATORY YOU WILL: ....................................................................... 2
ACCESSING THE HPLC CHEMSTATION ................................................................ 3
CONFIGURATION EDITOR ..................................................................................... 5
SCHEDULER ......................................................................................................... 6
MAINTAINING THE WINDOWS 2000 WORKSTATION ............................................ 8
Cleaning up Temporary Files ......................................................................... 8
Repairing the Disk .......................................................................................... 8
Disk Defragmentation..................................................................................... 9
Creating an Emergency Repair Disk .............................................................. 9
MAINTAINING THE WINDOWS XP WORKSTATION ............................................. 10
Cleaning up Temporary Files ....................................................................... 10
Repairing the Disk ........................................................................................ 10
Disk Defragmentation................................................................................... 11
Creating an Emergency Repair Disk ............................................................ 11
WINDOWS FEATURES (WINDOWS 2000 OR XP) ................................................. 13
Windows Explorer......................................................................................... 13
Wallpaper and Screen Savers ....................................................................... 14
Clipboard ...................................................................................................... 15
Closing a Program that is Not Operational ................................................. 15
LAB EXERCISE: ACQUISITION METHODS............................................. 17
IN THIS LABORATORY YOU WILL: ..................................................................... 18
PREPARATION .................................................................................................... 19
PREPARING THE HPLC ...................................................................................... 20
PRIMING THE AGILENT 1100/1200 SOLVENT DELIVERY SYSTEM ...................... 21
CREATING AN ACQUISITION METHOD ................................................................ 23
PRACTICE ENTERING GRADIENTS ...................................................................... 24
SNAPSHOT .......................................................................................................... 27
LAB EXERCISE: QUALITATIVE DATA ANALYSIS................................ 29
IN THIS LABORATORY YOU WILL: ..................................................................... 30
GRAPHICS - SIGNAL OPTIONS............................................................................. 31
GRAPHICS - ANNOTATION .................................................................................. 33
WINDOW FUNCTIONS ......................................................................................... 35
PEAK PURITY ..................................................................................................... 36
AUTOMATED PEAK PURITY ................................................................................ 38
DISPLAYING SPECTRA ........................................................................................ 39
ISOABSORBANCE PLOT ....................................................................................... 40
3D PLOT ............................................................................................................ 42
iii
LAB EXERCISE: SPECTRAL LIBRARIES .................................................. 43
IN THIS LABORATORY YOU WILL: ..................................................................... 44
BUILDING A LIBRARY ........................................................................................ 45
LIBRARY SEARCHING ......................................................................................... 47
LAB EXERCISE: OVERVIEW AND DIAGNOSTICS ................................. 49
IN THIS LABORATORY YOU WILL: ..................................................................... 50
Overview of Equipment and Tools Used....................................................... 50
Consumables ................................................................................................. 50
THE AGILENT 1200 FLOW PATH ........................................................................ 51
ERROR MESSAGES AND THE LOGBOOK .............................................................. 52
DIAGNOSIS VIEW ............................................................................................... 53
ROUTINE MAINTENANCE ................................................................................... 59
INSTRUMENT TESTS ........................................................................................... 60
LAB EXERCISE: OVERVIEW AND DIAGNOSTICS FOR THE
AGILENT-SL SERIES....................................................................................... 61
IN THIS LABORATORY YOU WILL: ..................................................................... 62
Overview of Equipment and Tools Used....................................................... 62
Consumables ................................................................................................. 62
THE AGILENT 1200 - SL FLOW PATH................................................................. 63
ERROR MESSAGES AND THE LOGBOOK .............................................................. 64
AGILENT LC DIAGNOSTIC TOOL ........................................................................ 65
INSTRUMENT STATUS REPORT ........................................................................... 69
ROUTINE MAINTENANCE ................................................................................... 70
INSTRUMENT TESTS ........................................................................................... 71
LAB EXERCISE: INTEGRATION................................................................. 73
IN THIS LABORATORY YOU WILL: ..................................................................... 74
INTEGRATION PREPARATION .............................................................................. 75
AUTO INTEGRATION........................................................................................... 76
INTEGRATE USING THE EVENTS TABLE ............................................................. 78
ADDING TIMED EVENTS ..................................................................................... 79
USING MANUAL EVENTS ................................................................................... 81
LAB EXERCISE: QUANTIFICATION.......................................................... 83
IN THIS LABORATORY YOU WILL: ..................................................................... 84
PREPARATIONS .................................................................................................. 85
CREATING THE ACQUISITION METHOD .............................................................. 87
DATA ACQUISITION-STANDARDS....................................................................... 89
INTEGRATION ..................................................................................................... 91
SETTING UP SIGNAL DETAILS............................................................................. 93
BUILDING A CALIBRATION TABLE ..................................................................... 94
SETTING UP LOW AND HIGH AMOUNT LIMITS FOR CALIBRATION STANDARDS . 96
SETTING UP QUALIFIERS (DIODE ARRAY ONLY) ................................................ 97
TESTING THE CALIBRATION TABLE.................................................................... 98
iv
LAB EXERCISE: RUNNING A SEQUENCE................................................ 99
IN THIS LABORATORY YOU WILL: ................................................................... 100
BUILDING A SEQUENCE .................................................................................... 101
SEQUENCE SUMMARY REPORTS ....................................................................... 104
STARTING THE SEQUENCE ................................................................................ 105
PAUSING A SEQUENCE ..................................................................................... 106
POST SEQUENCE............................................................................................... 107
LAB EXERCISE: SEQUENCE REVIEW AND REPROCESSING.......... 109
IN THIS LABORATORY YOU WILL: ................................................................... 110
REPROCESS WITH SEQUENCE FOLDER METHOD ............................................... 111
REPROCESS WITH DA METHODS ...................................................................... 117
BATCH REVIEW................................................................................................ 121
LAB EXERCISE: ADVANCED REPORTING ........................................... 123
IN THIS LABORATORY YOU WILL: ................................................................... 124
ADDING A REPORT HEADER............................................................................. 125
PERFORMANCE REPORTS (SYSTEM SUITABILITY) ............................................ 127
AUTOMATED PEAK PURITY .............................................................................. 129
AUTOMATED SPECTRAL LIBRARY SEARCH ...................................................... 130
LAB EXERCISE: CUSTOMIZED REPORT DESIGN ............................... 133
IN THIS LABORATORY YOU WILL: ................................................................... 134
PREPARATIONS ................................................................................................ 135
BUILDING A CUSTOMIZED REPORT .................................................................. 136
ADDING A GENERAL SECTION.......................................................................... 137
INSERTING TABLES .......................................................................................... 138
ADDING A CALIBRATION CURVE ..................................................................... 139
FINISHING UP ................................................................................................... 140
LAB EXERCISE: COMMANDS ................................................................... 141
IN THIS LABORATORYYOU WILL: .................................................................... 142
RETRIEVING INFORMATION ABOUT COMMANDS .............................................. 143
TRACKING ERRORS AND USING SOME COMMON COMMANDS .......................... 144
REGISTERS, OBJECTS, AND TABLES ................................................................. 145
LAB EXERCISE: MACRO WRITING ........................................................ 153
IN THIS LABORATORY YOU WILL: ................................................................... 154
WRITING AND EXECUTING A MACRO ............................................................... 155
WRITE YOUR OWN MACRO ............................................................................. 157
POSSIBLE ANSWER ........................................................................................... 158
APPENDIX: GETTING STARTED WITH NEW CHEMSTATION
WORKFLOW G2170-90041........................................................................... 159
v
vi
Lab Exercise: Introduction to the HPLC
ChemStation
Lab Exercise: Introduction to the HPLC ChemStation
In this Laboratory You Will:
In this Laboratory You Will:
•
Access the HPLC ChemStation software and understand the purpose of each
View.
•
Use the ChemStation Configuration Editor.
•
Learn about the Scheduler.
•
Learn how to maintain the HPLC ChemStation computer.
•
Become familiar with some Windows features (optional).
2
Lab Exercise: Introduction to the HPLC ChemStation
Accessing the HPLC ChemStation
Accessing the HPLC ChemStation
1)
Find the Start button in the bottom left hand corner of your screen. Click on
the Start button. Select the menu choice All Programs. You will find a list
of the software programs installed on this computer arranged in alphabetical
order. To launch the ChemStation for LC 3D Systems, find and select the
Agilent ChemStation menu item.
2)
The following items are available: Add Licenses, Configuration Editor,
Installation Qualification, Instrument 1 Online, Instrument 1 Offline,
readme.txt, and the Scheduler.
3)
Click on Instrument 1 Offline. The software program will begin loading.
Question:
What is the difference between the ChemStation's Online and Offline sessions?
_________________________________________________________________
_________________________________________________________________
4)
Open the View menu and select Full Menu, if this item is available.
5)
Access the View menu and examine the entries. Notice that there are several
main views starting with Method and Run Control. List the views below
and after exploring each view, list the primary function of each.
View
Function
3
Lab Exercise: Introduction to the HPLC ChemStation
Accessing the HPLC ChemStation
6)
You can also change views by utilizing the Navigation Buttons.
Switch Views Here
Minimize, Maximize
Close
7)
Click on the small triangle at the top of the ChemStation Explorer. Click
again. Notice that you can sort your files.
8)
Click on the Configure buttons icon at the bottom of the Navigation Pane –
you can use this to add or remove buttons.
9)
Find the Minimize, Maximize and Close buttons located in the upper right
hand corner of the Title Bar. Minimize the ChemStation software. Notice
that the ChemStation software now appears as a button to the right of the
Windows Start button in the Task Bar. The ChemStation software is still
running, but has been cleared from the Desktop.
10) Click on the Instrument 1 (offline) software button in the Task Bar to restore
the ChemStation window.
11) To close the ChemStation operating software you may either utilize the
Close button located in the upper right hand corner of the window; or, under
File select Exit. Close the ChemStation software now.
NOTE: You will access the Configuration Editor next. In order for the
Configuration Editor to open, the HPLC ChemStation software must be closed.
4
Lab Exercise: Introduction to the HPLC ChemStation
Configuration Editor
Configuration Editor
You customer engineer configured your instrument during installation. This
process includes configuring the instrument devices and setting the LAN IP
address so that the software can communicate with the instrument. After
installation, you may need to access the Configuration Editor to add another
instrument, change the default path for methods, sequences or data files, or
change the colors used for chromatograms, titles, and baselines.
1) Open the Configuration Editor from Start, All Programs, Agilent
ChemStation, then Configuration Editor.
2) Find the small window that contains your instrument configuration and click
on the title bar to activate the window.
3) Select Instruments… from the Configure menu. You should have a
Modular 3D System configured. Change the Instrument Name from
Instrument 1 to a name of your choice. The instrument name is printed in the
footer of all reports, and you will use this name to identify reports you print in
this course. You should have a
4) OK this dialog box and view the next dialog box showing the instrument type
and address. Do not make any changes here. OK this window as well.
5) Now select Configure > Colors.
6) Note the color changes that can be made and practice changing the signal
colors. When you are satisfied, set the elements back to their default values
and OK the dialog box.
7) Explore other areas of the Configure menu, but do not make any additional
changes.
8) Under File, Save the configuration so the new instrument name will be saved.
Exit the Configuration Editor (File, Exit).
5
Lab Exercise: Introduction to the HPLC ChemStation
Scheduler
Scheduler
The Scheduler may be used to make your ChemStation automatically execute
commands on a one time, daily, daily weekday, or weekly basis. You may
schedule events such as controlling a valve, loading a method or sequence,
starting a sequence, or initiating a blank run. As a command is processed, the
Scheduler records the completion status as accepted, rejected, not allowed, or
instrument not found in the Results column so that the progress can be reviewed
easily. Note that the events associated with running an analysis or sequence take
precedence over the execution of scheduler events.
1) From the Windows Start button find All Programs, Agilent ChemStation,
then Instrument 1 Offline. The ChemStation application must be open for
the Scheduler to work. Now select the Scheduler from Start, All Programs,
Agilent ChemStation.
2) Using the drop box in the upper left hand corner of the Scheduler, select the
instrument to be scheduled.
3) A blank schedule for that instrument will appear. The entries include:
Date:
Enter the first date this command should be sent, current date to
one year in advance.
Time:
Using the 24-hour clock format (13:00 is 1:00 PM), enter the Time
this command should be sent.
Command:
Enter the command you want to be sent. Command help is found
in the ChemStation under the Help menu.
Mode:
Select what day(s) this command should be sent.
Result:
Lists completion status of each command sent to an instrument.
4) For practice, you will make the ChemStation logbook appear on the screen
using the Scheduler. Enter the current date. Enter a time approximately 2
minutes from the current clock time (found in the lower right hand corner of
the screen). Type: Logbook On in the Command box. The mode will be Do
Once.
5) The selected commands will be stored for use once they are saved. Select the
Save tool from the toolbar.
6) Minimize the Scheduler and wait to see if the logbook appears automatically.
6
Lab Exercise: Introduction to the HPLC ChemStation
Scheduler
7) After you have witnessed the logbook display, return to the Scheduler to
check the Result column.
8) Close the Scheduler.
Note: The Scheduler can be useful for turning on the detector lamp prior to your
arrival each day to warm-up. The command is “Lampall On”. Do not include the
parentheses.
7
Lab Exercise: Introduction to the HPLC ChemStation
Maintaining the Windows 2000 Workstation
Maintaining the Windows 2000 Workstation
(Skip to Maintaining the Windows XP Workstation if you have
Windows XP)
Cleaning up Temporary Files
After the ChemStation has been used for some time, temporary files may
accumulate in the directory specified by the TEMP Variable. These files are
generally left open when Windows is abnormally terminated. The files generated
have a .tmp extension. These files should be deleted to maintain computer
efficiency.
1) Close all programs.
2) Go to the Start button then Search and select For Files or Folders….
3) In the Search for Files or Folders named: field, type *.tmp.
4) In the Look in: field, find your hard drive, C: or Local hard drives.
5) Select Search Now.
6) After a few moments, the files will be displayed. Highlight the first file.
Using the Shift key and the left mouse button, highlight the last file. Press the
Del key to remove the files. Remember that you will have to empty them
from the Recycle bin as well.
Repairing the Disk
Windows 2000 includes a utility to check disk integrity. Among the errors that
may be fixed are lost clusters and cross-linked files. Error-checking can move
your data from bad sectors as well. You must be logged on as part of the
administrator's group to run this utility.
1) Log on as the administrator. Press CTRL-ALT-DEL simultaneously. Log on
as the administrator. The password is agilent. Or check with your instructor
for the correct password.
2) Double click on the My Computer icon and right-click on the drive icon for
C:. Select Properties to display a selection box.
8
Lab Exercise: Introduction to the HPLC ChemStation
Maintaining the Windows 2000 Workstation
3) Click on the Tools tab. Find the Error-Checking tool and click Check
Now....
4) Select to Automatically fix file system errors and Scan for and attempt
recovery of bad sectors.
5) This process can take several minutes. If you don’t wish to do this in lab,
Cancel, otherwise select Start.
Disk Defragmentation
A defragmentation utility is included with Windows 2000. Several vendors sell
this utility as well such as DiskKeeper. Make certain you defragment your disk
drives on a periodic basis. You must close all programs before beginning. The
process can take a long time. We will not do this here.
Creating an Emergency Repair Disk
You should create an emergency repair disk as soon as possible. Use this disk if
your system files are damaged or your computer won't start. The disk includes
system settings such as registry files, disk partitions, and installed devices. Make
a new repair disk every time you make changes to your hardware or software.
Emergency Repair Disks are specific for each computer.
1) From the Start button, select Programs, Accessories, System Tools, then
Backup.
2) Click on the Emergency Repair Disk button.
3) Insert a floppy into drive A:.
4) Select to also back up the registry to the repair directory.
5) OK any dialog boxes that appear then close the Backup program.
9
Lab Exercise: Introduction to the HPLC ChemStation
Maintaining the Windows XP Workstation
Maintaining the Windows XP Workstation
(Skip this section if you completed Maintaining the Windows 2000
Workstation)
Cleaning up Temporary Files
After the ChemStation has been used for some time, temporary files may
accumulate in the directory specified by the TEMP Variable. These files are
generally left open when Windows is abnormally terminated. The files generated
have a .tmp extension. These files should be deleted to maintain computer
efficiency.
1) Close all programs.
2) Go to the Start button then select Search. On the right select All files and
folders.
3) In the All or part of the file name: field, type *.tmp.
4) In the Look in: field, find your hard drive, C: or Local hard drives.
5) Select Search.
6) After a few moments, the files will be displayed. Highlight the first file.
Using the Shift key and the left mouse button, highlight the last file. Press the
Del key to remove the files. Remember that you will have to empty them
from the Recycle bin as well.
Repairing the Disk
Windows XP includes a utility to check disk integrity. Among the errors that may
be fixed are lost clusters and cross-linked files. Error-checking can move your
data from bad sectors as well. You must be logged on as part of the
administrator's group to run this utility.
1) Log on as the administrator. The password is 3000hanover or, check with
your instructor for the correct password.
2) Double click on the My Computer icon and right-click on the drive icon for
C:. Select Properties to display a selection box.
10
Lab Exercise: Introduction to the HPLC ChemStation
Maintaining the Windows XP Workstation
3) Click on the Tools tab. Find the Error-Checking tool and click Check
Now....
4) Select to Automatically fix file system errors and Scan for and attempt
recovery of bad sectors.
5) This process can take several minutes. If you don’t wish to do this in lab,
Cancel, otherwise select Start.
Disk Defragmentation
A defragmentation utility is included with Windows XP. Several vendors sell this
utility as well such as DiskKeeper. Make certain you defragment your disk drives
on a periodic basis. You must close all programs before beginning. The process
can take a long time. We will not do this here.
Creating an Emergency Repair Disk
You should create an emergency repair disk as soon as possible. Use this disk if
your system files are damaged or your computer won't start. The disk includes
system settings such as registry files, disk partitions, and installed devices. Make
a new repair disk every time you make changes to your hardware or software.
Emergency Repair Disks are specific for each computer. The backup process for
Windows XP goes farther than Windows 2000. It consists of two of two parts: a
backup file, and a Recovery Disk. The backup file will be large (we cannot do
this here), and the Recovery Disk will be a floppy.
1) From the Start button, select All Programs, Accessories, System Tools, then
Backup.
2) The Backup Utility Wizard should start by default unless it is disabled. If the
Wizard has been disabled, select Tools then Switch to Wizard Mode.
3) Click the Advanced Mode button found in the text.
4) Click the Automated System Recovery Wizard button.
5) Click Next>. The Wizard will start and prompt you for the media to use for
the backup file. As we don’t have the proper media here, cancel out.
You can write this file to a tape drive, a hard disk or writeable CD or DVD. After
entering the destination for the backup file, click Next> again, and finally, Finish.
The Windows XP Backup utility will copy all important system files and settings
to the backup file. An estimate and status bar are provided. After this step is
11
Lab Exercise: Introduction to the HPLC ChemStation
Maintaining the Windows XP Workstation
complete, you will be prompted for a blank, formatted floppy disk. Several files
are written to the disk to complete the process.
12
Lab Exercise: Introduction to the HPLC ChemStation
Windows Features (Windows 2000 or XP)
Windows Features (Windows 2000 or XP)
Optional Section
Mastering WINDOWS could take a week long class in itself. This section of the
laboratory is simply a brief familiarization of some of the important aspects
frequently utilized in conjunction with ChemStations. If you are already familiar
with Windows, skip this section.
Windows Explorer
The Windows Explorer is a powerful file manager. All files are stored in folders
(directories) on your hard disk. The Explorer allows you to copy, move, and print
entire folders and individual files.
1) To open the Windows Explorer, click on the Start button and select All
Programs, Accessories, Windows Explorer. The program will load.
2) Take a look at the information displayed on the left. These are all the objects
found in your computer. If the object has a + sign next to the name, the
folder contains sub-items such as sub-folders (subdirectories) or files that are
not currently shown. Click on the + signs until you can open Chem32
(C:\Chem32). This opens the sublevels.
