Agilent 6100 Familiarization Guide

Agilent 6100 Series Quadrupole LC/MS Systems

Familiarization Guide

Agilent Technologies

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Revision A, September 2011

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Agilent 6100 Series Quadrupole LC/MS Systems Familiarization Guide

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.

If you have any comments about this guide, please send an e-mail to feedback_lcms@agilent.com.

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.

5Set 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.

Agilent 6100 Series Quadrupole LC/MS Systems Familiarization Guide 3

4 Agilent 6100 Series Quadrupole LC/MS Systems Familiarization Guide

Contents

1 Prepare for the Analysis 7

Exercise 1. Prepare the LC to run the sample 8

Task 1. Start up ChemStation 8 Tas k 2 . P ur ge t he pu mp 9 Task 3. Prepare the column for the analyses 10

Exercise 2. Prepare the samples for the analyses 12

Exercise 3. Check the current MS tune values and adjust if

necessary 14

2 Set Up and Run a Scan Method 15

Exercise 1. Set up a full-scan acquisition method 16

Task 1. Enter LC acquisition parameters 16 Task 2. Enter MS acquisition parameters 19

Exercise 2. Acquire data with the full-scan method 23

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 31

Exercise 3. Integrate the chromatogram 36

Exercise 4. Print a report 40

4 Set Up and Run a SIM Method 43

Exercise 1. Set up a SIM acquisition method 44

Agilent 6100 Series Quadrupole LC/MS Systems Familiarization Guide 5

Contents

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

Task 1. Enter sample information 49 Task 2. Acquire the data 50

5Set Up and Run a Sequence51

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

6 Agilent 6100 Series Quadrupole LC/MS Systems Familiarization Guide

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 12 Exercise 3. Check the current MS tune values and adjust if necessary 14

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.

Agilent Technologies

1 Prepare for the Analysis

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

1 Open the ChemStation window. • Click the ChemStation icon on the

desktop.

Alternate method:

• From the Start menu, select:

All Programs > Agilent ChemStation > Instrument 1 online.

8 Agilent 6100 Series Quadrupole LC/MS Systems Familiarization Guide

Prepare for the Analysis 1

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

1 Display the Method and Run Control

view.

2 Place the pump in standby mode. a Click More Pump > Control HPLC

3 Prepare solvents used in these

familiarization exercises.

• A – 5 mM ammonium formate in

water

• B – 5 mM ammonium formate in

methanol

4 Replace the solvent bottles with the

ones you just prepared.

5 Open the purge valve. a Turn the black purge valve on the

6 Enter a flow of 5 mL/min and

50% B, using water in channel A and methanol in channel B.

• In the view selection area in the lower left, click Method and Run

Control.

Pump on the Instrument menu to

open the Pump Control dialog box.

b Select Standby and click OK.

a Into a 1-liter reservoir of

HPLC-grade water, add 1 mL of 5 M ammonium formate.

b Into a 1-liter reservoir of

HPLC-grade methanol, add 1 mL of 5 M ammonium formate.

• Replace the bottles for channels A and B.

front of the pump counter-clockwise two turns.

b Place the tubing that exits the pump

into a 250-mL or larger beaker.

a Click the pump icon. b Select Set up Pump. c Enter the parameters in step 6 and

click OK.

Alternate method:

• Select Standby from the Pump context menu.

• The part number for ammonium formate is G1946-85021.

• Each ampoule contains 2.2 mL of ammonium formate solution.

• Be sure to use HPLC-grade solvents.

7 Turn the pump on and monitor the

tubing for bubbles.

a To turn the pump on, click the

little button to the lower right of the solvent delivery (pump) icon.

b Monitor 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.

Agilent 6100 Series Quadrupole LC/MS Systems Familiarization Guide 9

1 Prepare for the Analysis

Task 3. Prepare the column for the analyses

Steps Detailed Instructions Comments

8 After the bubbles are gone and the

purge is complete, enter a flow of 1 mL/min and 100% B.

9 Purge a short while longer, and then

close the purge valve.

a Right-click the pump icon. b Select Set up pump. c Enter the new parameters in step 8,

and click OK.

a Continue to purge for a short while. b Close the black valve.

For more information on purging the pump, see the reference manual that you received with your pump.

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.

Steps Detailed Instructions Comments

1 Disconnect the column from the

detector and MS.

a Turn the pump off by clicking

the little button to the lower right of the solvent delivery (pump) icon.

b Disconnect the column from the

detector and MS.

c Place 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.

2 Flush the column with 100%

methanol at 1 mL/min (5 to 10 min).

• ZORBAX SB-C18, 2.1 mm

3.5 µm, p/n 873700-902

× 30 mm,

a Tur n t h e pum p o n. b Flow 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 columnvolumes of 100% methanol (approximately 5 to 7.5 mL).

