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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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).

Prepare for the Analysis 1
Task 3. Prepare the column for the analyses
Agilent 6100 Series Quadrupole LC/MS Systems Familiarization Guide 11
3Condition the column as follows,
using the solvents made up in Task 2,
step 6:
•Flow rate – 0.4 mL/min
•100% B for 1/2 hour
•50% B for 1/2 hour
aClick Set up Instrument Method on
the Instrument menu to open the
Setup Method dialog box.
bClick the Binary Pump tab.
cEnter the flow rate in step 3.
dFor Solvent B, type 100 and click
Apply.
eWait 30 minutes.
fFor Solvent B, type 50 and click
Apply.
gWait 30 minutes.
•At a flow rate of 0.4 mL/min, the
checkout column should produce
about 70 to 80 bar pressure
(measured without any fittings at
the column exit).
•If, after you perform these steps,
the pump pressure through the
column is too high, order a
replacement SB-C18 column (p/n
873700-902).
•If your column is not new, you can
reduce the length of time that you
condition the column.
4Equilibrate the column at the analysis
conditions:
•12% B for 1/2 hour at 40 C
aFor Solvent B, type 12 and click
OK.
bClick the Column Comp. tab on the
Setup Method dialog box.
cFor Temperature, type 40 and click
OK.
•While you condition and equilibrate
the column, you may complete
step 5 in this exercise and then
work on the rest of the exercises in
this chapter. Be sure to complete
step 6 before you go on to the next
chapter.
Steps Detailed Instructions Comments

1 Prepare for the Analysis
Task 3. Prepare the column for the analyses
12 Agilent 6100 Series Quadrupole LC/MS Systems Familiarization Guide
5While the column equilibrates, set
parameters for the MS spray
chamber so it can heat and
equilibrate as well.
•Drying gas flow: 8 L/min
•Nebulizer pressure: 35 psig
•Drying gas temperature: 300 oC
•Capillary voltage: 3000 V
For 6150 with Agilent Jet Stream
technology:
•Sheath Gas Flow: 12 L/min
•Sheath Gas Temp: 360°C
•Nozzle Voltage: 0 V
aRight-click the MSD
icon on the system
diagram and select
Spray Chamber.
bEnter the parameters described in
step 5.
cClick OK.
dWait 10 minutes before you tune
the MS.
6Reconnect the column to the DAD
and MS.
•You can complete “Exercise 3.
Check the current MS tune values
and adjust if necessary” either with
or without the column connected to
the DAD and MS, but you do need
to reconnect prior to the exercises
in Chapter 2, “Set Up and Run a
Scan Method.”
Steps Detailed Instructions Comments

Prepare for the Analysis 1
Exercise 2. Prepare the samples for the analyses
Agilent 6100 Series Quadrupole LC/MS Systems Familiarization Guide 13
Exercise 2. Prepare the samples for the analyses
In the exercises in the next chapters, you analyze a mixture of four
sulfonamide compounds. The Electrospray LC Demo Sample (p/n
59987-20033), contains five ampoules with 100 ng/μL each of these
compounds:
•sulfamethizole (M+H)+= 271
•sulfamethazine (M+H)+= 279
•sulfachloropyridazine (M+H)+= 285
•sulfadimethoxine (M+H)+= 311.
To perform the analyses in the following chapters, you must first prepare the
sample at various dilutions. The final concentrations will be 1, 5 and 10 ng/μL.
You will also prepare a solvent blank.
Sulfamethizole Sulfamethazine Sulfachloropyridazine Sulfadimethoxine

