PerkinElmer FLEXAR SQ 300 MS User Manual

&+520(5$)/(;$56406

86(56*8,'(

Chromera

®

Chromatography Data System

Chromera and Flexar SQ 300 MS

User’s Guide

2 .

Flexar SQ 300 MS User’s Guide

Release History

Part Number Release Publication Date

09931001 A February 2011

Any comments about the documentation for this product should be addressed to:

User Assistance
PerkinElmer
710 Bridgeport Avenue
Shelton, CT 06484-4794
U.S.A.

Or emailed to: [e-mail]

Notices

The information contained in this document is subject to change without notice.

Except as specifically set forth in its terms and conditions of sale, PerkinElmer makes no warranty of
any kind with regard to this document, including, but not limited to, the implied warranties of
merchantability and fitness for a particular purpose.

PerkinElmer shall not be liable for errors contained herein for incidental consequential damages in connection with
furnishing, performance or use of this material.

Copyright Information

This document contains proprietary information that is protected by copyright.
All rights are reserved. No part of this publication may be reproduced in any form whatsoever or translated into any
language without the prior, written permission of PerkinElmer, Inc.

Copyright © 2011 PerkinElmer, Inc.

Trademarks

Registered names, trademarks, etc. used in this document, even when not specifically marked as such, are protected
by law.

PerkinElmer is a registered trademark of PerkinElmer, Inc.

Introduction. 3

Contents

Introduction ................................................................................................................... 5

Starting .......................................................................................................................... 7

Overview ................................................................................................................................ 8

Flexar SQ 300 MS Status LEDs ................................................................................................. 9

LED Functionality ............................................................................................................. 9

Starting the SQ 300 MS Detector ........................................................................................... 10

Checking the Position of the ESI Probe ......................................................................... 11

Starting Chromera ........................................................................................................ 13

Configuring Chromera ........................................................................................................... 14

Creating a System Database ........................................................................................... 14

Creating an LCMS Instrument.......................................................................................... 15

Setting the Operate Method ............................................................................................ 19

Auto Tune on the SQ 300 MS Detector ................................................................................... 22

About the Samples ......................................................................................................... 22

PKI Tune Mix .............................................................................................................. 22

Calibrants ................................................................................................................... 22

Calibrant Delivery Modes ............................................................................................. 22

Preparing the Calibration Vials for Auto Tuning................................................................. 23

Run an Auto Tune .......................................................................................................... 24

Running Auto Tune from the Calibration Vials .................................................................. 25

Running Auto Tune Using the Built-in Syringe Pump ......................................................... 33

Initial Process to Configure an Optimal Tune and Method on the SQ 300

MS Detector ............................................................................................................... 35

Process Overview .................................................................................................................. 36

Setting up a Sample Infusion ................................................................................................. 37

Creating a Peak Detection Mini-Method .................................................................................. 38

Ramping Parameters- Optimizing the Capillary Exit Voltage ..................................................... 46

Creating Methods and Sequences ................................................................................ 53

Creating an MS Method ......................................................................................................... 54

Creating a Chromera Method ................................................................................................. 61

Creating a Chromera Sequence .............................................................................................. 64

Starting Data Acquisition ............................................................................................. 67

Preparing for an Analysis ....................................................................................................... 68

Equilibrate the System........................................................................................................... 69

Running a Sequence ............................................................................................................. 71

Analyze Results in Post Run ......................................................................................... 75

Viewing the Results in Post Run ............................................................................................. 76

Importing Chromera Data and Methods .................................................................................. 80

Using the Data Selector ......................................................................................................... 83

Searching a Library to Identify Unknowns ............................................................................... 85

Viewing Spectra Results in the SQ 300 MS Driver Window ....................................................... 88

About the Total Ion Chromatogram (TIC) ............................................................................... 90

Creating an Extracted Ion Chromatogram (EIC) ...................................................................... 91

Using the EIC Dialog ....................................................................................................... 91

Creating a Base Ion Chromatogram (BIC) ............................................................................... 93

Processing of Ion Chromatograms .......................................................................................... 95

Displaying Statistics ........................................................................................................ 96

About Right Mouse Click Menus ....................................................................................... 96

Baseline Calculation ........................................................................................................ 96

Setting the Chromatogram Noise Calculation Preferences .................................................. 97

Chromatogram Threshold ............................................................................................... 97

4 .

Flexar SQ 300 MS User’s Guide

Chromatogram Smoothing .............................................................................................. 98

Chromatogram Peak Detection ........................................................................................ 98

Manual Peak Detection ................................................................................................ 98

Automatic Peak Detection ............................................................................................ 99

Evaluating Mass Spectra ............................................................................................ 103

Evaluating Acquired Mass Spectra ........................................................................................ 104

Creating an Average Mass Spectrum .................................................................................... 106

Using the Left Mouse Button Command ......................................................................... 106

Using the Generate Mass Spectrum Dialog ..................................................................... 106

Creating an EIC and BIC from a Mass Spectrum .................................................................... 108

Processing of Mass Spectra .................................................................................................. 110

Freezing and Thawing Mass Spectra .............................................................................. 110

Zooming In .................................................................................................................. 110

Displaying Statistics ...................................................................................................... 111

Using Right Mouse Click Menus .................................................................................. 111

Baseline Calculations .................................................................................................... 111

Setting Spectrum Noise Calculation Preferences ............................................................. 112

Setting the Mass Spectrum Threshold ............................................................................ 113

Mass Spectrum Smoothing ............................................................................................ 113

Mass Spectrum Peak Detection ...................................................................................... 114

Manual Peak Detection .............................................................................................. 114

Automatic Peak detection .......................................................................................... 114

Introduction

The Flexar SQ 300 MS combined with Chromera provides fast and reliable analysis of chemical
samples by liquid chromatography mass spectrometry. The system consists of the Flexar SQ 300 MS, LC
Pump, and Autosampler; and allows the addition of a column oven and alternate detector, such as a
UV/VIS, fluorescence or PDA if desired. These components are connected to a computer running
Chromera software that controls procedures and evaluation of results which runs under Windows.

This guide is intended to provide an overview of the workflow to run a SQ 300 MS analysis using
Chromera. Before beginning, the SQ 300 MS should be installed and connected to the LC instruments.

Chromera is a powerfully-easy data system for liquid chromatography. Any laboratory instrumentation
is only as good as the software behind it. For maximum productivity and long-term return on investment
(ROI), a Chromatography Data System (CDS) needs to be intuitive, application-focused and scalable. And
when chromatography is being used in combination with mass spectrometry, the software also needs to
provide complete control of both techniques and to allow the smooth integration of data from the two
systems. PerkinElmer’s Chromera
provide full mass spectrometer control and spectral data handling. This unparalleled integration enables
the software to smoothly transition from one analytical technique to the other and to seamlessly merge
data from the two instrument types. Chromera allows users to build and continually adapt a LC/MS
system to suit their specific needs. By using unique, patented Instrument Device Descriptors, users can
quickly and easily create custom configurations on the fly. It provides highly configurable and responsive
LC instrument control for multi-detector systems, combined with an elegantly simple user interface for
interactive processing, and flexible, multi-channel quantitation and reporting. Chromera is designed to
display all of the necessary information on the screen to give you complete control of your system. The
screen layout consists of a Navigation pane on the left that contains buttons and tree lists to select
various views within the software.