3) Click on the + sign next to the 1 folder. The data folder should now be
present. Similarly, open the Data and Demo folders. Click on the Demo item
itself not the + sign. Notice that the contents of the folder appear in the right
pane as a group of folders. The individual files in the Demo folder (directory)
are displayed after the sub-folders.
4) To copy files from the data folder to a floppy drive, simply click on the folder
or file which needs to be moved and drag it to the 3 1/2 Floppy (A:) item in
the left pane. Try this now with one of the displayed data files. Make sure
that you have a formatted disk in the drive.
NOTE: When you drag files between different drives they are copied, not moved.
An original copy remains on the source drive. When you drag files between
folders on the same drive, Windows assumes you want to move the files, not copy
them.
NOTE: To select multiple files, click on the first file to select it. Ctrl-Click on
any additional files. To select a group of consecutive files, click on the first file to
select it. Then, use a Shift-Click on the last item to be selected.
13
Lab Exercise: Introduction to the HPLC ChemStation
Windows Features (Windows 2000 or XP)
5) Check to make certain that you have copied the chosen file into drive A: by
expanding the 3 1/2 Floppy (A:) item.
6) To delete a file or folder (when you delete a folder, all the contents of the
folder will be deleted along with the folder itself) right click on the folder or
file in the left or right pane. From the File menu select Delete. Try this now
with one of the data files in the Data\Demo folder.
7) The contents of the file will be moved to the Recycle Bin. The Recycle Bin is
found as an icon on the Windows desktop. Open this now. This is a special
folder on your disk. The Recycle Bin holds your deleted files until you empty
the bin, just in case you made a mistake. To restore a file from the Recycle
Bin, click on the item or items you accidentally deleted. From the File menu
select Restore. Once the bin has been emptied however, the only way to
retrieve a file is with a special undelete program. Try to restore the file you
deleted in part 6.
NOTE: Remember you must empty the Recycle Bin to gain space on your hard
drive. The Empty Recycle Bin option is found under the File menu in the
Recycle Bin.
8) You may open any file in the Windows Explorer to display its contents.
Simply double click on the item in the right pane. Practice your skills. From
the File menu, Close the Windows Explorer.
Wallpaper and Screen Savers
The Wallpaper simply lets you decorate the background of your Desktop.
1) Right click on any empty area of the Desktop. Select Properties.
2) Click on the Desktop tab of the Display Properties box.
3) Scroll through the Background list and highlight the one you would like. OK
the panel.
4) A screen saver will prevent a static image from burning into your screen.
5) Right click on any empty area of the Desktop. Select Properties.
6) Click on the Screen Saver tab.
7) Select a file from the Drop box. Make any other changes you desire.
8) OK the panel when you have made your selections.
14
Lab Exercise: Introduction to the HPLC ChemStation
Windows Features (Windows 2000 or XP)
Clipboard
You may use the clipboard to capture the entire screen, an entire window or any
selected material within a document or graphic using the cut or copy commands
found in Windows based programs. The image is temporarily stored until you
paste it into the same or another application. For instance, say that you were
creating a written HPLC method. You can copy the Pump Settings window from
the HPLC ChemStation software then paste it into a word processing program
such as Microsoft Word or WordPad.
1) Open the Instrument 1 Offline session.
2) Go to the Method and RunControl view. Under the Instrument menu,
select Set up Pump....
3) Press the ALT and PrintScreen keys simultaneously to copy the window to
the clipboard. Cancel the Set up Pump dialog box.
4) Minimize the HPLC ChemStation.
5) From the Start button, go to Programs, Accessories. Select WordPad.
6) Click on the spot in the window where you would like to place the Pump
Parameter window.
7) From the Edit menu, select Paste.
The graphic is now part of the WordPad document.
Closing a Program that is Not Operational
Occasionally, you may find that the ChemStation or another Windows program
stops functioning or becomes "hung-up". You may try to cancel the hung-up
program rather than rebooting the entire computer. To do this, strike CTRL,
ALT and DEL simultaneously and abort the offending program.
1) Press CTRL, ALT, and DEL simultaneously. The Windows Security dialog
box will appear. For XP, select the Applications tab. Select Task Manager
for Windows 2000.
2) Select the program marked for termination. Here, select WordPad, then select
End Task. Note that WordPad is closed, while other software remains open.
15
Lab Exercise: Introduction to the HPLC ChemStation
Windows Features (Windows 2000 or XP)
If these actions do not clear your problem, you would have to reboot the
computer.
16
Lab Exercise: Acquisition Methods
Lab Exercise: Acquisition Methods
In this Laboratory You Will:
In this Laboratory You Will:
•
Prepare your instrument for acquisition.
•
Set-up an acquisition method.
•
Run an acquisition method.
•
Take a snapshot of the data.
18
Lab Exercise: Acquisition Methods
Preparation
Preparation
Before starting, make certain that the you have the following:
•
A sample vial labeled, sample, which contains the Test Mix, Part # 0108068704 (diluted).
•
HPLC grade water in channel A and HPLC grade acetonitrile in channel B.
•
A 4.0 x 100 mm, C-18 column installed in the column compartment.
•
An Agilent 1100/1200 with all modules powered on.
•
An HPLC ChemStation, B.03.xx, equipped with a spectral module.
19
Lab Exercise: Acquisition Methods
Preparing the HPLC
Preparing the HPLC
1) Enter the Method and Run Control view of the Agilent Online ChemStation.
2) From the View menu, select Instrument Actuals, the System Diagram and
Sampling Diagram. If a check mark exists beside the item, it is already on.
3) Load the default method, DEF_LC.M as a starting point for method creation
(Method, New Method) or double-click on DEF_LC.M in the ChemStation
Explorer.
4) Under the Instrument menu, select More DAD, Control. Turn on the UV
and Vis lamps. OK the window. Remember that you may also turn the lamp
on from the system diagram GUI.
20
Lab Exercise: Acquisition Methods
Priming the Agilent 1100/1200 Solvent Delivery System
Priming the Agilent 1100/1200 Solvent Delivery
System
You should prime the pump when you change mobile phases, when the
instrument has been sitting without flow for long periods of time, when you have
performed maintenance on the HPLC, or when you experience periodic
perturbations in the baseline.
1) If you are equipped with a vacuum degasser, make certain the degasser is on.
2) Ensure that the outlet tube is connected from the purge valve to the waste
container. Open the purge valve located on the pump module by turning the
knob counter clockwise several turns.
3) To prime the solvent delivery system, you should pump 100% at 5 mL/min
from each channel for several minutes. To accomplish this task, select the
following menu items: Instrument, Set up Pump...; or click on the pump in
the system diagram (shown below), then Set up Pump....
Click here
4) Type in a Flow of 5.000 mL/min and %A of 100. OK the window.
5) To turn the solvent delivery system on, you may use the menu items:
Instrument, More Pump, Control; or, access the tool shown in the diagram
below. Do this now.
21
Lab Exercise: Acquisition Methods
Priming the Agilent 1100/1200 Solvent Delivery System
6) Wait until a steady stream of solvent comes out of the purge valve waste tube.
7) Repeat steps 3 through 6 for the other channels of the pump. Change to the
composition for your next run, %B=70.
8) Turn off the pump.
9) Close the purge valve. Set the required composition and flow rate for your
next application. In this case: Flow 1.5 mL/min and %B 70.
10) Turn on the pump and allow the column to equilibrate.
Note: Always keep HPLC grade solvent in all pump channels to prevent valve
damage even when the channel is not being used. For a reversed phase system,
place water with 20% methanol in the unused channels.
22
Lab Exercise: Acquisition Methods
Creating an Acquisition Method
Creating an Acquisition Method
1) From the Method menu select Edit Entire Method... or select the Edit entire
Method tool.
2) Select only the Method Information and Instrument/Acquisition sections
for editing. OK this window.
3) The Method Information dialog box now appears. You may enter any
information here that pertains to the method. Enter a description now. OK the
window.
4) Fill in the following solvent delivery system parameters:
Question:
What is the function of the Min: Pressure Limit?
_______________________________________________________
23
Lab Exercise: Acquisition Methods
Practice Entering Gradients
Practice Entering Gradients
1) Select the Insert button from the Time Table group. Program a linear
gradient to 100%B over 4.0 minutes. In the Time column, type 4. In the %B
column type 100.
2) Go to the Display dropbox. Select Solvents.
3) Return to the Timetable display. Click on the 1 next to the timetable line,
then cut. Unfortunately, you do not need a gradient for this analysis, OK the
Pump Parameters window.
Back to the Acquisition Method
1) Select a Standard Injection with a 5 µl injection volume. Select More... to
view all injection options.
Question:
What is the slowest draw speed? (hint: type in 1µl)
________________________________________________________________
When would you reduce the draw speed?
________________________________________________________________
2) OK the window.
3) Fill in the DAD Signals as below. OK the window.
Note: If a VWD detector is being utilized, select the same wavelength as signal
A.
24
Lab Exercise: Acquisition Methods
Practice Entering Gradients
Question:
How is the threshold setting used?
________________________________________________________________
4) Set the column compartment temperature to 40°C. Make certain that the right
compartment has the same temperature as the left compartment. OK the
window.
5) From the Method menu, select Save Method As.... Name the method,
Class.m. The ChemStation will not let you overwrite DEF_LC.M; that way,
it will always be there as a template for new methods. This master acquisition
method is now prepared. Note that this method is saved in the directory:
C:\Chem32\1\Methods.
6) Add a comment to the method history. You can view the method history by
selecting Method, then Method Change History…. Close the window.
7) Go to the View menu and select Online Signals, Signal Window 1. Once the
Online Plot is displayed, select Change.
25
Lab Exercise: Acquisition Methods
Practice Entering Gradients
8) Add DAD A to Selected Signals. Fill in an x-axis range of 10 minutes.
Select the draw zero line box.
9) Click on the DAD A Signal in the Selected Signals box then set the y-axis
range to 100 mAU and the offset to 10%. OK the dialog box.
10) From the Method menu, select the Run Time Checklist. Select Data
Acquisition only. Save a copy of the Method with the data. OK the dialog
box. A report will not automatically print with this method.
11) Resave the Method.
12) Once you have a steady baseline and pressure, pull down the RunControl
menu and select Sample Info... The panel can be accessed by the tool shown
below. (hint: to stabilize the baseline use the Detector Balance Button)
13) Fill in the Operator Name, Filename (Class.d), the subdirectory (your
name), Location (Vial 1), sample name (test sample) and Comment
information.
Question:
What is the Sample Amount used for? Look in the Help screen for clues.
_________________________________________________________________
14) Start the run either from the Start tool or from the RunControl menu, then
Run Method.
26
Lab Exercise: Acquisition Methods
Snapshot
Snapshot
1) Once the first major peak has eluted, open an Offline Session of the Agilent
ChemStation.
2) Go to the Data Analysis view of the Offline Session.
3) Go to the File menu and select, Snapshot. The current chromatogram appears
in the chromatogram window of the Offline Session.
4) Click on the Data button in the ChemStation Explorer and find your
subdirectory.
5) Double-click on the Single Runs and find the SNAPSHOT.D in the navigation
table.
6) Return to the Online Session.
7) When the run is complete, turn the System Off. This control is found under
the Instrument menu.
You are finished with this laboratory.
27
Lab Exercise: Acquisition Methods
Snapshot
28
Lab Exercise: Qualitative Data Analysis
Ideally, use the data file generated from the isocratic test mix in the previous
laboratory exercise. If this is not available, use demo\005-0104.d and
demo\demodad.d.
Lab Exercise: Qualitative Data Analysis
In this Laboratory You Will:
In this Laboratory You Will:
1)
Use signal options to display data.
2)
Annotate chromatograms.
3)
Use the Window Functions.
4)
Use the peak purity algorithm to examine the purity of chromatographic
peaks.
5)
Overlay and view UV spectra.
6)
Use Isoabsorbance and 3D plotting.
30
Lab Exercise: Qualitative Data Analysis
Graphics - Signal Options
Graphics - Signal Options
1) Enter the Data Analysis view.
2) Load the method democal2.m. Either double-click on democal2.m in the
method ChemStation Explorer or select Method > Load Method.
3) In Data Analysis, just below the Navigation Table on the left, there are five
tools for five types of data analysis tasks: Integration, Calibration, Signal,
Purify and Spectrum.
The choice of data analysis task causes the tool bars to display appropriate
tools for the type of task. For this exercise, select the Signal Tool. The
chromatogram will then be displayed as large as possible, and the tool bars
will show the Signal Manipulation Tools.
4) In the Graphics menu, select Signal Options....
5) Use the Signal Options dialog box to define how chromatographic signals
will look when drawn to the screen or printer. For this example, select to
include: Axes, Baselines, Tic Marks, and Retention Times. Note that the
fonts are selectable. This example chromatogram will use the entire recorded
x and y axes, Full Ranges. For the Multi-Chromatogram output, select
Layout: Overlaid with the Scale: All the same Scale. OK this dialog box.
6) Expand the Demo files in the Data ChemStation Explorer. Find the Single
Runs and couble-click to load them into the Navigation Table.
7) Go to the Navigation Table. Position the mouse on the Method Name
heading. Click the right mouse button. Choose Group By This Column.
What happens?
8) Click on the ‘+’ on several of the methods listed to see the new layout of the
table. Right click anywhere in the heading row and choose Reset grouping.
What happens? Notice that you can customize the layout of the Navigation
table using the tools provided.
9) Find the data file C:\Chem32\1\data\demo\005-0104.d in the Navigation
Table (Demo – Single Runs) or find the file you acquired in the acquisition
lab. Double-click to load into the Navigation Table.
10) In the Navigation Table, click on the ‘+’ in the first column next to the
date/time stamp.
31
Lab Exercise: Qualitative Data Analysis
Graphics - Signal Options
11) A panel with 3 tabs will appear. The Signals tab should be highlighted and all
signals will be initially selected. Click off one of the signals.
12) Select the Inst. Curves tab. Click to make sure there is a check in the High
Pressure checkbox. Click the General Info tab to see what information is
available.
13) Click the right mouse button in the row containing the datafile. Choose Load
Selected Signals. The overlaid chromatograms and high-pressure plot will
appear.
14) An additional tool bar, the Graphics Tool Bar, can be displayed along the right
side of the chromatogram. This tool bar is turned on or off by clicking on the
Graphics Tool Do this now.
15) Now try to plot the signals separated, each in full scale. This time, you can
use the Overlay Separate Signals tool and the Each in Same Scale Tool.
You may need to scroll down to view all signals and the pressure plot.
16) Practice using the other tools in the Graphics Tool bar. You can see what the
names of the various tools are by positioning the cursor over that tool and
looking at the information line at the bottom of the Data Analysis Screen.
32
Lab Exercise: Qualitative Data Analysis
Graphics - Annotation
Graphics - Annotation
1)
Using the Signal(s) Displayed drop box, select DAD1A only.
2)
Select the Graphics too.
3)
From the Graphics menu, select New Annotation or select the Add
Annotation tool.
Position the cursor in the center-top of the chromatogram and click the left
mouse button.
4)
When the Text Annotation box appears, type in Test Mix. Select Options.
You may select a font and font size. OK the options and then OK the
annotation box.
5)
Click above the third major peak in the chromatogram with the left mouse
button. When the Text Annotation box appears, type in Target
Compound. Under Options, change the text rotation to 90. OK the options
and annotation box.
6)
From the Graphics menu, select Line Annotation or use the Draw Line in
Window Tool. Using the cursor, draw a line from the Target Compound
annotation to the chromatographic peak.
33
Lab Exercise: Qualitative Data Analysis
Graphics - Annotation
7)
To print this window, pull down the File menu and select Print, Selected
Window. Now Print the annotated chromatogram.
8)
Practice with the Delete Annotation and Move Annotation menu items
found under the Graphics menu or use the corresponding tools.
9)
When finished with these tools, you can turn off the Graphics Tool bar by
clicking the graphics tool again.
34
Lab Exercise: Qualitative Data Analysis
Window Functions
Window Functions
1) Use the Signal(s) Displayed drop box to view All Loaded Signals.
2) From the View menu, select Window Functions, List Content.
3) Position the cursor in the margin left of the Pump1, Pressure entry. Click
using the left mouse button. This line should now be highlighted.
4) Press the Delete Obj. button.
5) What happened to the pressure display?
_______________________________________________________________
6) Highlight one of the signals. Using the left mouse button, click on List Data.
The chromatographic data points are displayed. Close the windows created in
this exercise.
35
Lab Exercise: Qualitative Data Analysis
Peak Purity
Peak Purity
The Peak Purity algorithm can help you determine whether a chromatographic
peak is comprised of one or more components. In quality control, impurities
hidden behind the peak of interest can falsify results. In research, a hidden
component left undetected might lead to a loss of essential information.
1) In the Methods ChemStation Explorer, find DEF_LC.M and load the method
by double-clicking on the name.
2) In the Data ChemStation Explorer, double-click on the Single Runs found
under the Demo folder to load the files into the Navigation Table.
3) Load one signal from DEMODAD.D. Now go to the Spectra menu and
choose Select Peak Purity. In this exercise, you will use the default purity
options. Alternatively, use the tools.
4) Click on a major chromatographic peak. Examine the resulting displays.
Compare your results to that of the pure and impure peaks presented in the
lecture. Make sure you compare the Purity Factor and the calculated
Threshold found by selecting the Information on the LC Peak Purity Tool.
5) Select three major peaks and answer the questions below. To review another
chromatographic peak, simply click on the peak in the chromatogram located
in the upper portion of the purity display.
36
Lab Exercise: Qualitative Data Analysis
Peak Purity
Major Peaks
Peak 1
Peak 2
Peak 3
Retention Time
Calculated Threshold
Purity Factor
Spectral Options – Wavelength Range
Spectral Options – Spectra per Peak: Threshold
Does the ChemStation indicate purity or impurity?
In your opinion, does the spectral overlay indicate
impurity?
In your opinion, does the relationship between the
similarity and threshold curves indicate impurity?
Do the number of diamonds in the red band
indicate impurity?
Does the relationship between the purity factor and
calculated threshold indicate impurity?
Do you think this peak is impure?
6) Close the purity display with the tool found at the lower right.
37
Lab Exercise: Qualitative Data Analysis
Automated Peak Purity
Automated Peak Purity
The Short + Spectrum, Detail + Spectrum, and Full report styles will produce
automated peak purity analyses of integrated chromatographic peaks.
1) Make certain that you are in the Data Analysis view and demo\demodad.d is
loaded. Load the method, purity.m.
2) To print an automated peak purity report, open the Report menu and select
Specify Report.
3) Change the Report style to Short + Spectrum. You may send this report to
the Screen only to save time. OK the dialog box.
4) Under the Report menu, select Print Report. Examine the resulting report
and Close if you sent it to the screen.
Note: Purity Options may be saved as part of the method in order to produce the
desired peak purity report.
38
Lab Exercise: Qualitative Data Analysis
Displaying Spectra
Displaying Spectra
1) With demo\demodad.d still loaded, use the signals drop box to select only one
signal for the window. If necessary, enlarge the chromatogram window by
grabbing the bottom of the window and dragging to view the peaks more
easily.
2) Under the Spectra menu, select Spectra Options or select the Spectral Task
tool, then the Edit Spectra Display and Processing Options tool.
3) Select the type of background subtraction best suited for this analysis in the
Reference tab. Try Automatic or select your own spectra. OK this dialog
box.
4) Go to the Spectra menu and choose Select Spectrum or click on the Select
Spectrum tool.
5) Position the mouse pointer on a peak of interest at the upslope of the
chromatographic peak and click the left mouse button. The upslope is half
way up the left side. Notice that the Reference Spectra and the Original
Spectrum are displayed in one window (on the right). The subtracted
spectrum is displayed in the other window (on the left).
6) Now overlay the background subtracted downslope spectrum. The downslope
spectrum is on the right-hand side of the peak. To do this hold the CTRL key
down while clicking the left mouse button.
What information can you obtain by comparing the upslope and downslope
spectra?
7) From the Spectra menu, choose any of the other select spectra items and
practice with them. Practice with the tools as well.
39
Lab Exercise: Qualitative Data Analysis
Isoabsorbance Plot
Isoabsorbance Plot
In this section, you will try to optimize the data acquisition sample signal with the
emphasis on obtaining the best signal to noise ratio for one chromatographic peak.
