Agilent Technologies 6100 Series User manual

Agilent 6100 Series
Quadrupole LC/MS
Systems
Familiarization Guide

Agilent 6100 Series Quadrupole LC/MS Systems Familiarization Guide
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Revision A, March 2014
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pole LC/MS systems, until superseded.

Agilent 6100 Series Quadrupole LC/MS Systems Familiarization Guide 3
In this Guide...
This guide presents a series of exercises to help you learn the
basic operation of your Agilent 6100 Series LC/MS system.
1 Prepare for the Analysis
Use these exercises to prepare the LC, to dilute a sulfa
demonstration sample, and to check the tune on the MS.
2 Set Up and Run a Scan Method
Learn how to set up a scan method and acquire data for the
sulfa demonstration mix.
3 Qualitative Data Analysis
Learn how to examine chromatograms and spectra to identify
sample components. In these exercises, you review data from
the sulfa sample you analyzed in Chapter 2, or from a data file
that you received with your ChemStation software.
4 Set Up and Run a SIM Method
Learn how to set up a selected ion monitoring (SIM) method
and acquire data for the sulfa demonstration mix.
5 Set Up and Run a Sequence
Use these exercises to set up an automated sequence for SIM
analyses of the sulfa mix at various concentrations, and to
acquire data with that sequence.
6 Quantitative Data Analysis
Learn how to analyze data when you need to quantify sample
components. These exercises use caffeine data files that you
received with your ChemStation software.

4 Agilent 6100 Series Quadrupole LC/MS Systems Familiarization Guide

Agilent 6100 Series Quadrupole LC/MS Systems Familiarization Guide 5
Content
1 Prepare for the Analysis 7
Exercise 1. Prepare the LC to run the sample 8
Task 1. Start up ChemStation 8
Task 2. Purge the pump 9
Task 3. Prepare the column for the analyses 10
Exercise 2. Prepare the samples for the analyses 13
Exercise 3. Check the current MS tune values and adjust if necessary 15
2 Set Up and Run a Scan Method 17
Exercise 1. Set up a full-scan acquisition method 18
Task 1. Enter LC acquisition parameters 18
Task 2. Enter MS acquisition parameters 20
Exercise 2. Acquire data with the full-scan method 24
Task 1. Enter sample information 24
Task 2. Acquire the data 25
3 Qualitative Data Analysis 27
Exercise 1. Display and manipulate chromatograms 28
Exercise 2. Examine mass spectra 32
Exercise 3. Integrate the chromatogram 37
Exercise 4. Print a report 41
4 Set Up and Run a SIM Method 43
Exercise 1. Set up a SIM acquisition method 44
Task 1. Load the scan method you created previously 44
Task 2. Enter MS acquisition parameters 45
Exercise 2. Acquire data with the SIM method 48

6 Agilent 6100 Series Quadrupole LC/MS Systems Familiarization Guide
Contents
Task 1. Enter sample information 48
Task 2. Acquire the data 50
5 Set Up and Run a Sequence 51
Exercise 1. Set up a sequence 52
Task 1. Prepare to create a new sequence 52
Task 2. Edit sequence parameters 53
Task 3. Set up the sequence table 55
Task 4. Set up the sequence output 58
Exercise 2. Run the sequence 60
6 Quantitative Data Analysis 61
Exercise 1. Create a method for quantification 62
Task 1. Create a new method 62
Task 2. Set up the signal for quantification 63
Task 3. Integrate the low-level standard 65
Task 4. Set general calibration parameters 67
Task 5. Set up the calibration curve 68
Task 6. Explore options to refine the calibration 72
Exercise 2. Process a sample and print a report 73

7
Agilent 6100 Series Quadrupole LC/MS System
Familiarization Guide
1
Prepare for the Analysis
Exercise 1. Prepare the LC to run the sample 8
Task 1. Start up ChemStation 8
Task 2. Purge the pump 9
Task 3. Prepare the column for the analyses 10
Exercise 2. Prepare the samples for the analyses 13
Exercise 3. Check the current MS tune values and adjust if necessary 15
This chapter presents exercises to help you learn how to:
•Prepare the LC and column for an analysis
•Prepare the samples that you analyze in these exercises
•Check the tune settings of the MS and adjust if necessary.
Before you start
•Order the sample: Agilent Electrospray LC Demo Sample, p/n 59987-20033.
•Order the column: Agilent ZORBAX SB-C18, 2.1 mm x 30 mm, 3.5 μm, p/n
873700-902.
•You may use another similar column, but you may need to adjust the
HPLC conditions to obtain good separation.
•Make sure that the electrospray source is installed.
•Read the Agilent 6100 Series Quadrupole LC/MS Systems Quick Start
Guide and Chapter 2 of the Agilent 6100 Series Quadrupole LC/MS Systems
Concepts Guide.