®

CDS was specifically developed for chromatographers, but built to

6 .

Flexar SQ 300 MS User’s Guide

Starting

8 .

Sample Inlet:

Syringe Pump

Nebulizer Gas

Calibration/Sample

Flexar SQ 300 MS User’s Guide

Overview

This document provides basic operating instructions for the Flexar SQ 300 MS instrument including start-up,
introduction of samples, data acquisition, and shutdown.

The start-up instructions provided in this chapter assume that Chromera and the SQ 300 MS Driver
software, the Flexar SQ 300 MS instrument, and the PC have been correctly installed by a representative
of PerkinElmer.

NOTE: When planning analyses, bear in mind that the instrument needs a minimum of 12 hours from initial

installation power-on to establish the required vacuum. However, after venting for routine maintenance,
allow 1 hour after pump down and HV activation to allow equilibration of all electronics prior to
performing analyses.

Capillary tubing delivers sample infusion to the probe. Sample can be delivered from an LC system or
from the syringe pump.

Capillary Tubing
Connected to the
Probe

Connection

Vials 1 and 2

Starting. 9

Flexar SQ 300 MS Status LEDs

The Flexar SQ 300 MS detector status is displayed on the upper front panel through the following indicator
lights.

Power On

LED

Green Green/Red Green/Red

Ready/Error

LED

Vacuum

LED

LED Functionality

Power On LED Instrument Status

OFF OFF
ON (green) ON

Ready/Error LED Instrument Status

OFF OFF
ON (green) Ready/Running – No Error
ON (red) Error

Vacuum LED Instrument Status

OFF OFF
ON (green) At vacuum
ON (red) Not to vacuum

10 .

WARNING! High voltage is present within the source during

CAUTION!

Flexar SQ 300 MS User’s Guide

Starting the SQ 300 MS Detector

an experiment.

Do not move the instrument with the power on as

this may damage the vacuum pumps.

To start the Flexar SQ 300 MS system:

1. Switch the Power ON/OFF switch to position On (located on the right side panel). The Power
lamp should light.

Make sure the power switch on the roughing pump is on so that the Vacuum System will start when
Pumpdown is selected in the SQ 300 MS driver.

NOTE: Do not switch the electronics on at this stage.

2. Switch on power to the PC and login.

3. Check that the fans are operating. A cooling air flow should exit at the bottom of the instrument.

4. Double-click on the SQ 300 MS driver icon on the desktop.

5. Select Pumpdown from the Instrument menu.

6. Open the Status screen by selecting Status from the Instrument menu to display vacuum status
during pumpdown.

Starting. 11

7. Check the vacuum status displayed in the Status screen.

When ready, the Lamps (Power, Vacuum, and Ready) should all be green.
The Vacuum lamp changes from flashing to steady light at this stage.

NOTE: The vacuum state light will take several hours to become ready and the system will not be able to load a

Tune file.

Checking the Position of the ESI Probe

1. Observe the ESI probe through the inspection window and align to the center of the Capillary:
loosen the lock ring and move the manifold by turning the position adjustment screw. Tighten the
lock ring.

2. The marks on the probe assembly can be used for quick positioning. Observe the needle through
the inspection window and adjust the tip if necessary.

3. Loosen the lock ring.

4. Turn the needle assembly adjustment screw until the needle tip protrudes about 0.5 mm from the
nozzle.

5. Tighten the lock ring.

12 .

Flexar SQ 300 MS User’s Guide

Starting Chromera

14 .

Flexar SQ 300 MS User’s Guide

Configuring Chromera

The LC/MS system is configured in Chromera through the Chromera Manager; this acts as a control
panel for the system. Closing Chromera Manager will not affect data acquisition or processing on a
running instance of the Chromera.

If you have already configured a system in Chromera Manager you can skip this section.

Creating a System Database

The first time you install Chromera you must create a System Database.

To create a System Database:

1. Create a Chromera Manager shortcut on your desktop.
Click the Windows Start button, then click All Programs, locate then right-click on Chromera

Manager, then select Send To > Desktop (create shortcut).

2. Start Chromera Manager by double-clicking on it.

3. Click the System Database Management button.
The system database functions display in the Create tab.

Starting Chromera. 15

4. Click the Create Database button and observe the progress bar as the system database is created.

5. Upon successful completion, the next step is to configure “an instrument” for the system.
In Chromera, an instrument is defined as a collection of devices. For example, individual Devices

such as Flexar or Series 200 autosamplers, pumps and detectors are combined to create an
instrument. In addition, a Port Name (for communication to each device) must also be defined in
the Instrument configuration. Next create an LCMS instrument to use with the SQ 300 MS in a
system.

Creating an LCMS Instrument

NOTE: Prior to creating an Instrument Configuration, make sure all cables are connected between all devices

except

and the Edgeport box (
require Ethernet cable connections).

To create an LC instrument:

1. To create a new Instrument Configuration click on the Configuration button to
display the initial Configuration screen.

for the SQ 300 MS Detector and the Flexar PDA, since these devices

16 .

Flexar SQ 300 MS User’s Guide

2. Under the Instrument Configuration row, click in the box under Configuration Name and type
an instrument name (this example shows that LCMS was typed), then press the Enter key.

A next to the row with the name displays.

3. Click on the and the Device row displays.

4. Click on the drop-down button in Device Name box, and device choices appear. Select the
appropriate devices (modules) for the Instrument you are creating.

In this example, select Flexar SQ 300 MS Detector.

The Flexar SQ 300 MS Detector automatically fills in the Port Name field with COM DLL.

Starting Chromera. 17

5. Select your LC Pump from the Device Name drop-down list.
In this example, the Series 275 HRes Binary pump.

6. To determine a correct Port Name for the pump, open the Edgeport Configuration Utility from
the Start button > All Programs > Digi USB > Edgeport Configuration Utility.

7. The Edgeport Properties dialog displays. Click the plus sign to display a list of the physical
Ports (1 – 8) on the Edgeport with the corresponding COM port numbers.

8. In the Device Name pump row (in this example, Series 275 HRes Binary Micro Pump), click on the
drop-down button in the Port Name field.

9. Select COM4 from the Port Name drop-down list.
If your LC Pump is plugged into Port 1 on the Edgeport the corresponding COM port is COM4 as

shown in the Edgeport Properties dialog above.

18 .

Flexar SQ 300 MS User’s Guide

10. Select your LC Autosampler from the Device Name drop-down list.
In this example it is the Series 225/275 autosampler.

11. Select the Port Name for the autosampler.
If your autosampler is plugged into Port 2 on the Edgeport the corresponding COM port is COM5.

12. Observe the Database Name fields. This is where you define the database names.

The field Database Name is the name of the active database. The default database is Chromera.
The field Archive Database Name is the name of the archived database when an archive is created.
The default archive database is Chromera Archive. You can change the default names by typing
new names into these fields. If the names are changed, the you must click the Save button.

13. When all instrument components have been defined, click the Save button
located at the top of the screen.

Once you save, the Configuration Name (in this example LCMS) displays in the Configuration
pane.

14. Click Launch to launch Chromera.

Chromera starts and displays Device Connections as it connects to the devices.

Upon successful connection, the Run Time screen displays:

Starting Chromera. 19

Setting the Operate Method

At this time you must assign an Operate method. The Standby method not selectable; all you have to
do is apply it when necessary.

