MSQ: Getting Started - Thermo Fisher...

MSQMSQMSQMSQ™

Getting Started

Document No. 031870Revision 01July 2002

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Published by Technical Publications, Dionex Corporation, Sunnyvale, CA 94086.

____________________________ MSQ Getting Started______________________________ 1-i

Chapter 1 1. Preparing for Daily Operation

Preparing for Daily Operation................................................................................................. 1-i

Introduction.................................................................................................................................... 1-1 Starting Xcalibur............................................................................................................................ 1-2

Introducing the Home Page .............................................................................................. 1-2 Introducing the Server....................................................................................................... 1-4 Introducing the Tune Window .......................................................................................... 1-5

Checking the System...................................................................................................................... 1-7 Initial Setup.................................................................................................................................... 1-8

Configuring the MSQ for ESI/MS Operation ................................................................... 1-8 Preparing the Samples....................................................................................................... 1-8 Setting the Gas Flows and Probe Heater Temperature ..................................................... 1-9 Verifying Instrument Sensitivity..................................................................................... 1-12 Tuning and Calibrating the Instrument ........................................................................... 1-13 Standard Mass Scale Calibration .................................................................................... 1-14 Tuning in APCI Mode .................................................................................................... 1-17

Manual Tuning............................................................................................................................. 1-19 The Per Method Parameters............................................................................................ 1-20 The Scan Events Table.................................................................................................... 1-20 ESI Tune Controls........................................................................................................... 1-21 APCI Tune Controls ....................................................................................................... 1-22 Changing Ionization Modes in the Tune Window.......................................................... 1-23 Switching on Operate and Gas........................................................................................ 1-23

Preparing for Daily Operation ____________________________________________________________________________ Introduction

____________________________ MSQ Getting Started______________________________1-1

Introduction This chapter describes all the procedures you should follow when preparing the MSQ for daily operation. Refer to the chapter Shutting Down and Restarting the System in the MSQ Hardware Manual for information on starting the system and pumping down the MSQ.

Preparing for Daily Operation Starting Xcalibur _______________________________________________________________________

1-2 _____________________________MSQ Getting Started _____________________________

Starting Xcalibur The installation routine for Xcalibur® places an icon in the Start Menu Programs of the Windows Taskbar. To start Xcalibur, either: • Double-click on the Xcalibur short-cut icon shown on the Windows

desktop, or • Click on the Start button in the Taskbar and choose Programs |

Xcalibur | Xcalibur. Xcalibur displays the Home Page showing the Road Map view as shown in Figure 1-1.

Introducing the Home Page The Home Page opens to display a “road map” view of the data system.

Figure 1-1. The Xcalibur Home Page Road Map view

Preparing for Daily Operation ________________________________________________________________________ Starting Xcalibur


The icons shown on the road map provide you with an easy way to access all the major modules of the data system. These are: • Instrument Setup Use Instrument Setup to configure the MSQ and the LC equipment for acquisition. This information is saved as an instrument method. • Processing Setup Use Processing Setup to specify all parameters for processing, reporting and manipulation of acquired data. This information is saved as a processing method. • Sequence Setup Use Sequence Setup to enter the details of the samples to be examined and to control the acquisition of data. This can include instrument and processing methods. • Qual Browser Use Qual Browser to view acquired data, both chromatograms and spectra, in order to obtain more information about the compounds in the sample. • Quan Browser Use Quan Browser to examine acquired data in order to obtain an accurate determination of the amounts of individual components present in a sample. • Library Browser Use Library Browser to create your own libraries of spectra and to perform searches of those libraries.



Introducing the Server The Server is displayed as an icon in the Windows Taskbar. It is shown in Figure 1-2 just to the left of the time display.

Figure 1-2. The taskbar showing Xcalibur Home Page and Server

The Server is shown as one light: • When the light is yellow and flashing the instrument is pumping down. • When the light is yellow and not flashing the instrument is pumped

down but not in Operate. • When the light is green the instrument is pumped down and in Operate. • When the light is red the mains are on and the instrument is vented or an

error has occurred within the system. Right-click on the Server to display a menu: • Choose Manual Tune… to view the MSQ’s Tune page. • Choose Instrument Tune and Calibration… to view the MSQ

Instrument Tuning and Calibration Wizard. • Choose Pump to pump down the MSQ or Vent to vent the MSQ. • Choose Exit to close the server. It is only possible to close the Server if

Xcalibur is no longer running.

Preparing for Daily Operation ________________________________________________________________________ Starting Xcalibur


Introducing the Tune Window The Tune window allows you to manually tune the MSQ for optimum performance. The Tune window can be opened in any of the following ways: • Double-click on the MSQ Tune short-cut icon shown on the Windows

desktop, or • Click on the Start button and choose Programs | Xcalibur | MSQ

Tune, or • Double-click on the Server icon on the Windows Taskbar. • Click, with the right mouse button, on the Server icon on the Windows

Taskbar and choose Manual Tune… from the menu displayed. • From the MSQ method editor Welcome page. Click on the text below

the Tune bar (Access to tune allowing you to optimize or modify the system settings) to open the Tune window.

Figure 1-3. The Tune window in electrospray positive ion mode

The Tune window consists of three principle areas: the Per Method Parameters, the Scan Events table and the Peak Display.



Figure 1-4. The Scan Events table

Use this area of the Tune window to control the settings for parameters, which can be changed on a per scan basis for the MSQ in the current ionization mode. Also use this area to control the Peak Display when tuning.

Figure 1-5. The Per Method Parameters

Use this area of the Tune window to control the parameter settings for the MSQ in the current ionization mode. This dialog bar can be hidden or displayed as required. If it is not visible, click on the Show Per Method Parameters bar, at the right of the Graphs display. Click on the bar a second time to hide the parameters.

Preparing for Daily Operation ____________________________________________________________________ Checking the System


Checking the System Before you can begin preparing the MSQ for acquisition, the system must be switched on and under vacuum. If the MSQ system has not been in use recently, it may have been left in one of two possible conditions: stand-by or shutdown. To restart the system, use the following procedure: • Perform visual checks on the instrumentation. This includes:

• Checking that all power, communications and supply lines are correctly connected.

• Checking that solvent and gas supplies are adequate. • Check that the LC system is ready for use. If not, refer to the

information supplied by the manufacturer of the appropriate part of the LC system.

• Switch on the LC system, the MSQ, and the PC. Start Xcalibur. • Pump down the MSQ. Refer to the chapter Shutting Down and Restarting the System in the MSQ Hardware Manual for further information on restarting the system. If you encounter any problems when restarting the system, refer to the Troubleshooting Help.

Preparing for Daily Operation Initial Setup ___________________________________________________________________________


Initial Setup The MSQ only requires sensitivity verification, tuning and calibration when there is a noticeable loss of sensitivity or mass accuracy, although it can be performed on a daily basis if required. Initial setup involves the following steps: 1. Configure the instrument for ESI/MS acquisition. 2. Prepare the samples. 3. Set the gas flows and probe heater temperature. 4. Verify instrument sensitivity 5. Tune and calibrate the instrument 6. Tuning in APCI mode.

Configuring the MSQ for ESI/MS Operation Refer to the chapter Changing Ionization Modes in the MSQ Hardware Manual for instructions on how to configure the MSQ for ESI/MS operation. View the Tune window in positive ion electrospray mode. Refer to the topic Changing Ionization Modes in the Tune Window on page 1-23.

Preparing the Samples The instrument sensitivity verification, tuning and calibration samples are introduced into the source using the reference inlet system. The sample used is Sodium Iodide. This will calibrate up to around m/z 2000 using a cone voltage of 100 V. Peaks in the spectra have a characteristic spacing of 150 mass units. A sample is provided with your instrument.

Tuning Sample • Compound of interest – to tune the cone voltage on your specific

compound. Prepare a solution of the compound of interest at a concentration of approximately 1 ng/µL. (The concentration required depends on the ionization efficiency of the sample itself.) Use a concentration high enough to produce a strong signal at a gain of 20 or less, in the Peak Display area of the tune window.

