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HS 40 Control User's Guidefor TotalChrom Client Server and TotalChrom Workstation

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Release History

Part Number Release Publication Date

N515-6071 A May 2001

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

User Assistance

PerkinElmer Instruments LLC

710 Bridgeport Avenue

Shelton, Connecticut 06484-4794

U.S.A

Or emailed to: [email protected]

Notices

The information contained in this document is subject to change without notice. PerkinElmer makes no

warranty of any kind with regard to the material, 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.

NOTE: TotalChrom is the updated version of the software previously marketed as Turbochrom. This

product is compatible with TotalChrom and Turbochrom version 6.1.x. The term TotalChrom has been

used throughout this document to denote either system.

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 Instruments LLC.

Copyright 2001 PerkinElmer Instruments LLC.

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.

TotalChrom, AutoSystem, AutoSystem XL, and 600 Series Interface are trademarks of PerkinElmer

Instruments LLC.

Turbochrom is a trademark of Applera Corporation.

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i

Table of Contents

Introduction

Using this Guide.......................................................................................................................... 1

Conventions Used in this Guide..................................................................................................1

The Help System.........................................................................................................................2

HS 40 System Connections

Hardware Connections ................................................................................................................3

HS 40 Headspace AnalysisCreating an HS 40 Control Method.................. .......... ........... ........... .......... ........... ........... ......... 10

Creating a Sequence in TotalChrom.............. ........... .......... ........... ........... ........... ........... ........... 14

Setting up a Sequence and Displaying HS 40 Status............... ........... ........... ........... ............ ..... 14

Setting Up a Sequence from the HS 40..............................................................................15

Displaying HS 40 Status ....................................................................................................17

Multiple Headspace Extraction (MHE)

Overview of HS 40 MHE Analysis ...........................................................................................19

Plotting Regression (MHE) Curves........... .......... ........... .......... ........... .......... .......... ........... 20

Displaying and Printing Regression Curves........... ........... .......... ........... ........... .......... ....... 22

Managing MHE Files................................................................................................................25

MHE Theory and Calculations..................................................................................................27

Index

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Using This Guide

2

Terminology

The following terms are used to refer to program elements and the actions that you

perform to carry out tasks:

ChooseThe terms chooseand selectindicate actions that you perform. Choose

represents carrying out an action associated with a command or command button.

When you are likely to use the mouse to choose an action, mouse-specific terms

are used, such as click, right-click, and double-click. When you are likelyto use the keyboard to choose an action, the specific key is mentioned, such as

Enter or Tab.

SelectThe term selectrefers to highlighting an object or item or moving the cursor

focus. Selecting an item prepares it for an action: for example, when you select

text, it appears in reverse video. When you select a dialog box option, you

activate the option but the function is not carried out until you choose the OK

button, which closes the dialog box and sometimes completes another operation.

When you use the File Select or File Open dialog box to open a

file, the phrase enter or select is used to refer to the actions you can take to

open that file. When you entera filename, you type it in the File Name text box

exactly as it exists. When you selecta file, you browse for it on your computer or

your network. When you select a file, you will not introduce typographical errors.

Illustrations

Unless otherwise indicated, the values in the illustrations in this manual are examples

only. They are not intended to indicate the exact values you will see or to suggest the

values you should use.

Instrument Names

Throughout this manual, the nameHS 40refers to both the HS 40 and the HS 40XL

Headspace Samplers. Also,AutoSystem refers to both the AutoSystem and the

AutoSystem XL GC.

The Help SystemThe TotalChrom help system is a collection of help files; one for each TotalChrom

application. You access help in several ways:

Context sensitive help When you press F1, TotalChrom displays information

related to the dialog box or window you are currently using. Press Escape to close the

help.

When you choose Help Topics from the Help menu, TotalChrom

displays the list of help topics for the current application.

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3


This section describes how to connect the HS 40 hardware to the TotalChrom system.

This section does not cover the installation and configuration of your computer.

Hardware Connections

The TotalChrom computer communicates with the HS 40 through the PerkinElmer

600 Series LINK Interface or the built-in integral LINK. The LINK acquires and

buffers digital data from the AutoSystem GC and controls the GC and HS 40.

The following procedure explains how to make the HS 40, AutoSystem, and LINK

interface connections for TotalChrom control. It assumes a certain level of knowledge

of laboratory instrumentation installation and of your TotalChrom system.