NOTE: When you close Chromera you lose the Operate method settings. You must reassign it every time you

Launch Chromera.

1. In the row below Operate, click Browse for method.

20 .

Flexar SQ 300 MS User’s Guide

2. Select and open the ESI folder.

3. Select Operate method and click Open.

4. Look at the Manual Control section of the Run Time screen.

5. To verify the methods work with Chromera, in the Standby row click Apply.
Observe that in the Status Panel, the MS Detector State displays Standby.

6. Next, in the Operate row click Apply.

Starting Chromera. 21

Observe that in the Status Panel, the MS Detector State displays Operate.

7. Leave the SQ 300 MS Detector in the Operate mode.

22 .

Flexar SQ 300 MS User’s Guide

Auto Tune on the SQ 300 MS Detector

Tuning the Flexar SQ 300 MS sets ion source and ion lenses parameters to obtain optimal resolution and
sensitivity, and involves adjusting the probes.
and it will be checked and re-tuned if necessary during installation. Checking the SQ 300’s performance
by running a Check Tune or Auto Tune is a quick diagnostic procedure if changes in system performance
are observed. This Auto Tune procedure simplifies the tuning process.

Upon successful completion, Auto Tune will create calibrated Tunes at 3 different scan speeds, 1k/sec,
5k/sec and 10k/sec. These Tunes will be the foundation upon which you build an MS acquisition method.

Auto Tune is performed through the SQ 300 MS driver.

About the Samples

PKI Tune Mix

Negative Mode Ions: 92, 205, 531 1166, 1466, 2666

Positive Mode Ions: 123, 195, 506, 1022, 1422, 2222, 2522

The Flexar SQ 300 MS instrument is tuned at the factory

Calibrants

There are two calibration mixtures that can be employed at the present time. The PKI positive ion tune
mix which is good for positive ion tuning and the PKI negative ion tune mix for negative ion tuning.

Calibrant Delivery Modes

A calibrant can be introduced into the mass spectrometer by several methods including: using the
calibration vials that are located on the front end of the SQ 300 mass spectrometer, using the on board
syringe pump (if available) which is located on the front right hand side of the SQ 3000 or by using an
external syringe pump.

Starting Chromera. 23

Preparing the Calibration Vials for Auto Tuning

There are two calibration vials as shown below. Typically use Vial 1 for Positive Ion Tune mix and
Vial 2 for a Negative Ion Mix.

1. To remove calibration Vial 1, reach in and unscrew the left side calibration vial as shown below.

Vial 1

Vial 2

Calibration Vials on the SQ 300 MS

2. Remove the vial and fill the vial with approximately 15 mL of tune mix.

Unscrew the calibration vial

The calibration vial removed

3. Re-insert the calibration vial making sure the small draw tube is inserted into the 50 mL calibration
vial. Also make sure that the calibration vial is securely fastened into the blue cap to avoid leaks
after pressurization.

4. Connect the peak tubing that extends from the calibration compartment and use a Peak Finger tight
fitting to secure the tubing to the sprayer probe.

Filling the Calibration vial

24 .

Flexar SQ 300 MS User’s Guide

Run an Auto Tune

1. Click Method to open the Chromera Method screen.

Peak Tubing coming from the
calibration compartment

Finger tight
Peak Fastener

Sprayer Probe

2. Select Create/Edit MS Method/Tune from the File menu.

The SQ 300 MS driver screen displays.

Starting Chromera. 25

3. If an Acquisition Method displays, close the displayed Acquisition Method.

Running Auto Tune from the Calibration Vials

If you are tuning in positive ion mode open the tune Autotune_pos Tune or if you are tuning in the
negative ion mode open Autotune_neg Tune.

1. Select Open Tune from the File menu.

The Open Tune dialog displays.

2. Select a tune file and double-click on the tune file to open it.
The above example shows selecting Autotune_pos as the Tune file.

26 .

Flexar SQ 300 MS User’s Guide

3. Select Auto Tune from the Tune menu.

The Auto Tune dialog displays.

4. In the Auto Tune dialog, select a tuning compound reference file by clicking on Restore.
This displays the Load Calibration Parameters screen.

5. For Positive ion, highlight PKI tune mix positive up to 3000u and simply double-click on it. If
calibration is only required up to 2000u, then the PKI tune mix positive up to 2000u file is
available to use.

After running the PKI tune mix positive up to 3000u you will run Auto Tune again using the
PKI Tune mix negative up to 3000u.

Starting Chromera. 27

Start Pumping Button

Analyte Source

6. Select Vial 1 (with the Positive Ion Tune Mix) for the Analyte Source from the drop-down
menu.

7. Click on the Sample Prime button and the Tune Setup dialog displays.

.

8. Type in the System Name and the User Name.
Note that the Syringe Pump selections are grayed out.

9. Click the Start Pumping button.
A mid-range mass is displayed with the corresponding TIC. Keep priming the system until a steady

signal intensity is achieved. This generally takes about 30 seconds.

28 .

Depth of probe adjustment for maximum

NOTE:

Start Pumping

Mid-Range Mass

Flexar SQ 300 MS User’s Guide

A steady TIC is
achieved in around 30
seconds

10. While viewing the Mid-Range Mass, you can adjust the source position by turning the indicated
controls for maximum sensitivity.

sensitivity.

Locations of the Source Setting Adjustments.

After clicking the
button, make sure you are getting flow
through the PEEK tubing. If there is no flow,
remove the tubing connection to the probe,
cut off about ¼-inch of tubing with a tubing
cutter, and reconnect the tubing to the probe.
If this fails to work, replace the tubing.

Starting Chromera. 29

Set
Horizontal

 Set the source horizontal position so that the horizontal probe marking is positioned 10

spaces to the right side of the scale as shown below:

 Set the Probe Tilt position as shown below:

Set Tilt Position Here

11. To start auto tune, click on the Auto Tune button. Each Auto Tune takes approximately 20 min.
If the Auto Tune stops abruptly you can re-prime by clicking on the Sample Prime button and then

click the Start Pumping button and wait an additional 30 seconds before running Auto Tune again.

30 .

1. Pos_1K_04_Oct_2010_16h21m

where:

Flexar SQ 300 MS User’s Guide

The Status light on the Auto tune page will turn yellow and displays Status:
Executing auto tune and the Autotune Report screen displays.

As Auto Tune runs in the positive ion mode, it creates the following three master tunes: pos_1k,
pos_5k, and pos_10k upon completion.

It also creates a copy of each master tune with a date and time stamp appended to the file name.
For example:

2. Pos_5K_04_Oct_2010_16h21m

3. Pos_10K_04_Oct_2010_16h21m

Pos means: Positive Ion Mode
Neg means: Negative Ion Mode
1K means: a scan rate of 1000 u/sec
5K means: a scan rate of 5000 u/sec
10K means: a scan rate of 10,000 u/sec

The time and date stamp tunes should be used to create any Tunes to be used in a method for data
acquisition. The date and time stamp allows the newest Tune to be distinguished from older ones. If
Auto Tune runs to completion and passes all criteria, the Status light turns green and Status:
Auto tune Completed successfully displays.

Starting Chromera. 31

12. After auto tune is complete, click on Check Tune. The Status light will turn yellow and Status:
Executing check tune displays. If a significant amount of time passes between the completion of
Auto Tune and the execution of Check Tune, click the “Sample Prime” button to get the calibration
mixture flowing again.

