Agilent Technologies 6100 Series Familiarization Manual
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Agilent Technologies 6100 Series Familiarization Manual

Agilent Technologies 6100 Series Familiarization Manual

Quadrupole lc/ms systems
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  • Page 1 Agilent 6100 Series Quadrupole LC/MS Systems Familiarization Guide...
  • Page 2 FAR 12.211 (Technical Data) and 12.212 (Computer Software) and, for the Department of Defense, DFARS 252.227-7015 (Technical Data - Commercial Items) and DFARS 227.7202-3 (Rights in Commercial Computer Software or Com- puter Software Documentation). Agilent 6100 Series Quadrupole LC/MS Systems Familiarization Guide...
  • Page 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. 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.
  • Page 4 Agilent 6100 Series Quadrupole LC/MS Systems Familiarization Guide...

  • Page 5: Table Of Contents

    Set Up and Run a SIM Method Exercise 1. Set up a SIM acquisition method Task 1. Load the scan method you created previously Task 2. Enter MS acquisition parameters Exercise 2. Acquire data with the SIM method Agilent 6100 Series Quadrupole LC/MS Systems Familiarization Guide...
  • Page 6 Task 3. Integrate the low-level standard Task 4. Set general calibration parameters Task 5. Set up the calibration curve Task 6. Explore options to refine the calibration Exercise 2. Process a sample and print a report Agilent 6100 Series Quadrupole LC/MS Systems Familiarization Guide...

  • Page 7: Prepare For The Analysis

    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.

  • Page 8: Exercise 1. Prepare The Lc To Run The Sample

    Detailed Instructions Comments 1 Open the ChemStation window. • Click the ChemStation icon on the Alternate method: desktop. • From the Start menu, select: All Programs > Agilent ChemStation > Instrument 1 online. Agilent 6100 Series Quadrupole LC/MS Systems Familiarization Guide...

  • Page 9: Task 2. Purge The Pump

    Binary 3X the volume for the binary pump. Pump pane. • If you wish, you may purge each b Monitor for bubbles. channel individually first, to ensure that neither is air-locked. Agilent 6100 Series Quadrupole LC/MS Systems Familiarization Guide...

  • Page 10: Task 3. Prepare The Column For The Analyses

    ZORBAX SB-C18, 2.1 mm 30 mm, under the conditions used in Task 2, you flush with 20 to 30 column- 3.5 µm, p/n 873700-902 step volumes of 100% methanol (approximately 5 to 7.5 mL). Agilent 6100 Series Quadrupole LC/MS Systems Familiarization Guide...
  • Page 11 Setup Method dialog box. work on the rest of the exercises in c For Temperature, type 40 and click this chapter. Be sure to complete step 6 before you go on to the next chapter. Agilent 6100 Series Quadrupole LC/MS Systems Familiarization Guide...
  • Page 12 DAD and MS, but you do need to reconnect prior to the exercises Chapter 2, “Set Up and Run a Scan Method.” Agilent 6100 Series Quadrupole LC/MS Systems Familiarization Guide...

  • Page 13: Exercise 2. Prepare The Samples For The Analyses

    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. Agilent 6100 Series Quadrupole LC/MS Systems Familiarization Guide...
  • Page 14 Into the autosampler vial, transfer autosampler vial. 990 µL of 90:10 water:methanol that • You will use this sample for the contains 5 mM ammonium formate. SIM analysis in Chapter b Cap the vial. Agilent 6100 Series Quadrupole LC/MS Systems Familiarization Guide...

  • Page 15: Exercise 3. Check The Current Ms Tune Values And Adjust If Necessary

    MS settings are significantly out of standard scan speeds, so it is not adjustment, then run a full Autotune. necessary to tune for negative polarity or fast scan. Agilent 6100 Series Quadrupole LC/MS Systems Familiarization Guide...
  • Page 16 Prepare for the Analysis Exercise 3. Check the current MS tune values and adjust if necessary Agilent 6100 Series Quadrupole LC/MS Systems Familiarization Guide...