If you do not know how to perform some of the steps in this procedure, you will need

assistance in order to set up and install an HS 40 as part of your TotalChrom system.

Contact your local PerkinElmer representative to make an appointment for Service

Installation, or find a qualified person in your own organization.

This procedure assumes that you have done the following:

Installed the RS-232 communications board in your HS 40.

Installed current version of the firmware as described in the TotalChrom software

Release Notes.

Installed the TotalChrom software as described in yourTotalChrom Application

Managers Guide.

After installing the hardware, configure the LINK interface and the AutoSystem as

described in Chapter 3 of your TotalChrom Users Guide.

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4

To connect the HS 40 and the AutoSystem for TotalChromcontrol:

1. Install the HS 40 and any accessories as described in theHeadspace Sampler

for Gas Chromatography Instrument Manualand the accessorymanual for

each accessory.

2. Turn off and unplug all instruments, and disconnect the power cords from all

instrument rear panels.

3. Use one of the following procedures to connect an AutoSystem and HS 40 to

the LINK interface.

For an integral LINK:

Connect one end of the RS-232C cable (P/N 6560-9010, supplied with

the HS 40 IPM) to instrument port J2 on the LINK interface printed circuit

board (PCB) and the other end to the HS 40 RS-232 communication board.

The LINK interface PCB is located on the inside of the AutoSystem GC

right side panel.

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5

HS 40XL

OPTION-BOARD

SOFTWARE-BOARD BC

D

BOARD

RS-232

INTERFACE

TurbochromSerial/Host

orIEEE

Connection

AutoSystem XL GC

IEEE

Serial/Host

RS-232 Cable (P/N 6560-9010)

1. Remove this side panel.

2. Connect this cable to port J2 onthe LINK interface printed circuitboard located on the inside of

the side panel.

HS 40 to AutoSystem GC Using an integral LINK

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6

For a 600 Series LINK:

a) Connect one end of an RS-232C cable (P/N 6560-9010, available

separately) to Instrument port A on the LINK interface and the other end to

the communications port on the AutoSystem.

b) Connect one end of a second RS-232C cable (P/N 6560-9010,

supplied with the HS 40 IPM) to Instrument port B on the Series 600 LINK

interface and the other end to the communications port on the HS 40.

c) To attach a second AutoSystem and HS 40 to the LINK interface, connect

an RS-232C cable (P/N 6560-9010, available separately) from the GC

communications port to the LINK port C, and connect another RS-232C

cable (P/N 6560-9010) from the HS 40 communications port to the

LINK port D.

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7

610 LINK HS 40XL

Cable(P/N6

560-9010or6560-9025)

Cable (P/N6560-9010 or 6560-9025)

Inst AInst B

RS232

AutoSystem XL GC

OPTION-BOARD

SOFTWARE-BOARD B

CD

BOARD

RS-232

INTERFACE

HS 40 to AutoSystem GC Using a 600 Series LINK

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4. To make the ready/run signal connection, use the following procedure to

connect both Ready/Run cables (P/N B015-9984, supplied with the

HS 40) from the HS 40 to the AutoSystem GC.

HS 40 Run output to GC Ext. Start input:At the HS 40, connect the black

wire to HS 40 pin 4 and the red wire to HS 40 pin 5. At the AutoSystem,

connect the black wire to GC pin 3 and the red wire to GC pin 4.

HS 40 Ready input to GC Ready output:At the HS 40, connect the blackwire to HS 40 pin 11 and the red wire to HS 40 pin 12. At the AutoSystem,

connect the black wire to GC pin 10 and the red wire to GC pin 11.

Shielding:Connect the shield wire on each cable to the grounding screw on

the HS 40.

HS 40 to AutoSystem GC ready/run signal connection

5. Connect the LINK Interface to the TotalChrom system as described in

Appendix B of your TotalChrom Application Managers Guide.

6. Reconnect and plug in all power cords, and turn on all instruments.

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9

HS 40 Headspace Analysis

When you run an HS 40 analysis, you do the following:

Create a TotalChrom method (.MTH) in the TotalChrom Method

Editor that contains your AutoSystem GC control, processing, calibration, and

report parameters for the HS 40 method as described in Chapters 6-8 of the

TotalChrom Users Guide;

Create an HS 40 method (.HSM) in the HS 40 Method Editor that

contains HS 40 instrument control parameters.