When check tune is complete a Check Tune Results screen displays.
For each mass there are four evaluation criteria:

• The Mass criterion checks to see if the mass accuracy of a peak is within +/- 0.1 u of the
actual mass.

• The Width criterion checks the Full Width at Half Maximum is between 0.5 and 0.7 u.
Note that the Width criteria is expanded up to 0.85 u for high mass ions.

• The Area % criterion checks the relative intensities of each mass in the tune relative the
most intense peak in the tuning mix.

• The report also provides the Area counts for each mass after Check Tune is run.

32 .

Flexar SQ 300 MS User’s Guide

13. The Check Tune results above show that the tuning masses all passed the evaluation criteria.

NOTE: If check tune fails the Status light will turn red and display Status: Auto tune FAILED: masses –

(displays the masses which failed Auto tune) otherwise the status light will be green if all masses passed
Auto Tune and Check Tune.

This example shows the status light is Red and that many tune masses failed the Check Tune
criteria. Typically, one would just rerun Check Tune another time to see if it passes, since running
Check Tune takes considerably less time than running Auto Tune.

Starting Chromera. 33

Running Auto Tune Using the Built-in Syringe Pump

Running Auto tune using the built-in syringe pump is very similar to running Auto Tune using the
Calibration vials except that the syringe parameters and flow rate must be input as demonstrated in the
following screen:

1. Open Auto Tune and select Syringe from the Analyte Source drop-down menu.
Make sure the syringe is loaded with desired calibrant and that the peek line extends from the

syringe tip and connects to the probe sprayer with a peek finger tight connector.

2. Click the Sample Prime button.
The Tune Setup dialog displays.

3. Type in the System Name and the User Name.

4. In the Syringe Pump section, select the Make of the syringe from the drop-down menu.

When using a 500 uL Hamilton Syringe select Custom - user entered in the drop-down and the
set the syringe inner diameter to 3.26 mm and the flow rate to 15 µl/min.

5. Click the Start Pumping button and wait until the mid-range mass shown in the scan window has
stabilized just as you did before when using the calibration vial.

6. Run Auto Tune and Check Tune as described earlier in this section.

34 .

Flexar SQ 300 MS User’s Guide

Initial Process to Configure an

Optimal Tune and Method on the

SQ 300 MS Detector

36 .

Flexar SQ 300 MS User’s Guide

Process Overview

Once the SQ 300 is tuned and calibrated, the next step is typically to create a Method specific to the
analytes(s) that are to be measured or identified. The following pages describe a detailed process for
accomplishing this, specifically so that the operator will understand why the process is conducted and
how it will provide the best possible results. Note that the first time this process is followed, it will appear
to be rather complex and tedious. However, once it is completed, it can be used for all future analyses
with minimal modifications.

The process consists of the following steps:

• Infuse a target analyte into the SQ 300 MS. This will provide a constant signal to the MS, so the
analyte can be properly mass labelled and optimized for maximum sensitivity.

• Open a calibrated Tune (of a desired scan speed) and set a few parameters specific to the
analyte and analysis that will be run.

• Create a “mini-method” to identify and label ions of interest

Initial Process to Configure an Optimal Tune and Method on the SQ 300 MS Detector. 37

Syringe Pump

Disconnect

Setting up a Sample Infusion

Infusion is easy to accomplish using the SQ 300 MS Detector’s built in syringe pump. Typically, there are
two options for accomplishing this: infuse the standard at a low flow rate directly into the MS, or infuse it
into the LC stream running at a typical flow rate so as to also optimize “flow dependent” MS parameters
such as temperature and drying gas. The following example demonstrates how to infuse a reserpine
standard directly into the MS using a syringe. Reserpine was chosen because it is a widely used standard
in the LCMS community and it is readily available from a variety of commercial sources.

1. Fill a syringe with a ~100 pg/µl reserpine solution in LCMS grade methanol and water in a 3:1 ratio.

2. Disconnect the calibration line from the ESI sprayer on the SQ 300 MS and connect a Peek transfer
line from the syringe needle to the ESI sprayer.

3. With the syringe prepared for infusion, the next part of the process covered in the next section
involves creating a simple software evaluation routine to expedite the data evaluation process.

Capillary Tubing
to the Probe

38 .

Flexar SQ 300 MS User’s Guide

Creating a Peak Detection Mini-Method

The SQ 300 MS Driver software allows the creation of simple data evaluation routines called mini-methods.
Creating this routine will facilitate data evaluation for the process being described here, as well as for many
future evaluations.

To create a peak detection mini-method:

1. Click Method to open the Chromera Method screen.

2. Select Create/Edit MS Method/Tune from the File menu.

The SQ 300 MS driver screen displays.

3. If an Acquisition Method displays, close the displayed Acquisition Method.

Initial Process to Configure an Optimal Tune and Method on the SQ 300 MS Detector. 39

4. Select Open Tune from the File menu.

The Open Tune dialog displays.

5. Select the

most recent

pos_1k tune file and click OK to open it.

The above example shows selecting pos_1k_29_OCT_2010_14h30m as the tune file.

6. A scanning experiment is going to be run, so a few changes need to be made to the calibration
Tune created by Auto Tune. Reserpine has a molecular weight of ~608.2, so using electrospray a

m/

protonated molecular ion at

z ~609.2, and an isotopic distribution from m/z ~609-611 is

expected. Set the following parameters:

40 .

Flexar SQ 300 MS User’s Guide

In Global Variables: Set the Scan
from 100 to 700

• Low

• High

m/z

m/z

100

700

Turn off the Cal flow:

Set Cal Gas Valve and Cal Gas
Valve 2 On to:

• False (off)

Set Cal Sample 1 and 2 On to:

• False (off)

7. Enter the on-board Syringe Pump settings for the syringe that will be used.
In this example, since we are using a Hamilton 500 µl syringe that is not listed in the drop-down list,

we select CUSTOM – User Entered.

Syringe Make

• CUSTOM – User Entered

Set Syringe Diameter (mm)

• 3.26

Set Syringe Flow rate (µl/min)

• 15 (Note: Tune window shows 10)

Initial Process to Configure an Optimal Tune and Method on the SQ 300 MS Detector. 41

8. To download to the SQ 300 MS all the changes made to the Tune, we must “Apply the Tune” by
selecting Tune from the Apply menu. This will start the syringe flowing at the specified rate.

9. To start the data acquisition, click the green run button.
Acquire data for 1-2 minutes to allow enough time for the reserpine standard to be pumped all the

way to the ESI sprayer, so data can be collected on it.

10. Take a “snapshot” of the data by selecting Snapshot from the Collect menu. A snapshot allows
preliminary processing of the data acquired up to the point in time the snapshot was executed.

The TIC and Mass data evaluation screens appear.

42 .

Flexar SQ 300 MS User’s Guide

11. Close the Mass data evaluation screen.

Left-click and drag

12.

a box over the TIC area as shown below.

13. When you release the mouse button, select Average Spectra from the drop-down list.

Initial Process to Configure an Optimal Tune and Method on the SQ 300 MS Detector. 43

The Mass data evaluation screen appears.

14. Left mouse click and drag a box around the reserpine protonated molecular ion cluster at m/z 609
and select Zoom X-Y Axis from the drop-down list.