  • Page 17: Set Up And Run A Scan Method

    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...

  • Page 18: Exercise 1. Set Up A Full-Scan Acquisition Method

    Click Set up Instrument Method on injection. the Instrument menu to open the Setup Method dialog box. b Click the HiP Sampler tab. c In the Injection volume box, type 1 for a 1-µL injection. Agilent 6100 Series Quadrupole LC/MS Systems Familiarization Guide...
  • Page 19 10 Save the new parameters to the a Select File > Save > Method. method file, 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...

  • Page 20: Task 2. Enter Ms Acquisition Parameters

    Click OK. • Stepsize: 0.10 • Peakwidth: 0.05 min • Scan data storage: Condensed • Active signals: 1 only Set to 100 for 6120 Set to 125 for 6130 or 6150 Agilent 6100 Series Quadrupole LC/MS Systems Familiarization Guide...
  • Page 21 Sheath gas flow: 12 L/min • Capillary voltage: 4000 V • Nozzle voltage: 2000 V For all models except 6150 with Agilent Jet Stream technology For 6150 with Agilent Jet Stream technology Agilent 6100 Series Quadrupole LC/MS Systems Familiarization Guide...
  • Page 22 4 Save the method. a Select Method > Save Method to 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...
  • Page 23 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. Agilent 6100 Series Quadrupole LC/MS Systems Familiarization Guide...

  • Page 24: Exercise 2. Acquire Data With The Full-Scan Method

    Comments 1 Display the Single Sample toolbar. • In the top toolbar, click the single sample icon. 2 Display the Sample Information a Click Sample Info on the dialog box. RunControl menu. Agilent 6100 Series Quadrupole LC/MS Systems Familiarization Guide...

  • Page 25: Task 2. Acquire The Data

    2 Inject the sulfa mix sample. • Click the Single Sample button to This button is present only when you start the run. have selected Single Sample mode from the top toolbar. Agilent 6100 Series Quadrupole LC/MS Systems Familiarization Guide...
  • Page 26 During these initial injections, everything may be eluted from the column in the void volume. Repeat the process and separation will occur. Agilent 6100 Series Quadrupole LC/MS Systems Familiarization Guide...

  • Page 27: Qualitative Data Analysis

    • Read the Agilent 6100 Series Quadrupole LC/MS Systems Quick Start you generated in Guide. Chapter • Read the chapter on Data Analysis in the Agilent 6100 Series Quadrupole Alternatively, you LC/MS Systems Concepts Guide. can use the sulfa demo data file •...

  • Page 28: Exercise 1. Display And Manipulate Chromatograms

    C:\CHEM32\1\METHODS. loaded. c Select the method file and click OK. 3 Display the Signal Toolset. • Click the Signal icon, which is near the middle of the window. Agilent 6100 Series Quadrupole LC/MS Systems Familiarization Guide...
  • Page 29 Select the data file. file you generated in Chapter 2. You d Set other parameters as shown need the report from that data file below and click OK. to set up your SIM groups. Agilent 6100 Series Quadrupole LC/MS Systems Familiarization Guide...
  • Page 30 Check that you see the overlaid click the icon shown above to turn chromatograms, as shown below. on the display of the Graphics c Click the icon to display Toolset. separate signals. Agilent 6100 Series Quadrupole LC/MS Systems Familiarization Guide...
  • Page 31 • For other ways to remove signals, the method, click OK. see the chapter on Data Analysis in d Verify that the DAD window is gone the Concepts Guide. and only the TIC is displayed. Agilent 6100 Series Quadrupole LC/MS Systems Familiarization Guide...