10 Agilent 6100 Series Quadrupole LC/MS Systems Familiarization Guide

Prepare for the Analysis 1

Task 3. Prepare the column for the analyses

Steps Detailed Instructions Comments

3 Condition 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

4 Equilibrate the column at the analysis

conditions:

• 12% B for 1/2 hour at 40 °C

5 While 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

• 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

a Click Set up Instrument Method on

the Instrument menu to open the Setup Method dialog box.

b Click the Pump tab. c Enter the flow rate in step 3. d For Solvent B, type 100 and click

Apply. e Wait 30 minutes. f For Solvent B, type 50 and click

Apply. g Wait 30 minutes.

a For Solvent B, type 12 and click

OK. b Click the TCC tab on the Setup

Method dialog box.

c For Temperature, type 40 and click

OK.

a Right-click the MSD

icon on the system

diagram and select

Spray Chamber. b Enter the parameters described in

step 5.

c Click OK. d Wait 10 minutes before you tune the

MS.

• 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.

• 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.

6 Reconnect 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.”

Agilent 6100 Series Quadrupole LC/MS Systems Familiarization Guide 11

1 Prepare for the Analysis

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)

• sulfamethazine (M+H)

• sulfachloropyridazine (M+H)

• sulfadimethoxine (M+H)

= 271

= 279

= 311.

= 285

Sulfamethizole Sulfamethazine Sulfachloropyridazine Sulfadimethoxine

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.

12 Agilent 6100 Series Quadrupole LC/MS Systems Familiarization Guide

Prepare for the Analysis 1

Exercise 2. Prepare the samples for the analyses

Steps Detailed Instructions Comments

1 Prepare 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.

2 Prepare 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.

3 Prepare 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.

4 Prepare a solvent blank in a 1-mL

autosampler vial.

• You will use this sample for the SIM

analysis in Chapter 5.

a Transfer 100 µL of the sulfa mixture

into the autosampler vial.

b Add 900 µL of 90:10 water:methanol

that contains 5 mM ammonium formate (NH

c Cap the vial.

a Transfer 50 µL of the sulfa mixture

into the autosampler vial.

b Add 950 µL of 90:10 water:methanol

that contains 5 mM ammonium formate.

c Cap the vial.

a Transfer 10 µL of the sulfa mixture

into the autosampler vial.

b Add 990 µL of 90:10 water:methanol

that contains 5 mM ammonium formate.

c Cap the vial.

a Into the autosampler vial, transfer

990 µL of 90:10 water:methanol that contains 5 mM ammonium formate.

b Cap the vial.

HCO2).

• The original sulfa mixture is dissolved in a solvent mixture of 70% water and 30% acetonitrile.

Agilent 6100 Series Quadrupole LC/MS Systems Familiarization Guide 13

1 Prepare for the Analysis

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

1 Switch to the MSD Tune view. • In the view selection area in the

lower left, click MSD Tune.

2 Select the tune file. a In the Select Tune File dialog box,

select ATU NE S.TU N.

b Keep the default of Positive Polarity

(Standard). c Click OK. d In 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)

3 Run 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.

4 If Check Tune report suggests that

you adjust peak widths or mass axis, then do that.

5 If the Check Tune report shows poor

sensitivity, which indicates that your MS settings are significantly out of adjustment, then run a full Autotune.

a From the Tune menu, select Adjust

Mass Peak Width. b From the Tune menu, select

Calibrate Mass Axis.

• From the Tune menu, select

Autotune > Positive Polarity.

• Make sure that you use an

appropriate calibrant with an appropriate source.

• 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.

• 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.

14 Agilent 6100 Series Quadrupole LC/MS Systems Familiarization Guide

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 16

Task 1. Enter LC acquisition parameters 16 Task 2. Enter MS acquisition parameters 19

Exercise 2. Acquire data with the full-scan method 23

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.

Agilent Technologies

2 Set Up and Run a Scan Method

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 16

• “Task 2. Enter MS acquisition parameters” on page 19

Task 1. Enter LC acquisition parameters

Steps Detailed Instructions Comments

1 Display the Method and Run Control

view.

2 Open the method DEF_LC.M. a Select File > Load > Method.

3 Save the method under a new name,

SULFAMSSCAN1.M.

4 Enter a volume of

injection.

1 µL for the

• In the view selection area in the

lower left of the ChemStation window, click Method and Run

Control.

b If necessary, navigate to

C:\CHEM32\1\METHODS.

c Select DEF_LC.M and click OK.

a Select File > Save As > Method. b In the dialog box, for Name, type

SULFA MS SCAN 1.M.

c Click OK. d In the box for Comment for method

history, type a comment.

e Click OK.

a Click Set up Instrument Method on

the Instrument menu to open the Setup Method dialog box.

b Click the ALS tab. c Click Standard injection. d In the Injection volume box, type 1

for a 1-µL injection.