Create a TotalChromsequence that controls the HS 40, AutoSystem, data

acquisition, and analysis.

An HS 40 method mustsatisfy the following requirements for the TotalChrom

sequence to run:

The AutoSystem GC and HS 40 methods musthave thesame filename and must

be stored in the same directory. For example, a sequence that references the

methods HS40.MTH and HS40.HSM, both stored the directory

C:\MYMETHODS\, is valid.

An AutoSystem sequence will not run if the two method filenames do not match.

For example, a sequence that references the methods HS40.MTH and

HS40METH.HSM will not run.

The Cycle Time value in the HS 40 method must be greater than or equal to the

time from one injection to the time at which the GC is ready for the next

injection. The Cycle Time value must include the oven cool-down and GC

equilibration times to allow the HS 40 to properly calculate and execute theoverlapping thermostatting in the HS 40.

The HS40TEST method and sequence files in the TotalChrom Examples subdirectory

contain values that can be used as a starting point when creating HS 40 methods.

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Creating an HS 40 Control Method


After you have created an AutoSystem method, use the HS 40 Method Editor to

create HS 40 methods. The HS 40 Method Editor contains the following menus and

tabs:

File Lets you create, open, save, and print HS 40 method files and audit trail

information; enter descriptive information; and exit the HS 40 Method Editor.

Instrument Lets you upload the actual HS 40 configuration into the current HS

40 method when you install new options on your HS 40.

View Lets you toggle the display of the HS 40 toolbar and view HS 40 status

information.

Control Parameters Tab Lets you set the HS 40 instrument control method

parameters.

Documentation Tab Displays the last edit date of the current HS 40 method and

installed HS 40 options., and lets you enter or review information about your HS 40

method.

You can also enter descriptive text by choosing Description from the File menu.

To create a new HS 40 method:

1. From the TotalChrom Navigator Apps menu, choose HS 40 Method Editor

to display the HS 40 Method Editor dialog box with the Control Parameter

tab selected.

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The available text boxes reflect the options installed in your HS 40.

2. To upload the HS 40 configuration into the method, select an instrument if

prompted, and choose Update Method Config from the Instrument menu to

open the Instrument Configuration dialog box.

You need to update the instrument configuration so that the accessories

included in the HS 40 methods that you create match those installed in the

instrument.

If you try to run a sequence that references a method that has not been

updated, TotalChrom displays an error message and will not run the

sequence.

3. To include the Water Trap accessory in the instrument configuration, select

the Water Trap Installed check box, and choose OK.

This check box is available only if the CryoFocus accessory is installed.

4. Select the appropriate thermostatting mode from the Sampling Mode list:

Constant Select this mode if you want to use the same thermostatting

time for all samples that are analyzed by this method. Constant mode is the

standard mode of operation.

Progressive Select this mode if you want to automatically increase thethermostatting time for a series of samples. Sample 1 is thermostatted for the

value entered. Sample 2 is thermostatted for twice the entered value, Sample

3 for three times the entered value, and so on. This function is used for

initial method development to determine the thermostatting time required for

a specific application. The function can also be used for kinetic studies. Vial

position one must be filled. Subsequently, some vial positions can be empty

to increase the thermostatting time between some vials. For every vial

position after position one, the thermostatting time will be increased by the

length initial thermostatting time.

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MHE Select this mode if you want to perform multiple headspace

extractions. The MHE function uses from 2 to 9 extraction steps per sample

vial and pressurizes, injects, and vents each vial between extractions. This

function is used for method development and validation as well as for

quantitative analysis of difficult solid samples.

5. In the Injection group box, select whether you want to inject for a specific

length of time or inject a specific volume of sample.

If you select time injection,specify the desired injection time.

If you select volume injection, specify the column flow rate and the desired

injection volume. The HS 40XL will automatically calculate the

corresponding injection time.

6. Specify the time control parameters in the Times group box:

Cycle Time The length of time from sample injection to the time at

which the AutoSystem is ready for the next injection.

The Cycle Time value must be greater than or equal to the GC run time plus

the equilibration time and the oven cool-down time for the HS 40 to

correctly calculate the thermostatting time and process the vials.

Laboratory temperatures can strongly influence the GC oven cool-down time

and can vary (for example, overnight). The GC cycle time setting should take

into account the longest oven cool-down time expected during the analytical

sequence.

Thermostatting Time The length of time before pressurization/injection,

during which the sample vial is held in the thermostatting oven.