Note that to use the Isoabsorbance plot, data must have been acquired with the
spectra stored in the All format
1) Reload Signal B of DEMODAD.D. Use the Select Spectrum at Peak Apex
tool to display the spectrum of the peak at 4.85 minutes.
2) Before you make the Isoabsorbance plot, examine the spectrum of the peak of
interest. Convert the cursor to Trace Mode by pointing the cursor at the
spectrum and clicking the right mouse button. The cursor is converted to a
downward pointing arrow that follows the spectrum trace as you move the
mouse. If you find the cursor hard to control, you can also use the right and
left arrow keys on the keyboard to move the cursor. Notice that the cursor
position is identified on the information line at the bottom of the window.
This makes it easy to see where the absorbance maximum occurs.
3) Move the cursor along the spectrum and determine the wavelength to use for
the sample signal. Make a note of it below. Decide what the sample signal
bandwidth should be. Also, select an appropriate reference and reference
bandwidth. Remember the selection guidelines discussed in the lecture.
Sample Signal__________
Sample Signal Bandwidth__________
Reference Signal__________
Reference Bandwidth__________
4) Now we are ready to make the Isoabsorbance plot. Under the Spectra menu,
select Iso/3D Plot Options…. Set a time range of 4 to 14 minutes and the
Traditional color scheme. Select Make Isoplot.
5) Once the isoplot has been drawn, take a minute to familiarize yourself with
the display. Check that the Cursor mode is Quick View. Point the cursor at
the vertical cursor and move it right and left to select various spectra. The
spectra at the position of the cursor are shown in the window at the top right.
The blue spectrum that appears in this window is the spectrum you previously
40
Lab Exercise: Qualitative Data Analysis
Isoabsorbance Plot
selected in Data Analysis, that of the peak at 4.85 minutes. Convince yourself
that this is the case by moving the vertical cursor to a time of 4.85 minutes.
6) Now point the cursor at the horizontal line across your Isoabsorbance plot. As
you move this cursor up and down, watch the chromatogram in the lower
section of the screen. The black chromatogram changes as you change the
sample signal wavelength.
7) Change the cursor mode to Signal. To extract a signal corresponding to the
sample and reference wavelengths you determined earlier, you can simply
type the values you want in the box at the upper left. To access the reference
signal click in the check box next to Ref.
8) You can also select the sample and reference signals graphically. Press and
hold the CNTRL key while you move the sample signal band or the reference
signal band. You can keep the same bandwidth, but just adjust the position of
the band, by releasing the CNTRL key and then moving the signal band.
Note: If you want to start over, just exit the Isoabsorbance plot and redraw it
by selecting the Spectra menu, then Isoabsorbance plot. Don’t forget to
change the cursor mode to Signal.
9) When you are satisfied with your extracted signal, select the Copy button at
the lower left corner of the Isoabsorbance plot. Minimize the isoplot window
and view the extracted signal. Overlay the extracted signal with signal B.
You will have to select All Loaded Signals with the drop box.
Did you improve the signal to noise ratio of the peak?
10) You may continue to try other sample signals, bandwidths and references.
When you have finished, record the optimized values here.
Sample Signal__________
Sample Signal Bandwidth__________
Reference Signal__________
Reference Bandwidth__________
11) Close the IsoPlot.
If you have time, optimize the Diode Array signals for the 3rd and last major
peak in the acquisition data file. Record the results here and use in the next
laboratories.
Sample Signal__________
Sample Signal Bandwidth__________
Reference Signal__________
Reference Bandwidth__________
41
Lab Exercise: Qualitative Data Analysis
3D Plot
3D Plot
1) From the Spectra menu, select 3D Plot. Maximize the window.
2) Rotate the 3D plot. Notice that you can use the cursor on the wavelength axis
to change the wavelength and view the resulting chromatogram. Use the
cursor on the time axis to show a spectrum at different times.
3) Use the Data View button at the lower left to change the time range or
wavelength range.
4) Explore the other options.
5) Close the 3D plot.
42
Lab Exercise: Spectral Libraries
Lab Exercise: Spectral Libraries
In this Laboratory You Will:
In this Laboratory You Will:
•
Build a spectral library.
•
Perform a library search.
You will need Class.M and Class.D created in the acquisition laboratory. If you
don’t have either of these files, use Isocra.m and one of the demo sequence files
that was created with this method.
44
Lab Exercise: Spectral Libraries
Building a Library
Building a Library
1) From the View menu, select Data Analysis.
2) Load Class.D created in the acquisition exercise. (File, Load Signal). Load
the acquisition method as well, Class.M.
3) Your first task will be to create a library file. Under the Spectra menu, select
Library, then New Library. A new panel appears. Give the library a unique
name or call it Class.UVL. OK this window.
4) The Edit Library Header box appears. In the Info box, fill in the information
about the analysis conditions including column, flow rate, composition, and
instrumentation.
5) Click on Library name and type in Class Library.
6) Under Created by, type in your name. OK the Library Header.
7) Now you will put spectra into the library. First, set up automated background
subtraction to improve spectral quality. Under the Spectra menu, choose
Spectra Options or select the Edit Spectra Display and Processing Options
tool.
8) Find the Reference tab. Select Automatic. OK the window.
9) Pull down the Spectra menu and choose Select Peak Apex Spectrum or
click on the Select spectrum at peak apex position tool.
Obtain the spectrum of the last major peak by clicking on it. Holding down
the Ctrl key, select the apex spectrum for each of the three other major
chromatographic peaks in the chromatogram. Make certain that the order is
from the last peak to the first peak.
10) Go to the Spectra menu and select Library then Add Entries or use the Add
Spectra Entries to Current Library tool.
45
Lab Exercise: Spectral Libraries
Building a Library
11) Click on Name and type in Dimethylphthalate. (Do not press Enter, as
doing so will complete the entry.)
12) Other fields are present that you can use to customize your entries. Find the
Sample entry and fill in Test Mix.
13) Scroll down until you find Slit Width. This parameter may be found on the
Set up DAD Signals screen under the Instrument menu in the Method and
Run Control view. Fill in the applicable width: 1, 2, 4, 8, or 16 nm.
14) Explore the Edit Wavetable. Select Autom. Note that it labels lambda max.
This data is useful for spectral comparisons.
15) OK the Edit Wavetable dialog box and then Add the spectrum to the library.
The next spectrum you chose now appears.
16) Fill in the name of the compound, Diethylphthalate. Fill in the remaining
information requested. Select Add to add the spectrum to the library.
17) Now add the information for spectra for peaks 3 and 4 to the library. Peak 3 is
Biphenyl and peak 4 is o-terphenyl. You now have a library with 4 spectra.
OK the Add Entries dialog box.
18) View your library entries by pulling down the Spectra, Library, Manage
Entries… menu selections or click on the Change Spectra Entries of the
Current Library tool.
19) Select Biphenyl, then Show Spectra. The spectrum is displayed. OK this
dialog box. Note that you can delete or edit entries. You may also copy the
spectrum to the spectral window of Data Analysis (Copy Spectra). When
you have finished exploring, OK the Spectra Library Manager panel.
20) Save the library with the Save Current Library File tool or through the
menus: Spectra, Library, Save Library.
46
Lab Exercise: Spectral Libraries
Library Searching
Library Searching
In this section you will test your library with the same data file. Later we will use
the library with new files in an automated fashion. Check that the library file you
want to search from is currently loaded (look in the Library tool bar).
Normally, if you were going to perform a library search, you would have to open
the library first. Open Library is found under the Spectra, Library menus. You
could also use the Load a Library File tool.
1) From the Spectra menu, select Library, Edit Search Template. You will
perform the search within a retention time window. Select Left Window (%)
and type in 5. Give the Right Window (%) a value of 5 as well. You have
now defined a time range in the library within which spectra are searched, +
or - 5% of the retention time of the unknown.
2) Give the threshold a value of 2 mAU. Low absorbing portions of the spectra
will be removed and not used in the match calculation improving the
reliability of the match.
3) You do not need to put in a shift. This might be useful if your data and the
library data were acquired on different instruments. OK the template.
4) Under the Spectra menu, choose Select Peak Apex Spectrum or use the
Peak apex spectrum tool. Select the apex spectrum from the first
chromatographic peak. Under the Spectra menu, select Library, Search
Spectrum or use the Search the Spectrum in Current Library tool.
5) The Library Search Results are displayed in the lower right hand side of the
screen. Note that the best match is Dimethylphthalate. If other matches are
listed, they were found within the specified retention time window. Did you
get a perfect match of 1000?
6) Examine the Spectral Difference window. Because you have a perfect
match, the difference should be zero.
7) Notice also that you have the unknown (target) and the library spectrum
displayed in the window on the left.
8) Perform library searches on the other peaks in the chromatogram. Do not
delete this library file. You will use it later for automated library searching
later.
47
Lab Exercise: Spectral Libraries
Library Searching
48
Lab Exercise: Overview and Diagnostics
Lab Exercise: Overview and Diagnostics
In this Laboratory You Will:
In this Laboratory You Will:
•
Learn to correctly identify the hardware elements and flow path of the
1100/1200 Series Modules.
•
Learn how to track errors and use the logbook
•
Learn how the ChemStation can help you diagnose problems
•
Learn how to set EMF Limits
•
Become familiar with the Maintenance and Repair CD-ROM and some
maintenance procedures.
Overview of Equipment and Tools Used
•
Agilent 1100/1200 Series HPLC, HPLC-grade water in channel A and
HPLC grade methanol, acetonitrile, or isopropanol in channel B.
•
ChemStation equipped with HPLC ChemStation Software and speakers.
Consumables
•
50
Agilent 1100 Maintenance and Repair CD-ROM (01100-60007)
Lab Exercise: Overview and Diagnostics
The Agilent 1200 Flow Path
The Agilent 1200 Flow Path
Please locate the items listed below with your instructor and fill in the table
provided.
Component
Function
Solvent Reservoir
Solvent Inlet Filter
Vacuum Degasser
Multi-channel Gradient Valve
(Quaternary only)
Active Inlet Valve
Pump Head
Outlet Ball Valve
Damper
Purge Valve
Mixer (Binary)
Injection Valve in Autosampler
Metering Device
Sample Loop
Needle Seat
Needle Seat Capillary
Column Compartment
Column ID Tag and Sensor
Detector Lamps
Detector Flow Cell
51
Lab Exercise: Overview and Diagnostics
Error Messages and the Logbook
Error Messages and the Logbook
When an error (blockage, component failure, etc.) occurs on one of the Agilent
1200 Series modules, the tool in the Method and Run Control view that represents
that component will turn red and the Run Status window will display a Not Ready
message. Information about the error may be found in the bubble above the
module tool. To obtain more information, open the Logbook. In this exercise,
you will simulate a blockage by lowering the maximum pressure limit to
demonstrate these features.
1)
Enter the Method and Run Control view of the Online session. Select the
Instrument menu, then Setup Pump.... Lower the Max Pressure limit to
10 bar. Set the flow rate to 1.00 mL/min. OK the panel.
2)
Again, under the Instrument menu, select System On. In a few moments,
the instrument will shut down and the error will be displayed. Note the red
color and the information presented in the bubble.
3)
From the View menu, select Logbook, Current Logbook. Alternatively,
you can display the logbook using the Show/Hide Logbook tool on the tool
bar. The top entry in the logbook was the last entry, in this case, the error.
The information displayed is:
4)
To find out more about the error, double click on the logbook entry. The online help will appear. Scroll through the error listings until you have found
the above error. The on-line help can be a good starting point to diagnose an
error. Close the Help and the Logbook.
5)
Return the Max Pressure setting to 400 bar so that you may continue.
52
Lab Exercise: Overview and Diagnostics
Diagnosis View
Diagnosis View
The 1200 Series ChemStation is equipped with a view to help you: 1) diagnose
systemic problems such as poor retention time precision, 2) perform diagnostic
tests, such as the lamp intensity test, 3) record information pertaining to
instrument maintenance; and 4) provide you with a means to determine when
instrument maintenance should be performed, Early Maintenance Feedback
(EMF). This view is called Diagnosis. In this section, you will explore the
software to become acquainted with its layout and function. In later sections, you
will perform diagnostic tests and maintenance from this section of the software.
Early Maintenance Feedback (EMF)
The Early Maintenance Feedback system allows the user to schedule preventive
maintenance based upon instrument usage. For instance, seals may be replaced
based upon seal wear and the rotor seal may be replaced based upon the number
of valve switches. The analyst inputs usage limits based upon previous
maintenance experience. For instance, if the rotor seal previously failed after
20,000 injections, a limit of 18,000 valve switches might be inserted as the EMF
valve switch limit. When 18,000 injections have been made, the EMF indicator
found in the Method and Run Control or Diagnosis view would change from a
green check mark to a yellow question mark indicating that one of the EMF limits
had been reached. In this part of the laboratory, you will practice inserting EMF
limits and observe the EMF indicator.
1)
From the View menu, change to the Diagnosis view. Click on the EMF tool
located near the center of the window. The Memo Pad on the right hand side
of the window will display the EMF information.
2)
Select the pump in the drop-down box. To set a limit, highlight the limit
entry box. To test the EMF system, set the seal wear limit 2 units above the
actual seal wear. Note: The seal wear counter increases as a function of both
flow rate and pressure. If your system does not have a column installed, the
pressure will be quite low. In this case you may want to set the flow rate to
2-3 mL/min so that the seal wear counter will change sooner.
3)
Return to the Method and Run Control view. Notice that the check mark
on the EMF indicator is currently green. Under the Instrument menu, select
System On or turn the pump on at the System Diagram. In a few moments,
the EMF indicator will change from green to yellow, indicating that one of
the set limits has been exceeded (in this case the seal wear limit).
53
Lab Exercise: Overview and Diagnostics
Diagnosis View
Note: Exceeding an EMF limit will not shut down your system or abort any
method or sequence. It simply changes the appearance of the EMF indicator.
When the EMF indicator is yellow, the user should go to the Diagnosis view,
click on the EMF tool and examine the EMF Memo Pad to find out what
preventive maintenance is due by comparing the Limit and the Actual values.
4)
Return to the Diagnosis view.
Diagnosis Instrument Panel
In the left center of your Diagnosis view, a schematic of each instrument module
is shown. Information such as firmware revisions, the number of cycles a valve
has made, and the number of hours the lamp has burned can be accessed from
these graphical tools. A logbook, module tests, and module temperature history
are also available. In this section, you will explore these options.
1)
Click on the pump diagram, and then select Update Variables Display.
Note that pump module information such as the Serial Number and
Firmware Revision is displayed below the diagram.
2)
Click on the pump diagram again. This time, select Show Module Details.
A detailed schematic of the pump appears.
3)
In a box to the right, you will see a symbol for the Logbook, an open book
(representing the method) and a thermometer. Click on the Logbook
symbol, then Maintenance Logbook Entry. When you perform routine
maintenance, enter the maintenance activity into the Maintenance Logbook.
If an instrument counter is associated with a maintenance activity, you will
be prompted to reset the counter. Cancel the Maintenance Logbook.
4)
Again, click on the Logbook symbol, then Update Variables Display.
Below you will find access to the Error Logbook, Runs Logbook,
54
Lab Exercise: Overview and Diagnostics
Diagnosis View
Maintenance Logbook, and Date Changes Logbook. Examine each one of
these entries.
5)
Click on the open book tool and examine the menu choices available. Take
a look at the Solvent Fillings... option. This panel will help you keep track
of the amount of solvent in a reservoir. OK the panel.
6)
Now, click on the Thermometer, then Update Variables Display. Now
select, Pump Temp History. This plot shows the temperature history of the
ambient sensor on the main board over the past 12 hours of operation. The
sensor is able to respond to ambient temperature changes. The temperature
history data is useful when diagnosing problems related to large external
temperature fluctuations.
7)
Now drag the mouse over the detailed pump diagram. Notice that the arrow
changes to a hand when you cross over some parts of the schematic. Click
on one of the items associated with a hand, then select Update Variables
Display.
8)
Look at the detailed information provided. Select other items on the
diagram and view their detailed information.
9)
Now, click on the large blue arrow in the detailed instrument diagram box.
This arrow will take you back to the Toplevel Instrument Panel.
Diagnosing Systematic Problems
The Diagnosis view contains software to help you find the root cause of either a
systematic problem such as poor retention time precision or of an OQ/PV test
failure. You will explore this capability now.
1)
Find the Observed Symptoms part of the tool bar at the top center of your
window. There are two choices available, OQ/PV and Analytic. Click on
OQ/PV.
2)
NOTE: OQ/PV tests are found in the OQ/PV view. These tests are
designed to verify instrument performance.
3)
Click on the drop box arrow to access a list of the OQ/PV tests that may
have failed.
4)
Select Flow Test Failed.
5)
Go to the second drop box where you see Air in the System and click on the
drop box arrow. You will find a list of possible causes for the Flow Test
failure.
6)
Select Defective Seal or Piston. Notice that the Memo Pad displays the seal
wear as well as a Leak Test. The information presented is relevant to
diagnosis of the problem.
55
Lab Exercise: Overview and Diagnostics
Diagnosis View
7)
Click on the Start button located next to the Leak Test option. The Leak
Test is displayed. Click on the Explain button to find out more about the
test. At this point in your troubleshooting process, you could Start the Leak
Test, analyze the results, and then perform the required maintenance.
8)
Close the Leak Test and Leak Test Information. You will get a chance to
run an instrument test later.
9)
Now, select Analytic from the Observed Symptoms. Pull down the drop
box to find a list of common chromatographic problems. Select one of the
problems.
10) Go to the possible causes drop box and pull down a list of causes. Select
one of the causes and notice that instrument information is again provided
along with helpful tests to confirm the probable cause.
Instrument Tests
The Diagnosis view contains a complete selection of diagnostic tests such as the
Leak Test you encountered previously. The tests are a convenient resource to
track instrument performance. For instance, users often want to test the lamp
intensity. A test is available for that purpose.
1) Make certain that you are in the Diagnosis view. Select the Diagnosis menu,
then Tests….
2) The Test Selection panel is displayed. Select the detector in the drop-down
box. A list of tests available for the detector is shown.
3) Click on DAD Intensity Test. A description of the test is shown at the right
and the Start button is found below for beginning the test.
4) Explore the other available tests, but do not run any just now. In other lab
exercises you will perform some of the most important tests.
5) Close the Test Selection Panel.
Maintenance and Repair CD-ROM
When a diagnostic test procedure, Early Maintenance Feedback, or your own
experience indicates that maintenance should be performed, two resources can
help. First, each of the 1200 instrument modules has a detailed, systematic
reference manual. In addition, the Maintenance and Repair CD-ROM (part
number 01100-60007) can lead you through each maintenance procedure in a
multi-media format. The CD-ROM is easily accessed through the Diagnosis view
or can be viewed independently of the ChemStation software simply by accessing
your CD-ROM drive. In this part of the laboratory, you will learn how to use the
56
Lab Exercise: Overview and Diagnostics
Diagnosis View
maintenance CD-ROM and then learn to perform user maintenance procedures on
the 1200 modules.
1)
Insert the HPLC Maintenance and Repair CD into the CD-ROM drive. (The
CD may already be there.)
2)
When the contents of the CD-ROM are displayed, double-click the file
start.htm. Alternatively, you can get started by clicking the Windows Start
button, selecting Run…, then Browse to find the CD-ROM drive. View
Files of type: All Files, select start.htm, then Open, and OK. The program
has a routine to check for necessary software components on your PC, which
should already be present. Click the Start Application button when it
appears.
3)
The CD-ROM will now load. The Table of Contents appears in the lefthand part of the window. Find the chapter on the Quaternary Pump and
click to expand its contents. Next find Repairing the Pump, and expand its
contents to see the Simple Repair Procedures. Simple repairs are those
that can be done from the front of the module without removing the module
from the instrument stack.
4)
Under the Simple Repair Procedures, you will see a list of procedures.
The ones that have a video recorder icon next to them have a video clip as
part of the repair procedure. For practice, select the procedure for
Exchanging the Pump Seals and Seal Wear-in Procedure.