Preparing for Daily Operation ____________________________________________________________________________ Initial Setup


Setting the Gas Flows and Probe Heater Temperature With the MSQ under vacuum and configured for electrospray operation, the instrument can now be prepared for tuning and mass calibration. You will need to do the following: 1. Activate the gas flows 2. Set the probe heater temperature

Activating Gas Flows Electrospray operation requires two flows of nitrogen gas directed into the ion source. These flows are: • The nebulizing gas • The sheath gas The MSQ automatically splits a single source of nitrogen gas to form the two separate gas flows. The nitrogen gas enters the MSQ at the rear of the instrument at the connection marked GAS IN. The gas then passes through the MSQ to the API source enclosure and the probe. Before switching the gas on, check that the nitrogen supply head pressure is set to 75 psig (45 psig in APCI mode). To activate both gas flows, click on the Toggle Nitrogen Gas button in the Per Method Parameters part of the Tune window. The button will turn green and the text will change to On. If the Per Method Parameters are not visible, click on the Show Per Method Parameters bar, at the top right of the Tune window.

Note. The gas flows are automatically set and do not require adjustment.

To stop the gas flow, click on the Toggle Nitrogen Gas button again.


1-10____________________________MSQ Getting Started _____________________________

Heating the Probe The source enclosure contains a heater to evaporate the LC solvent. This is the probe heater. The temperature of the probe heater is controlled from, and read back to, the Tune window. Adjust the probe temperature depending on the liquid flow rate into the source (see Figure 1-7). In general, the greater the flow rate, the higher the required probe heater temperature. To change the probe temperature, click on the Probe Temperature setpoint box and enter a new value.

Caution. Take care not to heat the probe without the nitrogen gas flowing. The procedure is: gas on, heater on, and then LC flow on. When switching off, it is the reverse of the above; that is, switch off LC flow, switch off heater, let probe cool, and then switch off gas.

optimum temperature range v flow rate

0100200300400500600700

0.2 0.7 1.2 1.7flow rate ( ml/min)

oper

ating

temp

(oC)

max tempmin temp

Figure 1-6. Graph of recommended probe temperature versus ESI

flow rate.

For low flow rates, where the flow rate into the source is approximately 10 µL/min, set the probe temperature to between 100 and 150 °C.


____________________________ MSQ Getting Started____________________________ 1-11

Figure 1-7. The ESI tune controls showing the probe temperature set

at 149 °C



Baking out the Probe If the instrument has not been used recently or has been used in APCI mode, it is worth baking the probe before starting the liquid flow. To do this, set the probe heater (Probe Temperature (°C)) to 550 °C and leave with the gas flowing, but no liquid, for approximately 5 minutes.

Verifying Instrument Sensitivity Before tuning the instrument on the compound of interest, it is useful to go through the procedure of verifying the instrument sensitivity using a compound to which the instrument response is known. This example uses Sodium Iodide as the compound.

Note. Always perform this procedure in ESI mode.

1. Ensure that the API gas is turned on and that the probe heater is stable at its set point temperature.

2. On the Tune page click on the Show Per Method Parameters bar. 3. If the Inject from Ref. Inlet parameter is not visible select View |

Options. Select the Show advanced parameters check box and press OK.

4. Click on the Inject from Ref. Inlet toggle button. Sample will be drawn from the Reference bottle through the loop for 35 seconds and then a 6 minute injection will be performed.



Tuning and Calibrating the Instrument The phrase “tuning the instrument” refers to a process that optimizes several parameters to produce the strongest possible signal from a particular compound. Mass calibration of the instrument allows the MS detector to assign the correct mass values to the ion signals that it detects. It does this by acquiring data using a standard mass calibration sample and comparing this data to a reference file of the same compound. The reference file contains the correct mass for each peak and the acquired data file values are adjusted to match those in the reference file. These adjustments are then applied to all subsequent data acquisitions. The MSQ Full System Autotune feature automatically performs the tuning optimization and mass calibration.


To perform a full system autotune: 1. Ensure that the reference bottle has sufficient calibrant in it and that the

waste bottle is not full (see the Reference Inlet System section of the MSQ Hardware Manual for more details).

2. Ensure that the MSQ is set up for ESI + acquisition (see Changing Ionization Modes in the MSQ Hardware Manual for more details).

3. Set the LC flow to 0.2 mL/min. 4. Click, with the right mouse button, on the server icon and select

Instrument Tune and Calibration… from the menu displayed.

Figure 1-8. The Instrument Tuning and Calibration Wizard



5. Select Full System Autotune and press the Next button.


A series of messages is displayed informing you of the progress. If tuning or calibration fails an error message will be displayed. To accept the results of the autotune press the Finish button. The current tune and calibration files will be replaced with the ones generated by the autotune. Press the Print Report button to print the calibration report.

Standard Mass Scale Calibration A standard mass scale calibration is only required if there is a drift in the masses. It works in the same way as the calibration part of the Full Autotune, by acquiring data using a standard mass calibration sample and comparing this data to a reference file of the same compound. The reference file contains the correct mass for each peak and the acquired data file values are adjusted to match those in the reference file. These adjustments are then applied to all subsequent data acquisitions.


To perform a full standard mass scale calibration:



1. Ensure that the reference bottle has sufficient calibrant in it and that the waste bottle is not full (see the Reference Inlet System section of the MSQ Hardware Manual for more details).

2. Ensure that the MSQ is set up for ESI + acquisition (see Changing Ionization Modes in the MSQ Hardware Manual for more details).

3. Set the LC flow to 0.2 mL/min. 4. Click, with the right mouse button, on the server icon and select

Instrument Tune and Calibration… from the menu displayed.


5. Select Standard Mass Scale Calibration and press the Next button. A series of messages is displayed informing you of the progress. If calibration fails an error message will be displayed. To accept the results of the calibration press the Finish button. The current calibration files will be replaced with the ones generated. Press the Print Report button to print the calibration report.



Mass Calibration Schedule Perform a mass calibration every 3 to 6 months. Positive ion electrospray (ESI+) mass calibrations will be equally applicable for ESI- and APCI+/-, provided the low mass resolution, high mass resolution and ion energy settings remain constant. In addition, perform a mass calibration in the following instances: • If the mass ranges to be used in acquisition are significantly different to

those of the current mass calibration. • If the low mass resolution, high mass resolution or ion energy values are

changed.

Characteristic Peaks in Mass Calibration Spectra The following table shows the m/z values of the characteristic peaks in the calibration mixture mass spectrum. Table 1-1. Table of m/z values of the characteristic peaks in the

calibration mixture mass spectrum

172.8840 399.1948 440.2213 459.2523 472.6725 622.5667 772.4610 922.3552 1072.2494 1222.1437 1372.0379 1521.9321 1671.8264 1821.7206 1971.6149



Tuning in APCI Mode Before switching the gas on, check that the nitrogen supply head pressure is set to 45 psig. When working in APCI mode it is recommended that a Full System Autotune is performed in ESI mode to tune and calibrate the instrument. Follow the instructions for ESI mod e (see page 1-8), then change to APCI mode and perform a manual tune to fine tune/tweak the instrument.

Heating the Probe The source enclosure contains a heater to evaporate the LC solvent. This is the probe heater. The temperature of the probe heater is controlled from the Per Method Parameters. Set the probe temperature as appropriate for the flow rate (see Figure 1-11 and Figure 1-12). To change the probe temperature, click on the Probe Temperature setpoint box and enter a new value.

Caution. Take care not to heat the probe without the nitrogen gas flowing. The procedure is: gas on, heater on, and then LC flow on. When switching off, it is the reverse of the above; that is, switch off LC flow, switch off heater, let probe cool, and then switch off gas.