Pressurization Time The length of time during which the sample is

pressurized to the carrier gas pressure. During pressurization, the gas phase

in the vial is allowed to homogenize before injection.

Injection Time The length of time during which the carrier gas flow is

turned off and is replaced by sample gas flow from the pressurized vial.

Withdrawal Time The length of time after injection before the sample

needle is withdrawn from the sample vial or lowered into the vent position.

During this time the needle remains in the vial.

Vent Time The length of time during which the vial is vented to

atmosphere. If you select Vial Venting in the Options group box, venting

occurs immediately after the withdrawal time has elapsed.

7. Specify the required temperatures for the sample vial, sampling needle, and

transfer line (sample transfer tubing) in the Temperatures group box.

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13

For best reproducibility, set the needle and transfer temperatures to a value

5-10C higher than the sample temperature.

8. To backflush unwanted components from the pre-column after the

components of interest have passed, enter Backflush On and Off times.

Refer to theHS 40 Capillary BackflushAccessory Manualfor more

information.

9. To activate the CryoFocusing accessory, enter the Pre- and Post-CryoFocustimes.

To increase sensitivity, you can use the CryoFocusing accessory to

preconcentrate the injected analytes on the capillary column. The reference

point for the cryofocusing time is the end of the injection time. The PreCryo

time ends, and the PostCryo time starts, at the end of the injection. (Refer to

the HS 40 CryoFocusing accessory manual for more information.)

10. In the Options group box, select any accessories installed on your HS 40 that

you want to use during the run.

The Water Trap check box is available only when the CryoFocus check box

is selected. For more information on each HS 40 option, refer to the

appropriate accessory manual.11. To calculate the period of time between injections (PII), choose Update PII.

TotalChrom queries the HS 40 for the PII time based on the method

parameters.

The PII value lets you see the effects of your time parameter changes on vial

throughput.

12. Complete the description on the Documentation tab, and choose OK.

13. Save the HS 40 method.

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Creating a Sequence in TotalChrom

14

Creating a Sequence in TotalChrom

To create a sequence to be used with TotalChrom and HS 40 methods, follow the

procedures described Chapter 11 of the TotalChrom Users Guide, with the following

exceptions:

If you select One Per Row in the Method group box of the Global Parameters

dialog box, the Method column must contain the TotalChrom AutoSystem GC

method that has the same filename as the HS 40 method.

If you select Multiple Per Row in the Method group box, the Instrument method

column must contain the TotalChrom AutoSystem GC method that has the same

filename as the HS 40 method. The Processing and Calibration methods can be

different methods.

Setting up a Sequence and Displaying HS 40 Status

After you have created a sequence, you can set up and run that sequence, and display

the HS 40 instrument status from the HS 40 Status and Setup window. The HS 40

Status and Setup window contains a File and Instrument menu.

Do not use the Clear Setup command in TotalChrom to abort an HS 40 run.

If you want to abort (clear the TotalChrom setup) an HS 40 run after it has been set

up and is thermostatting, click the Stop button in the HS 40 Status and Setup window.

Choosing Clear setup from the TotalChrom Run menu will not stop the HS 40 from

thermostatting.

The Instrument menu contains the following commands:

Configuration Displays the currently installed accessories:

Advance Vial: Moves the tray to the next vial position. This command can be used

only when the HS 40 is in Standby mode.

Home Carousel Returns the carousel to the home position with vial 1 in the oven

load position. This command can be used only when the HS 40 is in Standby mode.

Set Up Lets you set up a sequence and download it to the LINK interface.

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Setting Up a Sequence from the HS 40

Once you have created your sequence, you can download it to the LINK interface.

To set up a sequence and start an HS 40/AutoSystem run:

1. Choose HS 40 Status and Setup from the Navigator Apps menu to display

the HS 40 Status and Setup window.

2. To display the Setup HS 40 dialog box, choose the Setup in the HS 40 Status

and Setup dialog box, or choose Set Up from the Instrument menu.

3. If there is more than one GC, select the desired GC from the Instrument list.

4. Enter or select the sequence in the Sequence list.

5. In the Raw Data Path text, enter or select the path to the directory where you

want TotalChrom to store your HS 40 raw file data.

6. In the Result Data Path text, enter or select the path to the directory where

you want TotalChrom to store your HS 40 result file data.