15. Move the cursor to the peak apex (it then turns into a hand), and right-click.

The peak table is displayed on the botton of the Mass data evaluation screen.

44 .

Flexar SQ 300 MS User’s Guide

16. A peak detection data evaluation mini-method will now be created to facilitate data evaluation for
this and all future analyses. Select Peak Detect... from the Evaluation menu.

The Mass Spectrum Peak Detect dialog box displays.

17. Enter the parameters shown in the above screen, then click OK.
A peak table is generated for all identified peaks at the bottom Mass data evaluation screen.

Initial Process to Configure an Optimal Tune and Method on the SQ 300 MS Detector. 45

18. Select Save from the Evaluation menu and the Save Spectrum Evaluation Method dialog box
displays.

19. Type Peak Detect to name the method, then click OK.

20. Close the Snapshot screens.

21. Stop the acquisition by clicking the red button.

46 .

Flexar SQ 300 MS User’s Guide

Ramping Parameters- Optimizing the Capillary Exit Voltage

The SQ 300 MS driver SW allows you to ramp certain Tune parameters to determine the best settings for
those parameters. However, the Auto Tune routine has eliminated the need to do this except for the one
parameter that is “compound dependent”, the Capillary Exit voltage. This is the one parameter that
should be checked in order to obtain the best sensitivity for quantitative analyses, or minimize or
maximize molecular fragmentation information for qualitative analyses, or both.

The Capillary Exit voltage operates in two different ways: the first is to set the voltage to a specific value,
and the second is to leave it set to “Calibrated”. In this case, the software will pick a value based on the
values used for the individual calibration ions analyzed during Auto Tune. This means a higher Capillary
Exit voltage will be applied to higher molecular weight species.

To Ramp the Capillary Exit voltage:

m/z

1. In the Global Variables section, set to Scan from Low

100 to High

m/z

700.

Note: the above window shows the Low

2. Select Save As ... from the File menu.

3. Save the Tune as Reserpine Ramp.

4. Select Ramp from the Tune menu.

5. Click on the Capillary Exit (Volts) and select the Ramp function from the drop-down menu.

m/z

set to 500, not 100.

Initial Process to Configure an Optimal Tune and Method on the SQ 300 MS Detector. 47

•

Set the following Ramp values:

• Start Value: 50

• End Value: 300

• Step Size: 2

• Spectra: 3

Note: The settings above are used to show the

process in greater detail than is typically
required. To expedite this process in the
future, the settings should typically be:

• Step Size: 10

Spectra: 1

6. Click on Capillary Exit (Volts) to apply the values.

7. Click the green run button to start acquiring.

Acquire about 3 to 4 minutes of data.

8. Take a snapshot of the data by selecting Snapshot from the Collect menu.

The TIC Snapshot screen displays.

9. Left-click and drag a box over the TIC area around 2.5 to 3.0 min. and select Average Spectra
from the drop-down list.

48 .

Flexar SQ 300 MS User’s Guide

10. Position the cursor at the apex of the 609 peak (the cursor turns to a hand) and right-click.

11. Position the cursor at the apex of the 195 ion (the cursor turns to a hand) and right-click. The 195
ion is a known fragment of reserpine, which will appear with higher capillary exit values.

When the Ramp completes, the Base Peak Preferences dialog displays.

In the Trace Selection section,
uncheck the following:

• Resolution

• m/z

• Width

In the Evaluation Method drop­down list, select Peak Detect.

12. Set the Base Peak Preferences as shown above then click OK.
The two Base Ion Chromatograms (BIC) display;

• the red curve 194.0 to 196.0 Amplitude – Peak Detect is a reserpine fragmentation ion

• the green curve 608.0 to 610.0 Amplitude – Reak Detect is the protonated molecular

ion for Reserpine.

Initial Process to Configure an Optimal Tune and Method on the SQ 300 MS Detector. 49

13. On the green curve, move the cursor to the apex of the highest point (it turns to a hand) and
left-click.

The Mass data evaluation window displays.

50 .

Flexar SQ 300 MS User’s Guide

14. Select Tune from the View menu.

Look at the Capillary Exit (Volts) value (the window below shows Single=152).
This means that the value of the Capillary Exit voltage at the cursor position, which was the

maximum of the curve, was equal to 152 volts. This is the Capillary Exit value that provides the
maximum intensity for the reserpine protonated molecular ion.

With a typical “bell shaped curve” as is demonstrated here, any value near the maximum will be
perfectly acceptable. So in this example, any value in between 150 to 160 volts will provide the
maximum signal intensity for reserpine.

15. Close all Snapshot screens except the BIC and main screen.

Initial Process to Configure an Optimal Tune and Method on the SQ 300 MS Detector. 51

16. Now we wish to examine our reserpine fragment ion a bit. In the BIC screen, click on the red line
(194 to 196 Amplitude – Peak Detect).

This makes the red line a solid line.

17. Left-click on the highest point of the red curve for the fragment ion.
The Mass window displays.

18. Select Tune from the View menu.
Note that the Capillary Exit value is Single=250.

52 .

Flexar SQ 300 MS User’s Guide

This indicates that to produce the maximum intensity
voltage of ~250 volts should be used.

19. Select Ramp from the Tune menu to deselect it.

20. Stop the analysis, close all Snapshot screens, and close the SQ 300 MS driver.
Obtaining fragmentation information on a compound can be very valuable for a number of reasons.

It gives insight into the structure of the compound, which is typically applicable to other compounds
with the same base structure (e.g., drugs and their metabolites). It also provides additional ions
associated with the analyte that can be measured instead of, or in addition to, the protonated
molecular ion. For example, in quantitative analyses, there is occasionally an issue with a
contaminant or mobile phase ion at the same nominal mass as the analyte to be measured. This
may have a significant effect on the detection limits of the analyte due to background noise. To
overcome this, fragments of the analyte can be monitored instead of, or in addition to, the
protonated molecular ion. The probability of the interfering ion having a

as a fragment from the analyte is extremely low (but still must be verified!)

value

m/

z 195 fragment ion, a Capillary Exit

fragment at the same

m/z

Creating Methods and Sequences

54 .

Flexar SQ 300 MS User’s Guide

Creating an MS Method

The foundation for an optimal MS Method is starting with a mass calibrated Tune (with a date/time
stamp). These are created every time Auto Tune is completed and under normal operating conditions,
they can remain stable and usable for
is still properly calibrated, the easiest thing to do is to run a Check Tune to verify that the MS is operating
to specification. If Check Tune indicates that retuning is required, simply run Auto Tune to recalibrate the
system.

The following example shows how to create an MS method that will acquire both a Scan and a SIM for a
given period of time, alternating between the two acquisition modes. For SIM acquisitions, the correct
mass calibrated Tune to optimize for the acquisition will always be the 1k Tune (with date/time stamp).
For Scan acquisitions on real world samples, selection of the correct mass calibrated Tune depends on

scan speed required to get enough sample points to properly profile the chromatographic

the
Typically, at least 10-15 data points (i.e., in this case, Scans) are required to properly profile a peak, and
the mass range required for the scan is another factor requiring consideration. The user should select
from the 1k, 5k and 10k Tunes (with date/time stamps) that will best meet the scanning requirements.

To create an MS method:

1. Click Method to open the Chromera Method screen.

months

. However, if there is any doubt regarding whether the MS

peak.

