PerkinElmer FLEXAR SQ 300 MS User manual

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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]
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Copyright © 2011 PerkinElmer, Inc.
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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®
CDS was specifically developed for chromatographers, but built to
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.

6 . Flexar SQ 300 MS User’s Guide

Starting

8 . 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.
Sample Inlet:
Capillary Tubing
Connected to the
Probe
Syringe Pump
Nebulizer Gas
Connection
Calibration/Sample
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.
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
Power On
LED
Ready/Error
LED
Vacuum
LED
Green Green/Red Green/Red

10 . Flexar SQ 300 MS User’s Guide
Starting the SQ 300 MS Detector
WARNING! High voltage is present within the source during
an experiment.
CAUTION!
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.
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.
If you have already configured a system in Chromera Manager you can skip this section.

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
and the Edgeport box (except
To create an LC instrument:
for the SQ 300 MS Detector and the Flexar PDA, since these devices
require Ethernet cable connections).
1. To create a new Instrument Configuration click on the Configuration button to
display the initial Configuration screen.

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.