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7. In the Starting Row text box, you may change the row number with which

you want to begin the sequence operation.

8. In the Ending Row text box, you may change the sequence row number with

which you want to end the sequence operation.

9. Select Suppress Processing if you do not want to process the data

immediately after they are collected.

This automatically suppresses the data analysis process, including the

printing of reports and plots, and the creation of a result file.

You may want to select Suppress Processing if you plan to use your

computer for another task and do not want to be interrupted by data

processing at the end of each run. Since data processing is a compute-

intensive task, it can cause your computer to be unresponsive for several

seconds as the various data analysis functions are carried out.

If you suppress processing, you can use Batch Reprocessing later to obtain

results from the analyses.

10. Select Suppress Report/Plots if you do not want to print a report and plot.

11. To initiate the sequence setup and HS 40 run, choose OK.

TotalChrom verifies that the vials indicated in the sequence are in sequential

order with the correct number of injections indicated, downloads the

sequence to the LINK, and starts the HS 40 run.

12. To stop the run before the sequence is complete, choose Stop from the

HS 40 Status and Setup dialog box.

Do not use the Clear Setup command in TotalChrom to abort an HS 40 run.

If you want to abort (clear the TotalChrom setup) an HS 40 run after it has been set

up and is thermostatting, click the Stop button in the HS 40 Status and Setup window.

Choosing Clear setup from the TotalChrom Run menu will not stop the HS 40 from

thermostatting.

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Displaying HS 40 Status

During a run the HS 40 Status and Setup dialog box displays the HS 40 status,

current parameter values, and current sequence.

To display the HS 40 status information:

Choose HS 40 Status and Setup from the Navigator Apps menu to display

the HS 40 Status and Setup window.

TotalChrom may display any of the following messages:

Initializing The HS 40 is checking for vials in the thermostatting oven after

power-up before switching to Standby mode and getting ready for operation.

Standby The HS 40 is ready and waiting for the next user input.

ExternalDevice The HS 40 is waiting for the GC to become Ready or data

handling operations to end.

Searching The HS 40 is looking for the next vial, and the carousel is in motion.

Equilibrating The thermostatting oven has reached the programmed temperature

and is going through temperature equilibration.

Stopping The HS 40 has been stopped by the user and is in the process of

unloading vials to the sample tray before returning to Standby mode.

Finishing The HS 40 has finished processing the last vial in the programmed

sequence and is in the process of unloading vials and returning to Standby mode.

Indexing The HS 40 is moving the sample tray to locate a particular position.

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Extended Thermostatting The sample vial is undergoing a longer-than-

programmed thermostatting while waiting for the GC to become ready for injection.

Pressurizing The sample vial is being pressurized to carrier gas pressure.

Injecting Sample gas is being introduced onto the column.

Withdrawing The sampling needle remains in the vial after injection has taken

place before being withdrawn or lowered into the vent position. The withdrawal time

period is designed to allow transfer of the injected sample into the separation system .

Venting The sample vial is being vented to atmosphere.

CryoFocus The sample is being focused cryogenically on the column over an

extended injection period to improve analytical sensitivity.

BackFlushing The HS 40 is backflushing the precolumn to remove higher boiling

analytes from the separation system and to shorten analysis time.

Analyzing GC analysis of a headspace sample is in progress. Depending upon the

method parameters used, the next sample in the sequence is most likely undergoing

thermostatting. The HS 40 is ready to pressurize and inject the next sample after the

thermostatting time has elapsed.

To print an HS 40 report or audit trail:

1. To print an HS 40 report, select Print from the HS 40 Files menu in the HS

40 Method Editor to open the Print Options dialog box, and select Print

Method Parameters, and choose OK.

2. To print the audit trail for an HS 40 file, select Print from the Files menu in

the HS 40 Method Editor to open the Print Options dialog box, select Audit

Trail, and choose OK.

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19

Multiple Headspace Extraction

(MHE)

This section describes how to set up a method using multiple headspace extraction

and how to perform a regression analysis of HS 40 data using the TotalChrom HS 40

MHE application.

The Multiple Headspace Extraction (MHE) application lets you analyze and report

data and results obtained by performing multiple extractions and injections from a

headspace vial. You can also calculate and report MHE calibration and analysis

results. The MHE application generates semilogarithmic regression curves (MHE

curves that display the peak area as a function of the injection number.) MHE curves

can be displayed and printed for each analyte, including supporting statistical data.