5)
The instructions for this procedure will appear on the right side of the
program window. The information provided includes: When required,
Tools required, Parts required, Preparation and Completion Steps, and
the step by step directions. You can scroll down and read all the directions
or you can run the video clip by clicking the start button at the lower left side
of the video area. Once started, the start button becomes a pause button.
Buttons to stop the video clip and to adjust the volume are also present.
6)
Note: You must have speakers or the headphones installed in order to use
the audio portion of the presentation.
7)
Now try to find the procedure for changing the needle assembly in the
autosampler and play the video for this maintenance procedure.
NOTE: Some of the maintenance procedures will require you to use the
ChemStation software to move a part of the instrument. For instance, to change
the needle, the needle position must be moved up and down. In order to perform
this function, you must go to the Diagnosis view, select the Maintenance menu,
then ALS, and Maintenance Positions. When you are ready to begin the
maintenance procedure, you would select Start in the Change Needle box.
Changing the piston, changing the gripper and parking the arm for transport are
also performed from this window.
57
Lab Exercise: Overview and Diagnostics
Diagnosis View
Other important information found on the Maintenance and Repair CD includes
Part and Materials, where you can find diagrams of instrument assemblies and
part numbers, and information such as Optimizing Performance for each of the
modules. Browse the contents of the CD as time allows and familiarize yourself
with its contents.
58
Lab Exercise: Overview and Diagnostics
Routine Maintenance
Routine Maintenance
Using the CD-ROM, perform the instrument maintenance listed below. If you
have any questions during this part of the laboratory, please contact your
instructor. Please reuse old parts for the purposes of this laboratory unless
otherwise instructed.
Pump Maintenance
•
Replacing the Outlet Ball Valve
•
Replacing the Purge Valve or Frit
•
Removing and Disassembling the Pump Head
•
Replacing the Pump Seals
•
Reassembling the Pump Head
Autosampler
•
Replacing the Needle Assembly
•
Replacing the Needle Seat Assembly
•
Replacing the Rotor Seal
•
Replacing the Metering Seal and Plunger
Diode Array
•
Exchanging the Lamp
•
Repairing the Standard Flow Cell (View this procedure, but do not perform)
59
Lab Exercise: Overview and Diagnostics
Instrument Tests
Instrument Tests
The tests have been saved for the end of the laboratory in order to make certain
the instrument is still functioning well.
1) In the Diagnosis view, select Diagnosis, then Tests....
2) Perform the following tests using the step-by-step instructions provided on the
ChemStation:
•
Pressure Test
•
Leak Test
•
Injector Steps
•
DAD Self-Test.
60
Lab Exercise: Overview and Diagnostics
for the Agilent-SL Series
Lab Exercise: Overview and Diagnostics for the Agilent-SL Series
In this Laboratory You Will:
In this Laboratory You Will:
•
Learn to correctly identify the hardware elements and flow path of the
Agilent 1200 SL Series modules.
•
Learn how to track errors and use the logbook.
•
Learn how the LC Diagnostic Tool can help you diagnose problems.
•
Learn how to set EMF Limits.
•
Become familiar with the Maintenance and Repair CD-ROM .
•
Become familiar with some maintenance procedures and tests.
Overview of Equipment and Tools Used
•
Agilent 1200 Series HPLC- SL, HPLC-grade water in channel A and
HPLC grade methanol, acetonitrile, or isopropanol in channel B.
•
ChemStation equipped with HPLC ChemStation Software and speakers.
•
The Agilent LC Diagnositc Tool.
Consumables
•
Agilent 1100 Maintenance and Repair CD-ROM (01100-60007)
The procedures on this CD apply to some components on the SL. A new
CD will be available sometime in 2007.
62
Lab Exercise: Overview and Diagnostics for the Agilent-SL Series
The Agilent 1200 - SL Flow Path
The Agilent 1200 - SL Flow Path
Please locate the items listed below with your instructor and fill in the table
provided.
Component
Function
Solvent Reservoir
Solvent Inlet Filter
Vacuum Degasser
Active Inlet Valve
Pump Head
Absorber Capillary
Outlet Ball Valve
Damper
Purge Valve
Mixer
Injection Valve in Autosampler
Metering Device
Sample Loop
Needle Seat
Needle Seat Capillary
Column Compartment
Column ID Tag and Sensor
Detector Lamps
Detector Flow Cell
63
Lab Exercise: Overview and Diagnostics for the Agilent-SL Series
Error Messages and the Logbook
Error Messages and the Logbook
When an error (blockage, component failure, etc.) occurs on one of the Agilent
1200 Series modules, the tool in the Method and Run Control view that represents
that component will turn red and the Run Status window will display a Not Ready
message. Information about the error may be found in the bubble above the
module tool. To obtain more information, open the Logbook. In this exercise,
you will simulate a blockage by lowering the maximum pressure limit to
demonstrate these features.
1)
Enter the Method and Run Control view of the Online session. Select the
Instrument menu, then Setup Pump.... Lower the Max Pressure limit to
10 bar. Set the flow rate to 1.00 mL/min. OK the panel.
2)
Again, under the Instrument menu, select System On. In a few moments,
the instrument will shut down and the error will be displayed. Note the red
color and the information presented in the bubble.
3)
From the View menu, select Logbook, Current Logbook. The top entry in
the logbook was the last entry, in this case, the error. The information
displayed is:
__________________________________________________________
4)
To find out more about the error, double click on the logbook entry. The online help will appear. Scroll through the error listings until you have found
the above error. The on-line help can be a good starting point to diagnose an
error. Close the Help and the Logbook.
5)
Return the Max Pressure setting to 600 bar so that you may continue.
64
Lab Exercise: Overview and Diagnostics for the Agilent-SL Series
Agilent LC Diagnostic Tool
Agilent LC Diagnostic Tool
The Agilent 1200 Series HPLC- SL modules are shipped with a tool to help you:
1) troubleshoot problems such as high pressure errors, 2) perform diagnostic tests,
such as the pressure test, 3) perform calibrations, 4) provide you with a means to
determine when instrument maintenance should be performed, Early Maintenance
Feedback (EMF), and 5) provide instrument status reports. In this section, you
will explore the software to become acquainted with its layout and function. In
later sections, you will perform diagnostic tests and maintenance from this
software.
Early Maintenance Feedback (EMF)
The Early Maintenance Feedback system allows the user to schedule preventive
maintenance based upon instrument usage. For instance, seals may be replaced
based upon seal wear and the rotor seal may be replaced based upon the number
of valve switches. The analyst inputs usage limits based upon previous
maintenance experience. For instance, if the rotor seal previously failed after
20,000 injections, a limit of 18,000 valve switches might be inserted as the EMF
valve switch limit. When 18,000 injections have been made, the EMF indicator
found in the Method and Run Control view would change from a green check
mark to a yellow question mark indicating that one of the EMF limits had been
reached. In this part of the laboratory, you will practice inserting EMF limits and
observe the EMF indicator.
1)
Go to the Diagnosis view. Double click on the Agilent LC Diagnostic tool
found in the Explorer and then Start the tool.
2)
Access the Tools button.
3)
Highlight the EMF Editor, then Start.
4)
In the Instrument/Module Selection tree, select the pump.
5)
Hightlight the Seal Wear line. To test the EMF system, set the seal wear
limit 2 units above the actual seal wear then Set Limit. Note: The seal wear
counter increases as a function of both flow rate and pressure. If your
system does not have a column installed, the pressure will be quite low. In
65
Lab Exercise: Overview and Diagnostics for the Agilent-SL Series
Agilent LC Diagnostic Tool
this case you may want to set the flow rate to 2-3 mL/min so that the seal
wear counter will change more quickly.
6)
Return to the Method and Run Control view. Notice that the check mark
on the EMF indicator is currently green. Under the Instrument menu, select
System On or turn the pump on at the System Diagram. In a few moments,
the EMF indicator will change from green to yellow, indicating that one of
the set limits has been exceeded (in this case the seal wear limit).
7)
Note: Exceeding an EMF limit will not shut down your system or abort any
method or sequence. It simply changes the appearance of the EMF
indicator.
8)
When the EMF indicator is yellow, the user should go to the Agilent LC
Diagnostic tool and examine the EMF counter to find out what preventive
maintenance is due by comparing the Value and the Limit values.
9)
Reset the Limit to its original value.
10) Back out of the software until you are on the main LC Diagnostic page.
Diagnosing Common Problems
The Guided Diagnostic tool contains software to help you find the root cause of a
problem such as Unstable pressure or excessive pressure ripple.
1)
Click on the Guided Diagnositc tool.
2)
Select a symptom from the drop box.
3)
Follow the troubleshooting guide to see how you can use this tool.
4)
Back out to the main menu of the LC Diagnostic tool.
Instrument Tests
The LC Diagnsotic tool contains a complete selection of diagnostic tests such as
the pressure test. The tests are a convenient resource to track instrument
performance. For instance, users often want to test the lamp intensity. A test is
available for that purpose.
1)
66
Select the Tests tool.
Lab Exercise: Overview and Diagnostics for the Agilent-SL Series
Agilent LC Diagnostic Tool
2)
The Test Selection panel is displayed. Select the detector in the drop-down
box. A list of tests available for the detector is shown.
3)
Click on Intensity Test. A description of the test is shown by clicking on
the More… button. The Start button is found below for beginning the test.
4)
Explore the other available tests, but do not run any just now. You will run
some of these tests later.
5)
Select Back to leave the Test module.
Maintenance and Repair CD-ROM
When a diagnostic test procedure, Early Maintenance Feedback, or your own
experience indicates that maintenance should be performed, two resources can
help. First, each of the 1200 instrument modules has a detailed, systematic
reference manual. In addition, the Maintenance and Repair CD-ROM (part
number 01100-60007) can lead you through each maintenance procedure in a
multi-media format. The CD-ROM is easily accessed through the Diagnosis view
or can be viewed independently of the ChemStation software simply by accessing
your CD-ROM drive. In this part of the laboratory, you will learn how to use the
maintenance CD-ROM and then learn to perform user maintenance procedures on
the 1200 modules. A new CD-Rom for the 1200- SL modules will be available in
2007.
1)
Insert the HPLC Maintenance and Repair CD into the CD-ROM drive. (The
CD may already be there.)
2)
When the contents of the CD-ROM are displayed, double-click the file
start.htm. Alternatively, you can get started by clicking the Windows Start
button, selecting Run…, then Browse to find the CD-ROM drive. View
Files of type: All Files, select start.htm, then Open, and OK. The program
has a routine to check for necessary software components on your PC, which
should already be present. Click the Start Application button when it
appears.
3)
The CD-ROM will now load. The Table of Contents appears in the lefthand part of the window. Find the chapter on the Quaternary Pump and
click to expand its contents. Next find Repairing the Pump, and expand its
contents to see the Simple Repair Procedures. Simple repairs are those
that can be done from the front of the module without removing the module
from the instrument stack.
4)
Under the Simple Repair Procedures, you will see a list of procedures.
The ones that have a video recorder icon next to them have a video clip as
67
Lab Exercise: Overview and Diagnostics for the Agilent-SL Series
Agilent LC Diagnostic Tool
part of the repair procedure. For practice, select the procedure for
Exchanging the Pump Seals and Seal Wear-in Procedure.
5)
The instructions for this procedure will appear on the right side of the
program window. The information provided includes: When required,
Tools required, Parts required, Preparation and Completion Steps, and
the step by step directions. You can scroll down and read all the directions
or you can run the video clip by clicking the start button at the lower left side
of the video area. Once started, the start button becomes a pause button.
Buttons to stop the video clip and to adjust the volume are also present.
6)
Note: You must have speakers or the headphones installed in order to use
the audio portion of the presentation.
7)
Now try to find the procedure for changing the needle assembly in the
autosampler and play the video for this maintenance procedure.
8)
NOTE: Some of the maintenance procedures will require you to use the
ChemStation software to move a part of the instrument. For instance, to
change the needle, the needle position must be moved up and down. In
order to perform this function, you must go to the Diagnosis view, select the
Maintenance menu, then ALS, and Maintenance Positions. When you are
ready to begin the maintenance procedure, you would select Start in the
Change Needle box. Changing the piston, changing the gripper and parking
the arm for transport are also performed from this window.
9)
Other important information found on the Maintenance and Repair CD
includes Part and Materials, where you can find diagrams of instrument
assemblies and part numbers, and information such as Optimizing
Performance for each of the modules. Browse the contents of the CD as
time allows and familiarize yourself with its contents.
68
Lab Exercise: Overview and Diagnostics for the Agilent-SL Series
Instrument Status Report
Instrument Status Report
You can print a status report for your own use or for a Service Engineer.
1) Select the Instrument Status Report tool.
2) Fill in the required information and Generate Report.
3) Examine the report details.
4) Press the Back button to return to the main menu.
69
Lab Exercise: Overview and Diagnostics for the Agilent-SL Series
Routine Maintenance
Routine Maintenance
Using the CD-ROM, perform the instrument maintenance listed below. If you
have any questions during this part of the laboratory, please contact your
instructor. Please reuse old parts for the purposes of this laboratory unless
otherwise instructed.
Pump Maintenance
•
Replacing the Outlet Ball Valve
•
Replacing the Purge Valve or Frit
•
Removing and Disassembling the Pump Head
•
Replacing the Pump Seals
•
Reassembling the Pump Head
Autosampler
•
Replacing the Needle Assembly
•
Replacing the Needle Seat Assembly
•
Replacing the Rotor Seal
•
Replacing the Metering Seal and Plunger
Diode Array
•
Exchanging the Lamp
•
Repairing the Standard Flow Cell (View this procedure, but do not perform)
70
Lab Exercise: Overview and Diagnostics for the Agilent-SL Series
Instrument Tests
Instrument Tests
The tests have been saved for the end of the laboratory in order to make certain
the instrument is still functioning well.
1) Open the LC Diagnostic tool go to the Tests section and Tools section
(injector steps).
2) Perform the following tests using the step-by-step instructions provided on the
ChemStation:
•
Pressure Test
•
Pump Test
•
Injector Steps
•
Intensity Test.
71
Lab Exercise: Overview and Diagnostics for the Agilent-SL Series
Instrument Tests
72
Lab Exercise: Integration
Lab Exercise: Integration
In this Laboratory You Will:
In this Laboratory You Will:
•
Perform autointegration.
•
Integrate using the Events Table.
•
Add Timed Events.
•
Use Manual Events.
74
Lab Exercise: Integration
Integration Preparation
Integration Preparation
ChemStation revisions B.01 and higher have a new, more efficient and accurate
integrator than previous revisions. During this lab exercise, practice integration
using the data file, Integration.d.
1) Enter the Data Analysis view. Using the Load Method Tool, load the
method, DEF_LC.M.
2) From the File menu, select Load Signal…. Under the Data folder, find the
file Integration.d. Load the data file.
You are now ready to begin integration.
75
Lab Exercise: Integration
Auto Integration
Auto Integration
One of the easiest ways to obtain initial integration events is through Auto
Integration. Auto Integrate determines initial values for the slope sensitivity,
peak width and area reject. First, however, you will display the default events.
1) Click on the Integration tool, then the Edit/Set Integration Events Table tool
or from the Integration menu, select Integration Events.
Record the Default initial events here:
Initial Slope Sensitivity__________
Initial Peak Width
__________
Initial Area Reject
__________
Initial Height Reject
__________
Initial Shoulders
__________
2) The chromatogram in front of you was integrated using these values.
3) Examine the chromatogram for improper integration. Check for placement of
tic marks and position of the baseline.
Question: Were the default values appropriate?
_________________________________________________________________
4) You may print a copy of the chromatogram by choosing the Display Graphics
Task tool then the Printer tool, and Print. (File menu, Print, Selected
Window, Print).
76
Lab Exercise: Integration
Auto Integration
5) Using the Events Table drop box, select signal DAD1 B Specific.
6) To Auto Integrate, select the Integration tool then the Auto Integrate tool
or select the Integration menu, then Auto Integrate. When asked if you
want to save the changed events table, reply No.
7) View the events and chromatogram that were created for Signal B.
8) Now see what happens when you add the recommended For All Signals
settings:
Tangent Skim Mode: New Exponential
Tail Peak Skim Height Ratio: 3.00
Front Peak Skim Height Ratio: 6.00
Skim Valley Ratio: 20.00
Baseline Correction: Advanced
Peak to Valley Ratio 500.00
Note: Auto Integrate does not set the For All Signals Integration Events. You
must set these manually.
10) Use Auto Integrate again. Again, do not save the changed events table.
Print the chromatogram to notice any differences. You may want to print
this chromatogram for comparison and record the event settings below.
Did you see any improvement?
Initial Slope Sensitivity__________
Initial Peak Width
__________
Initial Area Reject
__________
Initial Height Reject
__________
Initial Shoulders
__________
77
Lab Exercise: Integration
Integrate Using the Events Table
Integrate Using the Events Table
Now, you will set up integration events for the Integration.d chromatogram using
the approach discussed in lecture.
1)
Click on the Integration tool, then the Edit Integration Events tool.
2)
Using the signal drop box, select DAD1B Specific.
3)
Set the Slope Sensitivity to 50.
4)
Estimate the peak width from the initial integration. Use the smallest peak
width from a real chromatographic peak, not noise.
5)
Set the initial height and area reject to zero. Set the Shoulders to OFF.
6)
Set the Tail Peak Skim Height Ratio to 3, the Front Peak Skim Height Ratio
to 6, and the Skim Valley Ratio to 20.
7)
Set the Baseline Correction mode to Advanced with the Tangent Skim Mode
set as New Exponential.
8)
Integrate using the integration tool and view the results.
9)
Print the chromatogram and notice any differences in integration.
78
Lab Exercise: Integration
Adding Timed Events
Adding Timed Events
At times, the initial events are not able to properly construct baselines or place tic
marks for integration. Timed events may be added to improve integration. In this
section, you will learn how to add timed integration events to your method.
1) Open the Select an event drop box.
2) To practice with timed events, select the timed event, Integration.
3) Move the line that appears in the chromatogram to time=0 and click. Note
that you have turned the integrator off at the beginning of the run.
4) Move the line to time=3 minutes and click. Note that you have turned the
integrator on at 3 minutes (the events appear in the Events Table).
5) Reintegrate the chromatogram. This may be accomplished by selecting the
Integrate current Chromatogram(s) tool
or by selecting the Integration menu, then Integrate.
79
Lab Exercise: Integration
Adding Timed Events
6) Practice adding other timed events.
Note, you can also add and remove timed events with the tools shown below.
Practice this now.
7) When finished, delete all timed events by selecting the event then clicking the
remove event tool shown above.
8) Exit the Integration Events using the Exit and save events to method tool.
NOTE: Once the appropriate events are found, they must be saved as part of the
method. Save to the method with the Save current Method tool, or from the File
menu, select Save, Method. You do not need to do this here.
80
Lab Exercise: Integration
Using Manual Events
Using Manual Events
Manual integration allows you to integrate specific peaks or groups of peaks with
complete control. For example, you may set the integration start and stop points
of any chromatographic peak yourself. This type of integration is convenient, but
may not be applicable from run to run. It also may not be as reproducible from
operator to operator.
1) Select the Manual Integration: Remove integrated Peak(s) from the
Integration Results tool. Note: Integration Events must be closed to manually
integrate.
2) Draw a rubber band box around one of the chromatographic peaks including
the integration start and stop tic marks. Notice that the peak is no longer
integrated.
3) Magnify an area of the chromatogram.
4) Now select the Manual Integration: Draw Peak Baselines(s) and integrate tool
or select the Draw Baseline selection from the Integration menu. Draw in
the baseline using the left mouse button. The Area is displayed to the side
after the integration is accomplished.
Other manual integration events include: Negative Peaks, Tangent Skim, Split
Peak, and All Valleys. Practice with these manual events.
Copying Manual Integration Events to a Method
The software allows you to copy the integration events you defined using manual
integration to the currently loaded method using the Copy Manual Events to
Method item from the Integration menu. These manual events are then stored
separately in the method. They cannot be reviewed in the Integration Events
dialog box. When a signal is loaded they are only applied when the Apply
Manual Integration option is checked in the Integration Events or when you
select Apply Manual Events from method in the Integration menu. To see
how this task works, do the following.