  • Page 32: Exercise 2. Examine Mass Spectra

    If there is not room under your chromatogram window to display spectra, use your mouse pointer to reduce the height of the chromatogram window. Agilent 6100 Series Quadrupole LC/MS Systems Familiarization Guide...
  • Page 33 If you cannot see the spectra, adjust the size and location of the window labeled Reference Mass Spectrum(a). b Note the two background spectra — one before the peak and one after. Agilent 6100 Series Quadrupole LC/MS Systems Familiarization Guide...
  • Page 34 • Near the middle of the Data selected are specified there. Analysis window, you can view and e Click OK. change the setting for background subtraction. Agilent 6100 Series Quadrupole LC/MS Systems Familiarization Guide...
  • Page 35 In the chromatogram average spectrum is usually more window, click and drag the mouse accurate. across the peak, as shown below. c View the average spectrum in the window labeled MS Spectrum. Agilent 6100 Series Quadrupole LC/MS Systems Familiarization Guide...
  • Page 36 Qualitative Data Analysis Exercise 2. Examine mass spectra Steps Detailed Instructions Comments 9 Be sure to see step 6 “Exercise 3. Integrate the chromatogram” for an easier, faster way to display spectra. Agilent 6100 Series Quadrupole LC/MS Systems Familiarization Guide...

  • Page 37: Exercise 3. Integrate The Chromatogram

    Click the icon to zoom out. 2 Display the Integration Toolset. • Click the Integration icon, which is near the middle of the window. Agilent 6100 Series Quadrupole LC/MS Systems Familiarization Guide...
  • Page 38 • If you do not see the pink integration baseline, in the graphics tools, click the icon to display baselines. Agilent 6100 Series Quadrupole LC/MS Systems Familiarization Guide...
  • Page 39 ChemStation: Understanding Your table, for Baseline Correction, ChemStation. select Advanced. c For Height Reject, type 500000. d Click the Integrate current Chromatogram icon. e Verify that your results are similar to those shown below. Agilent 6100 Series Quadrupole LC/MS Systems Familiarization Guide...
  • Page 40 Verify that the spectrum is similar to the one shown below. Agilent 6100 Series Quadrupole LC/MS Systems Familiarization Guide...

  • Page 41: Exercise 4. Print A Report

    In the Specify Report dialog box, screen. under Destination, mark the check box for Screen. c For Report Style, select LCMS Qualitative. d Check that other settings are as shown below. e Click OK. Agilent 6100 Series Quadrupole LC/MS Systems Familiarization Guide...
  • Page 42 SULFA MS SCAN 1.M. spectral display options, report b In the box for Comment for method settings, and other data analysis history, type a comment. settings become part of the c Click OK. method. Agilent 6100 Series Quadrupole LC/MS Systems Familiarization Guide...

  • Page 43: Set Up And Run A Sim Method

    Agilent 6100 Series Quadrupole LC/MS System Familiarization Guide Set Up and Run a SIM Method Exercise 1. Set up a SIM acquisition method Task 1. Load the scan method you created previously Task 2. Enter MS acquisition parameters Exercise 2. Acquire data with the SIM method Task 1.

  • Page 44: Exercise 1. Set Up A Sim Acquisition Method

    In the dialog box, for Name, type inadvertent overwrites of your scan method. SULFA MS SIM 1.M. c Click OK. d In the box for Comment for method history, type a comment. e Click OK. Agilent 6100 Series Quadrupole LC/MS Systems Familiarization Guide...

  • Page 45: Task 2. Enter Ms Acquisition Parameters

    (to the nearest 0.1) for the 271 ion. d Click Add Ion, and type the mass for the sulfamethizole 156 ion. Agilent 6100 Series Quadrupole LC/MS Systems Familiarization Guide...
  • Page 46 • If the retention time difference between sulfachloropyridazine and sulfamethazine is less than 0.3 minutes, merge these ions into one group. • The sulfachloropyridazine additionally includes the chlorine isotope at m/z 287. Agilent 6100 Series Quadrupole LC/MS Systems Familiarization Guide...
  • Page 47 4 Save the method. a Select Method > Save Method to overwrite the method SULFA MS SIM 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...