The MHE technique is a tool for method development and for quantitative analysis of

volatile analytes in difficult solid sample matrices, or solid samples for which no

method has been developed.

Overview of HS 40 MHE Analysis

To run an HS 40 MHE analysis, you will create an AutoSystem method, an HS 40

method, and a TotalChrom sequence. You will then set up and run the sequence, and

perform the regression analysis on the TotalChrom result (.RST) files.

To create an HS 40 MHE Method:

Enter your MHE GC and HS 40 method and sequence parameters as you wouldfor a standard HS 40 application, with the following exceptions:

TotalChrom Method Use Calibration Factor is not a recommendedcalibration type for MHE calibration calculations because the user-

supplied calibration factor (rather than the total peak area of the

standard) is used to calculate component concentration, which

effectively overrides the MHE regression analysis calculations.

MHE method Specify MHE as the Sampling Mode in the HS 40Method Editor.

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MHE sequence An MHE sequence must contain at least two rowsand must have between 2 and 9 injections (extractions) from the same

vial.

MHE Results Window Develop calibration and sample lists ofTotalChromresult files (.RST) in the MHE Results window, perform

the regression analysis, and view the component results.

Plotting Regression (MHE) Curves

The MHE application lets you calculate and plot regression curves (MHE Curves) for

TotalChrom result files or for manually entered peak areas. The supporting statistical

information is obtained and displayed for each MHE curve.

When you open an MHE file, the MHE Results window opens.

The MHE Results File window contains the following:

Result Files Window Lets you build sample and calibration lists of TotalChrom

result files for regression analysis.

Side-By-Side Plots Window Lets you view individual components within the

calibration or sample lists.

File Menu Lets you open and save MHE files (.MHE), and print sample and

component information and regression results.

Samples Menu Lets you select TotalChrom result files to build sample and

calibration lists in the Result Files window, and manage file lists.

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Component Menu Lets you select components for display in the Side-by-Side

Plots window and manually enter peak areas for extractions. The component selection

choices on this menu are available only when the Side-By-Side Plots window is

active.

To plot an MHE regression curve:

1. Select HS MHE Application from the TotalChrom Apps menu to display the

MHE Results window.

2. To create an MHE results file, do one of the following:

Choose New from the File menu to create a new MHE file.

Choose Open from the File menu and select an existing MHE file.

TotalChrom displays the Result Files and Side-by-Side Plots windows.

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3. To open one or more file(s) for the Calibration or Sample list, position the

cursor in the list in which you want to enter a result file, select Open Sample

Result (.RST) Files from the Samples menu, select the desired file(s), and

choose Open.

The files that you enter into a sample list should be consecutive runs

(extractions) from the same vial.

Once you have added at least two entries to a sample list, the MHEapplication generates a regression analysis of those entries and continues to

update the analysis as you add more samples to the list.

4. To save the regression results, choose Save or Save As MHE Results from

the File menu, and enter the MHE result file name .

To calculate the MHE curve for manually entered peak areas:

1. To enter the peak areas manually, select Manual Area Entry from the

Component menu to display the Manual Area Entry dialog box.

2. Enter the peak area for each extraction for which you want to calculate an

MHE curve.

After you have entered two peak areas, the MHE application calculates anddisplays the MHE curve along with the total area (TA), the Y-intercept (A1),

and the slope of the regression curve. The total area is calculated as the total

area underneath the curve extrapolated to the X-intercept. TotalChrom

continues to update the curve as you enter new peak areas.

Displaying and Printing Regression Curves

The MHE application displays the regression curves, component, and sample

information for each sample that is displayed in the Side-By-Side Plots

window.

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23

General Information

Component The name of the component that is displayed.

The name of the study as entered in the Sequence Editor.

Sample Name The name of the sample as entered in the Sequence Editor.

The number of the sample vial that contains the component

that is displayed.

The amount of sample in the sample displayed.

The value used to account for the dilution of the sample

before injection.

Sample and Calibration Results

The MHE application displays the following information about the

calibration and sample results.

Calibration Amount The amount of component in the calibrationsample.

Concentration The concentration of the sample component as calculated

by the MHE application software. The MHE calculation relates the areas

under the calibration and sample MHE curves, both extended to their x-axis

intersection. It then divides the amount of analyte determined in the sample

by the sample amount to determine the analyte concentration.