81
Lab Exercise: Integration
Using Manual Events
1)
Select Copy Manual Events to Method in the Integration menu.
2)
Select the Edit/Set Integration Events Table tool to display the integration
events.
3)
Integrate the chromatogram using the Integrate Current Chromatograms tool.
4)
Notice that the manual integration events have not been applied.
5)
Click in the Manual Events box and re-integrate the chromatogram (this box
is located integration events section of the window on the left side).
6)
Notice that the manual events are now applied to the integration.
7)
Deselect the checkbox and re-integrate. Note that the events are no longer
applied.
82
Lab Exercise: Quantification
Lab Exercise: Quantification
In this Laboratory You Will:
In this Laboratory You Will:
•
Run three external standards to create a calibration table.
•
Select appropriate integration parameters and save them to your method.
•
Build a three level calibration table.
•
Test the calibration table
•
Print a basic report.
84
Lab Exercise: Quantification
Preparations
Preparations
Obtain the low, medium and high standards. Insert them into vial positions 1, 2
and 3 of the autosampler. Turn on the detector lamp. Prime your solvent delivery
system.
Priming the Agilent 1100 or 1200 Solvent Delivery System
1) If you are equipped with a vacuum degasser, make certain the degasser is on.
2) Ensure that the outlet tube is connected from the purge valve to the waste
container. Open the purge valve located on the pump module by turning the
knob counter clockwise several turns.
3) To prime the solvent delivery system, you should pump 100% at 5 mL/min
from each channel for several minutes. To accomplish this task, select the
following menu items: Instrument, Set up Pump...; or click on the pump set
up GUI in the system diagram (shown below), then Set up Pump....
Click
4) Type in a flow of 5.000 mL/min and %B of 100. OK the dialog box.
85
Lab Exercise: Quantification
Preparations
5) To turn the solvent delivery system on, you may use the menu items:
Instrument, More Pump, Control; or access the tool shown in the diagram
below. Do this now.
6) Wait until a steady stream of solvent comes out of the purge valve waste tube.
7) Repeat steps 3 through 6 for the other channels of the pump. Complete the
priming process by setting the composition to %B=65 and pump at 5 mL/min.
8) Change the flow rate to 1.5 mL/min and then close the purge valve. Allow the
column to equilibrate.
86
Lab Exercise: Quantification
Creating the Acquisition Method
Creating the Acquisition Method
1) Load the default method, DEF_LC.M as a starting point for method creation
(Method, New Method) or double-click on DEF_LC.M in the ChemStation
Explorer. From the Method menu select Edit Entire Method.… Select
only the Instrument/Acquisition section for editing. OK this dialog box.
2) Fill in the following parameters for the solvent delivery system:
OK the dialog box.
3) Select a Standard Injection with a 5 µl injection volume. OK the dialog
box.
4) Fill in the DAD Signals as below. OK the dialog box.
87
Lab Exercise: Quantification
Creating the Acquisition Method
Note: If a VWD detector is being utilized, select the same wavelength as signal
A.
5) Set the column compartment temperature to 40°C (both left and right). OK
the dialog box.
6) From the Method menu, select Save Method As.... Name the method
Quant.m.
7) Type in a short comment for the Method History and OK.
8) Turn on the pump and equilibrate the HPLC column for several minutes with
30% water/70% acetonitrile.
88
Lab Exercise: Quantification
Data Acquisition-Standards
Data Acquisition-Standards
In this example, you will run each sample individually, not as a sequence. Of
course, it is possible to run the three standards in sequence mode.
1) From the RunControl menu, select Sample Info.... The same panel may be
accessed from the tool:
Fill in the information for the low level standard and OK the dialog box.
89
Lab Exercise: Quantification
Data Acquisition-Standards
2) Check that your baseline and pressure are stable.
3) Start the run either from the Start tool or from the RunControl menu, then
Run Method.
4) Now acquire data for the medium standard and the high standard as well. Call
the data files, med.d and high.d, respectively. When you have finished, turn
off the pump.
90
Lab Exercise: Quantification
Integration
Integration
In this section you will optimize the integration events for the analysis and save
them to the method.
1) Go into the Data Analysis view.
2) From the View menu, select Preferences.
3) Select the Signal Options tab. Make certain that integrate after load is the
only box checked.
4) Find your data analysis directory in the ChemStation Explorer. Double-click
on the Single Runs to place the files in the Navigation Table. Find the data
file, Low.d, and load all signals. The data file is initially integrated using the
default integration events. Make certain both signals A and B are present.
5) After loading, go to the Integration menu and select Integration Events or
select the Select Integration Task tool, then the Edit/Set Integration Events
Table tool.
6) Select DAD Default for the Events Table and insert the following integration
events:
Tangent Skim Mode: New Exponential
Tail Peak Skim Height Ratio: 3
Front Peak Skim Height Ratio: 6
Skim Valley Ratio: 20
Baseline Correction: Advanced
Peak to Valley Ratio: 500
Slope Sensitivity: 50
Peak Width: Set to the narrowest real chromatographic peak
Area Reject: 0
Height Reject: 1
Shoulders Off
7) Reintergrate the chromatogram using the new events. Either select the
Integration menu then Integrate, or the Integrate tool.
8) Examine the current integration. Change settings appropriately. Remove
undesirable peaks (there should be four main peaks) using either the area
reject or timed events.
91
Lab Exercise: Quantification
Integration
9) Check the integration for signal B as well. Make certain that both signals are
mapped to the DAD Default Events Table by selecting the tool, or if you
choose, create different events for each signal.
10) Exit and save the events to the method using the Exit and save events to
method tool. Then, resave the method, Quant.m.
92
Lab Exercise: Quantification
Setting up Signal Details
Setting up Signal Details
The Signal Details dialog box describes which signals will be evaluated during a
method run. Signals that are not in the list box will not be integrated or reported
if all defined signals can be found in the current data file. Whenever the defined
signals cannot be found the system loads all available signals from the data file
and tries to generate the report.
1) From the Calibration menu, select Signal Details…. You may also access
the Signal Details dialog box by first selecting the Calibration Task tool, then
the Edit current method signals tool.
2) Pull down the Available Signals and select DAD1A, Sig=250, 100
Ref=360,100. Now select the Add to Method button. Repeat with signal B.
OK this dialog box.
3) Resave the method.
93
Lab Exercise: Quantification
Building a Calibration Table
Building a Calibration Table
You have already loaded and integrated the lowest level standard. The first step
toward building your calibration table will be to select the calibration settings.
1) In the Calibration menu, select Calibration Settings...
2) Fill in the Title of the Calibration Table, e.g., Class.
3) You will Use Sample Data From Data File.
4) Type in µg/mL for Amount Units.
5) The Default RT Windows should be set at 5%.
6) Select No for Calculate Uncalibrated Peaks.
7) If you do not understand any of these entries, read the online help. OK this
dialog box.
8) Under the Calibration menu, select New Calibration Table… or, select the
New Calibration Table from current chromatogram tool.
9) Select Automatic Setup Level 1. You may select a default amount for all
peaks or fill in the amount after entering the calibration table. For your
current purposes, leave the Default Amount as 0.00. Do not check the box to
Calculate Signals Separately because we will be using qualifiers. OK the
dialog box.
10) The calibration table now appears. You will be calibrating the last two of four
major chromatographic peaks. Locate the third major chromatographic peak
at approximately 1.9 minutes (for a mobile phase of acetonitrile). Use the
signal that provides the most area counts.
11) Click in the Compound column, and type in Biphenyl; selecting the signal
that gives you the most area counts. In the Amt[µg/ml] column, type in 0.8.
Scroll to the right and make this a reference peak. If you do not see the
reference peak as an option, make certain that you are currently in the
Overview portion of the calibration table. This selection can be found in the
pull down box in the middle of the window.
94
Lab Exercise: Quantification
Building a Calibration Table
12) Now find the last major peak at approximately 3.3 minutes. The compound
name is o-terphenyl and the amount is 2.4 µg /mL. Select the signal that
provides the most abundant area counts. You will also make this a reference
peak.
13) OK this initial Calibration Table to remove lines with zero amounts. This
step will make building your table easier.
14) From the Calibration menu, select Calibration Table to redisplay the
Calibration Table.
15) Save the method.
16) Now, load all signals for data file, Med.d, the medium standard. The
integration will be performed automatically using the integration events
chosen for the last data file.
17) Under the Calibration menu, select Add Level or select the Add new level
from current chromatogram tool. This time, Level 2. OK the box.
18) Fill in the amount fields only. For level 2, Biphenyl is 2.4 µg/mL and the oTerphenyl is 7.2 µg/mL.
19) Now load the high-level standard, high.d. Select Add Level. This data file is
for level 3.
20) The amounts for the third level are: Biphenyl, 8.0 µg/mL, and o-Terphenyl,
24 µg/mL.
21) You may click on the chromatographic peak in the chromatogram to view the
corresponding calibration curve in the lower right hand corner. You may
change the curve fit by selecting Peak Details from the Calibration Table
Option Set drop box found in the middle of the window. Now the Calibration
Table contains a Curve Type and Origin column. Select a Linear curve type
and Ignore the origin.
95
Lab Exercise: Quantification
Setting up Low and High Amount Limits for Calibration Standards
Setting up Low and High Amount Limits for
Calibration Standards
You may specify low and high amount limits in the calibration table. If the
calculated amount is outside the limits, a warning appears in the report. The
amount limits can be used as qualifiers to confirm the peak identification.
Compounds that are outside of the amount limits, are not used to recalibrate the
Calibration Table. The minimum and maximum amounts limits do not consider a
multiplier value that you may have specified.
1) From the Calibration menu, select Calibration Table Options..., then
Compound Details or change from Overview to Compound Details in the
drop box located in the middle of the window.
2) Click in the box presented to the left of the Low Limit column while in the
biphenyl row. Now click in the Low Limit cell. A lower limit is
automatically calculated. This is a lower limit of -20%.
3) Click in the box presented to the left of the High Limit column while in the
biphenyl row. An upper limit will appear in the cell. This is an upper limit of
+20%. The default is +/- 20%, but you may set any desired limits.
4) Repeat this procedure for the o-terphenyl peak.
5) Resave your method.
96
Lab Exercise: Quantification
Setting up Qualifiers (Diode Array only)
Setting up Qualifiers (Diode Array only)
A qualifier peak is used to confirm that the main peak within the retention time
window belongs to the expected compound. It is assumed that the peaks of one
compound have a constant response ratio at two different wavelengths. The
qualifier peak response is a percentage of the main peak response.
1) Change from Compound Details to Identification Details either in the drop
box or through the menus: Calibration, Calibration Table Options...,
Identification Details.
2) Identify the Pk Usage column. Note that one signal for the biphenyl peak is
designated as the Main peak.
3) Notice the other signal. Change the peak usage from Ignore to Qualifier for
one of the peaks. The expected ratio is shown with a +/- 20% window.
4) Set up a qualifier for the o-terphenyl compound as well.
5) Resave the method. Type in a comment for method history.
You have now set up two curves with qualifiers. In the next section you will test
your calibration table and print a simple report.
97
Lab Exercise: Quantification
Testing the Calibration Table
Testing the Calibration Table
1) Load the medium standard's data file, med.d. Go to the Report menu, then
Specify Report... or select the Specify Report Calculation and Print Style
tool.
2) Make the following selections:
Destination = Printer and Screen
Calculate = ESTD
Based on = Area
Report Style = Detail
Select chromatogram output.
3) OK the report specifications and save this to your method.
4) From the Report menu, select Print Report or select the Identify Peaks,
calculate Results & print Report tool.
5) Study the resulting report and modify retention time windows or qualifiers if
necessary. Remember to resave the method if you are satisfied with
report style.
98
Lab Exercise: Running a Sequence
Lab Exercise: Running a Sequence
In this Laboratory You Will:
In this Laboratory You Will:
•
Build a sequence with automated recalibration of the existing calibration
table.
•
Learn how to pause and restart the sequence.
•
Print a Sequence Summary report.
•
Access the Sequence Logbook.
Sequence operation allows you to perform unattended analysis of more than one
sample, using more than one method. In this exercise you will utilize the method,
including the calibration table, created in the last exercise. Place the low,
medium, and high calibration standards in vial positions 1, 2, and 3, respectively.
Place the isocratic test mix in position 4.
100
Lab Exercise: Running a Sequence
Building a Sequence
Building a Sequence
1) Go to the Method and Run Control view.
2) Under the Sequence menu, select New Sequence. This menu selection loads
the default sequence template. You may also access this option by clicking on
the sequence tray setup tool located on the left hand side of the screen.
3) Under the Sequence menu, find Sequence Parameters….
4) Fill in the Operator Name. Create a subdirectory using your last name. Do
not change the Path. Select Auto for the data file naming system.
5) For Part of methods to run, select According to RuntimeChecklist.
Question:
What is the purpose of the WaitTime?
________________________________________________________________
6) Select the Post-Sequence Cmd/Macro checkbox, then from the drop box,
STANDBY. Enter a nRdy Timeout of 30 minutes. OK the dialog box.
Question:
What is the difference between the STANDBY option and Shutdown.mac?
________________________________________________________________
101
Lab Exercise: Running a Sequence
Building a Sequence
7) Now enter the Sequence Table from the Sequence menu or from the
sequence tray setup tool. You would like to do the following:
a) Replace all existing calibration table response factors with new response
factors at all three calibration standard levels.
b) There after, perform cyclic recalibrations every 2 sample injections
averaging the response factors and retention times.
c) Perform four sample injections, in this case utilize the vial containing your
isocratic test mix and make four injections from this vial.
Fill in the table. Each time you wish to add a new line to the bottom of the table,
highlight the last line by clicking on the line number, then use the Append Line
button. To add a line above the currently highlighted line, use the Insert button.
The table should look like the one below. You can also try to use the
Insert/FillDown Wizard.
102
Lab Exercise: Running a Sequence
Building a Sequence
8) OK the Sequence Table after completing the entries and save the sequence
either through the Save Current Sequence tool or through the Sequence menu.
9) Select Sequence>Partial Sequence to view the injection order that will be
followed by your Sequence Table. Cancel out.
103
Lab Exercise: Running a Sequence
Sequence Summary Reports
Sequence Summary Reports
The ChemStation can print a variety of standard reports for individual sample
analyses as directed in the Specify Report panel. These styles are stored as part
of the method and are printed at the end of each analysis if desired. Sequence
summary reporting is an additional, optional way of reporting. You may calculate
and report parameters across a number of different analyses, for instance, you
may report the standard deviation of the retention time for calibration standards.
These calculations are useful, for example, to test instrument performance or the
stability of a new method. You will add a sequence summary report to this
sequence. This type of report will print at the end of the entire sequence.
1) To begin setting up your sequence summary report, select Sequence Output
from the Sequence menu or from the sequence tray setup tool.
2) Select the Print Sequence Summary Report option along with the Report to
Printer and Print individual reports for each run as well, options.
Question:
If you had not selected Print individual reports for each run as well, would you
receive an individual report after each sample had been run?
________________________________________________________________
3) Specify the destination for individual reports as the Printer, as specified
here, not as specified with each method.
4) Click on the Setup... button. Select items 4-9. For items 7 and 8, select
Extended Statistic. For item 9, select Sample Summary. OK this dialog
box and the Sequence Output dialog box.
5) To specify the Extended Statistics, go to the Sequence menu and select
Extended Statistics…. Select any item you would like calculated by double
clicking the Active box. Note that you can place limits in the field to the right
that will be marked in the report if exceeded. OK this dialog box.
6) Resave the sequence.
104
Lab Exercise: Running a Sequence
Starting the Sequence
Starting the Sequence
1) Make certain that Quant.M is currently loaded.
2) View the RunTime Checklist (under the Method menu). Make certain that
both Data Acquisition and Data Analysis are selected. In previous exercises,
data acquisition was the only item selected.
3) Turn on the pump, column thermostat, and detector. Allow the column to
equilibrate.
4) When you have a stable baseline, start the sequence either from the Start tool
or RunControl, RunSequence.
105
Lab Exercise: Running a Sequence
Pausing a Sequence
Pausing a Sequence
1) Pausing a sequence to run a priority sample is quite easily accomplished. Pull
down the Run Control menu and select Pause Sequence.
The current sequence will pause after the current method has completed. You
can make changes in the sequence for those samples that have not yet been
injected.
2) To resume the sequence, select Resume Sequence from the Run Control
menu. The sequence continues with the next run.
106
Lab Exercise: Running a Sequence
Post Sequence
Post Sequence
1) Examine the printed reports. How did your sequence go?
2) If you have errors during your sequence, it may be helpful to print the
Sequence Logbook. To do this, go to the View menu. Select Logbook, then
Sequence Logbook. To print this logbook, select Print Logbook after you
have displayed the logbook on the screen. The logbook can be displayed
with the Sequence Logbook tool, as well.
3) Alternatively, open My Computer and find the sequence folder and the
sequence logbook. Examine the sequence folder entries created by your
sequence.
107
Lab Exercise: Running a Sequence
Post Sequence
108
Lab Exercise: Sequence Review and
Reprocessing
Lab Exercise: Sequence Review and Reprocessing
In this Laboratory You Will:
In this Laboratory You Will:
•
Load a sequence into the sequence navigation table.
•
Reprocess a sequence after changing the folder method.
•
Reprocess a sequence after updating DA methods.
•
Use Batch Review to review a sequence.
You will need the following data:
•
Batch.B
•
SEQUENCECLASS 2006-03-20 13-48-40, found in the folder
Chem32\1\Data\03-20-06
•
CLASSSEQUENCE 2006-03-15 10-46-09 found in the folder,
Chem32\1\Data\03-15-06.
Please note that the sequence container names are similar! One starts with
SEQUENCECLASS and the other startw with CLASSSEQUENCE. .Be sure to use the
correct file.
110
Lab Exercise: Sequence Review and Reprocessing
Reprocess with Sequence Folder Method
Reprocess with Sequence Folder Method
You will reprocess a sequence after updating the Folder Method. This sequence
has two problems. First, calibrated compounds fell outside the method’s retention
time windows, and therefore, no amounts were calculated. The retention time
windows are set in the Calibration Settings.
In addition, the 4th level of the calibration is out of specifications and needs to be
dropped from the analysis. See the report below.
111
Lab Exercise: Sequence Review and Reprocessing
Reprocess with Sequence Folder Method
You will update the Sequence Folder Method with new retention time windows
and drop the 4th level calibration data point, then reprocess the entire sequence.
Note that only the method (.M) and sequence file (.S) in the sequence folder will
change. The master method stored in Chem32\1\Methods and the sequence
template stored in Chem32\1\Sequence will not change.
1)
Go to Data Analysis.
2)
In the Data Explorer, find the sequence folder: 03-20-06 and expand. Find
the sequence folder: Chem32\1\Data\03-20-06\SequenceClass 2006-03-20
13-48-40. Double-click to load into the Navigation Table.
3)
Load the container method. The DA Method for the first file will load
automatically as stated in the Preferences. In this part of the exercise,
however, you are changing the folder method so the changes apply to all
data files. The path for the sequence folder method is: Chem32\1\Data\0320-06\SequenceClass 2006-03-20 13-48-40\CLASSQUANT.M. Load the
sequence folder method now.
112
Lab Exercise: Sequence Review and Reprocessing
Reprocess with Sequence Folder Method
4)
First, let’s change the retention time windows so that the amounts will be
calculated.
5)
Go to the Calibration menu and then Calibration Settings.
6)
Change the Default RT Windows as below. OK the dialog box.
113
Lab Exercise: Sequence Review and Reprocessing
Reprocess with Sequence Folder Method
7)
Now, take a look at the calibration curve. Select the Calibration menu and
then Calibration Table.
8)
Click next to the 4th level row to highlight the line and press the Delete
button.
9)
Remove all level 4 rows from the table.
10) Notice that the Correlation is now improved.
11) Save the sequence folder method.
Always check to make certain you are in the correct directory path. Fill in the
change history stating the the retention time window was updated and the 4th level
calibrator was removed.
12) During the original sequence acquisition, there was quite a large sequence
summary report as well as individual data files printed. We do not want to
print that much information here. Therefore, we will change the sequence
output. This is a sequence, .S file, parameter. The tools shown will allow
you to make changes to this file without going to the Method and Run
Control view.
