User Information Plan
Document Information:
Filename
Current Owner
Product Identifier
Project Identifier
Current Lifecycle Phase
Form Rev. Apr. 10, 2000
USERINFO.DOC
Deborah Perkins
H8966A Agilent 1100 Series LC/MSD Trap Operation
SS020138
Release
Revision Log:
Version
Date
A.00
January 6,
2003
A.01
January 8,
2003
A.02
October 6,
2005
Reason For Update
Initial user information plan.
Added Course Timing Considerations provided by John
Hughes
Update for XCT Series and Revision 6.0 Software
Expected Product Availability Date on EPI Warehouse: October 2005.
Localization- 2005 English only
Product Delivery Requirements
Agilent 1100 Series LC/MSD Trap Techniques and Software Operation
H8966A
4 days
This course is designed for those who want to enhance their skills in the techniques and software
operation of the Agilent 1100 Series LC/MSD Trap, SL and VL. The course utilizes a lecture format
with laboratories using a laptop or ChemStation computer. An LC/MSD Trap instrument is not present.
Current software revision is 4.2.
Student Prerequisites
A basic liquid chromatography course such as H1186A, Techniques of HPLC, is required. A basic mass
spectrometry course is desirable as well as one month on the instrument.
2. Course Outline with correlation to file names:
Ion Trap Overview……………………..……………………………………………01_overview.ppt/.doc
Understanding API…………….…………………………………………………………..02_API.ppt/doc
Understanding Ion Trap Mass Spectrometry…………………………………………….03_itms.ppt/doc
Basics of LC/MS/MS …………………………………………………………….04_basicslcmsms.ppt/doc
Agilent Technologies
For Internal Use Only
1
LC/MSD TRAP Sample Introduction and HPLC Configurations…….………05_sampleintro.ppt/doc
Optimizing ESI and APCI for On-Line LC/MS Analysis……………………...06_optimization.ppt/doc
Laboratory: Understanding API Processes and Ion Trap MS/MS………..……07_labapilcms.ppt/doc
LC/MSD ChemStation and Windows Overview………………………………..08_chemstation.ppt/doc
Laboratory Exercise: Chemstation and Windows.………………………….09_chemstationlab.ppt/doc
Starting and Shutting Down………..….……………………………………10_start_shutdownw.ppt/doc
Tuning and Calibrating…………………..…………………………………………11_tune_calib.ppt/doc
Laboratory Exercise: Tuning and Calibrating………..……………………………….12_tuninglab.doc
Flow Injection Analysis (FIA)………………………………………………..13_Flow_inj_analy.ppt/doc
Laboratory Exercise: Flow Injection Analysis……………………….………………14_lab_flowinj.doc
Data Acquisition…………………………………………………………………… 15_acquisition.ppt/doc
Developing Acquisition Methods…………………………………………… 16_acquistion_meth.ppt/doc
Laboratory Exercise: Acquisition Methods………………………………..…..17_lab_aqumethods.doc
Qualitative Data Analysis………………………………………………………18_qual_Anaysis.ppt/doc
Laboratory Exercise: Qualitative Data Analysis………..………………………………19_quallab.doc
Sequences……………………………………………………………………..………20_sequence.ppt/doc
Laboratory Exercise: Sequences………………………………………….…………21_labsequence.doc
Quantitation Using the LC/MSD TRAP………………………………………22_quant_theory.ppt/doc
Laboratory Exercise: Quantitation Theory………………………………………..23_quantlab.ppt/doc
Integration…………………………………………………………….……………24_integration.ppt/doc
Laboratory Exercise………………………………………………………………...25_integrationlab.doc
QuantAnalysis…………………………………………………………………….26_quantitation.ppt/doc
Laboratory Exercise: QuantAnalysis…………………………………………….27_quantitationlab.doc
Reporting………………………………………………………………………………28_reports.ppt/doc
Laboratory Exercise:Reporting…………………………………………………………29_labreport.doc
Diagnosis and Maintenance……………………………………………30_diagnosis_maintenace.ppt/doc
Laboratory Exercise: Diagnosis and Maintenance………………………………31_labmaintenace.doc
Libraries…………………………………………………………………………………32_library.ppt/doc
Agilent Technologies
For Internal Use Only
2
Laboratory Exercise: Libraries…………………………………………….33_lablibrarysearch.ppt/doc
Deconvolution……...…………………………………………………………….34_deconvolution.ppt/doc
Laboratory Exercise: Deconvolution …………………………………………..35_deconvolutionlab.doc
Tools for Peptide Analysis ………………………………………………….………..36_proteinid.ppt/doc
Basics of Mass Spectral Information Obtained by the LC/MSD TRAP………..37_spectinterp.ppt/doc
The whole course manual is contained in two volumes found in the following files:
Volume 1: H8966Ast1.doc
Volume 2: H8966Ast2.doc
To view the instructor notes, load the Word document and click on the Show/Hide icon. To print an
instructor module: File, Print, Options, select Hidden text, OK, Print.