  • Page 48: Exercise 2. Acquire Data With The Sim Method

    Comments 1 Display the Single Sample toolbar. • In the top toolbar, click the single sample icon. 2 Display the Sample Information a Click Sample Info on the dialog box. RunControl menu. Agilent 6100 Series Quadrupole LC/MS Systems Familiarization Guide...
  • Page 49 • Prefix: Sulfa_SIM below. from one run to the next. • Location: Vial 1 b Click OK. • Sample Name: Sulfas 10 ng/µL • Comment: SIM familiarization exercise Agilent 6100 Series Quadrupole LC/MS Systems Familiarization Guide...
  • Page 50 • If you need help, follow the general results. b Load the data file you just created. procedure in “Exercise 1. Display c Examine the DAD and MS and manipulate chromatograms. chromatograms” on page 28 in Chapter Agilent 6100 Series Quadrupole LC/MS Systems Familiarization Guide...

  • Page 51: Set Up And Run A Sequence

    You also run a solvent blank. Before you start • Read 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. • Complete the previous exercises in this manual.

  • Page 52: Exercise 1. Set Up A Sequence

    • Select Sequence> Sequence Table. 5 Save the sequence under a new a Select Sequence > Save Sequence name, SULFA MS SIM 1.S Template As. b For Name, type SULFA MS SIM 1.S. c Click OK. Agilent 6100 Series Quadrupole LC/MS Systems Familiarization Guide...

  • Page 53: Task 2. Edit Sequence Parameters

    Subdirectory: Sulfas sequence. The directory will be • Prefix: Sulfa_seq created if it doesn’t already exist on your computer. • Unique file names are automatically created for each data file within the subdirectory. Agilent 6100 Series Quadrupole LC/MS Systems Familiarization Guide...
  • Page 54 MS state to standby. the event of an error. See the online Help for commands. • SHUTDOWN.MAC is a macro that will shut down the system, but you must customize it before using it. Agilent 6100 Series Quadrupole LC/MS Systems Familiarization Guide...

  • Page 55: Task 3. Set Up The Sequence Table

    Run a SIM Method”. • Sample Type=Sample ways — use of the Insert/Filldown c Select Line 1. Wizard. d Click the Insert/Filldown Wizard button. e Complete the Insert/Filldown Wizard as shown in the next figure. Agilent 6100 Series Quadrupole LC/MS Systems Familiarization Guide...
  • Page 56 • Your results will likely differ, but in have created so far. below. the next step you can recreate the b Note any differences, such as table format below. columns that are included and column widths. Agilent 6100 Series Quadrupole LC/MS Systems Familiarization Guide...
  • Page 57 Vial 1 – blank b Click OK. • Remaining vials – sulfa mix at 1, 5 and 10 ng/µL 5 Save the sequence. • Click the Save Sequence button in the Sequence toolset. Agilent 6100 Series Quadrupole LC/MS Systems Familiarization Guide...

  • Page 58: Task 4. Set Up The Sequence Output

    Click OK in the Sequence Summary Understanding Your ChemStation. Parameters dialog box. • The setup shown in the dialog box g Click OK in the Sequence below prints the simplest summary Parameters dialog box. report. Agilent 6100 Series Quadrupole LC/MS Systems Familiarization Guide...
  • Page 59 If you click the Print All button, you Template. print all the parts of the sequence b Mark the check boxes as shown in rather than the items you just the figure below. specified. c Click the Print button. Agilent 6100 Series Quadrupole LC/MS Systems Familiarization Guide...

  • Page 60: Exercise 2. Run The Sequence

    • When you analyze your own other data files. samples, you can set up your method to automatically generate a data analysis report for each sample in the sequence. Agilent 6100 Series Quadrupole LC/MS Systems Familiarization Guide...

  • Page 61: Quantitative Data Analysis

    • Read the Agilent 6100 Series Quadrupole LC/MS Systems Quick Start Guide. • Read the chapter on Data Analysis in the Agilent 6100 Series Quadrupole LC/MS Systems Concepts Guide. • Make sure that you have the caffeine data files on your ChemStation. Check for the files under C:\CHEM32\1\DATA\MSDEMO.