APCI POS temperature range v flow rate

0100200300400500600700

0.2 0.4 0.6 0.8 1 1.2 1.4 1.6 1.8 2flow rate (ml/min)

prob

e tem

p (oC

)

Figure 1-11. Graph of recommended probe temperature versus APCI

(positive) flow rate



APCI NEG temperature range v flow rate

0

100

200

300

400

500

0.2 0.4 0.6 0.8 1 1.2 1.4 1.6 1.8 2flow rate (ml/min)

prob

e tem

p (oC

)

Figure 1-12. Graph of recommended probe temperature versus APCI

(negative) flow rate

Preparing for Daily Operation _________________________________________________________________________ Manual Tuning


1.2 Manual Tuning This section introduces the concept of manual tuning. Once the parameters are familiar, most can be left at their default settings for all acquisitions.

Figure 1-13. The MSQ Tune window in electrospray positive ion mode

View the peaks in the Tune window and optimize each parameter as indicated in ESI Tune Controls on page 1-21 or APCI Tune Controls on page 1-22, which details the controls, their descriptions, and typical associated values.

Preparing for Daily Operation Manual Tuning ________________________________________________________________________


The Per Method Parameters

Figure 1-14. The Per Method Parameters

Use this area of the Tune window to control the parameter settings for the MSQ. This dialog bar can be hidden or displayed as required. If it is not visible, click on the Show Per Method Parameters bar, at the right of the Peak Display. Click on the bar a second time to hide the parameters. To change a value click on the Setpoint box and enter a new value.

The Scan Events Table

Figure 1-15. The Scan Events table

Use this area of the Tune window to control the settings for parameters, which can be changed on a per scan basis for the MSQ in the current ionization mode. Also use this area to control the Peak Display when tuning.



ESI Tune Controls Table 1-2. Table of the ESI tune controls

Control Description Typical Value Needle (kV) The voltage that is placed on the

stainless steel insert capillary. 3 - 5 kV. The value obtained will be the optimum for all compounds.

Probe Temperature (°C)

The probe heater temperature. Dependent on the flow rate. See Heating the Probe on page Heating the Probe.

Cone (V) The voltage that is placed on the source block, including the entrance and exit cones.

This value is compound dependent. Increasing this value gives rise to source fragmentation, which provides structural information on the compound of interest.

RF Lens Bias (V)

The voltage that is placed on the hexapole RF lens.

0.3 V. This is the default value.

LM Res Low Mass resolution. Determines peak width at low mass. The peak should be no wider than 1 amu.

12.5. This is the default value.

HM Res High Mass resolution. Determines peak width at high mass. The peak should be no wider than 1 amu.


Ion Energy (V)

The ion energy determines the transmission and resolution of ions through the quadrupole analyzer. The optimum value, 0 – 2 V, is a compromise between peak shape and sensitivity. If the analyzer becomes contaminated then the optimum value tends to increase.

1 V. This is the default value.

Needle and Probe Temperature are in the per method parameters part of the window. If this is not visible click on the Show Per Method Parameters bar to display them. Cone is in the Scan Events table part of the window. To view RF Lens Bias, LM Res, HM Res and Ion Energy values select View | Options, check the Show advanced parameters box and press OK. The advanced parameters will be displayed in the Per Method Parameters part of the window.



APCI Tune Controls Table 1-3. Table of the APCI tune controls

Control Description Typical Value Corona (µA) The voltage that is placed on the

corona pin. 3 - 3.5 kV (+ve ion), 2 - 3.0 kV (-ve ion). The value obtained should be the optimum for all compounds.

Probe Temperature (°C)

The probe heater temperature. Set at 250 °C. This value in APCI is independent of the flow rate. See Heating the Probe on page 1-17.

Cone (V) The voltage that is placed on the source block, including the entrance and exit cones.

This value is compound dependent. Increasing this value gives rise to source fragmentation, which provides structural information on the compound of interest.

RF Lens Bias (V)

The voltage that is placed on the hexapole RF lens.

0.3 V. This is the default value.

LM Res Low Mass resolution. Determines peak width at low mass. The peak should be no wider than 1amu.


HM Res High Mass resolution. Determines peak width at high mass. The peak should be no wider than 1amu.


Ion Energy (V)

This determines the transmission and resolution of ions through the quadrupole analyzer. The optimum value, 0-2V, is a compromise between peak shape and sensitivity. If the analyzer becomes contaminated then the optimum value tends to increase.

1 V. This is the default value.

Corona and Probe Temperature are in the per method parameters part of the window. If this is not visible click on the Show Per Method Parameters bar to display them. Cone is in the Per Scan Parameters part of the window. To view RF Lens Bias, LM Res, HM Res and Ion Energy values select View | Options, check the Show advanced parameters box and press OK. These parameter will be displayed in the Per Method Parameters part of the window.



Changing Ionization Modes in the Tune Window To change between ionization modes: If the Per Method Parameters are not visible, click on the Show Per Method Parameters bar, at the top right of the Tune window. Click on the Ionization Mode, Setpoint box and then click on the required ionization mode in the context menu displayed.

Note. To change ionization modes, you must first remove the current probe and then install the probe for the ionization mode you want to work in. This is described in the chapter Changing Ionization Modes in the MSQ Hardware Manual.

Switching on Operate and Gas If the Per Method Parameters are not visible, click on the Show Per Method Parameters bar, at the top right of the Tune window. To switch the MSQ into Operate mode, click on the Toggle Operate button. The button will turn green and the text will change to On.

To switch the nitrogen gas flow on, click on the Toggle Gas button. The button will turn green and the text will change to On.


Chapter 2 2. Acquiring and Viewing Data from a

Single Sample

Acquiring and Viewing Data from a Single Sample .............................................................. 2-i

Introduction.................................................................................................................................... 2-1 Setting up the Instrument Method.................................................................................................. 2-2 Setting up the MSQ........................................................................................................................ 2-3

The Welcome page ........................................................................................................... 2-3 The MSQ Method Editor .................................................................................................. 2-5

Setting up the LC System............................................................................................................. 2-14 Saving the Instrument Setup Method........................................................................................... 2-15 Setting up the Sequence ............................................................................................................... 2-16

Step 1 – Opening Sequence Setup .................................................................................. 2-16 Step 2 – Creating the Sequence....................................................................................... 2-17 Step 3 – Saving the Sequence ......................................................................................... 2-19

Starting Data Acquisition............................................................................................................. 2-20 Step 1 – Specifying the Sequence and Sample to be Run............................................... 2-20 Step 2 – Specifying any Special Acquisition or Processing ........................................... 2-22 Step 3 – Starting an Acquisition ..................................................................................... 2-22

Viewing the Data as it is Acquired .............................................................................................. 2-23

Acquiring and Viewing Data from a Single Sample Introduction ___________________________________________________________________________

2-ii _____________________________MSQ Getting Started ____________________________

Acquiring and Viewing Data from a Single Sample ____________________________________________________________________________ Introduction


Introduction This chapter describes how to acquire and view data for a single sample. It is divided into the following sections: • Configuring the MSQ and the other instruments using Instrument Setup. • Entering the sample details in a sequence using Sequence Setup. • Starting data acquisition. • Viewing data as it is acquired.

Acquiring and Viewing Data from a Single Sample Setting up the Instrument Method __________________________________________________________

2-2 _____________________________MSQ Getting Started ____________________________

2.1 Setting up the Instrument Method Before starting to acquire data, do the following: 1. Set up the MSQ (see page 2-3). 2. Set up any LC equipment (see page 2-14). 3. Save the instrument setup method (see page 2-15). Setting up instruments involves using the Xcalibur Instrument Setup facilities to create a “method” file. This file holds the details of the parameters that are to be used during the acquisition. Click on Instrument Setup on the Road Map view of the Home Page to display the Instrument Setup window. The Instrument Setup window contains method editors for all the equipment that is configured with the instrument.

Acquiring and Viewing Data from a Single Sample ______________________________________________________________________Setting up the MSQ


Setting up the MSQ Click on the MSQ button in the Instrument Setup window to display the MSQ method editor. This method editor consists of two pages, the Welcome page and the Method Editor.