All laboratories are conducted with software revision 6.0.
Data, method, and sequence files are essential for the laboratory exercises. These are found in
Datafiles.exe. You will also need some of demonstration files that come with the software. If acquiring
the data files from a CD-ROM, remove the read-only designations.
Four pdf files are available for professional printing:
H8966Ast1.pdf
H8966Ast2.pdf
H8966Ainst1.pdf
H8966Ainst2.pdf
The instructor pdf files include the userinformation plan (set up directions).
Time on topics
The following are suggestions concerning the time and importance of each section of the course provided
by John Hughes with inputs from other instructors.
Suggested time schedule for topics
Day 1 Survey the class to determine how much time should be spent on API theory. This is covered
several times in the course. Spend the amount of time appropriate to the student’s need on this initial
section, and avoid repetition later in the course.
Section
Suggested time
Overview
Module 1
1 hr
API
Module 2
1hr
Ion Trap MS
Module 3
1.5hr
Transport CID
Module 4
minimum time: just make the point Don’t do
transport CID on the trap.
Agilent Technologies
For Internal Use Only
3
Tandem MS
Module 4
good overview, minimize time
(Spl Introd, LC Method Conv
Module 5
See specific topics below)
Inlet modes
15”
Capillary LC, CE/MS
5-10”, VERY brief overview
Column and mobile phase
15-30”, depending on questions; VERY
important material
Optimization/Solution Chem
Module 6
1.5 hr Some of this is previously covered.
Focus on new material and not revisiting
ionization mechanisms. This is good method
development information.
Lab Exercise- Check your…
Module 7
Time as needed for discussion. Recommend
ending Day 1 with this, or beginning Day 2
with this as a review.
Day 2
30”, If not done on Day 1
Lab exercise
ChemStn/Windows overview
Module 8
WinNT, 15” max; course must assume some
Windows familiarity. Maintaining computer
is important.
5-10”, important for new ChemStn users
Chemstation
Lab Exercise
Module 9
30” max; Windows portion optional
Starting up/shutting down
Module 10
30”. Important topics: starting up from
shutdown; using Standby; shutting down;
checking status (vacuum, helium, etc). Do
not read thru EVERY possible combination
of state change!
Calibration and Tuning
Module 11
Explain differences between “tuning”
(optimizing parameters for a particular
analysis) and calibrating (ensuring the
instrument gives a correct result).
Suggest calling 800# if calibrations fail,
rather than creating calibration problems.
Lab exercise
Module 12
15” if live instrument available for
demonstration
FIA, Lab Exercise
Module 14
15”; primarily ChemStn instructions for
doing flow injection with autosampler.
Clarify FIA Series on LCMSD only if
brought up by student.
Data Acquisition
Module 15
30”. Most of the time should be spent on the
different modes of MSMS.
Agilent Technologies
For Internal Use Only
4
VOLUME 2
Developing Acquisition Methods
Module 16
1 hr cover differences between LC, MSonly, LCMS methods (xxx.M, xxx.ms,
xxx.M containing MS part (Msmethod.yep).
Emphasize making template method from the
write-protected method def_lcms.m, making
all new methods from that.
After setting up MS method and LC method,
demonstrate adding MS part to LC-only
method.
Ensure that students understand that they do
not have to make parallel .M and .ms
methods!!
Module 17
30”, to include practice on creating LC/MS
methods from LC-only and LC/MS or MSonly methods.
Qualitative analysis
Module 18
1 hr. Important topics which must be
illustrated:
-autoscaling axes
–zooming, unzooming
–use of left and right mouse buttons on axes
to scroll and expand
–use of Ctl key with mouse to select spectra
and rapidly scroll across chromatogram
–use of context-sensitive menu (R-click in
Chro window) to rapidly add TICs and
BPCs, edit chros, change # of ListWindows.
Lab exercise, Qualitative analysis
Module 19
1 hr. Ensure that students have learned the
essential skills for browsing the analysis tree,
selecting files, creating chros and spectra.