1 Prepare for the Analysis
Exercise 1. Prepare the LC to run the sample
8 Agilent 6100 Series Quadrupole LC/MS Systems Familiarization Guide
Exercise 1. Prepare the LC to run the sample
For the following tasks, try the steps in the first column. If you need more
help, follow the detailed instructions in the middle column.
Task 1. Start up ChemStation
Steps Detailed Instructions Comments
1Open the ChemStation window. •Click the ChemStation icon on the
desktop.
Alternate method:
•From the Start menu, select:
All Programs > Agilent
ChemStation > Instrument 1
online.

Prepare for the Analysis 1
Task 2. Purge the pump
Agilent 6100 Series Quadrupole LC/MS Systems Familiarization Guide 9
Task 2. Purge the pump
Use these instructions with the binary and quaternary pumps. See the
ChemStation online Help for instructions for the capillary and nanoflow
pumps.
Steps Detailed Instructions Comments
1Display the Method and Run Control
view.
•In the view selection area in the
lower left, click Method and Run
Control.
2Place the pump in standby mode. aClick More Binary Pump > Control
on the Instrument menu to open
the Pump Control dialog box.
bSelect Standby and click OK.
Alternate method:
•Select Standby from the Pump
shortcut menu.
3Prepare solvents used in these
familiarization exercises.
•A – 5 mM ammonium formate in
water
•B – 5 mM ammonium formate in
methanol
aInto a 1-liter reservoir of
HPLC-grade water, add 1 mL of 5 M
ammonium formate.
bInto a 1-liter reservoir of
HPLC-grade methanol, add 1 mL of
5 M ammonium formate.
•The part number for ammonium
formate is G1946-85021.
•Each ampoule contains 2.2 mL of
ammonium formate solution.
4Replace the solvent bottles with the
ones you just prepared.
•Replace the bottles for channels A
and B.
5Open the purge valve. aTurn the black purge valve on the
front of the pump
counter-clockwise two turns.
bPlace the tubing that exits the
pump into a 250-mL or larger
beaker.
6Enter a flow of 5 mL/min and
50% B, using water in channel A and
methanol in channel B.
aRight-click the pump icon.
bSelect Method.
cEnter the parameters in step 6 and
click OK.
•Be sure to use HPLC-grade
solvents.
7Turn the pump on and monitor the
tubing for bubbles.
aTo turn the pump on, click the
green button in the Binary
Pump pane.
bMonitor for bubbles.
•Purge for about 3 minutes to pass
3X the volume for the binary pump.
•If you wish, you may purge each
channel individually first, to ensure
that neither is air-locked.

1 Prepare for the Analysis
Task 3. Prepare the column for the analyses
10 Agilent 6100 Series Quadrupole LC/MS Systems Familiarization Guide
Task 3. Prepare the column for the analyses
In the exercises in the next chapters, you analyze a mixture of four
sulfonamide compounds. To perform the analyses in the following chapters,
you must first condition and equilibrate your column.
8After the bubbles are gone and the
purge is complete, enter a flow of
1 mL/min and 100% B.
aRight-click the pump icon.
bSelect Method.
cEnter the new parameters in step 8,
and click OK.
9Purge a short while longer, and then
close the purge valve.
aContinue to purge for a short while.
bClose the black valve.
For more information on purging the
pump, see the reference manual that
you received with your pump.
Steps Detailed Instructions Comments
Steps Detailed Instructions Comments
1Disconnect the column from the
detector and MS.
aTurn the pump off by clicking
the red button in the Binary
Pump pane.
bDisconnect the column from the
detector and MS.
cPlace the open end of the tubing
that exits the column into the
beaker.
•To prevent detector contamination,
allow the column effluent to go
directly to the waste beaker.
2Flush the column with 100%
methanol at 1 mL/min (5 to 10 min).
•ZORBAX SB-C18, 2.1 mm 30 mm,
3.5 µm, p/n 873700-902
aTurn the pump on.
bFlow methanol through the column
under the conditions used in Task 2,
step 8.
•The data sheet shipped with the
column cartridge recommends that
you flush with 20 to 30 column-
volumes of 100% methanol
(approximately 5 to 7.5 mL).