2. Select Create/Edit MS Method/Tune from the File menu.

The SQ 300 MS driver screen displays.

3. Select New from the File menu.

The following dialog displays.

Creating Methods and Sequences. 55

4. Select Method then click OK.
The following method screen displays.

5. Click the plus sign to expand the method row and display Periods.

56 .

Flexar SQ 300 MS User’s Guide

6. Give the method a name by selecting Save as from the File menu.
In this example, the method name is MS Method 1.

7. Click on the green Time Period dot and set the run Duration to equal that in the Chromera HPLC
method.

NOTE: At this point in time, it is up to the user

specified in Chromera. Otherwise, the timing issues may arise during the running of a sequence.

Since you have not yet created a Chromera HPLC method, set the run duration to 4 minutes then
set your HPLC method to a 4 minute run duration time.

8. Click on Scan (the blue dot).

to insure that the MS Time Period equals the the run Duration

9. Select Import from the Edit menu.

The Import Tune dialog displays.

Creating Methods and Sequences. 57

10. Select the most recent pos_1k tune file (with a date/time stamp), then click OK.
This example shows pos_1k 29 Oct 2010 is selected as the most recent pos_1k tune file.

11. In the Global Variables section, click on Acquisition Function, select SIM from the drop-down list,
and then press Enter. This will convert the Tune from a scanning acquisition to a SIM acquisition.

12. To set the specific SIM mass, select Low m/z, type your value, and press Enter. This example

m/

uses

z 609.3.

13. Set the Pulse Counting Time (µs) to 300000.
This is a 300 msec dwell time, which will give excellent signal to noise. It is possible to acquire

data with much shorter dwell times to achieve higher sampling rates, such as those required for
UHPLC separations or if the acquisition requires monitoring many SIM ions “simultaneously”
(current limit is 38).

14. Set the Drying Gas Flow Rate (L/min) to 12.
The optimal drying gas flow is dependent on a number of factors, such as the LC flow rate,

composition of the mobile phase and the temperature setting of the Drying Gas.

15. Click on Capillary Exit (Volts) and from the drop-down list and select Single Valve. Enter a
value of 150.

58 .

Flexar SQ 300 MS User’s Guide

NOTE: This is a pre-determined value known to be optimal for the compound used in this example. The

alternative selection for

Capillary Exit (Step Function…) uses a calculated value for the mass selected
that is derived from the optimal values determined for the masses in the calibration mix during the Auto
Tune procedure.

16. Left-click on Capillary Exit (Volts) again to set this value.

17. Select Save from the File menu to save the method.

Notice that in Time Period 1, the Sim (+) displays the set values.

18. Click on the blue dot next to Sim ( )

19. Select Copy from the Edit menu, click on the Time Period (green dot), then select Paste from the

Edit menu.
This makes both blue dots Sim (+).

Creating Methods and Sequences. 59

NOTE: This is a quick way to build a Method requiring multiple acquisition experiments. Simply copy previously

defined Tunes as this minimizes the number of changes required for each Tune in the Method.

20. Click on the top Sim blue dot and change it to Scan in Acquisition Function of the Global Variables

section.

21. Set the Low

22. Set the Pulse Counting Time (µs) to 100.

m/ z

to 500 and the High

m/z

to 625.

NOTE: The pulse counting (dwell) time entered here applies to each data point within the scan. In this example,

the scan range is m/z 500-625, and there are 10 samples per mass, resulting in 1251 total data points

each

acquired per scan,

23. Set the Drying Gas Flow Rate (L/min) to 12.

of which will be measured for 100 µs.

60 .

Flexar SQ 300 MS User’s Guide

24. Click on Capillary Exit (Volts) and from the drop-down list select Step Function Calibrated to

Q1 m/z.

25. Left-click on Capillary Exit again to set this value.

26. Select Save from the File menu.

This example MS acquisition Method, comprised of two Tunes (1 SIM and 1 Scan) that will cycle
continuously until the end of Period 1, is now defined and saved.

27. Close the SQ 300 MS driver.

Creating Methods and Sequences. 61

Creating a Chromera Method

After creating the MS method, open Chromera to create a Chromera method. The Chromera method will
define all the operating requirements for all the other components in the Chromera configuration.

To create a Chromera method:

1. Click Method to open the Method screen.

2. Select New Method from the File menu.

3. Type a Method Name and a Group.
Optionally, you can also enter a Description.

62 .

Flexar SQ 300 MS User’s Guide

This example shows a Method Name of Demo Method and a Group of Reserpine Group.

4. Select Save Method from the File menu.

5. Enter your instrument parameters by clicking on each instrument.
Click on BPump-1 and enter the pump parameters. Click Advanced to show additional parameters.

6. Click on AS275CO-2 and enter the autosampler parameters. Click Advanced to show additional
parameters.

7. Click on QMS-3 in order to link the MS Method previously defined in the SQ MS driver software to
the Chromera method being defined.

8. Click Browse for Method.

Creating Methods and Sequences. 63

The Select Method dialog displays.

9. Select the method you created in the SQ 300 MS driver, then click Open.
This example shows MS Method 1.sqm

10. Select Save Method from the File menu.

64 .

Flexar SQ 300 MS User’s Guide

Creating a Chromera Sequence

After creating a Chromera method, create a simple Chromera Sequence to run the method.

To create a Chromera sequence:

1. Click Sequence to open the sequence screen.

The Sequence screen opens with the last run sequence displayed.

2. Select New Sequence from the File menu.

A blank sequence screen displays.

3. Set the sequence identifiers.

• Click in the Name box and type a name for this sequence. This example shows Reserpine.

• Select the Group from the drop-down list. This example shows New Method Group.

• Select the Sample Tray Type of your autosampler. This example shows 100-Position

Tray.

4. Click the plus sign to display the sequence parameters.

Creating Methods and Sequences. 65

5. Enter the Sequence Parameters.

• Select the Sample Type from the drop-down list. This example shows Sample.

• Type a Sample Name. This example shows Reserpine.

• Type the number of Injections. This example shows 1 injection.

• Type the Injection Volume (µL). This example shows 5 µL.

6. Select the Method for this sequence by clicking the button in the Method field.
The Data Selector – Single Method dialog displays.

7. Since you saved the method in the Reserpine Group, click the plus sign to expand the Method
Group: Reserpine Group.

This displays all methods saved in the Reserpine Group.

8. Click in the Select box to select the method. This example shows Demo Method is selected.

9. Click Open to enter this method in the sequence.

10. Select Save Sequence from the File menu.

66 .

Flexar SQ 300 MS User’s Guide

Starting Data Acquisition

68 .

Flexar SQ 300 MS User’s Guide

Preparing for an Analysis

Prepare the system with the mobile phase, column, and sample listed below for the analysis. The analysis
conducted for the example shown on the following pages utilizes an isocratic HPLC method, which is

mobile phase reservoir (“A” in the example).

delivered from a

• Mobile Phase: 5 mM ammonium formate in 75/25 Methanol/Water

• Sample: 100pg/µL reserpine

• Column: 3x3 CR C18 column and column holder

single

Starting Data Acquisition. 69

Equilibrate the System

Before running an analysis, the LC system must be equilibrated to achieve a stable chromatographic
baseline and to properly condition the LC column.