• You save the method now with a new name to avoid inadvertently overwriting the default method later.

16 Agilent 6100 Series Quadrupole LC/MS Systems Familiarization Guide

Set Up and Run a Scan Method 2

Task 1. Enter LC acquisition parameters

Steps Detailed Instructions Comments

5 Enter pump parameters. a Click the Pump tab on the Setup

Method dialog box.

b Set the parameters as follows:

Flow=0.400mL/min StopTime=7.00 min PostTime=3.00min

Solvent A=Water 88% Solvent B=Methanol 12%

6 Set up the gradient timetable as it

appears in the figure below.

7 Enter a column compartment

temperature of 40

8 Enter parameters for the diode-array

detector (DAD).

a Open the Timetable area in the

lower part of the tab, click Insert, and type the first line.

b Click Append and type the second

line.

c Repeat step b for lines 3 and 4.

a Click the TCC tab on the Setup

Method dialog box.

b Click the option button for oC. c Ty p e 40.0 for

a Click the DAD tab on the Setup

Method dialog box.

b Enter 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: > 0.1 min

c Click OK to close the Setup Method

dialog box with the new setpoints.

Set the timetable parameters as follows:

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

• The DAD is used in this example, but the variable wavelength detector (VWD) may be used analogously.

Agilent 6100 Series Quadrupole LC/MS Systems Familiarization Guide 17

2 Set Up and Run a Scan Method

Task 1. Enter LC acquisition parameters

Steps Detailed Instructions Comments

9 Select Data Acquisition only in the

Run Time Checklist.

10 Save the new parameters to the

method file, SULFA MS SCAN 1.M.

a Click Run Time Checklist on the

RunControl menu.

b Mark the Data Acquisition check

box.

c Click OK.

a Select File > Save > Method. b In the box for Comment for method

history, type a comment.

c Click 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.”

18 Agilent 6100 Series Quadrupole LC/MS Systems Familiarization Guide

Set Up and Run a Scan Method 2

Set to 100 for 6120 Set to 125 for 6130 or 6150

Task 2. Enter MS acquisition parameters

Steps Detailed Instructions Comments

1 Enter 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

a Right-click the MSD

icon on the system diagram and select Set

up MSD Signals.

b Enter the parameters described in

step 1 and shown in the figure

below. Take care to enter the appropriate Fragmentor voltage for your MS model.

c Click 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).

Agilent 6100 Series Quadrupole LC/MS Systems Familiarization Guide 19

2 Set Up and Run a Scan Method

Task 2. Enter MS acquisition parameters

Steps Detailed Instructions Comments

2 Enter parameters for the spray

chamber of the ion source:

• Drying gas flow: 9 L/min

• Nebulizer pressure: 40 psig

• Drying gas temperature: 300

• Capillary voltage: 3000 V

For 6150 with Agilent Jet Stream technology:

• Nebulizer pressure: 30 psi

• Drying gas flow: 7 L/min

• Drying gas temperature: 350°C

• Sheath gas flow: 12 L/min

• Sheath gas temperature: 360 C°

• Capillary voltage: 4000 V

• Nozzle voltage: 0 V

• Fragmentor: 200 V

• Multiplier gain: 3

a Right-click the MSD

icon on the system diagram and select

Spray Chamber.

b Enter the parameters described in

step 2 and shown in the figure

below.

c Click OK.

For all models except 6150 with Agilent Jet Stream technology

For 6150 with Agilent Jet Stream technology

20 Agilent 6100 Series Quadrupole LC/MS Systems Familiarization Guide

Set Up and Run a Scan Method 2

Task 2. Enter MS acquisition parameters

Steps Detailed Instructions Comments

3 Set up to store the fragmentor voltage

throughout the run.

4 Save the method. a Select Method > Save Method to

a Right-click the MSD

icon on the system diagram and select

Data Curves. b Select Fragmentor - 1. c Click the Add button. d Click OK.

overwrite the method

SULFA MS SCAN 1.M. b In the box for Comment for method

history, type a comment. c Click OK.

Agilent 6100 Series Quadrupole LC/MS Systems Familiarization Guide 21

2 Set Up and Run a Scan Method

Task 2. Enter MS acquisition parameters

Steps Detailed Instructions Comments

5 Print the method. a Select Method > Print Method.

b Mark the check boxes as shown in

the figure below.

c Click the Print button.

22 Agilent 6100 Series Quadrupole LC/MS Systems Familiarization Guide

Set Up and Run a Scan Method 2

Exercise 2. Acquire data with the full-scan method

Now you are ready to acquire data for the sulfa mix with the method you just created. This exercise consists of the following tasks:

• “Task 1. Enter sample information” on page 24

• “Task 2. Acquire the data” on page 25

Agilent 6100 Series Quadrupole LC/MS Systems Familiarization Guide 23

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