The Welcome page The Welcome page: • Displays a list of templates available for creating new methods. • Allows existing method files to be opened. • Gives access to the Tune window.

Figure 2-1. MSQ Welcome page

Acquiring and Viewing Data from a Single Sample Setting up the MSQ_____________________________________________________________________


Creating a New Method To create a new method, click on the description closest to the type of acquisition you are to perform. A basic method of the type selected is created.

Editing a Method To edit an existing method, click on the text Open an existing file and locate the .meth file from the dialog displayed.

Opening the Tune Window To open the Tune window, click on the text Access to tune to allow you to optimize or modify the system settings.



The MSQ Method Editor The MSQ Method Editor is split into 5 main areas. • The Method Options. • The Per Method Parameters table. • The Chromatogram View. • The Full Scan Events and SIM Scan Events tables. • The menus.

Figure 2-2. Method Editor

Basic Operation For basic operation you need to: • Select whether to use current or stored tune file. • Set ionization mode and probe temperature in the Per Method

Parameters table. • Add full and SIM scans using the Full Scan Events and SIM Scan

Events tables. • Save Method.



The Method Options The Method Options allow you to: • Select a chromatogram for display in the Chromatogram View. • Select a tune file for use with this method. • Stop acquiring data when LC run has completed. To display the Method Options click on the Show Method Options bar, to hide them again click on the Hide Method Options bar.

Figure 2-3. Method Options

Displaying a file in the Chromatogram View To display a raw data file in the Chromatogram View, click on the Rawfile:

button and specify or browse for the appropriate file name and path.

Selecting a Tune File To use the current tune file select the Use current tune file check box. To use a previously created tune file, clear the Use current tune file check box, click on the Use selected tune file: button and specify or browse for the appropriate file name and path. To open the Tune window and view the tune settings, click with the right mouse button on the server icon on the task bar and select Manual Tune… from the menu displayed. If the check box is clear, or there is no file in the text box, the Tune window displays the current tune settings.



Note. If you do not specify a tune file, the current tune settings are used.

Event Control To stop acquiring data at the end of the LC run, select the Stop after LC finishes check box. If this box is not checked data acquisition will stop at the end of the time range.

The Per Method Parameters Table The Per Method Parameters allow the Ionization Mode and Probe Temperature to be set. If the Per Method Parameters are not visible, click on the Show Per Method Parameters bar. To hide the parameters click on the Hide Per Method Parameters bar.

Figure 2-4. Per Method Parameters table

To change the Ionization Mode, click on the Setpoint box and select ESI or APCI from the menu displayed. To change the Probe Temperature, click on the Setpoint box and type in a new value.



The Chromatogram View • The Chromatogram view allows you to display a previously acquired

file. For SIM scans you can right click on the chromatogram to display a spectrum, which will show the most abundant ion at the selected retention time. Enter this value in the mass column in the SIM Scan Events table.

Figure 2-5. Chromatogram View

Displaying a Spectrum To display a mass spectrum for a retention time, click with the right mouse button on a point in the chromatogram and hold the button down.



Editing the Retention Time in the Chromatogram View The retention time of a scan event can be edited in the Chromatogram View. Click on the scan event in the Full Scan Events or SIM Scan Events table to select it. To change the start or end time of an event: 1. Place the mouse cursor at either end of the colored bar, it will change to

or depending on which end of the bar you are on. 2. Click and drag the cursor to the retention time required. A pop up will

appear showing the event Name, Start time, End time and Duration to assist in the positioning of the cursor.

3. The new values will automatically be updated in the Time Range

column of the events table. To move the scan window of an event:

1. Place the mouse cursor on the colored bar, it will change to . 2. Click and drag the bar to the retention time required. A pop up will

appear showing the event Name, Start time, End time and Duration to assist in the positioning of the bar.

3. The new values will automatically be updated in the Time Range column of the events table.


2-10____________________________MSQ Getting Started ____________________________

The Full Scan Events and SIM Scan Events Tables Only one of the tables will be visible at a time. If you created a full scan method then the Full Scan Events table will be visible, if you created a SIM scan method then the SIM Scan Events table will be visible. To switch between tables click on the Show Full Scans or Show SIM Scans bars.

Full Scan Events

Figure 2-6. Full Scan Events table

Table 2-1. Full Scan Events parameters

Parameter Range Name Alphanumeric

characters Click on the field and enter the name of the scan

Mass Range 0.00 – 2000.00 Click on the field and enter the mass range to acquire over

Time Range 0.00 – 9999.00 Click on the field and enter the time range

Peak Format Centroid or Profile Click on the field and select Centroid or Profile from the pop up menu displayed

Scan Time 0.04 – 4.00 Click on the field and enter the time for each scan

Polarity +ve; -ve Click on the field and select +ve or -ve from the pop up menu displayed

Cone 0-200 Click on the field and enter the cone voltage to use



Source Fragmentation To perform source fragmentation create two identical functions then increase the cone voltage on the second function. The data acquired at the higher cone voltage will show increased fragmentation relative to that acquired at the lower cone voltage. This fragmentation is characteristic of the compound being analyzed and can yield diagnostic fragment ions for structural determination. Polarity switching To perform polarity switching create two identical functions one with a –ve polarity and the other –ve. Cone voltage ramping To perform cone voltage ramping enter a range in the Cone field.

SIM Scan Events In Selected Ion Monitoring (SIM) mode, you configure the MSQ to monitor a limited number of m/z values, characteristic of a target compound or compounds. The voltages applied to the quadrupole analyzer switch between the selected m/z values, each value being monitored for a programmed “dwell” time before averaging and moving on to the next. Xcalibur automatically calculates the dwell times. The dwell time is the factor that most affects the signal-to-noise in SIM acquisitions. SIM offers the following: • Improved sensitivity because more time is spent monitoring the ions of

interest rather than scanning across the complete mass range. • A wide and linear dynamic range (typically 3-4 orders of magnitude

without modification of tuning parameters). This is important in isotope dilution techniques that use co-eluting labeled standards and in the analysis of trace components.

• Better definition of a LC peak profile because more scans (data points) can be recorded across it.

• Reduced raw file sizes compared to full scan operation because SIM records only a limited amount of data relating to a particular m/z value of interest.

SIM is ideally suited to trace analysis and quantitation.



Figure 2-7. SIM Scan Events table

Table 2-2. SIM Scan Events parameters

Set the rate at whi

Parameter Range Name Alphanumeric

characters Click on the field and enter the name of the scan

Mass 0.00 – 2000.00 Click on the field and enter the mass range to acquire over

Span 0.00 – 2000.00 Click on the field and enter the span to acquire over

Time Range 0.00 – 9999.00 Click on the field and enter the time range

Peak Format SIM Scan Time 0.04 – 4.00 Click on the field and enter

the time for each scan Polarity +ve; -ve Click on the field and

select +ve or -ve from the pop up menu displayed

Cone 0-200 Click on the field and enter the cone voltage to use



The Menus The menus allow you to: • View scan events, defined in the Full Scan Events and SIM Scan Events

tables, in the Chromatogram View. • View and print a report of the method parameters. • Add and delete full and SIM scan events. Click on Preview | All Scans to display scan events, defined in both the Full Scan Events table and the SIM Scan Events table, in the Chromatogram View. Each scan event is displayed as a colored bar, a full scan event is orange and a SIM scan event is green. If you hover over a bar with the mouse cursor a tooltip showing the event Name, Start time and End time is displayed Click on Preview | Full Scans to display scan events, defined in the Full Scan Events table, in the Chromatogram View. Each scan event is displayed as an orange bar. If you hover over a bar with the mouse cursor a tooltip showing the event Name, Start time and End time is displayed Click on Preview | SIM Scans to display scan events, defined in the SIM Scan Events table, in the Chromatogram View. Each scan event is displayed as a green bar. If you hover over a bar with the mouse cursor a tooltip showing the event Name, Start time and End time is displayed Click on Preview | Parameters to display a report of the method parameters. Click on Print to print the report or Close to return to the Method Editor. Click on Scans | Add Full to add a full scan event to the Full Scan Events table. If a full scan event is selected (the row in the table will be green) the new scan will be created using the values of the selected event, if no scan is selected then the values of the last row in the table are used. Click on Scans | Add SIM to add a SIM scan event to the SIM Scan Events table. If a SIM scan event is selected (the row in the table will be green) the new scan will be created using the values of the selected event, if no scan is selected then the values of the last row in the table are used. Preview I SIM Scans command Choose Preview I SIM Scans (from the Menus on the Method Editor page) to preview all the currently defined SIM Scan events (that is, all the entries in the SIM Scan Events table). The SIM Scan events are displayed as green scan bars in the Chromatogram View. Use the mouse to hover over a scan bar to display a tooltip providing the Name, Start time and End time of the scan.