114
Lab Exercise: Sequence Review and Reprocessing
Reprocess with Sequence Folder Method
The Sequence folder .S file is currently loaded. If you want to check this, go to
Method and Run Control and check the path.
13) Open the Sequence Output dialog box by clicking on the tool shown below.
14) Deselect Print individual reports for each run as well. OK the dialog box.
15) Select the Sequence Summary tool shown below.
Make certain only 5 and 9 are selected. Make section 9, Summary, is a
Compound Summary. OK the dialog box.
115
Lab Exercise: Sequence Review and Reprocessing
Reprocess with Sequence Folder Method
16) To reprocess the sequence, select the Start sequence reprocessing tool
outlined below.
The sequence will reprocess. You will notice that the status bar turns blue to
indicate the sequence is reprocessing. The method and a Compound Summary
should print at the end of the sequence.
Examine the method. You should see the record of your action in the Change
History. Find the calibration table and note that only four data points are now
used for the analysis.
The Compound Summary should now calculate amounts for all compounds.
Did you successfully reprocess the data using the sequence folder method ?
NOTE: If you reload the sequence from the Explorer, the all DA Methods will
update with the folder method changes.
116
Lab Exercise: Sequence Review and Reprocessing
Reprocess with DA Methods
Reprocess with DA Methods
In this section, you will see how to change the individual DA methods. You will
add manual integration events to two runs and reprocess.
1)
Go to the View menu, then Preferences… >Signal Options.
2)
Make certain that Integrate after load is selected under Load Signal
Options and Individual Method from Data File is selected under Method
used for Review of Sequence Data. OK the dialog box.
Note that this can also be specified directly from the user interface at the top of
the Navigation Table. If you change which method is used via this interface, does
it take effect immediately?__________________ Is this method choice used for
data review or reprocessing?_____________________________
3)
Find the Data folder 03-15-06 and expand. Double-click on the sequence,
Chem32\1\Data\03-15-06\ClassSequence 2006-03-15 10-46-09 to load .
117
Lab Exercise: Sequence Review and Reprocessing
Reprocess with DA Methods
4)
You can review the data to check integration by stepping through the files
manually or automatically. To step through the files manually, press the tool
below .
5)
To step through the files automatically, press the button outlined below.
6)
To stop, press the Pause button. Practice navigation for a moment.
7)
Now we will change individual DA Methods. Load 001-0101.D.
8)
Press the Inegration button. The manual events become available.
Use the Draw peak baseline and integrate icon to manually integrate both
signals of the last peak. Do something significant so that you can trace the
result.
9)
Go to Integration > Copy Manual Events to Method.
10) Open the Integration Events Table and select Manual Events.
11) Save and Close the Events Table.
118
Lab Exercise: Sequence Review and Reprocessing
Reprocess with DA Methods
12) Resave the DA method. Make certain you have saved the desired changes to
the correct method by checking the path. Insert a comment for the method
history.
13) Perform the same exercise on 003-0301.D and save its DA method.
In order to use the DA methods for these data files instead of the sequence folder
method during reprocessing, you must change the methods in the Sequence Table.
14) Open the Sequence Table using the tool below.
15) You want to use the DA method for samples 001-0101.D, and 003-0101.D .
Go to the line in the sequence table that is responsible for the first data file
and change the method as below by browsing to the DA method.
16) Note that you will go to Chem32\1\Data\ClassSequence 2006-03-15 10-4609\001-0101.D\DA.M.
17) Change the method for 003-0301.D as well.
18) Check the Sequence Output and Summary to print only the Compound
Summary.
119
Lab Exercise: Sequence Review and Reprocessing
Reprocess with DA Methods
19) Now, reprocess the sequence. Take a look at the resulting Compound
Summary page and compare it to the one at the end of this lab to make
certain your changes took affect.
120
Lab Exercise: Sequence Review and Reprocessing
Batch Review
Batch Review
If you generate a large number of samples on a daily basis and would like a
convenient way to view the results and check the quality of integration then Batch
Review may help. Batch Review allows you to step through calibration
standards, control samples, and unknown samples adjusting the integration
parameters and reviewing the results of sequence runs.
1)
In the Data Analysis view, select Demo\Batch. Find the .b batch file
created during the sequence and load demo\batch.b. Every sequence
creates a .b batch file.
2)
Process the data using Batch.M. You would typically process the batch data
using the sequence container method.
3)
Select all runs for batch processing. OK the dialog box.
4)
Examine the integration for the first data file. If you would like to view a
new data file every 10 seconds, press Start.
5)
If you want to change the integration events, then Pause the batch review.
6)
Try changing the slope sensitivity on one of the data files and reintegrating.
7)
Use the Marked changes to current run for save with batch file tool to mark
those changes you would like to keep. If you do not want to keep an
integration change, select the discard tool.
Note the following additional features before leaving the batch review:
Update the calibration table from all calibrated batch runs
Batch Preview and Batch Report (found under the batch menu), and
Saving the batch.
121
Lab Exercise: Sequence Review and Reprocessing
Batch Review
122
Lab Exercise: Advanced Reporting
Lab Exercise: Advanced Reporting
In this Laboratory You Will:
In this Laboratory You Will:
Preview the following report styles:
•
GLP
•
Performance (System Suitability)
•
Automated Peak Purity
You will also learn how to add your own header to report styles.
124
Lab Exercise: Advanced Reporting
Adding a Report Header
Adding a Report Header
You may add an individualized header to many of the report styles including:
Detail, Header + Short, GLP + Short, GLP + Detail, and Full. The directions
presented below will guide you through this process.
1) Go to the Data Analysis view. Load the method, Advrep.M. Try using the
Load Method tool.
2) From the Start button, select All Programs, then Accessories.
3) Open Notepad.
4) After Notepad loads, type in information to test the header. Try your name
and company address or similar information.
5) Under the File menu, select Save As…. The header must be saved as
rpthead.txt to the specific method you will use. For example:
C:\Chem32\1\methods\advrep.m\rpthead.txt
Save the file as a text document. Make certain the entire path is correct.
6) Exit Notepad (File, Exit).
7) Make certain that you are in the Data Analysis view. Load the data file
Lab4m.d by double clicking on the file in the Navigation Table or by
selecting File, Load Signal..…
8) From the Report menu, find Specify Report. You may also access this panel
from the Select Integration Task tool followed by the Specify Report
Calculation and Print Style tool.
9) Select the Printer destination, ESTD by Area with a GLP + Detail report
style. You may also add a chromatogram output. OK the dialog box.
10) Now from the Report menu, select Print Report or use the Print Report tool.
11) Find your personalized header information on the printed report. The GLP
report style was utilized in this example. Note that this report style includes a
printout of the run logbook.
125
Lab Exercise: Advanced Reporting
Adding a Report Header
Note: The GLP report style is not in any way connected to the Save GLP data
selection found in the Run Time Checklist. It is simply a report style that
includes a printout of the logbook, firmware, serial numbers and some
other information.
126
Lab Exercise: Advanced Reporting
Performance Reports (System Suitability)
Performance Reports (System Suitability)
Many chromatographers utilize the Performance report styles to help evaluate
the analysis system before and during routine analyses. The information
generated for each calibrated peak includes the retention time, k', symmetry, peak
width, plate number, resolution, signal-to-noise ratio, and compound name. You
may also determine the noise and drift using a Performance + Noise report style.
The results can be compared to expected results automatically.
Note: To evaluate the performance of both the analytical instrument or of an
analytical method before routine use, utilize the Sequence Summary function.
Some of the system suitability calculations require the void time or volume. You
may also desire column identification within reports. This information must be
input prior to sample injection and cannot be input after the fact. The first part of
this exercise will demonstrate this process.
1) Go to the Method and Run Control view.
2) Load Quant.M from you previous exercises.
3) Pull down the Instrument menu and select Columns.
4) Fill in the column description, length, diameter, and particle size. Examine
previous runs to determine the void time of the column. If you don't know
how to find the void time, ask your instructor. Scroll to the right and fill in
the value. Select min as the Unit.
5) The Installed field specifies which LC columns are being used. Highlight
this field and select YES for the column in your system.
6) OK this window. Resave your method.
7) Return to the Data Analysis menu. Under Report, select System Suitability,
then Edit Performance Limits....
8) Fill in low and high limits for chosen criteria in absolute units, for instance,
retention times in minutes. The ChemStation will calculate parameters for all
the calibrated peaks in your method. If actual values of any one peak exceed
the defined limits, this will be noted in the report. You can get an idea of the
upper and lower limits for some of the criteria from your Sequence Summary
report from the previous laboratory exercise. Fill in this screen now.
9) Noise can be calculated three different ways. First, as six times the standard
deviation of the linear regression of the drift; second, as peak-to-peak(drift
corrected); and third, as determined by the ASTM method(ASTM E 685-93).
127
Lab Exercise: Advanced Reporting
Performance Reports (System Suitability)
Select System Suitability from the Report menu, followed by Edit Noise
Ranges.
10) Examine prior chromatograms and select several time ranges for the noise
calculation. The ranges should be greater than one minute in duration in a
region where there are no chromatographic peaks. Fill in the table and OK
the dialog box.
11) Now, go to the Report menu and select Specify Report or use the Specify
Report tool.
Select an Extended Performance Report Style. OK the dialog box and
resave the method. Resave Quant.m.
12) Return to the Method and Run Control view. A complete system suitability
report cannot be printed from previously acquired data unless the column
information (void time) was filled in prior to the actual data acquisition. Turn
on the pump and lamp. Equilibrate the column. Make certain that the
method loaded is Quant.M. Under Method, RunTime Checklist, make
certain that both Data Acquisition and Data Analysis are selected.
13) From the RunControl menu, select Setup Sample Info. Give this data file
the name, syss.d. Set up the autosampler to inject one of the standards or the
original test mix. Run the Method.
14) Wait for the report to print and examine the report. Note the < or > sign that
appears when the calculated values exceed the specified limits.
Note: The equations for each performance test can be found in your manual,
Understanding Your ChemStation.
15) Return to the Data Analysis window. Under Report, select Specify Report.
Now select Performance + Noise as the Report style. Resave the method
and print the report.
128
Lab Exercise: Advanced Reporting
Automated Peak Purity
Automated Peak Purity
The Short + Spectrum, Detail + Spectrum, and Full report styles will produce
automated peak purity analyses of calibrated chromatographic peaks.
1) Make certain Quant.M is still loaded. Make certain the last data file you
collected is still loaded, Syss.d.
2) To print an automated peak purity report, open the Report menu and select
Specify Report or use the Specify Report tool.
3) Change the Report style to Short + Spectrum. OK the dialog box.
4) Resave the method under the File menu or use the Save Method tool.
5) Load a data file from your last sequence run.
6) Under the Report menu, select Print Report. Examine the resulting report.
Note: Purity Options may be saved as part of the method in order to produce the
desired peak purity report.
129
Lab Exercise: Advanced Reporting
Automated Spectral Library Search
Automated Spectral Library Search
Make certain that Quant.M and Syss.D are still loaded.
Quantification based solely on retention time correlation can lead to erroneous
identification if:
a) Compounds other than the calibrated ones appear in the specified retention
time window.
b) More than one peak elutes in the specified retention time window.
c) The compound is retained on the column longer than the specified window
because of solvent flow rate changes or changing column characteristics.
The ChemStation can automate a library search process to positively identify
chromatographic peaks based upon their UV spectra. In addition, the software
can perform a peak purity check using the settings specified in the Purity Options
dialog box.
1) To set up a library search report, first go to the Report menu and select
Automated Library Search. The Parameters for Automated Identification
panel will appear.
2) The libraries you specify in this window must already exist. You created a
library for this test mix earlier in the course. Select the checkbox for library 1
and click on the Selection box. Load your library.
3) Click on the Edit Search Parms button to the right. Create a search window
of + or - 10%. Set a Threshold of 2 mAU. OK this dialog box.
4) You may perform a purity check. This procedure is recommended before a
library search, since a match is only as reliable as the quality of the separation.
It calculates and reports a purity factor. Purity can be defined by a threshold,
below which questionable results are tagged with an "x" in the report. Select
the Purity check? checkbox.
5) Three search modes can be applied to your application. First, select Identify
by spectral library search.
This mode is the most general search mode. The ChemStation compares the peak
spectra of all those peaks found by the integrator and characterized by their
retention times, with spectra from up to four specified libraries. The name of the
compound with the best match is assigned to the peak. You can specify how good
this match must be with your own threshold value. The ChemStation then refers
130
Lab Exercise: Advanced Reporting
Automated Spectral Library Search
to the calibration table for an identically named entry. If one is found, the data are
used to calculate and report the amount.
6) OK this dialog box. Open the library you intend to use in the report and
check that the names of the calibrated peaks are identical with the library
names. In other words, check your calibration table as well as the entries in
the library.
7) Under Specify Report, select Library Search. OK the dialog box. Resave
your method. Print the report.
8) Now, set up a method where the search mode is Target compound analysis
using a calibration table.
This search mode is only for those compounds in the calibration table. Peaks are
only reported as identified if confirmed by spectral comparison.
9) Print the report.
131
Lab Exercise: Advanced Reporting
Automated Spectral Library Search
132
Lab Exercise: Customized Report Design
A Customized Report Layout view is included in the ChemStation for users who
want to define the exact content of their reports. This laboratory will help you
develop a customized report. For further information, study the customized report
files that are included with the software.
Lab Exercise: Customized Report Design
In this Laboratory You Will:
In this Laboratory You Will:
•
Build your own customized report template.
•
Save the template as part of the standard report options.
You will need the Demo File, 005-0101.D and Democal1.M.
134
Lab Exercise: Customized Report Design
Preparations
Preparations
1) Enter the Report Layout view. Load the method Democal1.m (Double-click
on the file name in ChemStation Explorer, or choose File > Load, Method).
2) Load Signals from the file Demo\005-0101.d. using File > Load > Signals.
135
Lab Exercise: Customized Report Design
Building a Customized Report
Building a Customized Report
1) Under the File menu, select New Template.
First, add a header and footer to the report style. The header would be reproduced
on each page of the report at the top while any footer would appear on each page
at the bottom.
2) Next to the Header designation you will see two gray horizontal borders
which represent the section start and stop points. Right now, the borders are
adjacent with no space between indicating no existing header. Adjust the
length of the Header section by clicking on the bottom gray border and
dragging about one inch down the page.
3) You will create a header consisting of the data file name and sample name.
Click on the Text Tool.
Click in the white header space and draw a rubber band box the width of a
single text line. Release the mouse. The New Element selection box will
appear. Select Raw Data File Name then OK. The Raw Data File will be
left aligned. OK this selection.
4) Resize the Raw Data Filename box by dragging the borders to fit the size of
the actual element.
NOTE: To delete an element you have already added, click once with the left
mouse button within the borders of the element. Go to the Edit menu and select,
Delete Element.
5) Repeat steps 4 and 5, but this time add the Sample Name.
6) Reposition the bottom Header border by clicking and dragging the bottom
border up to the bottom of the Sample Name.
136
Lab Exercise: Customized Report Design
Adding a General Section
Adding a General Section
1) Click on the blue triangle. The color will change to red.
2) Select Edit, New Section, General.
3) Expand the General section by dragging the bottom border down.
4) Click on the Text Tool and position a rubber-band box at the top. Select
Constant Text, then OK. Type in any address.
NOTE: Use the CTRL and Enter keys simultaneously to move to the next line
within a text box. Striking Enter alone is equivalent to selecting OK.
5) Click on the Text Tool again and outline a single line below the Agilent
Technologies logo and address. Select Constant Text and type: Injection
Data, then OK. Resize the text box appropriately when finished. This text
will appear on the report to describe the next field which you will add. Click
on the Text Tool again. This time outline a single line to the right of the text:
Date of Injection.
When the New Element box appears, select
Injection Date. OK the dialog boxes.
6) Add the Acquisition Operator in the same manner as above.
7) At this point, you may need to enlarge the white space. Scroll down then
click on the border and drag it down the page.
8) Perform the following steps to add a chromatogram. Click on the Graphics
Tool.
Outline a rather large box, perhaps 80 x 40. These dimensions can be found
in the message line.
9) When the New Element box appears, select Chromatogram, then OK.
Select the desired preferences for chromatographic display and click OK.
137
Lab Exercise: Customized Report Design
Inserting Tables
Inserting Tables
1) Below the chromatogram, expand the size of the general white space.
2) Select the Table Tool,
and outline a space approximately 80 x 20.
3) Select Cal. Peaks Sorted by Ret. Time. The Setup Cal. Peaks sorted by Ret.
Time table should appear. From the Printed Columns box select Response
Factor then <. Note that you have removed this item from the Printed
Columns list. Check that the table layout lies within the bounding box length.
Remove Expected Retention Time, as well. OK the table and adjust the size
of the box, if necessary.
Creating a Table Footer
1) Double click on the table box. Click on the Edit Format button in the Set-up.
2) When the Edit Format of Cal. Peaks panel is displayed click on Footer.
3) Click on the button labeled 2 Lines. In the Amount row, Line 2 column,
type: @sum. In the first row (#), Line 2 column, type: Total. OK this panel.
In the Edit Format panel, change the width of the # row to 6. OK this panel
and the subsequent panel.
Inserting a Page break
1) Click on the Page Break Tool,
then click in the white space below the table to insert a page break.
2) Reposition the bottom General Section border by dragging the border up to
the page break (the blue band).
138
Lab Exercise: Customized Report Design
Adding a Calibration Curve
Adding a Calibration Curve
1) Click on the blue triangle found between the General section and the Footer.
The triangle will change from blue to red.
2) Pull down the Edit menu and select New Section, Cal. Compounds.
3) Drag the Cal. Compounds borders to create free white space in this new
section.
4) Click on the Graphics Tool.
5) Draw in a rubber band box for the calibration table.
6) When the New Element panel is displayed, select Calibration Curve, then
OK. OK the Set-up panel as well.
139
Lab Exercise: Customized Report Design
Finishing Up
Finishing Up
1) From the File menu, select Save Template As. Give the template a unique
name.
2) Now test the template. Use the Report Preview tool to display the report.
Review the report and make any necessary modifications. Resave the
template as needed. You may print the report now.
3) You can add your report to the list of report styles by selecting Add to Report
Styles under the File menu. Try this now.
4) Go to the Data Analysis view and select Report, Specify Report. Find your
report style by scrolling to the top of the list of report styles. In order for this
style to become part of a method, select this report style and then resave the
method.
NOTE: You may also remove report styles with the Remove from Report Styles
menu item found in the Report Layout view, under the File menu.
140
Lab Exercise: Commands
Lab Exercise: Commands
In this LaboratoryYou Will:
In this LaboratoryYou Will:
•
Become familiar with some of the more common commands on the HPLC
ChemStation.
•
Understand the use of variables.
•
Understand the concept of data storage in registers and tables.
•
Become familiar with some of the commands used to access/manipulate data.
During this laboratory exercise you will be using the HPLC ChemStation
equipped with version B.02.xx software. The data file, demodad.d, from the
demo directory will be utilized.
142
Lab Exercise: Commands
Retrieving Information about Commands
Retrieving Information about Commands
Several resources are available to help you utilize the ChemStation commands.
These resources include Online Help and the Show command. Start the exercise
in the Data Analysis view.
1) Check to make certain that the command line is displayed at the bottom of the
ChemStation software window. If the line is not available, select View then
Command Line.
2) Type Show<enter> on the command line. Select the Commands domain and
find the File command. Double click on the File command. Notice that the
syntax for this command is now printed below the command line.
3) Type in: File, type a space then:
"c:\chem32\1\data\demo\demodad.d".
Note that the path may be different for your ChemStation. The busy indicator
should be displayed as the file is loaded into memory.
4) Look up the following commands and determine their function and syntax.
•
LoadSpectrum
•
IdentifyPeaks
•
QuantifyPeaks
•
PumpAll
143
Lab Exercise: Commands
Tracking Errors and Using some Common Commands
Tracking Errors and Using some Common
Commands
A window can be displayed which will help you track software errors while your
are using commands or writing/using macros. The window is accessed with the
ListMessages command.