Ensure that students know the mouse
shortcuts listed above, and the different uses
of the four windows.
Coupled LCMSD Trap Opns-Seq’s
Module 20
15”. This time is primarily for setting the
stage for the lab exercise. Emphasize
checking all parts of the method, especially
the various MS tabs.
Lab Exercise Acquisition
END OF DAY 2
Day 3
Illustrate how to use the shutdown macro in
Agilent Technologies
For Internal Use Only
5
the Sequence parameters panel.
Lab Exercise, Sequences
Module 21
up to 30” depending on student needs
Quantitative Parameters
Module 22
approx 30”. Survey the class to determine
need to cover this topic. Spend the
appropriate amount of time, given the
primary use of the trap as a qualitative tool
by the majority of users.
Lab exercise, Quant Parameters
Module 23
30” max. Instructor’s discretion depending
on class needs.
Integration
Module 24
30”. Emphasize the importance of adding
DA part to the acq method before acquiring
data, setting good data analysis parameters in
the default or template method, and saving
the method for future use. Clarify for
students that the datafile contains embedded
acq and DA parameters. This will make their
lives easier and minimize frustration with
trap DA.
Lab exercise, Integration
Module 25
30” or more. This is one of the most
important labs, as all users will need the Find
tools. Ensure that students are clear on
method parameters, saving the DA part of the
method once optimized, and saving
processed results upon closing datafiles.
Quantitation
Module 26
30” max. The students will need to do the
lab exercise to really learn how to do this.
Survey the class to determine need to cover
this topic. Spend the appropriate amount of
time, given the primary use of the trap as a
qualitative tool by the majority of users.
Lab exercise, Quantitation
Module 27
1 hr. This can be done from the
Familiarization manual also by the students
in their own labs; the data files used here are
on the software CD.
Reports
Module 28
30” max. This is another topic that requires
learning by doing.
Lab exercise, reports
Module 29
1 hr. FIRST the students should try the
standard reports for different kinds of data.
Emphasize NOT overwriting any of the
standard reports (they may be write-protected
or not).
Agilent Technologies
For Internal Use Only
6
Maintenance and Troubleshooting
Module 30
Lab exercise, Maint/Troubleshooting Module 31
1 hr (perhaps on Day 4). It is very important
for students to learn what THEY can do to
prevent and correct problems before placing
a service call. These same skills can help
remote engineer support provide effective
solutions to problems if the customer do
diagnosis properly.
15-30” max. Much of this topic is difficult to
do without a live instrument.
Day 4
Note: Day 4 may need to contain Maintenance and Troubleshooting depending on class needs for
optional topics like quantitation. Ensure that
the essential topics are covered early on Day
4.
Library Searching
Module 32
15’. Really an introduction to the lab.
Survey the class to determine the amount of
time to be spent on this topic.
Lab exercise, Library Search
Module 33
30”. Previous students have indicated that
selecting good spectra is crucial to this lab
working well. Ensure that students can set
Find Parameters properly to do this (e.g.
spectra background subtracted).
Deconvolution
Module 34
30” if class composition determines the need
for this material.
Lab exercise, Deconvolution
Module 35
15”. This lab requires a datafile not found on
the software CD. Ensure that instructors
have all the datafiles for the labs in this
course.
Peptide Tools for Protein ID
Module 36
15” overview of protein id solution. Cover
if student interest, refer students to software
Familiarization manual for details and
practice.
Interpretation of Spectra
Module 37
30-60” if time permits. optional nature of
the material.
3. For marketing purposes of this product see the Product Release Bulletin (if available):
Agilent Technologies
For Internal Use Only
7
Product Release Bulletin available? Yes, upon release
Product Release Bulletin location:
EPI warehouse
4. Instructor or Delivery Agent skills and training required to deliver the product:
The Instructor must be expert in the field of HPLC Applications.
The Instructor must be expert in the field of mass spectrometry.
The Instructor must take the recommended training (listed below).
The instructor must understand and be able to explain the theory of both electrospray and atmospheric
pressure chemical ionization.
The instructor must know the product structure of the Agilent 1100 Series LC/MSD Trap VL and SL.
The instructor must be able to explain electrospray theory.
The instructor must be able to convert common HPLC methods into LC/MSD methods.
The instructor must understand the principles of collision-induced dissociation.
The instructor must be able to explain the isotopic ratios in (M+H)+ clusters.
The instructor must understand how to maintain and work with Windows NT and 2000.
The instructor must understand electrospray solution chemistry, i.e. proper additives to promote
ionization, cationization, etc.