To equilibrate the system:

1. In Chromera click Run Time then click Manual Control for the Control Mode.

2. Make sure the SQ 300 MS in the Operate mode.
Observe that in the Status Panel, the MS Detector State displays Operate. If not, Look at the

Manual Control section of the Run Time screen.

3. In the Operate row click Apply.

70 .

Flexar SQ 300 MS User’s Guide

4. Make sure the chromatographic tubing is connected between the LC system and the SQ 300 MS
detector.

5. Enter your Pump Settings (0.4 mL/min and 100% A).

6. Click the Apply button to start the pump.
Monitor the pump pressure in the Status Panel.

Starting Data Acquisition. 71

Running a Sequence

Once the system has reached equilibration, you can load and run the sequence.

To run a sequence:

1. Click Sequence for the Control Mode.

2. Select Open Sequence from the File menu.

The Data Selector – Single Sequence screen displays.

3. Since you saved the sequence in New Method Group, click the plus sign to expand the
Sequence Group: New Method Group. This displays all sequences saved in this group.

4. Click in the Select box to select the sequence. This example shows the Sequence named
Reserpine is selected.

72 .

Flexar SQ 300 MS User’s Guide

5. Click Open to open this sequence.

The sequence displays and is ready to run, indicated by the green Start button.

6. Click on the green Start button. The sequence starts to run.

Starting Data Acquisition. 73

The running sequence is displayed as a green line. Observe the two chromatographic traces. The
Total Ion Chromatogram or TIC, which is the sum of intensities for all ions observed in each scan is
is displayed as a black line, and the SIM of

m/

z 609.3 is displayed as a blue line.

7. Observe the Plots pane on the left side. Click the plus signs to expand the plots.

8. During an analysis, you can view the SIM or Scan individually by unchecking the PER
(0.0:4.0)PSIM(6... box. This example shows only the Scan.

74 .

Flexar SQ 300 MS User’s Guide

When the run completes the display clears. You can review the results in Post Run.

Analyze Results in Post Run

76 .

Flexar SQ 300 MS User’s Guide

Viewing the Results in Post Run

Whether reprocessing existing data or acquiring new data, the completed samples will be displayed in the
Post Run environment, and can be inspected by navigating through the Sample tree and interacting
graphically with the chromatographic display.

• Data can be treated as view only from the standard Post Run display.

• Individual results can be optimized graphically.

• The current version of the method can be graphically modified (GME, Graphic Method Editing) using

the selected sample data.

• Data can be viewed in Single Plot mode, Stacked Plot mode, Matrix mode for multiple channels
and replicate injections, or in Overlay and 3D mode (3D mode is only available for PDA data at
present).

To view results in Post Run:

1. Click the Post Run button in the navigation pane.

The data trees for Reserpine are displayed in the Data pane.

Analyze Results in Post Run. 77

You could also search for previously stored data by selecting Open Data from the File menu.

This displays the Data Selector. Search and select
the data you want to analyze then click Open.

2. Uncheck the pump pressure (BPump-1).

3. Click on SIM.
A SIM chromatogram is displayed in the top plot window and the SIM data are displayed in the

Results pane.

4. Click on Scan.
The TIC (total ion chromatogram) is displayed in the top plot window and the TIC chromatographic

data are displayed in the Results pane.

78 .

Flexar SQ 300 MS User’s Guide

5. Move the mouse pointer to the apex of the peak (it turns to a hand) at retention time ~1.2 min. and
then right-click.

6. Select Examine Mass Spectra from the menu.

Analyze Results in Post Run. 79

The spectrum from the selected retention time opens in the lower portion of the SQ 300 MS driver
window, and a copy of the TIC is displayed in the top portion of the window.

m/z

7. Move the mouse pointer to the apex of the

609.3 peak (it turns to a hand) then right-click to
display the peak table on the bottom of the window. The peak table provides some statistical data
on the identified peak including absolute intensity, the peak width, etc.

80 .

Flexar SQ 300 MS User’s Guide

Importing Chromera Data and Methods

To import the data into Chromera:

1. Select Import from the Tools menu then select Chromera Results…

The Import Results dialog appears:

2. Click the browse button

to the right of Source file name.

The Open dialog appears:

3. Navigate to the directory containing your data.

Analyze Results in Post Run. 81

4. Select the data file then click Open.
This example shows data file name (External std-results.chxb), that appears in the Select

batches to import list.

82 .

Flexar SQ 300 MS User’s Guide

5. Select External stds-UTM-2 then click the Import button.
The progress bar shows the import progress. Upon completion, the message Import of results

successful appears in the Messages box.

6. Click the Close button.

Analyze Results in Post Run. 83

Using the Data Selector

Now that data has been imported into Chromera you can open it using the Data Selector. To open a
data file using the Data Selector follow this procedure:

1. Click on the Post Run button

The Post Run environment opens:

2. Select Open Data… from the File menu.

The Data Selector opens.

84 .

Flexar SQ 300 MS User’s Guide

3. Click on the plus sign to expand the batch and choose one or more samples from a batch.
The data are stored in Batches, which are, in turn, stored in groups. Use the Data Selector to

choose data for guiding the method editing. The operation of the Data Selector is the same
throughout the software.

4. Click in the check box to Select the Batch of data (this example shows 092810 seq 4-quant-
3again), then click the Open button.

This selects all of the samples under the batch. The Post Run screen displays the Example Data.

Analyze Results in Post Run. 85

Searching a Library to Identify Unknowns

It is important to note that both electrospray (ESI) and atmospheric pressure chemical ionization (APCI) are
very soft ionization techniques, especially when compared to more traditional techniques, like electron impact
used for GC/MS analysis. Consequently, API mass spectra typically have very little information beyond the
protonated molecular ion isotopic cluster, especially after proper background subtraction is performed to
remove chemical noise. However, additional “characteristic” fragmentation information can be obtained by
increasing the capillary exit voltage, as demonstrated in the

Capillary Voltage

of success when trying to compare unknown spectra to previously acquired library spectra.

This example assumes there are previously acquired and stored spectra in the library which can be
searched for comparison to the unknown spectra. The following steps summarize the procedure for
searching a library to identify unknowns.

section of this guide. Providing additional fragmentation ions will increase the probability

 Display the mass chromatogram of the sample run.

 Select the peak of interest representing the unknown.

 Display the mass spectrum in the SQ Driver.

 Perform a library search.

Ramping Parameters- Optimizing the

To search a library to identify unknowns:

1. In Chromera, open the chromatogram of interest in Post Run.

2. Move the cursor to the apex of the peak of interest.

3. Right-click on the apex of the unknown peak of interest and select Examine Mass Spectra from
the pop-up menu.

The SQ 300 MS driver window is displayed with the Mass spectrum window selected.

86 .

Flexar SQ 300 MS User’s Guide

4. Select Save Spectrum to Library from the Evaluation menu.

5. Run a library search by selecting Run Library Search from the Evaluation menu.

6. The search results are then displayed.

Unknown vs. Match Difference

Unknown Spectrum

Best Library Match

Analyze Results in Post Run. 87

For additional information on library searching, refer to the NIST manual which should be installed
on the hard drive and is also available on line.

88 .

Flexar SQ 300 MS User’s Guide

Viewing Spectra Results in the SQ 300 MS Driver Window

NOTE: Some of the following examples show data from a time-of-flight (TOF) mass spectrometer. However, the

commands demonstrated may be applied to the quadrupole data generated by the SQ 300 MS detector.