1) On the command line, type ListMessages On. Notice that a white message
box is now displayed. The box can be sized differently by dragging the edges.
Cryptic explanations regarding syntax or other errors will be displayed here as
they occur.
2) Type the following sequence of commands onto the command line. After
each command line, you may either use the Enter key or use the semicolon,;,
and the Enter key at the very end of the list. Note the message display.
Delreg chromreg
Loadsignal dad,b,,chromreg
IntegrateObj chromreg[1]
Draw 2,chromreg,6:8
3) Move back through the previous commands by using the up and down arrow
keys. These keys are useful if a typing error has been made and you don't
want to type the entire command over again.
144
Lab Exercise: Commands
Registers, Objects, and Tables
Registers, Objects, and Tables
A register is a place to store data such as chromatograms and signals. The
ChemStation uses pre-defined registers such as ChromReg for chromatograms
and SpecReg for spectra. The chromatograms and spectra are objects in the
registers. If you load a data file containing signals A, B, and C; the ChromReg
register will contain three objects. To access data for a specific signal, specify the
number of the object in square brackets after the name of the register. For
example, ChromReg[2], accesses the second object in ChromReg.
A register is made up of a contents list, a header and one or more objects. Each
object has a header, a data matrix and possibly one or more tables.
In this exercise, you will learn how to obtain information stored in the pre-defined
registers and objects.
Registers
1) Get the contents of the register as follows: Type on the command line
Contents$=RegCont$(ChromReg) <enter>
Print Contents$ <enter>.
2) To find out how may objects are in a register, type:
Size=RegSize(ChromReg)<enter>
Print Size <enter>
3) To delete the register as well as the contents, type:
DelReg ChromReg <enter>
145
Lab Exercise: Commands
Registers, Objects, and Tables
Objects
Objects are the data you work with such as chromatograms or spectra. Each
object has a header which contains a description of the object. For instance, if the
object is a chromatogram, the header contains the injection time and date, the
operator name and vial number.
Objects will also have a data block. The data block is the actual x and y data
points that describe the chromatogram or spectrum.
Objects may also comprise tables. Tables are made of a series of rows and
columns which typically hold information about the object. An example of a
table would be the IntResults table created after chromatograms are integrated.
The rows indicate the number of integrated chromatographic peaks and the
column names include retention time, area, peak height, and etc…. Tables also
have an associated table header. The IntResults table header will tell you how
many rows are in the table, thus how many integrated peaks.
Use the following commands to load objects into registers, access the information
from the object header, access data points from the data block, and retrieve
information from tables. In the following example, you will load a
chromatographic signal into ChromReg, integrate the chromatogram, then access
information from the object header, data block and IntResults table.
Loading a chromatogram into a Register as an Object
1) Load a data file into memory:
File c:\chem32\#\data\demo\demodad.d <enter>
where # is your instrument number.
2) Load the chromatographic signals into the ChromReg register by typing:
LoadSignal ,,,ChromReg <enter>
3) To view the first of three chromatograms loaded type:
Draw 2,ChromReg[1] <enter>
146
Lab Exercise: Commands
Registers, Objects, and Tables
4) To integrate the chromatogram, use the command IntegrateObj. Type:
IntegrateObj ChromReg[1] <enter>
Draw 2,ChromReg[1]<enter>
5) Access scalar variables(numbers) from the object header using ObjHdrVal.
To obtain the vial number which is stored in the object header type:
VialNum=ObjHdrVal(ChromReg[1],"vial") <enter>
Print VialNum <enter>
6) To add more clarity in a macro you may modify the above to:
Print "The vial number is",VialNum <enter>
Note: The vial number is zero in this case. The data was originally acquired
on a 1090 which does have a zero vial position.
7) Access string variables (text) in the object header with the function
ObjHdrText$. Note that the variable name for a string variable must end in
the $ sign. Try obtaining the signal description.
Signal$=ObjHdrText$(ChromReg[1],"signalDesc") <enter>
Print Signal$
8) To obtain the information as above, you must know the name of the text or
scalar variable. There are two functions which can help you obtain this
information:
ObjHdrName$(Register,Index)
retrieves the item name from the object and
ObjHdrType(Register, Item$)
determines if the object is a string, scalar, or table variable.
147
Lab Exercise: Commands
Registers, Objects, and Tables
Try typing:
Item$=ObjHdrName$(ChromReg[1],3) <enter>
Obtype=ObjHdrType(ChromReg[1],item$) <enter>
Print "Item 3 in object 1 is called",Item$,"and is
a",Obtype,"variable" <enter>
Note: The answer to your print appears above the command line. 0 is a
string variable.
Data Matrix
To complete the next section, you will need to load the method, democal1.m, load
the data file, demo\005_0101.d, and print a report. Do these tasks now.
1) To obtain the actual x and y coordinates of a chromatogram or spectrum, use
the Data(Register,axis,datapoint) function. Type:
Timeof3rddatapoint=Data(ChromReg[1],0,3) <enter>
where 0 indicates the x-axis
Print Timeof3rddatapoint <enter>
Absorbanceof20thdatapoint=Data(ChromReg[1],1,20) <enter>
where 1 specifies the y-axis
Print Absorbanceof20thdatapoint <enter>
2) Reference a time value in the chromatographic signal directly using the
DataIndex function. You can use the index number to retrieve the absorbance
data at a given time.
Index=DataIndex(ChromReg[1],5.0)
Absorbance=Data(ChromReg[1],1,Index)
The above commands obtain the data index at time 5.0.
Use the print command to access the values obtained.
148
Lab Exercise: Commands
Registers, Objects, and Tables
Tables
1) You have already integrated the chromatogram with the IntegratObj
command. When each signal is integrated, the integration results are stored in
a table called Intresults. One IntResults table is created for each integrated
signal and the table is stored in the same object as the corresponding signal.
When you quantify the peaks, the integration tables in ChromReg are
identified and quantified. The quantification results are stored in tables found
in the ChromRes register. ChromRes holds the compound and quantification
data for the entire analysis in the first object.
Some of the information you need may be stored in the table header, for instance,
the number of integrated peaks in the chromatogram. Two functions can help you
access information in table headers:
TabhdrVal(RegObj,TabName,ItemName)
gets a single numeric item from the table header and
TabhdrText$(RegObj,TableName,ItemName)
gets a single text item from the table header.
Type:
pks=tabhdrval(ChromReg[1],"intresults",numberofrows)<enter>
Print pks <enter>
The number of rows in the intresults table indicates the number of
integrated chromatographic peaks.
2) To obtain information directly from the table use:
TabText$(RegObj,TabName,RowIndex, ColName)
to get a single text element from a table.
Or
TabVal(RegObj,TabName,RowIndex,ColName)
149
Lab Exercise: Commands
Registers, Objects, and Tables
to obtain a single numeric element from a table.
Type:
pk_width=tabval(ChromReg[1],"intresults",2,"width") <enter>
Print pk_width <enter>
You obtained the peakwidth of the second integrated chromatographic peak.
3) Use the commands to obtain the name of the 4th compound from the
quantification results table "compound" found in the ChromRes register. To
get this information, you must print a report first. By doing this, the variables
will be stored in ChromRes. Print a report now if you haven’t already done
so.
Name$=Tabtext$(ChromRes,"compound",4,"name")<enter>
Print Name$ <enter>
To obtain the quantified amount for the compound use adifferent column
name to access the same table.
Amount=TabVal(ChromRes,"compound",4,"amount") <enter>
Print Amount <enter>
4) To access the compounds retention time, you will need to reference the "peak"
table in ChromRes. You cannot simply use reference index "4" because there
are more integrated chromatographic peaks than in the "compound" table.
Type the following to obtain the retention time:
FirstPeak=TabVal(ChromRes,"compound",4,"FirstPeak") <enter>
RT=TabVal(ChromRes,"peak",FirstPeak,"MeasRetTime") <enter>
Print RT <enter>
150
Lab Exercise: Commands
Registers, Objects, and Tables
Viewing Tables
Often, you will need to display the contents of a table to understand what row or
column to access. EdTab is a useful command.
1) Type:
EdTab 3,ChromRes,Peak<enter>
where 3 is the window number, ChromRes is the Register and Peak is the
table. Scroll to view all entries.
151
Lab Exercise: Commands
Registers, Objects, and Tables
152
Lab Exercise: Macro Writing
Lab Exercise: Macro Writing
In this Laboratory You Will:
In this Laboratory You Will:
•
Write and execute a macro.
•
Learn how to find errors in a macro.
•
Add a macro to your method and execute that macro.
154
Lab Exercise: Macro Writing
Writing and Executing a Macro
Writing and Executing a Macro
During this first exercise, you will simply follow the directions to create and
execute a simple macro.
1) Minimize the ChemStation. From the Start button, select All Programs then
Accessories, and Notepad.
2) Type in the following simple macro:
Name Class
File “c:\chem32\1\data\demo\005-0101.d”
If RegSize(ChromReg)>0 then
DelReg ChromReg
Endif
LoadSignal dad,a,,ChromReg
IntegrateObj ChromReg
SetWinTitle 1, "Class Chromatograms"
Draw 2, ChromReg
EndMacro
3) Under the File menu, select Save As. Save the file as a text document
c:\chem32\core\class.mac.
Note: Your path statements may be different than the example depending on
the drive used for Chem32 and the instrument number.
4) Minimize Notepad and restore the ChemStation.
5) Now, set error detection. On the command line type:
ListMessages On
Question:
What are other ways you can debug a macro?
____________________________________________________
6) To test the macro, type on the command line:
155
Lab Exercise: Macro Writing
Writing and Executing a Macro
Macro class.mac,go
The macro should begin executing. If a problem is found, correct the macro and
re-execute.
Note: If the ChemStation cannot open the file, class.mac, go to the directory and
check the file extension. Notepad may put a .txt after the .mac extension by
default. Rename the file to class.mac.
156
Lab Exercise: Macro Writing
Write Your Own Macro
Write Your Own Macro
Write a macro to do the following. Use your own registers and windows 2-10:
•
Get a file from your sequence.
•
Obtain signal B.
•
Get another file from your sequence.
•
Draw the overlaid signal to a full page window.
•
Send to the printer with the message: "Overlaid Chromatographic Signals".
Hint: You will need to access the _Config register.
157
Lab Exercise: Macro Writing
Possible Answer
Possible Answer
name overlaid
file "c:\chem32\1\data\demo\005-0101.d"
if regsize (myreg) >0
delreg myreg
endif
loadsignal dad,a, ,myreg
file "c:\chem32\1\data\demo\005-0102.d”
loadsignal dad,a, ,myreg
draw 2,myreg
settabtext _config [1] ,window,3,"destination","printer"
settabval _config [1],window,3,defwyhigh,0.6
settabval _config [1],window,3,defwylow,0.1
opendevice "printer" as #5
print #5,"Overlaid Chromatograms"
draw 3,myreg
Close #5
settabtext _config [1] ,window,3,"destination","screen"
settabval _config [1] ,window,3,defwyhigh,1
settabval _config [1] ,window,3,defwylow,0
endmacro
Note: Any two data files may be used in the example.
158
Appendix: Getting Started with New
ChemStation Workflow G2170-90041
Agilent ChemStation
Getting Started with New
ChemStation Workflow
A
Notices
© Agilent Technologies, Inc. 2006, 2007
Warranty
No part of this manual may be reproduced in
any form or by any means (including electronic storage and retrieval or translation
into a foreign language) without prior agreement and written consent from Agilent
Technologies, Inc. as governed by United
States and international copyright laws.
The material contained in this document is provided “as is,” and is subject to being changed, without notice,
in future editions. Further, to the maximum extent permitted by applicable
law, Agilent disclaims all warranties,
either express or implied, with regard
to this manual and any information
contained herein, including but not
limited to the implied warranties of
merchantability and fitness for a particular purpose. Agilent shall not be
liable for errors or for incidental or
consequential damages in connection with the furnishing, use, or performance of this document or of any
information contained herein. Should
Agilent and the user have a separate
written agreement with warranty
terms covering the material in this
document that conflict with these
terms, the warranty terms in the separate agreement shall control.
Microsoft ® is a U.S. registered trademark
of Microsoft Corporation.
Manual Part Number
G2170-90041
Edition
02/07
Printed in Germany
Agilent Technologies
Hewlett-Packard-Strasse 8
76337 Waldbronn, Germany
Software Revision
Technology Licenses
This guide is valid for B.03.xx revisions of
the Agilent ChemStation Getting Started
with New ChemStation Workflow software,
where xx refers to minor revisions of the
software that do not affect the technical
accuracy of this guide.
The hardware and/or software described in
this document are furnished under a license
and may be used or copied only in accordance with the terms of such license.
Restricted Rights Legend
If software is for use in the performance of a
U.S. Government prime contract or subcontract, Software is delivered and licensed as
“Commercial computer software” as
defined in DFAR 252.227-7014 (June 1995),
or as a “commercial item” as defined in FAR
2.101(a) or as “Restricted computer software” as defined in FAR 52.227-19 (June
1987) or any equivalent agency regulation or
contract clause. Use, duplication or disclosure of Software is subject to Agilent Technologies’ standard commercial license
terms, and non-DOD Departments and
Agencies of the U.S. Government will
receive no greater than Restricted Rights as
defined in FAR 52.227-19(c)(1-2) (June
1987). U.S. Government users will receive
no greater than Limited Rights as defined in
FAR 52.227-14 (June 1987) or DFAR
252.227-7015 (b)(2) (November 1995), as
applicable in any technical data.
Safety Notices
CAUTION
A CAUTION notice denotes a hazard. It calls attention to an operating procedure, practice, or the like
that, if not correctly performed or
adhered to, could result in damage
to the product or loss of important
data. Do not proceed beyond a
CAUTION notice until the indicated
conditions are fully understood and
met.
WA R N I N G
A WARNING notice denotes a
hazard. It calls attention to an
operating procedure, practice, or
the like that, if not correctly performed or adhered to, could result
in personal injury or death. Do not
proceed beyond a WARNING
notice until the indicated conditions are fully understood and
met.
Data Organization
In This Guide…
In analytical laboratories, chromatography data need to be acquired efficiently
in a short time. Clarifying ambiguous results can be time-consuming, and may
result in high administrative costs. Since ChemStation Revision B.02.01, data
storage and data browsing capabilities have been improved to enable fast
review and reprocessing of result data.
In this manual, the efficient use of the new data storage and retrieval
functions in ChemStation B.02.01/B.03.01 to boost your lab’s productivity are
described.
1
ChemStation Data Structure
This chapter gives an overview of the differences between the data structure
used in ChemStation revisions prior to B.02.01 and the new data structure in
revision B.02.01 and its successors.
2
Data Acquisition
This chapter explains how the new data structure influences the workflow for
the acquisition of data for sequences and single runs.
3
Data Analysis
This chapter outlines the data analysis and review options that are available,
and explains how consideration of the data structure affects your choice of
options.
4
Workflow with Unique Folder Creation switched off
This chapter provides information on working with Unique Folder Creation
switched off which allows you to store data as in ChemStation revisions
B.01.03 or earlier. This mode does not take full advantage of the latest data
review and reprocessing functionality in ChemStation.
Data Organization
4
Data Organization
Contents
1
ChemStation Data Structure
7
ChemStation prior to B.02.01
8
ChemStation B.02.01/B.03.01
2
Data Acquisition
9
13
Data Acquisition 14
Data Acquisition in a Sequence
Data Acquisition of Single Runs
3
Data Analysis
19
Data Analysis
20
Data Analysis: Data Review
15
17
23
The ChemStation User Interface during Data Review
Data Analysis: Reprocessing data
4
31
Workflow with Unique Folder Creation switched off
Working with Unique Folder Creation on or off?
35
36
Workflow with “Unique Folder Creation” switched off
Data Organization
29
37
5
Contents
6
Data Organization
Agilent ChemStation
Getting Started with New ChemStation Workflow
1
ChemStation Data Structure
ChemStation prior to B.02.01 8
ChemStation B.02.01/B.03.01 9
This chapter gives an overview of the differences between the data structure
used in ChemStation revisions prior to B.02.01 and the new data structure in
revision B.02.01 and its successors.
Agilent Technologies
7
1
ChemStation Data Structure
ChemStation prior to B.02.01
ChemStation prior to B.02.01
In ChemStation revisions prior to B.02.01, sequences, methods and the
generated data files and results were stored in fixed, specified and separated
locations. For example, methods were referenced by name in a sequence and it
was the user’s responsibility to maintain the integrity of methods, sequences,
and data files. Because of this, the long-term archiving of data, and
reproduction of results was a tedious task. Users had to document the
chromatogram, results, and associated method; this was the case not only for
regulated labs, but also for some areas of unregulated labs (such as
environmental labs). In ChemStation prior to B.02.01, this could be achieved
only by printing everything in a report.
8
Data Organization
ChemStation Data Structure
ChemStation B.02.01/B.03.01
1
ChemStation B.02.01/B.03.01
In order to strengthen the association between data files and methods, the
following new data organization scheme has been implemented with
ChemStation B.02.01/B.03.01. When used with the ChemStation, the Agilent
Enterprise Content Manager (ECM) also makes use of the new data concept,
since the complete data set (sequence/methods/data files) can now be
transferred (archived) to ECM as one entity.
Figure 1
Sequence Acquisition B.02.01 / B.03.01
The methods in the folder Chem32\1\methods serve as master methods, i.e.
during acquisition and data analysis, they remain unchanged.
Data Organization
9
1
ChemStation Data Structure
ChemStation B.02.01/B.03.01
Similarly, the sequences in the folder Chem32\1\sequence serve as sequence
templates that can be used to rerun (but not reprocess) a sequence several
times.
The data storage pattern varies depending on whether single run data or
sequence data is acquired:
1 When a sequence is executed, a new folder is automatically created
(“sequence container”) with a unique name in the specified subdirectory.
When a single sample is run, the data file (*.d) is written to the specified
subdirectory.
2 For sequence data, the executed sequence template (*.s) and all the
methods (*.m) involved are copied into the sequence container. The copies
of the methods are called the “sequence methods” in order to distinguish
them from the original master methods.
All sequence-related tasks (e.g. acquisition and data analysis) are
performed on the copies of the sequence and the methods. Therefore, the
sequence template and the master methods remain unchanged for future
sequence execution.
While executing the sequence, all generated data files (*.d) are stored in the
sequence data folder, along with the corresponding batch file (*.b) and
sequence log file (*.log).
3 Each data file contains two copies of the method used to create the run.
• The first one, called ACQ.M, is saved directly after the acquisition part of
the method is completed.
• The second copy, called DA.M, is saved after completion of the data
analysis part.
The preservation of the state of the method at acquisition of the specific
data file is thus ensured. The acquisition parameters can be viewed and
printed in Data Analysis view. The DA.M can be modified during data
analysis. In this case, these two methods may differ if, for example, the
calibration table is updated.
10
Data Organization
ChemStation Data Structure
ChemStation B.02.01/B.03.01
1
The following chapters explain the impact of this structure on typical
workflows in more detail. The corresponding settings in the ChemStation
dialogs are also shown.
Data Organization
11
1
12
ChemStation Data Structure
ChemStation B.02.01/B.03.01
Data Organization
Agilent ChemStation
Getting Started with New ChemStation Workflow
2
Data Acquisition
Data Acquisition 14
Data Acquisition in a Sequence 15
Data Acquisition of Single Runs 17
This chapter explains how the new data structure influences the workflow for
the acquisition of data for sequences and single runs.
Agilent Technologies
13
2
Data Acquisition
Data Acquisition
Data Acquisition
Starting with ChemStation B.02.01, flexible data storage for single runs and
sequences allows you to specify various saving locations without
reconfiguration. The Paths tab in the Preferences dialog box in the View menu
gives you the opportunity to add multiple paths in addition to the default path
C:\chem32\x\DATA (where x is the instrument number). Using the Add and
Remove buttons, existing paths can be simply deleted, or you can navigate to a
selected location and add the path to the new location into the Preferences.
The default path cannot be removed from the list, but it can be changed in the
Configuration Editor.