Acquiring and Viewing Data from a Single Sample Setting up the LC System ________________________________________________________________


Setting up the LC System Xcalibur can control LC equipment from the following manufacturers: • Thermo Finnigan • Hewlett Packard • Gilson • Waters Refer to the Online Help and other documentation, for information on setting up the LC system. Each module in the LC system has a button in the Instrument Setup window. Click on each button to open the method editor and set up the module.

Acquiring and Viewing Data from a Single Sample ________________________________________________________ Saving the Instrument Setup Method


Saving the Instrument Setup Method Once you have entered details of the MSQ and the LC equipment, save them in a method file. You specify the method file to be used when preparing samples for data acquisition. 1. Choose File | Save As. 2. Save the instrument method details under an appropriate method file

name.

Acquiring and Viewing Data from a Single Sample Setting up the Sequence_________________________________________________________________


Setting up the Sequence To setup a sequence to record details of the sample and instrument method: 1. Open Sequence Setup. 2. Create the sequence. 3. Save the sequence.

Step 1 – Opening Sequence Setup Click on the Sequence Setup toolbar button on the Home Page to open the Sequence Setup view. A view similar to that in Figure 2-8 is displayed.

Figure 2-8. Sequence Setup view of Home Page

Acquiring and Viewing Data from a Single Sample __________________________________________________________________Setting up the Sequence


Step 2 – Creating the Sequence Use Sequence Setup to create the sequence, by entering the details of your sample. Sequence Setup works just like a spreadsheet, with each row representing a single sample, and each column representing an item of information required for a sample. To alter the current column arrangement, click on the Column Arrangement toolbar button to display the Column Arrangement dialog box (see Figure 2-9).

Figure 2-9. Column Arrangement dialog box

To add a column to the sequence, select the column from the Available Columns list and click on Add. To remove a column from the sequence, select the column from the Displayed Columns list and click on Remove. To alter the position of the columns in the sequence, select the column from the Displayed Columns list and click on either Move Up or Move Down as appropriate.

Acquiring and Viewing Data from a Single Sample Setting up the Sequence_________________________________________________________________


You can choose the items to supply for each sample, but typically, you may need to enter some or all of the following items, depending on how the system has been configured. Table 2-3. Subset of columns available in a sequence

Item Description Position The sample’s vial number. The format of the entry

depends on the configured autosampler, for example, Gilson 215: “6:b10”; AS800: “97”.

Sample Type Type of sample, selected from the following: Unknown (the normal choice for qualitative analysis; all other types are only normally used for quantitative analysis) Blank QC (quality control) Standard Clear Standard Update Start Bracket End Bracket Standard Bracket

File Name Name of the file to contain the sample data. Double-click on this field to select a file.

Path The path to the raw file that Xcalibur creates for the sample data. Xcalibur creates this file with extension .raw. Double-click on this field to select a directory.

Sample ID An identifier unique to the sample. This field can also be used to import a barcode identifier.

Inst Meth The path and file name of the Instrument Method to be used for acquisition.

Inj Vol The volume of sample to be injected in microliters. For full details of all the items that you may need to enter to record a sample, refer to the Online Help or Xcalibur Getting Productive: Qualitative Analysis or Quantitative Analysis. For the purposes of this procedure, the sequence contains just one sample. However, if the sequence is to hold details of more than one sample, you must add further rows to the spreadsheet.

Acquiring and Viewing Data from a Single Sample __________________________________________________________________Setting up the Sequence


When you start to complete a row, a new blank row is automatically inserted below it at the end of the sequence. So, to add an additional sample to the sequence, simply start to complete this blank row. To insert a row within a sequence entered, click anywhere in the row below where you would like the new sample to be inserted and choose Edit | Insert Row. You are asked to confirm the insertion, as shown in Figure 2-10.

Figure 2-10. Insert Row dialog box

Click on Yes or No, as appropriate.

Step 3 – Saving the Sequence To save the sequence: 1. Choose File | Save As. 2. Save the sequence under an appropriate name.

Acquiring and Viewing Data from a Single Sample Starting Data Acquisition_________________________________________________________________


Starting Data Acquisition To start acquiring chromatogram data and mass spectra for a sample in a specified sequence: 1. Specify the sequence and sample to be run. 2. Specify any special acquisition or processing. 3. Start the acquisition.

Step 1 – Specifying the Sequence and Sample to be Run To specify the sequence and sample to be run: 1. Open the sequence that contains the sample you want to run in the

Sequence Setup view of the Home Page. 2. Highlight the sample within the sequence. Do this even if the sequence

contains just one sample (see Figure 2-11).

Figure 2-11. A sequence containing one sample

Acquiring and Viewing Data from a Single Sample __________________________________________________________________Starting Data Acquisition


3. Choose Actions | Run This Sample, or click on the Run Sample toolbar button, to open the Run Sequence dialog box.

Figure 2-12. Run Sequence dialog box

To run a single sample from a sequence containing more than one sample, do one of the following: • Choose Actions | Run Sequence, or click on the Run Sequence

toolbar button. In the Run Sequence dialog box, specify the sample or range of samples in the Run Rows text box. For example, to run samples 1 to 3 inclusive, type 1-3. To run just sample 3, type 3.

• Highlight the sample in the sequence. Choose Actions | Run This Sample, or click on the Run This Sample toolbar button. Xcalibur enters the requested sample row number in the Run Rows text box.

Acquiring and Viewing Data from a Single Sample Starting Data Acquisition_________________________________________________________________


Step 2 – Specifying any Special Acquisition or Processing Use the other fields in the Run Sequence dialog box to specify particular acquisition or processing requirements to be used in the subsequent run, overriding details of method files, specified in the sequence. For full details of these facilities, refer to the Online Help or Xcalibur Getting Productive: Qualitative Analysis or Quantitative Analysis.

Step 3 – Starting an Acquisition There are two ways to start an acquisition run. These are: • Automatic start • Manual start

Automatic Start For the system to start acquiring data as soon as it is ready, select the Start When Ready check box in the Run Sequence dialog box (see Figure 2-12).

Manual Start To manually start an acquisition, clear the Start When Ready check box and choose Actions | Start Analysis from the Sequence Setup menu. To subsequently control the acquisition, choose Actions | Pause Analysis or Actions | Stop Analysis. Alternatively, go to the Home Page, and use the following control icons: • Start acquisition

• Stop acquisition

• Pause acquisition

Acquiring and Viewing Data from a Single Sample ___________________________________________________________Viewing the Data as it is Acquired


Viewing the Data as it is Acquired To view the data from the sample as it is acquired, use the Real Time Plot facilities. Click on the Real Time Plot View toolbar button on the Home Page. A view similar to that in Figure 2-13 is displayed.

Figure 2-13. Real Time Plot view of Home

View TIC chromatogram and spectral data as it is acquired.