Total Area (TA) For each component,the total area under the curve is

calculated from the MHE regression analysis by extrapolating the MHE

curve to its intercept with the x-axis. The cumulative peak area (TA) for all

extractions is a measure of the total amount of analyte in the sample and is

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24

used for quantitative analysis to compare TA from a sample MHE curve to

TA from a calibration standard MHE curve. (Refer to MHE Theory and

Calculations.)

Slope (-k) The slope of the regression line.

A1 The Y-intercept of the regression line, equal to the logarithm of the

peak area of the first injection.

Correlation The correlation coefficient for the MHE curve. A correlation

coefficient is displayed for each calibration and sample MHE curve.

To view sample and component information:

1. With the Side-By-Sides window active, choose Select from the Component

menu to open the Select Component dialog box.

2. To view the plot for a component in a sample list, select the desired list from

the options to the right of the component list, then select the desired

component, and choose OK.

TotalChrom displays the component data and plot in the Side-By-Sides Plot

window.

3. To scroll for the selected component in another sample within the same list,

choose Next/Previous in the Side-by-Side Plots Component menu.

4. To clear the MHE files, choose Abandon from the MHE Results File menu.

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Managing MHE Files

25

To print sample and component information:

1. To print a report of the MHE analysis results, select Print from the Files

menu to open the Print Options dialog box.

2. From the Print Option dialog box, choose the sample list(s), the desired

component(s), and the data you want to print.

Sample File List: Lets you print the list of TotalChrom result files for each

sample list selected in the Samples group box.

Component Table:Lets you print the table of statistical data on thecomponent that is currently on display, or for all components, for each

sample list that is selected in the Samples group box.

Component Plot(s): Lets you print the side-by-side plot(s) for the

component that is currently on display, or for all components for each

sample list selected in the Samples group box.

Managing MHE Files

Use the Samples menu commands in the MHE Results window to change the paths to

the result files, delete, and sort result files

To change the default result file paths:

1. To change the paths to the Calibration and Sample Lists, choose Set File

Paths from the MHE Results Samples menu to open the Set File Paths dialog

box.

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26

2. Enter or select the path(s) for the TotalChrom result files, and choose OK.

To delete all files from a sample list:

1. Choose Erase File Lists from the File menu to open the Select Sample(s) To

Erase dialog box.

2. Select the check box for each sample list that you want to erase, and choose

OK to clear all result files from the selected list.

To sort files by injection date and time:

1. Choose Sort Files from the File menu to open the Select Sample(s) To Sort

dialog box.

2. Select the check box for each sample list that you want to sort, and choose

OK.

TotalChrom arranges the result files by injection date and time.

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MHE Theory and Calculations

27

MHE Theory and Calculations

The most comprehensive quantitative analysis technique for headspace GC is the

Multiple Headspace Extraction (MHE) technique. Even for samples, for which no

complete analysis method has been developed, MHE analysis can give the analyst

information about the total amount of the given analytes in the sample. In addition,

the MHE curve will yield useful information on whether equilibrium between the

phases in the headspace vial has been established during the thermostatting time and

whether the analysis system has a linear response. All this makes the MHE technique

an invaluable tool for method development and validation, especially in the analysis

of solid samples. In solid samples, analyte migration may be hindered by the sample

matrix and analyte adsorption may occur on the surface of the sample matrix. Such

effects will prevent the analyte from reaching a true equilibrium (partitioning)

between the sample phase and the headspace phase in the vial. Using MHE analysis,

it is possible for the analyst to prove whether equilibrium is actually reached before

the GC analysis takes place.

The MHE technique, of course, requires multiple extractions from the sample vial,

which makes the technique time-consuming and limits its usefulness for routine

analysis. If equilibrium is being reached during thermostatting for the type of sample

being analyzed, a single injection analysis will yield the correct results and there will

be no need for further injections.In MHE analysis, the gas-phase concentration of the volatile compound of interest is

determined at each extraction step. Following each extraction / injection, the vial

pressure is vented to atmospheric pressure to remove a percentage of the gas phase.

Reequilibration then occurs between the two phases (liquid/solid and gas) in the

sample vial, and although the concentration (by volume) of the particular compound

in both phases will be smaller than it was originally, their ratio (partition coefficient)

remains the same.