Figure 2
Preferences Dialog / Paths Tab
All newly specified Data Paths are then available for selection in the Sample
Info/Sequence Parameters dialog boxes when performing runs.
14
Data Organization
Data Acquisition
Data Acquisition
Figure 3
2
Data Path Selection
Data Acquisition in a Sequence
In order to run a sequence, appropriate pre-defined methods must be
available. These are the master methods as outlined above. Typically, master
methods and sequence templates are worked on in the Method and Run
Control view of the ChemStation. For this reason, in Method and Run Control
view, the ChemStation Explorer provides access to master methods and
sequence templates.
Data Organization
15
2
Data Acquisition
Data Acquisition
The sequence template references these methods in the sequence table.
As explained previously, when a sequence is run with sequence template
<sequence_name>.S, and the master method <method_name>.M is used, a new
folder is created that contains all resulting files from the sequence run
(“sequence container”).
The location of this folder is determined by the settings in the Sequence
Parameters dialog box; the naming of this folder is determined by the
Sequence tab of the Preferences dialog box. By default, the name is
<sequence_name> <acquisition_date> <acquisition_time>, but it can be
configured by using the tokens Operator, Instrument, Counter, and PC Name,
or you can manually enter any name. If the Name Pattern would not result in
unique names for the sequence containers, the ChemStation will append a
counter to ensure uniqueness.
Figure 4
Preferences Dialog / Sequence Tab
At the start of an acquisition sequence, the method specified in the sequence
table is copied from the master methods folder into the sequence container. In
addition, a copy of the sequence is created and placed with the sequence log
and the batch (*.b) file in the sequence container. All updates of the method
(e.g. updates of the calibration table) are written to this sequence method in
16
Data Organization
Data Acquisition
Data Acquisition
2
the container. All necessary files are now available for future data review and
reprocessing, without changes that were applied to the master method or
sequence template for other sequence runs.
During acquisition, the data files are stored to the sequence container. Within
each data file (*.D), two additional methods, ACQ.M and DA.M, are saved for
this specific run. These two methods are copies of the sequence method,
preserving the state of the method as it was at the time of acquisition of the
specific data file. In the case of e.g. calibration table updates the DA.M
methods differ for each of the runs.
The individual acquisition method ACQ.M is intended to preserve the
acquisition parameters, therefore it is recommended that you do not change
this method during future data review activities. In Data Analysis view, the
acquistion parameters of this method can be viewed and printed.
With these files saved in the sequence folder, all data review and reprocess
activities can be performed without altering the master method or the
sequence template. If needed, method changes can also be saved to the master
method again.
Data Acquisition of Single Runs
The new data concept is also introduced for single runs. In this case the data
file is saved directly into the respective subdirectory. Since only one method is
employed for a single run, this method does not need to be copied into the
subdirectory; all actions are performed directly with the master method. After
the acquisition part of the method is completed a copy of the master method is
saved into the data file directory (ACQ.M). Another copy (DA.M) is saved after
the data analysis part of the master method has been executed.
Data Organization
17
2
18
Data Acquisition
Data Acquisition
Data Organization
Agilent ChemStation
Getting Started with New ChemStation Workflow
3
Data Analysis
Data Analysis 20
Data Analysis: Data Review 23
The ChemStation User Interface during Data Review 29
Data Analysis: Reprocessing data 31
This chapter outlines the data analysis and review options that are available,
and explains how consideration of the data structure affects your choice of
options.
Agilent Technologies
19
3
Data Analysis
Data Analysis
Data Analysis
Once the data have been acquired, they can be analyzed in ChemStation Data
Analysis view. When selecting the Data tab of the ChemStation Explorer, you
can load all the runs of a sequence or all single runs in a specific folder by
double-clicking the corresponding symbol. The corresponding data set is then
available in the Navigation Table.
Figure 5
Loading a Sequence from the ChemStation Explorer into Navigation Table
The main body of the Navigation Table consists of a list of all runs of the set.
Instead of loading a run via the File / Load Signal menu, a run can now be
loaded into ChemStation memory by double-clicking the relevant line in the
Navigation Table. Additionally, a right-click on a run offers several options, e.g.
to load or overlay specific signals from the file, to export the data, or to view
the acquisition method parameters.
Once the run is loaded, you can review it, i.e. adjust data analysis parameters,
integrate the signals and finally print a report. In this case you analyze the run
as a single run without taking the sequence context into account.
This way of data analysis is called “Data Review”. The Navigation Table
provides the tool set shown in Figure 6, which makes data review more
convenient.
Figure 6
20
Data Review Toolset of the Navigation Table
Data Organization
Data Analysis
Data Analysis
3
With this toolset, you can jump to the beginning or end of the Navigation
Table, step on to next or previous run, automatically step through the runs,
and stop automatic stepping.
A different way to analyze your data is to “Reprocess” a complete sequence.
During this process, all runs are reanalyzed in the sequence context, i.e. the
calibration tables of the sequence methods are updated in the case of
calibration runs, multipliers, amounts etc. can be changed in the sequence
table, new methods can be added to the sequence container, etc. For
reprocessing, the Navigation Table provides the following toolset:
Figure 7
Sequence Reprocessing Toolset of the Navigation Table
Note that the reprocessing icons in the Navigation Table are available only for
sequence data generated with ChemStation B.02.01 and higher. For Single Run
data, for data generated prior to B.02.01, and for data acquired while Unique
Folder Creation is switched off (see Chapter 4, “Workflow with Unique Folder
Creation switched off”), reprocessing in Data Analysis is not accessible.
Sequences acquired prior to B.02.01 need to be reprocessed in “Method and
Run Control”, defining the sequence parameter “parts of method to run” to
“reprocess only”. For sequences generated with ChemStation B.02.01 and
higher, the reprocessing option in Method and Run Control has been removed
(see Figure 8 on page 22), and the Navigation Table offers reprocessing as a
Data Analysis Task.
Data Organization
21
3
Data Analysis
Data Analysis
Figure 8
22
Sequence Parameters in Method and Run Control view of ChemStation
B.02.01 and higher
Data Organization
Data Analysis
Data Analysis: Data Review
3
Data Analysis: Data Review
Data Review means analyzing on a run-per-run base. ChemStation allows you
to specify default actions that are performed automatically when a data file is
loaded from the Navigation Table. These include data analysis tasks like
integrating the chromatogram directly after loading, and also specifying the
method that is to be loaded.
The corresponding options for reviewing (not used for reprocessing) are set up
on the “Review/Signal Options” tab of the Preferences dialog box.
Figure 9
Preferences Dialog / Signal/Review Options Tab
The first section, “Load Signal Options”, specifies which of the signals in a run
are loaded, and if the chromatograms are to be integrated and the results
reported directly after loading.
In the second section, “Data Review Options”, you have the possibility to
configure the interval for stepping through the runs in the Navigation Table
automatically.
Data Organization
23
3
Data Analysis
Data Analysis: Data Review
The remainder of this section specifies which method is loaded during data
review when a run is loaded from the Navigation Table. They only apply to
data review, but not to reprocessing. The following separate option sets are
available for sequence runs and single runs:
Table 1
Data Review Options for Sequence and Single Run Data
Method used for review of sequence data
Method used for review of single run data
Current Method
Current Method
Sequence Method
Individual method from data file (DA.M)
Individual method from data file (DA.M)
Keep “Current Method”
The review setting “Current Method” should always be used when you want to
use the method that is currently loaded. In this respect, for data review the
current method remains irrespective of which single run data file or sequence
container file is loaded. This setting should be used for method development,
to create a calibration table or other updates etc. For example, you run a
sequence with runs of different concentrations in order to optimize
integration parameters, and afterwards build the calibration table. As these
changes in data analysis parameters must be available for all future
acquisition runs, you have to apply them to the master method. In order to do
this, you select the option “Current Method” in the Preferences dialog, see
Figure 10. This ensures that, for each run loaded, the same method is always
kept in memory.
24
Data Organization
Data Analysis
Data Analysis: Data Review
Figure 10
3
Keep the Current Method for Data Review
You load the master method, most conveniently from the Method tab of the
ChemStation Explorer.
Now, you optimize the integration parameters and use the runs to construct
the calibration table. All future acquisitions using this method will apply these
optimized data analysis parameters.
Load “Sequence Method”
It may also occur that sequence-specific events require changes in all the
methods employed with the sequence. Once every run has been reviewed and
the sequence methods have been improved, the complete sequence can be
reprocessed with the updated methods.
In this scenario, you review the data using the option “Sequence Method” (see
Figure 11).
Data Organization
25
3
Data Analysis
Data Analysis: Data Review
Figure 11
Load the Sequence Method for Data Review
This means that each time you load a run from the Navigation Table, the
sequence method corresponding to the run’s sequence line is loaded.
Once the data review has been completed, it may be necessary to reprocess the
complete sequence in order to apply the method changes not only on a
run-per-run base, but also in the complete sequence context (including
calibration table updates etc.).
Load “Individual Method from Data File (DA.M)”
The review setting “Individual Method from Data File (DA.M)” (see Figure 12),
should be used, if you want to load the individual DA.M automatically along
with the corresponding data file, when this file is loaded using the navigation
table. When you change a method and then load the next run, you will be
asked to save your method changes, because you load a new method: the DA.M
of the next run.
Various workflows may require that you always review data with the
individual data analysis method (DA.M) loaded. For example, when all runs
are loaded with DA.M, the analysis results are the same as during acquisition
26
Data Organization
Data Analysis
Data Analysis: Data Review
3
or last reprocessing. Additionally, it is possible to perform run-specific
changes and save them in the individual data analysis method of the run. This
is especially useful for manual integration events.
Figure 12
Load the Individual Method from Data File for Data Review
Using the individual data analysis method (DA.M) a convenient means is
available to store manual integration events to a specific data file only. So,
when you use the automatic review and print the reports, the specific manual
integrations are reported in one shot.
Use the following steps to copy manual integration events to a method:
1 Perform manual integration until the results are satisfactory.
2 Select Integration / Copy Manual Events to Method
Data Organization
27
3
Data Analysis
Data Analysis: Data Review
Figure 13
Copy Manual Integration Events to a Method
When the manual events have been saved to the individual method (instead of
e.g. the corresponding master method), it is probably most useful always to
apply those events when the chromatogram is integrated:
3 Check the “Manual Events” option in the Integration Events dialog (see
Figure 14).
Figure 14
Apply Manual Integration Events each time the chromatogram is integrated
4 Save the method.
NOTE
28
When a sequence is reprocessed, all actions are performed on the sequence methods and
the DA.M of each data file is overwritten, including the manual integration events.
Data Organization
Data Analysis
The ChemStation User Interface during Data Review
3
The ChemStation User Interface during Data Review
The ChemStation user interface provides a number of features to facilitate
working with the different methods available for data analysis (Figure 15).
Figure 15
User Interface in Data Analysis
• The method modification status is displayed in Data Analysis view, so you
can easily follow if there are unsaved method changes. The user interface
always displays the name of the currently loaded method (together with the
information whether it is an individual data analysis method of a data file
or a sequence method).
• When you move the mouse pointer over this field, a tool tip additionally
displays the complete path and name of the method.
• A dropdown box provides a “shortcut” to the method options of the
Preferences dialog. You can directly enable any of the available options and
it will be applied the next time you load a run from the Navigation Table.
Moreover, it is also very convenient to see which option is currently active.
Note that these options only apply to data review, but not to reprocessing.
Update master method feature
During working on the individual data analysis method, you may decide that
you want to have the data analysis parameters you developed for the
individual method available for the sequence or master method. A right-click
in the Navigation Table on the corresponding run allows you to update your
sequence or master method with the data analysis parameters.
Data Organization
29
3
Data Analysis
The ChemStation User Interface during Data Review
Figure 16
Update the Master or Sequence Method
This feature is available in the following situations:
Table 2 Availability of the Update … Method Functionality
Loaded Method
Available Options
Individual data analysis method (DA.M)
Update Master Method
Update Sequence Method
Sequence method
Update Master Method
Master method
—
It is important to note that this feature only updates data analysis parameters
of the target method, and that it overwrites all data analysis parameters.
NOTE
30
For technical reasons, in addition to the data analysis parameters, the Method Change
History of the target method is also overwritten with History of the source method.
Data Organization
Data Analysis
Data Analysis: Reprocessing data
3
Data Analysis: Reprocessing data
In contrast to data review, sequence reprocessing means that all the runs of a
sequence are reanalyzed in the sequence context, i.e. including calibration
table updates, parameters changes in the sequence table, additions of new
methods to the sequence, etc.
With the new data organization concept, the sequence container includes all
files needed for reprocessing: the data files, a copy of the sequence file, and all
the sequence methods originally employed with the acquisition. Thus, in order
to reprocess a sequence you simply have to load it into the Navigation Table
and the required tool set is available.
Figure 17
Toolset for Sequence Reprocessing
Note the following rules with regard to reprocessing:
• All actions are performed on the sequence methods. If changed analysis
parameters are to be applied, you have to change the sequence methods.
• The method loading settings of the Preferences dialog have no influence on
reprocessing; it always works on the sequence methods or updated
sequence methods. This feature set is valid for reviewing only.
• During reprocessing, the Batch (*.b) file, the sequence/single run log (*.log),
the Navigation Table, and the individual data analysis method (DA.M) of
each processed data file are updated.
• If you want to add new methods from one of the master method directories
to the sequence table, you have to use the Browse item in the list of
methods to browse to any specified method directory (only the methods
already in the sequence container are available without browsing). The new
method is also copied to the sequence container during reprocessing. This
implies that you cannot select a method with the same name as a method
already present in the container.
Data Organization
31
3
Data Analysis
Data Analysis: Reprocessing data
Figure 18
Browse to the Master Methods directory in the Sequence Table
• In the sequence table, it is not possible to add or remove lines.
• In the Sequence Parameters dialog, only the operator name, the sequence
comment, and the usage of sequence table information can be changed. All
other fields have to be set during data acquisition or do not apply to
reprocessing.
32
Data Organization
Data Analysis
Data Analysis: Reprocessing data
Figure 19
Data Organization
3
Sequence Parameters in Data Analysis
33
3
34
Data Analysis
Data Analysis: Reprocessing data
Data Organization
Agilent ChemStation
Getting Started with New ChemStation Workflow
4
Workflow with Unique Folder Creation
switched off
This chapter provides information on working with Unique Folder Creation
switched off which allows you to store data as in ChemStation revisions
B.01.03 or earlier. This mode does not take full advantage of the latest data
review and reprocessing functionality in ChemStation.
Agilent Technologies
35
4
Workflow with Unique Folder Creation switched off
Working with Unique Folder Creation on or off?
Working with Unique Folder Creation on or off?
The new data concept as outlined in the previous chapters provides a number
of advantages:
• Sequence data are not overwritten. Each sequence acquisition stores the
resulting data files in its own sequence container with unique name.
• With the sequence container concept, the data are stored with all necessary
information needed for data analysis, i.e. copies of the sequence file and of
all methods employed with the sequence. These methods can be changed
with sequence specific input and do not influence the original master
method. The container concept thus strengthens the meaning of a sequence
as a set of data files and methods belonging together for result creation.
• Data review and reprocessing are both available in Data Analysis view via
the Navigation Table.
• The data container concept provides the optimal preconditions for the
ChemStation Integration with the Agilent Enterprise Content Manager
(ECM).
However, there may be situations where users may want to store their data as
in ChemStation B.01.03 or earlier and work according to the corresponding
workflows:
• During method development it may be more convenient to have only one
method for both acquisition and data analysis to automatically have
changes available for future acquisition and reanalysis of already acqired
data.
• Data from several acquisitions have to be in one folder, e.g. in case of partial
acquisition.
• Customized macro solutions on a ChemStation system that have been
designed for older revisions may require the data, methods, or sequence to
be stored according to the old data organization scheme.
• When ChemStation B.03.01 runs in a lab where there are also system still
running on ChemStation revisions B.01.03 or earlier, it may be more
convenient to use the same data organization mode on all systems.
36
Data Organization
Workflow with Unique Folder Creation switched off
Workflow with “Unique Folder Creation” switched off
4
Workflow with “Unique Folder Creation” switched off
In order to allow working with a data storage concept as in ChemStation
revisions before B.02.01, the Sequence tab of the Preferences dialog box a Data
Storage section. Here you can choose between “Unique Folder Creation ON”
and “Unique Folder Creation OFF” (Figure 20). Per default, “Unique Folder
Creation ON” is selected. “Unique Folder Creation ON” enables the data
storage concept as outlined in the three chapters above.
Figure 20
NOTE
Data Organization
Preferences Dialog / Sequence Tab
Switching Unique Folder Creation on or off only affects future acquisitions, but does not
change the data organization of already acquired data.
37
4
Workflow with Unique Folder Creation switched off
Workflow with “Unique Folder Creation” switched off
NOTE
We recommend to decide between the two modes at the beginning of your work and not to
switch between them.
Switching Unique Folder Creation off is not supported with the ChemStation ECM
Integration or ChemStore/Security Pack installed.
Selecting “Unique Folder Creation Off” has the following impact on data
storage:
• Sequence data are not acquired into a sequence container, but directly into
the subdirectory as specified in the Sequence Parameters (Figure 3 on
page 15). Therefore, the sequence name pattern is grayed out on the
Sequence tab of the Preferences dialog (Figure 20 on page 37).
• This means that for two or more sequence acquisitions the data may be
acquired into the same subdirectory. This implies the risk to overwrite
existing data, but on the other hand allows to split sequences using partial
sequence execution and still combine the results in one folder (which would
not be possible with Unique Folder Creation switched on).
• No sequence methods (.M) or copies of the sequence file (.S) are stored with
the data, but only the sequence logfile and the batch file (.B). This means
only the methods and sequences in the paths specified in the Preferences
dialog (Figure 2 on page 14) are available. They have to be used for
acquisition as well as for data review and reprocessing. Sequence or data
file specific method changes can only be stored by saving the method with
different name. Otherwise these changes are also applied to the acquisition
method. On the other hand, this may be desired behavior during method
development.
38
Data Organization
Workflow with Unique Folder Creation switched off
Workflow with “Unique Folder Creation” switched off
4
• There are no data file specific methods ACQ.M and DA.M stored. Saving
information about the original acquisition is only possible by including this
information in the report or by selecting “Save Method with Data” from the
method’s runtime checklist (Figure 21). With this option the acquisition
method will be stored as RUN.M in each data file.
Figure 21
Data Organization
Run Time Checklist: Save Method with Data
39
4
Workflow with Unique Folder Creation switched off
Workflow with “Unique Folder Creation” switched off
The enhanced ChemStation user interface as introduced with ChemStation
B.02.01 is also available when Unique Folder Creation is switched off.
However, there are functions you can not take advantage of in this mode. The
same limitations also apply to any run acquired with ChemStation prior to
B.02.01.
• When a sequence is loaded into the Navigation Table, the reprocessing
toolset is grayed out (Figure 22). Sequences that have been acquired in this
data storage mode can only be reprocessed in Method and Run Control view
using the “Reprocessing only” option in the Sequence Parameters
(Figure 23).
40
Figure 22
Navigation Table for Sequences acquired with Unique Folder Creation
switched off
Figure 23
Reprocessing of sequence data acqired with “Unique Folder Creation” off
Data Organization
Workflow with Unique Folder Creation switched off
Workflow with “Unique Folder Creation” switched off
4
• With the method usage options “Use method from data file” and “Use
sequence method” (see “Data Analysis: Data Review” on page 23), a
warning message will be displayed each time a run is double-clicked in the
Navigation Table that the individual method/sequence method does not
exist. As outlined above, these methods are not stored with the data. In this
case, the only meaningful option for data review is “Use current method”.
Data Organization
41
4
42
Workflow with Unique Folder Creation switched off
Workflow with “Unique Folder Creation” switched off
Data Organization
www.agilent.com
In This Book
With revision B.02.01 of ChemStation,
data review and data reprocessing
capabilities have been significantly
improved to enable fast review of result
data.
The new data storage functions in
ChemStation help to efficiently organize
sequence data and methods.
©
Agilent Technologies 2006, 2007
Printed in Germany
02/07
*G2170-90041*
*G2170-90041*
G2170-90041
Agilent Technologies