1 Prepare for the Analysis
Exercise 2. Prepare the samples for the analyses
14 Agilent 6100 Series Quadrupole LC/MS Systems Familiarization Guide
Steps Detailed Instructions Comments
1Prepare a 1:10 dilution of the original
sample in a 1-mL autosampler vial.
•Final concentration is 10 ng/µL
•You will use this sample for the
scan analysis in Chapter 2, and for
the SIM analyses in Chapter 4 and
Chapter 5.
aTransfer 100 µL of the sulfa mixture
into the autosampler vial.
bAdd 900 µL of 90:10 water:methanol
that contains 5 mM ammonium
formate (NH4HCO2).
cCap the vial.
•The original sulfa mixture is
dissolved in a solvent mixture of
70% water and 30% acetonitrile.
2Prepare a 1:20 dilution of the original
sample in a 1-mL autosampler vial.
•Final concentration is 5 ng/µL
•You will use this sample for the
SIM analysis in Chapter 5.
aTransfer 50 µL of the sulfa mixture
into the autosampler vial.
bAdd 950 µL of 90:10 water:methanol
that contains 5 mM ammonium
formate.
cCap the vial.
3Prepare a 1:100 dilution of the
original sample in a 1-mL
autosampler vial.
•Final concentration is 1 ng/µL
•You will use this sample for the
SIM analysis in Chapter 5.
aTransfer 10 µL of the sulfa mixture
into the autosampler vial.
bAdd 990 µL of 90:10 water:methanol
that contains 5 mM ammonium
formate.
cCap the vial.
4Prepare a solvent blank in a 1-mL
autosampler vial.
•You will use this sample for the
SIM analysis in Chapter 5.
aInto the autosampler vial, transfer
990 µL of 90:10 water:methanol that
contains 5 mM ammonium formate.
bCap the vial.

Prepare for the Analysis 1
Exercise 3. Check the current MS tune values and adjust if necessary
Agilent 6100 Series Quadrupole LC/MS Systems Familiarization Guide 15
Exercise 3. Check the current MS tune values and adjust if necessary
The MS is very stable and does not need to be tuned very often. You can
usually tune just once a month, or once a week at most. You can use the Check
Tune program described in this exercise to confirm that the MS is in
adjustment.
Steps Detailed Instructions Comments
1Switch to the MSD Tune view. •In the view selection area in the
lower left, click MSD Tune.
2Select the tune file. aIn the Select Tune File dialog box,
select ATUNES.TUN.
bKeep the default of Positive
Polarity (Standard).
cClick OK.
dIn the status bar near the top of the
MSD Tune view, verify that you see
the following:
•Mode is API-ES
•Source is ESI (electrospray)
•Make sure that you use an
appropriate calibrant with an
appropriate source.
3Run a Check Tune. •From the Tune menu, select Check
Tune.
Note that Check Tune requires
values for comparison that are
determined from a previous
Autotune. Autotune is normally run
during installation.
•Check Tune is normally all that you
need to do to confirm that the MS
settings are correct.
•If Check Tune indicates a problem
with your MS settings, then
proceed to step 4 and/or step 5.
4If Check Tune report suggests that
you adjust peak widths or mass axis,
then do that.
aFrom the Tune menu, select Adjust
Mass Peak Width.
bFrom the Tune menu, select
Calibrate Mass Axis.
5If the Check Tune report shows poor
sensitivity, which indicates that your
MS settings are significantly out of
adjustment, then run a full Autotune.
•From the Tune menu, select
Autotune > Positive Polarity.
•The exercises in this manual use
only the positive ion mode and
standard scan speeds, so it is not
necessary to tune for negative
polarity or fast scan.

1 Prepare for the Analysis
Exercise 3. Check the current MS tune values and adjust if necessary
16 Agilent 6100 Series Quadrupole LC/MS Systems Familiarization Guide

17
Agilent 6100 Series Quadrupole LC/MS System
Familiarization Guide
2
Set Up and Run a Scan Method
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
These exercises show you how to set up a scan data acquisition method for the
demonstration sample (sulfa mix) and to acquire data with that method.
The LC parameters that you enter in these exercises are appropriate for the
standard Agilent 1100/1200/1260/1290 Series liquid chromatography (LC)
systems. You must enter LC parameters that are appropriate for your LC
model.
To view the results of these exercises, see Chapter 3, “Qualitative Data
Analysis.”
Before you start
•Review the Agilent 6100 Series Quadrupole LC/MS Systems Quick Start
Guide and Chapter 3 of the Agilent 6100 Series Quadrupole LC/MS Systems
Concepts Guide.
•Prepare the LC, column and sample as described in Chapter 1, “Prepare for
the Analysis.”
For the tasks on the following pages, try the steps on the left without the
detailed instructions. If you need more help, follow the detailed instructions
on the right.