The instructor must understand the principles of chemical ionization (proton affinity principles).
The instructor must be familiar with post-column addition methods for API.
The instructor must understand the autotune and manual tuning processes of the Agilent 1100 LC/MSD
trap.
The instructor must understand how to use FIA for optimization.
The instructor must understand how data is filtered in the Agilent 1100 LC/MSD trap.
The instructor must understand how data is acquired.
The instructor must be thoroughly familiar with typical spray chamber settings and conditions.
The instructor must have a thorough knowledge of the Data Analysis software including background
subtraction, peak purity, and library searching.
The instructor must understand the deconvolution algorithm.
The instructor must understand the best parameters for a deconvolution.
The instructor must understand how to use the software to integrate, build calibration tables, and perform
sequencing with automated recalibration.
The instructor must be able to demonstrate the diagnosis view.
Agilent Technologies
For Internal Use Only
8
The instructor must be able to provide routine maintenance information for the Agilent 1100 LC/MSD
Trap. The instructor must be able to answer questions from customers about Agilent 1100 maintenance.
The instructor must be able to troubleshoot common LC/MSD trap problems as presented in the manual.
The instructor must be able to use the trap to analyze a protein digest as well as peptide tools as an aide
in that process.
The instructor must be able to build a user-contributed library and search the library.
Instructor Training:
Those new to the Agilent 1100 LC/MSD Trap are required to take the following training:
A fundamentals of HPLC and MS course or equivalent experience.
An HPLC method development course or equivalent experience.
Ion Trap Internal Training - (ANCE-MS-II-066)
Gain experience on the equipment. Attend ASMS or equivalent scientific meetings for application
knowledge.
5. Equipment (Instrumentation and Software) required for product delivery:
This course does not require the use of an Agilent 1100 LC/MSD Trap instrument. Customers prefer an
instrument present.
This course only requires ChemStations. Current ChemStation Configuration is:
600 MHz, 128 MB RAM, CD-ROM drive/Sound Card
Speakers (required for listening to the Maintenance and Repair CD-ROM)
Windows 2000
Laserjet with 4 MB RAM
17” display monitor, 1024 X 768 resolution
Agilent 11000 Series LC/MSD Trap software 6.0
HPLC ChemStation software B.01.02
Bioanalysis software (optional)
Note that you cannot demonstrate the dissect feature without the proper license and software (not
required for course).
No more than two students should share a ChemStation. No more than two ChemStations should share a
printer.
Agilent Technologies
For Internal Use Only
9
A high resolution projection system is required for the electronic slide presentation. The instructor is
encouraged to demonstrate software in front of the class with the projection system. The instructor is
also encouraged to obtain show and tell parts for demonstration.
6. Consumables required for product delivery:

Classroom supplies: flip chart, markers, etc.

Floppy disk 3 ½” per ChemStation

7. Samples required for product delivery:
Samples are not required for this course.
8. Laboratory Preparation and Notes
The laboratories will require one ChemStation with the following software for every two students:
HPLC ChemStation B.01.03
IonTrap Software 6.0 or higher
Windows 2000 or Windows XP.
Load the HPLC ChemStation software, B.01.03. The licenses are:
LC2D – TL04041172
Spectral Module – TN040411B9
LC/MSD Trap Control and DataAnalysis OFFLINE Installation.
Note, offline installation will be somewhat different than an online installation. Most notable, the links
between the ChemStation and Ion Trap software are not in place. This can present some problems during
class. It cannot be resolved without the presence of an ion trap instrument.
Note: This installation does not include the Compliance Software. The Compliance software is
only mentioned as an option in this class due to volume.
Offline Installation of Ion Trap 6.0 Software – stand-alone PC’s
1. First, remove any older versions of the ion trap software. Run the Clean PC from MSD Software
batch file.
Agilent Technologies
For Internal Use Only
10
2.
Install the HPLC ChemStation software, revision B.01.03. Licenses appear above.
3.
Go to the Install folder and double-click on Setup.exe.
4.
Click Next> until you get to the license agreement. Accept the license agreement and click Next>.
5.
Select the following Setup Type: Offline and Custom. Select Next>.
6.
Expand the LC/MSD Trap node as shown below and select “This feature will be installed on the local hard drive. Click
Next>.
7.
Click Install.
8.
Finish when prompted.
9.
Install the Supplemental files by launching Setup.exe in the Supplemental Files folder.