  • Page 62: Exercise 1. Create A Method For Quantification

    Navigate to the folder C:\CHEM32\1\METHODS. c In the dialog box, for Name, type CAFFEINE CAL.M. d Click OK. e In the box for Comment for method history, type a comment. f Click OK. Agilent 6100 Series Quadrupole LC/MS Systems Familiarization Guide...

  • Page 63: Task 2. Set Up The Signal For Quantification

    For Ion 1, type 195.1. c For Ion 2, type 195.1. d Click OK. 3 Display the Calibration Toolset. • Click the Calibration icon, which is near the middle of the window. Agilent 6100 Series Quadrupole LC/MS Systems Familiarization Guide...
  • Page 64 (CAFFEINE CAL.M). b In the box for Comment for method method frequently, but you could history, type a comment. wait instead until you had c Click OK. established all the method settings. Agilent 6100 Series Quadrupole LC/MS Systems Familiarization Guide...

  • Page 65: Task 3. Integrate The Low-Level Standard

    Click the Auto Integrate icon. d When you are prompted to save the events table, click Yes. e Verify that your results are the same or very similar to those shown below. Agilent 6100 Series Quadrupole LC/MS Systems Familiarization Guide...
  • Page 66 5 (Optional) Save the method under the a Select File > Save > Method. same name (CAFFEINE CAL.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...

  • Page 67: Task 4. Set General Calibration Parameters

    2 (Optional) Save the method under the a Select File > Save > Method. same name (CAFFEINE CAL.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...

  • Page 68: Task 5. Set Up The Calibration Curve

    Select Calibration > invalid. New Calibration Table. b Click Automatic Setup Level 1. c Click OK. d In the Calibration Table pane (shown below), under Compound, type caffeine and under Amt (amount), type 0.5. Agilent 6100 Series Quadrupole LC/MS Systems Familiarization Guide...
  • Page 69 Mark the check box for Load using Signal Details. d Mark the check box for Integrate after load. e Check that your dialog box looks like the one below. f Click OK. Agilent 6100 Series Quadrupole LC/MS Systems Familiarization Guide...
  • Page 70 Repeat step a through step f until you have added all the standards. h Confirm that your calibration table and calibration curve look like the ones below. Agilent 6100 Series Quadrupole LC/MS Systems Familiarization Guide...
  • Page 71 7 (Optional) Save the method under the a Select File > Save > Method. same name (CAFFEINE CAL.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...

  • Page 72: Task 6. Explore Options To Refine The Calibration

    +-: A window for the expected percentage. 3 Display the original options for the a Select Calibration Table Options > calibration table. Overview. b Verify that the calibration table looks the same as in step 5 page Agilent 6100 Series Quadrupole LC/MS Systems Familiarization Guide...

  • Page 73: Exercise 2. Process A Sample And Print A Report

    External standard (ESTD) icon. calculation, based on area b Enter parameters as • Report style: Detail described in step 1 and shown in the next two figures. c Click Quantitation settings. d Click OK. Agilent 6100 Series Quadrupole LC/MS Systems Familiarization Guide...
  • Page 74 In the box for Comment for method history, type a comment. c Click OK. 3 Load the standard of medium a Select File > Load Signal. concentration. b Under File name, select CAFCAL03.D. Agilent 6100 Series Quadrupole LC/MS Systems Familiarization Guide...
  • Page 75 (Optional) At the bottom of the report window, click the Print button so you get a hard copy. g At the bottom of the report window, click the Close button. Agilent 6100 Series Quadrupole LC/MS Systems Familiarization Guide...
  • Page 76 Quantitative Data Analysis Exercise 2. Process a sample and print a report Agilent 6100 Series Quadrupole LC/MS Systems Familiarization Guide...
  • Page 78 • Set up methods for scan and selected ion monitoring analyses • Acquire data • Set up sequences for automated sample analyses • Perform qualitative and quantitative analyses.  Agilent Technologies, Inc. 2014 Printed in USA Revision A, March 2014 *G1960-90084* G1960-90084...

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