Acquiring and Viewing Data from a Single Sample Viewing the Data as it is Acquired__________________________________________________________



Chapter 3 3. Analyzing Acquired Data

Analyzing Acquired Data ......................................................................................................... 3-i

Introduction.................................................................................................................................... 3-1 Cursor Actions ............................................................................................................................... 3-2 Viewing Data in Qual Browser...................................................................................................... 3-4

Step 1 - Accessing Qual Browser ..................................................................................... 3-4 Step 2 - Displaying a Chromatogram and a Mass Spectrum in the Same Window.......... 3-5 Step 3 - Zooming in on the Time Period of Interest ......................................................... 3-8 Step 4 - Viewing the Mass Spectrum for a Specific Chromatogram Retention Time .... 3-10 Step 5 - Subtracting Background Contamination From the Spectrum............................ 3-11 Step 6 - Displaying Mass Range Chromatograms and Looking for Related Compounds ..................................................................................................................... 3-13

Determining Peak Heights and Areas .......................................................................................... 3-15 Step 1 - Switching on Peak Detection............................................................................. 3-15 Step 2 - Resolving Valleys into Separate Peaks ............................................................. 3-17 Step 3 - Switching on Peak Height/Area Labels............................................................. 3-19 Step 4 - Manually Adding and Deleting Peaks ............................................................... 3-21

Analyzing Acquired Data Introduction ___________________________________________________________________________

3-ii _____________________________MSQ Getting Started ____________________________

Analyzing Acquired Data ____________________________________________________________________________ Introduction


Introduction This chapter provides an introduction to using the Xcalibur Qual Browser facilities. Qual Browser enables you to analyze the raw data obtained from an acquisition run that has now finished. The aim is to answer the questions, “Which compounds are in sample ‘X’?” and “Does this sample contain compound ‘Y’?” This chapter also looks at how to determine chromatogram peak heights and areas. The chapter begins with information on the use of the cursor in chromatogram and spectrum cells.

Analyzing Acquired Data Cursor Actions_________________________________________________________________________


Cursor Actions Within the chromatogram and spectrum cells you can use the cursor in three ways: • A click picks a point on the cell. • A line dragged parallel to any axis picks a range. • A line dragged in any diagonal direction selects an area. The effect of these actions depends on the state of the cell: • Inactive • Active and unpinned (each cell has a pin icon in its top right corner) • Active and pinned Only one of the cells can be active at any one time. The active cell is highlighted with a gray border. In Figure 3-8, for example, the upper chromatogram cell is active, but not pinned. Pinning fixes the active status of a cell. To make a cell active: 1. Make sure the currently active cell is not pinned. If it is, click on the pin

icon to unpin it. 2. Click anywhere within the cell you want to be active. Xcalibur

highlights it with a gray border. Click on its pin icon if you want to fix it as the active cell.

Cursor actions in an active cell cause the cell to be scaled according to the dimensions of the dragged line or area (see Table 3-1). The same actions in the unpinned or inactive cell have a very different effect. In this case, the cursor actions affect the active, pinned cell (see Table 3-2). Important points to note are: • The cursor action is always applied to the pinned cell. • Within an active cell, cursor actions rescale the plot.

Analyzing Acquired Data _________________________________________________________________________ Cursor Actions


Table 3-1. Cursor action in an active, unpinned, cell

Cursor Action Effect Drag parallel to X-axis Rescale graph showing selected X

range only, same Y range Drag parallel to Y-axis Rescale graph showing selected Y

range only, same X range Dragged area Rescale graph showing both the

selected X and Y ranges

Table 3-2. Cursor action in an inactive or unpinned cell

Pinned cell Cursor action Effect spectrum Click in a

chromatogram cell

The spectrum cell displays the mass spectrum at that retention time.

spectrum Drag across a time range in a chromatogram cell

The spectrum cell displays the combined mass spectrum from that retention time range.

chromatogram Click in a spectrum cell

The chromatogram cell displays the mass chromatogram of the selected mass. If the Plot Type is TIC or Base Peak, it is changed to Mass Range.

chromatogram Drag across an m/z range in a spectrum cell

The chromatogram cell displays the mass range chromatogram of the selected mass range. If the Plot Type is TIC or Base Peak, it is changed to Mass Range.

Analyzing Acquired Data Viewing Data in Qual Browser_____________________________________________________________


Viewing Data in Qual Browser This section introduces you to viewing data in Qual Browser. It describes how to open a raw data file, and view mass chromatograms and spectra. The steps are: 1. Accessing Qual Browser. 2. Displaying a chromatogram and a mass spectrum in the same window. 3. Zooming in on the time period of interest. 4. Viewing the mass spectrum for a specific chromatogram retention time. 5. Subtracting background contamination from the spectrum. 6. Displaying mass range chromatograms and looking for related

compounds.

Step 1 - Accessing Qual Browser To access Qual Browser, follow the steps described below: 1. Click on the Qual Browser icon on the Home page roadmap or choose

GoTo | Qual Browser to display the empty Qual Browser window (see Figure 3-1).

Figure 3-1. The empty Qual Browser window

2. Choose File | Open. 3. Select the .RAW file that contains the data for the sample you want to

work on. The appearance of the Qual Browser screen will vary, depending on the default layout definition; see Xcalibur Getting Productive: Qualitative Analysis for more information on layouts. The simplest layout is shown in Figure 3-2.

Analyzing Acquired Data _____________________________________________________________ Viewing Data in Qual Browser


Figure 3-2. Qual Browser window showing selection of a raw data file

Here, the Qual Browser window shows a single chromatogram trace: the TIC chromatogram.

Step 2 - Displaying a Chromatogram and a Mass Spectrum in the Same Window To identify the compound that is eluting at approximately 4.35 minutes, you must look at the mass spectrum recorded at that time in the chromatogram. Click on one of the Insert Cells toolbar buttons. This allows you to add a new cell to the existing window, either above or to the left of the current cell. Initially, the new cell will contain the same information as the existing cell. The result of inserting a new cell above the existing one is shown in Figure 3-3.



Figure 3-3. Displaying two chromatograms

The same chromatogram is displayed in both cells. To change the lower cell so that it displays spectral data, click on it to select it, and then right-click to display a context sensitive menu. Choose View | Spectrum or click on the View Spectrum toolbar button.

Figure 3-4. Qual Browser context-sensitive menu

Spectral data is displayed in the lower cell similar to Figure 3-5.



Figure 3-5. Displaying a chromatogram cell and a mass spectrum cell

The lower cell displays a graph of Relative Abundance vs. m/z; that is, a mass spectrum. When opening a mass spectrum cell, the first mass spectrum of the corresponding total ion chromatogram is displayed. See page 3-10 for details of how to look at a mass spectrum for a particular retention time.



Step 3 - Zooming in on the Time Period of Interest Although the full TIC chromatogram is currently displayed in the upper cell, only the retention time around 4.35 minutes is of particular interested. It therefore makes sense to zoom in on that part of the chromatogram and view it in more detail, as shown in Figure 3-6.

Figure 3-6. Zoomed chromatogram

There are two ways of doing this: • Using the mouse • Using the ranges facility Before zooming ensure that the chromatogram is pinned.

Using the Mouse To use the mouse to zoom in on a time period: 1. Position the cursor at the starting time you are interested in, at a position

close to or on the baseline. 2. Click and drag the cursor along the time line to the end of the period

required. Notice that a line is drawn on the screen as you do this.



3. When you have defined the period of the chromatogram you are interested in, release your mouse button. The cell is redisplayed, showing only that part of the chromatogram between the ends of the line you have just drawn.

Using the Ranges Facility To use the ranges facility to zoom in on a time period: 1. Right-click on the chromatogram to display the context-sensitive menu,

and choose Ranges. The Chromatogram Ranges dialog box is displayed (see Figure 3-7).

Figure 3-7. The Chromatogram Ranges dialog box

2. Enter the required time range in the Time Range text box. 3. Click on OK.



Step 4 - Viewing the Mass Spectrum for a Specific Chromatogram Retention Time At this point, a selected time period of a chromatogram is displayed in one cell and an empty mass spectrum in another cell. To select a particular retention time on the chromatogram and view the corresponding mass spectrum, do the following: 1. Locate the “pin” icon shown in the top right corner of the mass

spectrum cell and click on it. 2. The icon changes from this:

to this:

Note. Pinning is a very important concept in the use of Qual Browser. The pinned cell displays the result of any action performed on any other cell within the window. If a number of cells are open in a window, it is important that you keep track of which cell is currently pinned.