In typical MHE analysis, it is not practical or possible for extractions from a sample

vial to continue until all of the volatile compound is removed so that the sum of the

resulting peak areas for the compound for each extraction step corresponds to the total

amount of compound present in the sample. Using the MHE technique, however, it is

unnecessary to perform exhaustive extraction of the volatile compound , as the

decrease in concentration of the compound in subsequent extraction steps follows the

mathematical relationship of a first-order reaction. That is, the decrease in

concentration over time is proportional to the prevailing concentration:

dC

dtkC= (1)

wheret is time, Cis concentration, and kis a constant. Since the concentration at any

time, C, depends on the initial concentration C0and the exponentk, the equation

becomes

C C e kt= 0 (2)

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28

Since the extractions are performed in steps, the time tmay be replaced by the

number of extraction steps, n. The initial concentration C0is replaced by the peak area

from the first extraction step,A1, which occurs at time t= 0 orn= 1. The constant k,

which now includes instrument parameters, becomes k*:

A A enk n= 1

1* ( )(3)

This can be expressed in the form of a linear equation:

ln ( ) ln*A k n An = +1 1 (4)

A simple regression analysis by the MHE application arrives at a straight line plotted

through the values of the peak areas derived from three or four extraction steps. This

line gives the values for the slope (k) and y intercept (A1). If the MHE curve is notlinear, equilibrium is not established inside the headspace vial under the given

thermostatting conditions. In this case, neither MHE analysis nor a standard single

injection analysis with external or internal calibration will yield the correct results.

The total peak area of the volatile compound in the sample is then determined by

summing the partial peak areas from each extraction step. Using equation (3) as a

geometric progression, the total area Anbecomes

A A e e enk k k

= + + + 12 31 * * .. . (5)

Written in a more general form, the equation for the sum of the areas is

AA

en k=

1

1* (6)

Applying the values determined by the regression analysis, the application arrives at

an extrapolated total peak area number that directly corresponds to the total amount of

the compound present in the sample.

Once the software has calculated the total area for a particular analyte in the sample

the equivalent concentration of the analyte in the sample is also calculated. Using

total peak areas derived from previous MHE analyses of a calibration standard, theamount of analyte can be calculated and if the sample amount has been entered, the

concentration is subsequently displayed.

For more information about MHE theory and techniques, refer to the following:

Kolb, Chromatographia, Vol. 15 No. 9, September 1982.PerkinElmer order number: GC-319.

Bruno Kolb, Leslie S. Ettre; Static Headspace-Gas Chromatography, Theoryand Practice, Wiley-VCH, 1997. PerkinElmer order number: N101-1210.

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i

Index

6

600 series LINK, HS 40 connections 6

A

audit trail 18

AutoSystem connections

HS 40 4

interfaces 4

ready/run signal 8

C

component information, MHE

about 23

viewing/printing 24

constant thermostatting 12

conventions, manual 1

cycle time 9, 12

D

data processing, delaying 16

E

equations, MHE 27

G

ground 8

H

hardware connections 3

HS 40 analyses

about 9

creating a method 11

creating a sequence 14

displaying status 17

printing audit trail 18

printing method parameters 18

setting up a sequence 15

starting 15

HS 40 connections

AutoSystem 4

interface 4, 6ready/run signal 8

shield wire 8

HS 40 Method Editor 10

HS 40 methods

creating 11

printing audit trail 18

printing parameters 18

requirements 9

HS 40 MHE analyses SeeMHE analyses

HS 40 status 17

I

instrument configuration, updating 12

integral LINK, HS 40 connections 4

M

manual conventions 1

messages, HS 40 status 17

methods

GC 9

HS 40 11

HS 40 MHE 19

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ii

MHE

about 27

files, managing 25

mode 12

regression curves 21

MHE analyses

about 19

creating a method 19creating a result file 21

printing/viewing results 24

regression curves

displaying 22

entering peak areas 22

plotting 21

sequence 20

P

peak areas, entering 22

PII time 14

progressive thermostatting 12

R

ready/run signal 8

regression curves, plotting 21

S

sample information

about 23

viewing/printing 24

sample lists

creating 21

managing 26sampling modes 12

sequence

creating 14

HS 40 14

MHE 20

setting up 15

shield wire 8

side-by-side plots 22

starting a run 15

status, HS 40 17

stopping a run 16

suppress processing 16

T

theory, MHE 27

thermostatting

extended 17

mode 12

TotalChrom sequence Seesequence

W

Water Trap accessory 12

Documents

HS40_IPM_N515-6071a