1. As demonstrated earlier, moving the mouse pointer to a point on the chromatogram in Chromera
and then right-clicking and selecting Examine Mass Spectra from the drop-down list will open the
SQ 300 MS driver processing window.

2. Another way to enter the mass spectral processing domain (demonstrated on a different data file) is
to select Examine Spectra from the Chromera Actions menu.

The spectra open in the SQ 300 MS driver window.

Analyze Results in Post Run. 89

3. The SQ 300 MS driver window displays a Total Ion Chromatogram (TIC) in the upper half of the
window. If the mouse was right-clicked in the Chromera chromatogram (as in the first example
above), the spectrum from that retention time will be displayed. If no point in the Chromera
chromatogram is selected, then the first spectrum from the acquisition will be displayed.

90 .

Flexar SQ 300 MS User’s Guide

About the Total Ion Chromatogram (TIC)

The TIC is a chromatogram where each data point represents the sum
for each scan. Consequently, each data point in a TIC has a scan associated with it. The TIC mirrors a
typical chromatogram displayed in an LC analysis where the amplitude is UV absorbance.

However, "all ions" are only those that were within the sampled mass range, which was determined by
the Method used for the data acquisition and the Tune contained within that Method.

of intensities of all ions detected

Analyze Results in Post Run. 91

Creating an Extracted Ion Chromatogram (EIC)

The EIC is a form of a limited mass range TIC. An EIC is extracted from scan data, and typically used
where specific ion(s) of interest, or small mass ranges are being searched for. It is possible to display
several mass ranges in the same EIC chromatogram.

NOTE: If specific ions of interest are being monitored for quantitative purposes, the best sensitivity (i.e., lowest

detection limits) will be obtained by monitoring the ions in SIM
them from scan data acquired over a large mass range.

An EIC can be generated in two ways:

• Using the EIC dialog.

• Using a "box" procedure in a generated mass spectrum.

Using the EIC Dialog

To create an Extracted Ion Chromatogram (EIC):

acquisition

mode, rather than extracting

1. Select the TIC in the SQ 300 MS driver window

2. Select EIC from the View menu.

3. Enter the first mass range in the first row of the
and entering a value.

m/z

Range Selection table by clicking in the cell

4. Enter the second mass range in the second row of the

5. Decide how to display the curves in the Curve Definition table.
This allows you to perform simple algebraic operations (addition or subtraction) on the mass ranges

selected. Enter the row identification (A,B) to display the curves in a simple way.

6. Select the Range Limit dialog.

m/ z

Range Selection table.

92 .

Flexar SQ 300 MS User’s Guide

7. For the Range Selection, select Full Range, Time Range or Spectrum Range.

8. Select how to display the created EIC.

NOTE: An EIC cannot be displayed in the same window as a TIC.

9. Click OK to close the Range Limit dialog and click OK to close the Extracted Ion
Chromatogram dialog.

The EIC displays.

Analyze Results in Post Run. 93

Creating a Base Ion Chromatogram (BIC)

The BIC is a form of a limited mass and time range ion chromatogram where mass, peak intensity/area
and resolution are displayed as a function of time or spectrum number.

A BIC can be generated in two ways:

• Using the BIC dialog.

• Using a "box" procedure in a generated mass spectrum.

To create a Base Ion Chromatogram (BIC):

1. Select a TIC in the SQ Driver window

2. Select BIC from the View menu.

3. In the

4. In the Display section, select how to display the chromatogram.

5. In the Trace Selection section, select the Traces to be displayed.

6. Select the Evaluation Method from the drop-down list.

7. Click the Range Limits search button . The Range Limit dialog displays.

m/z

Range Selection section enter the

m/z

range to cover the mass peak to be studied.

94 .

Flexar SQ 300 MS User’s Guide

8. In the Range Selection section, select to display Full Range, Time Range, or Spectrum Range.

• If you select Time Range, enter the number of the first and last spectrum.

• If you select Spectrum Range, enter the number of the first and last spectrum.

9. Click OK to close the Range Limit dialog, then click OK to close the Base Peak Preference
dialog.

The BIC will display mass, intensity and resolution for the selected base peak as a function of
retention time.

10. To display intensity only, right click and select Customization Dialog from the menu to display the
Customization dialog. In the Subsets tab, select Intensity and click OK.

Analyze Results in Post Run. 95

Processing of Ion Chromatograms

To zoom in:

1. Left-click and drag a box around the area of interest, release button and select Zoom X-axis.

The zoomed area appears.

2. Left-click and drag a box somewhere in the chromatogram, release the button and select Zoom
Out.

3. Use the right mouse button command, Undo Zoom.

96 .

Flexar SQ 300 MS User’s Guide

Displaying Statistics

 Left-click and drag a box around a peak of interest, release the button and select Display

Statistics.

About Right Mouse Click Menus

Each graph view contains a graphical package which includes functions to modify and export graphs.
Individual functions can be selected or the Customization Dialog can be used.

Baseline Calculation

1. When a TIC, EIC, or BIC window is selected, select Baseline from the Evaluation menu.

2. To calculate an Auto baseline with the morphological function, select Auto and the Function

morph.

3. Enter shortest Baseline Segment Size, and Baseline Noise Window.

APB

Analyze Results in Post Run. 97

NOTE: An increased Baseline segment value will flatten the baseline. A decreased value may lead to a baseline

which interferes with the peaks.

4. The level of the calculated baseline is found in the Peak Information box when Manual peak
detection is used.

5. A Manual baseline can be created by entering a value resulting in a straight line as a baseline.

6. The baseline can be hidden or displayed with the Baseline from the View menu.
The baseline can be subtracted from the chromatogram with the menu command Subtract

Baseline from the Evaluation menu.

Setting the Chromatogram Noise Calculation Preferences

1. When a TIC, EIC or BIC window is selected, select Noise from the Evaluation menu.

2. To detect the noise manually, select Manual and enter a mass range where there are not any peaks.

3. Select

• If the noise is Peak to peak, the amplitude of the peak top or the centroid is subtracted by the

4. The level of the calculated noise is found in the Peak Information box when Manual peak

Peak to peak or Root mean square

In the default version of the signal to noise calculation the following is done:

low value of the noise and divided by the peak-to-peak difference. Click OK.

detection is used.

.

Chromatogram Threshold

1. Activate a TIC, EIC or BIC and select Threshold from the Evaluation menu.

98 .

Flexar SQ 300 MS User’s Guide

2. Enter a Threshold value and decide if the ion chromatogram will be subtracted with this value.

3. The threshold can be hidden or displayed with the Threshold from the Chromatogram View
menu.

NOTE: If a new chromatogram has not been created, the "undo" function is not available. To return to the

original chromatogram, the data file has to be closed and opened again.

Chromatogram Smoothing

1. Select a TIC, EIC, or BIC.

2. Select Smooth from the Evaluation menu.

3. Enter number of smooths (1-10), window size (0.01-100) and select a Function from the drop-
down list.

0-Mean: For each data point in the source curve the processed curve is calculated as the average
of the data points within the specified window.

1-Median: The processed curve is calculated as the median of the data points.

Chromatogram Peak Detection

Manual Peak Detection

1. Using the right-mouse button select Mark Data Points.