3. Move the cursor over the chromatogram until it is positioned at the retention time you are interested in, and then click on the chromatogram. The mass spectrum for the retention time selected is shown in the pinned cell. The screen below shows the result of selecting the retention time 2.07 minutes in Figure 3-8.

Figure 3-8. Displaying the mass spectrum for a specific retention time



Step 5 - Subtracting Background Contamination From the Spectrum All peaks eluting from an HPLC column will contain chemical noise due to constituents of the mobile phase, resulting in background contamination. These constituents will be superimposed on the sample compound. The mass spectrum of a chromatogram peak will therefore be made up of the mass peaks resulting from the compound of interest plus mass peaks resulting from background contamination. In order to obtain the mass spectrum for the compound that can be used in a library search, you must subtract the mass spectrum of the background contamination: 1. Ensure that the mass spectrum cell is pinned. 2. Click in the chromatogram cell. 3. Select Actions | Subtract Spectra. 4. You can choose between subtracting the background from one side of

the peak you are interested in, or from both sides. To get the best results, you will usually want to subtract from both sides, so choose 2 Ranges.

5. Move the cursor back into the chromatogram cell and position it slightly to one side of the peak you are interested in; retention time 2.07 in the example.

6. Click and drag the cursor away from the peak, making sure to stop before reaching the next discernable peak on the chromatogram; 3.33 in the example. A line is drawn on the screen as you do this.

7. Repeat step 6 to mark the subtraction range on the other side of the peak.

The result of background subtraction on the example data is shown in Figure 3-9.



Figure 3-9. Background subtraction

Compare the mass spectrum shown in Figure 3-9, after background subtraction, with that shown earlier in Figure 3-8, before background subtraction. Most of the larger mass peaks are unchanged; these are ‘true’ peaks, due to the sample compound. Some of the minor peaks, for example at m/z 113,158, 181 and 216, have been removed; these were due to background contamination. The removal of such contaminant peaks will help you to obtain a more accurate result from a library search. The Info Bar to the left of the window contains details of the background subtraction.



Step 6 - Displaying Mass Range Chromatograms and Looking for Related Compounds Having viewed the mass spectrum of a particular peak in the TIC (total ion current) chromatogram, you may now want to view the mass range chromatogram of a particular ion of interest in that mass spectrum. From the mass range chromatogram, you can see if any compounds eluting at a different time contain the same significant ion. To look for related compounds in this way, do the following: 1. Pin the new chromatogram cell and click on a significant peak in the

mass spectrum. A typical result, for m/z 216 in the example, is shown in Figure 3-10.

Figure 3-10. Displaying a mass range chromatogram

2. To investigate another significant ion, click on one of the Insert Cells toolbar buttons to insert another cell, and pin it. Then click on another peak in the spectrum. Figure 3-11 shows the result for both m/z 232 (top cell) and m/z 216 (middle cell).



Figure 3-11. Displaying two mass range chromatograms

Further investigation would involve returning to the chromatogram and looking at each elution peak in turn, and then generating a full mass spectrum for each peak.

Analyzing Acquired Data ________________________________________________________Determining Peak Heights and Areas


Determining Peak Heights and Areas In a chromatogram, the area of an isolated peak is directly proportional to the amount of material present; for peaks that overlap, height may give a more accurate determination. Determining the area and/or height of a peak is therefore an important feature of quantitation. To make a peak height and area determination using Qual Browser, do the following: 1. Switch on peak detection. 2. Resolve valleys into separate peaks. 3. Switch on peak height/area labels. 4. Manually add and delete peaks.

Step 1 - Switching on Peak Detection To switch on peak detection, do the following: 1. Access Qual Browser and open the .RAW file you are interested in. 2. Zoom in on the part of the chromatogram that is of particular interest. A

typical example is shown in Figure 3-12.

Figure 3-12. Zoomed chromatogram

Analyzing Acquired Data Determining Peak Heights and Areas _______________________________________________________


3. To switch on peak detection, right-click on the chromatogram and choose Peak Detection | Toggle Detection in This Plot. The window shown in Figure 3-13 is displayed.

Figure 3-13. Peak detection switched on

Individual peaks are shaded and a block is displayed on the baseline, at the start and end of the peak (shown blue on the screen).



Step 2 - Resolving Valleys into Separate Peaks Compare the two chromatograms shown in Figure 3-12 and Figure 3-13, before and after switching on peak detection. The two peaks at 3.33 and 3.75 minutes are treated as a single peak when peak detection is switched on. This is because the drop after the first peak does not fully reach the baseline; instead, it forms a valley. To resolve valleys into separate peaks: 1. Right-click on the chromatogram and choose Peak Detection |

Settings. The Peak Detection settings tab will be displayed dialog box shown in Figure 3-14 is displayed.

Figure 3-14. Peak detection dialog box



2. Select the Valley Detection Enabled check box and click on OK. You are returned to the chromatogram display. See Figure 3-15.

Figure 3-15. Resolved valleys

The valleys are resolved into separate peaks.



Step 3 - Switching on Peak Height/Area Labels To change the peak labels shown on a chromatogram to display peak area and height: 1. Right-click on the chromatogram and choose Display Options. The

Display Options dialog box is displayed as shown in Figure 3-16.

Figure 3-16. Display Options dialog box



2. Click on the Labels tab to display the Labels page.

Figure 3-17. Options dialog box, Label page

3. Clear the Retention Time check box and select both the Area and Height check boxes.

4. Click on OK to return to the chromatogram display.



Figure 3-18. Area and height labeled peaks

Step 4 - Manually Adding and Deleting Peaks Qual Browser also allows you to make manual adjustments to peaks: • To add peaks, or to separate adjacent peaks that the automatic peak

detection facility treats as a single peak. • To delete peaks, or to combine adjacent peaks that the automatic peak

detection facility treats as two separate peaks. In the example chromatogram, there are two adjacent peaks at retention time 3.33 and 3.75 minutes. When valley detection is enabled, these are shown as separate peaks. Suppose, however, that a mass spectrum analysis of these two peaks shows them to contain the same compounds, and that the peak separation is simply the result of poor injection technique at the start of the LC run. In fact, an ideal chromatogram would show them as a single peak. This has important consequences for quantitation, because the total amount of compound present is represented by the sum of both peaks. To delete a peak and combine two peak areas, do the following:



1. Right-click on the chromatogram and choose Peak Detection | Delete Peaks, or click on the Delete Peaks toolbar button.

2. Click the cursor on the peak to be deleted. The deleted peak is shown as unshaded (see Figure 3-19).

Figure 3-19. Peak deletion - marking the peak to be deleted

3. Right-click on the chromatogram and choose Peak Detection | Delete Peaks, or click on the Delete Peaks toolbar button again. The cursor will return to a cross hair.

4. Position the cursor on the blue block at the base of the area to be included in the remaining peak. Click and drag the block along the baseline to include the area of the deleted peak. The peak area is recalculated and shown on the chromatogram in Figure 3-20.



Figure 3-20. Peak deletion - recalculation of peaks

Note. A similar procedure can be performed to add a peak.