10. Select A4(Europe) or Letter(US) as appropriate to your geography.
11. Select the XCT Ultra as the instrument.
12. Finish this installation and reboot the PC.
13. IMPORTANT: The revision 6.0 installs the classic instrument as the default. You must run
the file provided, Offline XCT Ultra Settings.reg to change the Ion Trap Control View from
the Classic to an XCT Ultra. This file changes the registry settings appropriately. Reboot the
PC. This file is available with the course on the EPI Warehouse.
14. Select Start| All Programs| LCMSD Trap| Administration| License Manager .
15. Enter the following license numbers:
Agilent Technologies
For Internal Use Only
11
Laboratory Requirements
Datafiles are found in the EPI Warehouse with this course. Load all the data files
and methods found in Datafiles.exe.
07 – Understanding the API Process and Ion Trap MS/MS
PreLaboratory – nothing needs to be loaded, no supplies required.
Use the Powerpoint slides provided and lead the class through the laboratory. Pause at the
questions and allow the students to try to answer them themselves or in small groups. Then,
review the answers.
09-ChemStation and Windows Overview
PreLaboratory: Floppy disk required. Tell students log in password for administrator.
The students will need to go to their ChemStations. They will do this laboratory by reading the
instructions and executing them with their lab partner. A floppy disk is required for each
ChemStation. Tell students that if they want to skip some sections because they are already
familiar with Windows functions that they can do this.
12-Tuning the LC/MSD
PreLaboratory - No files or supplies are required unless you have an online instrument.
The students can go to the ChemStation with their partner to execute this laboratory. This
laboratory does not have much meaning without an instrument. You may want to go to the lab
and lead the students through the lab and describe your experiences with each step.
Agilent Technologies
For Internal Use Only
12
14-Flow injection Analysis
PreLaboratory – Load the following method and data files:
Caff fia.m
Caffeine data files
The students can go to the ChemStations, but again the lab has little meaning without an
instrument. Walk them through the lab with commentary on your experiences.
17-Acquisition Method
PreLaboratory – Load Sulfms01.d
Students will go to the ChemStations to complete the lab with their partner.
Sulfmso1.d
19-QualAnalysis
PreLaboratory – Load sulflms01.d and sulfmsms.d. Tell students where you have placed these
needed files.
Students can execute this laboratory with their lab partner on the ChemStation without
intervention.
21-Sequences
PreLaboratory – Load Quantms3.m
Students can execute this laboratory with their lab partner on the ChemStation without
intervention.
23-QuantTheory
PreLaboratory – no files or supplies are required
Use the Powerpoint slides to lead the class through this exercise. Allow them to answer the
questions by themselves or in groups before reviewing the answers.
25- Integration
PreLaboratory – You will need to load coeh0006.d and coeh0008.d. Tell your students where you
have placed these files.
Students can perform the exercise on the ChemStations with their lab partner without
intervention.
27-QuantAnalysis
PreLaboratory – Load all the files contained in the Quantitation folder. Tell the students where
you have placed these files.
Students can perform the exercise on the ChemStations with their lab partner without
intervention.
29-Report
PreLaboratory – Load sulfms01.d and logo.bmp. Tell students where you have placed the logo file.
Agilent Technologies
For Internal Use Only
13
Students can perform the exercise on the ChemStations with their lab partner without
intervention.
31-Maintenance
PreLaboratory – no files or supplies needed.
Students will perform exercises and answer questions with their lab partner. At the end of the
exercise, the instructor should go over the questions and answers in the laboratory.
33-Library
PreLaboratory – Load the file, sulfmsms.d. Tell students where to find this file.
Students can perform this lab exercise with their partner without intervention.
35-Deconvolution
PreLaboratory – Load the file, cytc_000.d. Tell students where to find this file.
Students can perform this lab exercise with their partner without intervention.
9. Tools required for product delivery:
Tools are not required to complete this course.
10. Activities required prior to product delivery.
1. Set up ChemStations
2. Set-up and Configure printers.
3. Load all course files required into appropriate directories.
4. Set-up projection system and slide presentations. Set-up instructor software for demonstrations.
5. Place out student manuals, tablets, pencils.
6. Obtain any show-and-tell pieces.
Agilent Technologies
For Internal Use Only
14
11. Activities required during product delivery.
Response to students during laboratory sessions.
Show-and-tell where possible during lectures.
12. Activities required after product delivery.
Ensure that students fill out the evaluation form.
Shut-down equipment.
Report any significant feedback to Deborah Perkins, Deborah_perkins@agilent.com.
Agilent Technologies
For Internal Use Only
15