2 Set Up and Run a Scan Method
Exercise 1. Set up a full-scan acquisition method
18 Agilent 6100 Series Quadrupole LC/MS Systems Familiarization Guide
Exercise 1. Set up a full-scan acquisition method
This exercise changes the default method and saves it as a new method. This
exercise consists of the following tasks:
•“Task 1. Enter LC acquisition parameters” on page 18
•“Task 2. Enter MS acquisition parameters” on page 20
Task 1. Enter LC acquisition parameters
Steps Detailed Instructions Comments
1Display the Method and Run Control
view.
•In the view selection area in the
lower left of the ChemStation
window, click Method and Run
Control.
2Open the method DEF_LC.M.aSelect File > Load > Method.
bIf necessary, navigate to
C:\CHEM32\1\METHODS.
cSelect DEF_LC.M and click OK.
3Save the method under a new name,
SULFA MS SCAN 1.M.
aSelect File > Save As > Method.
bIn the dialog box, for Name, type
SULFA MS SCAN 1.M.
cClick OK.
dIn the box for Comment for method
history, type a comment.
eClick OK.
•You save the method now with a
new name to avoid inadvertently
overwriting the default method
later.
4Enter a volume of 1 µL for the
injection.
aClick Set up Instrument Method on
the Instrument menu to open the
Setup Method dialog box.
bClick the HiP Sampler tab.
cIn the Injection volume box, type 1
for a 1-µL injection.

Set Up and Run a Scan Method 2
Task 1. Enter LC acquisition parameters
Agilent 6100 Series Quadrupole LC/MS Systems Familiarization Guide 19
5Enter pump parameters. aClick the Binary Pump tab on the
Setup Method dialog box.
bSet the parameters as follows:
Flow=0.400mL/min
StopTime=7.00 min
PostTime=3.00min
Solvent A=5mM ammonium
formate in water
Solvent B=5mM ammonium
formate in methanol
6Set up the gradient timetable:
Line 1 Time 1:00, %B=12, Flow=0.4
Line 2 Time 3:00, %B=100, Flow=0.4
Line 3 Time 6:00, %B=100, Flow=0.4
Line 4 Time 7:00, %B=12, Flow=0.4
aOpen the Timetable area in the
lower part of the tab, click Add, and
type the first line.
bClick Add and type the second line.
cRepeat step b for lines 3 and 4.
7Enter a column compartment
temperature of 40 oC.
aClick the Column Comp. tab on the
Setup Method dialog box.
bClick the option button for oC.
cType 40.0 for oC.
8Enter parameters for the diode-array
detector (DAD).
aClick the DAD tab on the Setup
Method dialog box.
bEnter the parameters shown below:
•Use Signal A: Wavelength 272nm,
Bandwidth16 nm
•Reference Wavelength: 360 nm,
Reference Bandwidth 100 nm
•Spectrum Store: All in peak
•Peakwidth: > 0.10 min
cClick OK to close the Setup Method
dialog box with the new setpoints.
•The DAD is used in this example,
but the variable wavelength
detector (VWD) may be used
analogously.
9Select Data Acquisition only in the
Run Time Checklist.
aClick Run Time Checklist on the
Method menu.
bMark the Data Acquisition check
box.
cClick OK.
•While it is common to include Data
Analysis in the Run Time Checklist,
for these exercises, you will view
the results in Chapter 3,
“Qualitative Data Analysis.”
10 Save the new parameters to the
method file, SULFA MS SCAN 1.M.
aSelect File > Save > Method.
bIn the box for Comment for method
history, type a comment.
cClick OK.
Steps Detailed Instructions Comments

2 Set Up and Run a Scan Method
Task 2. Enter MS acquisition parameters
20 Agilent 6100 Series Quadrupole LC/MS Systems Familiarization Guide
Task 2. Enter MS acquisition parameters
Steps Detailed Instructions Comments
1Enter parameters for the quadrupole
mass spectrometer (MS):
•Signal 1, scan mode, positive
polarity
•Scan range: 100 to 500
•Fragmentor:
100 V for the Agilent 6120;
125 V for the 6130 or 6150
•Gain: 1.00
•Threshold: 150
•Stepsize: 0.10
•Peakwidth: 0.05 min
•Scan data storage: Condensed
•Active signals: 1 only
aRight-click the MSD
icon on the system
diagram and select Set
up MSD Signals.
bEnter the parameters described in
step 1 and shown in the figure
below. Take care to enter the
appropriate Fragmentor voltage for
your MS model.
cClick OK.
•To save disk space you usually
acquire line spectra (Scan Data
Storage = Condensed). However,
when you acquire spectra from
intact proteins or protein
digests/peptides, you must acquire
and deconvolute profile spectra.
(Scan Data Storage = Full).
Set to 100 for 6120
Set to 125 for 6130 or 6150