_________________________________________________________________________________ Index

____________________________ MSQ Getting Started________________________________ I

Index

A acquiring data

acquiring and viewing data from a single sample, 2-i

analyzing acquired data, 3-i preparing for daily operation, 1-i preparing for LC/ESI/MS acquisition, 1-8 setting up an instrument method, 2-2

APCI tune controls, descriptions and typical values

(table), 1-22 APCI mode

tuning, 1-17

B baking out the probe, 1-12

C calibration

full system autotune, 1-13 introduction, 1-13 sample, 1-8 schedule, 1-16 standard mass scale calibration, 1-14

calibration mixture reference file, 1-16 Cautions

heating the probe, 1-10, 1-18 changing ionization modes

Tune window, 1-23 changing ionization modes (Note), 1-23 chromatogram

cursor actions, 3-2 cursor actions (table), 3-4 zooming in on period of interest, 3-9

Chromatogram View, 2-6, 2-8 editing the retention time, 2-9

creating a method, 2-4 cursor actions

active, 3-2 pinning, 3-2

D data acquisition

acquiring and viewing data from a single sample, 2-i

analyzing acquired data, 3-i preparing for daily operation, 1-i setting up an instrument method, 2-2

dialog box

Chromatogram Ranges (figure), 3-10 Display Options (figure), 3-20 Peak Detection Settings (figure), 3-18

displaying a spectrum, 2-8

E editing a method, 2-4 editing the retention time, 2-9 ESI

configuring MSQ, 1-8 preparing for acquisition, 1-8 tune controls, descriptions and typical values

(table), 1-21

F file name, 2-18 full scan acquisition

mass spectrum, 2-11 scan range, 2-11

Full Scan Events Table (figure), 2-11 full system autotune, 1-13

G Gas

switching on, 1-23 gas flow

activating, 1-9 flow rate (Note), 1-9 nebulizing gas, 1-9 sheath gas, 1-9

graph probe temperature against flow rate, APCI

(negative), 1-18 probe temperature against flow rate, APCI

(positive), 1-18 probe temperature against flow rate, ESI, 1-10

H heating

probe (APCI), 1-17 probe (ESI), 1-10 probe heating procedure (Caution), 1-10, 1-18

high mass resolution, 1-21, 1-22 HM Res, 1-21, 1-22 Home Page, 1-2

I I Energy (ion energy), 1-21, 1-22 inj vol, 2-18

Index ________________________________________________________________________________

II _______________________________MSQ Getting Started _____________________________

Inst Meth, 2-18 Instrument Setup, 1-3

method file, 2-2 opening, 2-2 setting up MSQ, 2-3 the Welcome page, 2-3

Instrument Setup, MSQ Welcome page (figure), 2-3

Instrument Tuning and Calibration Wizard (figure), 1-14

Instument Tuning and Calibration Wizard (figure, 1-15 ion energy (I Energy), 1-21, 1-22 Ionization Mode, 2-7

L Library Browser, 1-3 LM Res, 1-21, 1-22 low mass resolution, 1-21, 1-22

M manual tuning, 1-19 mass calibration. See calibration. See calibration mass range chromatograms, 3-14 method

setting up an instrument method, 2-2 Method Editor, 2-5

Chromatogram View, 2-8 editing the retention tume, 2-9 selecting files, 2-6

displaying a spectrum, 2-8 Full Scan Events Table, 2-10 Full Scan Events Table (figure), 2-11 Menus, 2-13 Method Options, 2-6 Preview

All Scans, 2-13 Full Scans, 2-13 Parameters, 2-13 SIM Scans, 2-13

Scans Add Full, 2-13 add SIM, 2-13

selecting a tune file, 2-6 SIM Scan Events Table, 2-10 SIM Scan Events Table (figure), 2-12

Methods creating, 2-4 editing, 2-4

Methods Editor Per Method Parameters Table, 2-7

N nebulizing gas, 1-9 nitrogen head pressure, 1-9

APCI, 1-17 Notes

changing the ionization mode, 1-23

gas flow rate, 1-9 Qual Browser, pinning, 3-11 verifying instrument sensitivity, 1-12

O Operate

status, 1-4 switching on, 1-23

P path, 2-18 peak detection

adding and deleting peaks, 3-22 Display Options dialog box (figure), 3-20 Display Options dialog box, Labels page (figure),

3-21 Peak Detection Settings dialog box (figure), 3-18 peak height/area labels, 3-20 performing in Qual Browser, 3-16 resolving peaks, 3-18 valley detection, 3-18

Per Method Parameters Table, 2-7 pinning, 3-2 polarity switching, 2-11 preparing for daily operation, 1-i probe

baking out (ESI), 1-12 heating (APCI), 1-17 heating (ESI), 1-10 heating procedure (Caution), 1-10, 1-18

Probe (°C) APCI probe temperature, 1-22 ESI probe temperature, 1-21

Probe (kV), 1-21 Probe Temperature, 2-7 Processing Setup, 1-3 pump down, 1-4

Q Qual Browser, 1-3

Chromatogram Ranges dialog box (figure), 3-10 cursor actions, 3-2 cursor actions (table), 3-4 determining peak areas, 3-16 determining peak heights, 3-16 Display Options dialog box (figure), 3-20 Display Options dialog box, Labels page (figure),

3-21 displaying a chromatogram and spectrum in the

same window, 3-6 displaying mass range chromatograms, 3-14 Insert Cells, 3-6 opening, 3-5 opening a raw file, 3-5 peak detection, 3-16 Peak Detection Settings dialog box (figure), 3-18 peak height/area labels, 3-20

_________________________________________________________________________________ Index

____________________________ MSQ Getting Started_______________________________ III

pinning, 3-2 pinning (Note), 3-11 Qual Browser window (figure), 3-5 subtracting background contamination from a

spectrum, 3-12 valley detection, 3-18 viewing a mass spectrum for a specific retention

time, 3-11 Quan Browser, 1-3

R Ranges

subtracting spectra, 3-12 raw file, opening in Qual Browser, 3-5 reference file

characteristic peaks in calibration mixture (table), 1-16

resolution high mass (HM Res), 1-21, 1-22 low mass (LM Res), 1-21, 1-22

RF Lens (V)(hexapole RF lens), 1-21, 1-22

S sample

calibration, 1-8 tuning, 1-8

sample ID, 2-18 sample type, 2-18 selecting a tune file, 2-6 Sequence Setup, 1-3

file name, 2-18 inj vol, 2-18 Inst Meth, 2-18 path, 2-18 position

sample type, 2-18 sample ID, 2-18 vial number, 2-18

server figure, 1-4 introduction, 1-4 menu, 1-4 Operate status, 1-4 vacuum status, 1-4

sheath gas, 1-9 SIM Scan Events Table (figure), 2-12 simultaneous acquisitions

polarity switching, 2-11 source fragmentation, 2-11

Source (V) (source voltage), 1-21, 1-22 source fragmentation, 2-11 spectrum

cursor actions, 3-2 cursor actions (table), 3-4 displaying a spectrum, 3-6 subtracting background contamination, 3-12

standard mass scale calibration, 1-14

starting Xcalibur, 1-2 MSQ

checking the system, 1-7 Method Editor, 2-5 pump down and vent, 1-4 restarting the system, 1-7 server, 1-4 setting up a method, 2-3 starting Xcalibur, 1-2

MSQ Per Method Parameters (figure), 1-6, 1-20 MSQ Scan Events table (figure), 1-6, 1-20 system

checking, 1-7 restarting, 1-7

T Tune file (Note), 2-7 Tune window

APCI controls and typical values (table), 1-22 changing ionization modes, 1-23 ESI controls and typical values (table), 1-21 ESI positive (figure), 1-5 introduction, 1-5 opening, 1-5, 2-4 MSQ Per Method Parameters (figure), 1-6, 1-20 MSQ Scan Evenets table (figure), 1-20 MSQ Scan Events table (figure), 1-6 switching on Operate and Gas, 1-23 tuning in ESI (figure), 1-19 verifying instrument sensitivity (figure), 1-19

Tune window (figure), 1-19 tuning

manual, 1-19 sample, 1-8

tuning and calibrating the instrument, 1-13 tuning in APCI mode, 1-17 tuning sample, 1-8

V vacuum status, 1-4 valley detection, 3-19 vent, 1-4 verifying instrument sensitivity, 1-12

introduction, 1-12 perform in ESI (Note), 1-12

vial number, 2-18

X Xcalibur

Home Page, 1-2 Home Page (figure), 1-2 server, 1-4 starting, 1-2 Tune window, 1-5

MSQ: Getting Started - Thermo Fisher...

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