HS 40 Control User's Guide

for TotalChrom Client Server and TotalChrom Workstation

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: AI.UserAssistance@perkinelmer.com

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.
 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 Analysis
Creating 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

i
 Introduction

The HS 40 Control application is used on a TotalChrom system to set parameters and

obtain status reports from the HS 40 or HS 40XL Headspace Sampler. The HS 40
control application comprises the HS 40 Method Editor, HS 40 Status and Setup, and
HS MHE commands, which are on the TotalChrom Navigator Apps menu.
The Multiple Headspace Extraction (MHE) software application that is included with
the TotalChrom HS 40 Control software lets you analyze and report data and results
obtained by performing multiple extractions and injections from a headspace vial.

Using this Guide

The TotalChrom HS 40 Control User's Guide describes how to connect your HS 40,
AutoSystem GC, and LINK interface for TotalChrom control. This guide also
describes how to create and run HS 40 methods and sequences by using the
TotalChrom HS 40 Control and MHE software.
The procedures in this manual assume that you are familiar with the TotalChrom
software and know how to configure instruments, use the TotalChrom Navigator, and
create and run TotalChrom methods and sequences. Before beginning this manual,
you may want to review Chapters 3-9 and 11 in the TotalChrom User’s Guide.

Conventions Used in this Guide

This guide assumes that you will be using a mouse or similar device. Many shortcut
keys are listed on the menus, and the documentation for your operating system can
provide information about equivalent keyboard procedures.

Capitalization
All menus, commands, and dialog box option names appear with initial capital letters
whether or not they are completely capitalized in the user interface. The names of
keyboard items, such as the Enter key, are capitalized. This will help you to
distinguish these items from narrative or procedural text.

1
Using This Guide I

Terminology
The following terms are used to refer to program elements and the actions that you
perform to carry out tasks:
Choose  The terms choose and select indicate 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 likely
to use the keyboard to choose an action, the specific key is mentioned, such as
Enter or Tab.
Select  The term select refers 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.
³(QWHURUVHOHFW´ 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 enter a filename, you type it in the File Name text box
exactly as it exists. When you select a 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 name HS 40 refers 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 System

The 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.
+HOSPHQX When you choose Help Topics from the Help menu, TotalChrom
displays the list of help topics for the current application.

2
 HS 40 System Connections

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 your TotalChrom Application
Manager’s Guide.
After installing the hardware, configure the LINK interface and the AutoSystem as
described in Chapter 3 of your TotalChrom User’s Guide.

3
 HS 40 System Connections

½ To connect the HS 40 and the AutoSystem for TotalChrom control:

1. Install the HS 40 and any accessories as described in the Headspace Sampler
for Gas Chromatography Instrument Manual and the accessory manual 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.

4
HS 40 System Connections

1. Remove this side panel.

2. Connect this cable to port J2 on

the LINK interface printed circuit
board located on the inside of
the side panel.
Serial/Host

IEEE

AutoSystem XL GC
Turbochrom
Serial/Host
or
IEEE
Connection

'<1+6   %)287&5<2287

&$55, 1   +3,1   %),1    &5< 2,1

BOARD

INT ERFAC E

1&
12
7 ,0 ('
SOFTWARE-BOARD

(9(17   &
OPTION-BOARD

1&
7 ,0 ('
12
(9(17   &
1&
7 ,0 ('
12
(9(17   &
1&
12
7 ,0 ('

(9(17   &

7 ,0 ('

(9(17  
1&
12
&
RS-232 Cable (P/N 6560-9010)
1&
BCD

RS-232

7 ,0 ('
12
(9(17   &

HS 40XL

HS 40 to AutoSystem GC Using an integral LINK

5
 HS 40 System Connections

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.

6
HS 40 System Connections

RS232

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

AutoSystem XL GC

'<1+6 %)287&5<2287

Inst A &$55,1+3,1%),1&5< 2,1

Inst B

INTE RFACE
BOARD
1&
7 ,0('
12

SOFTWARE-BOARD
(9(17  &

OPTION-BOARD
1&
7 ,0('
12
(9(17  &
1&
7 ,0('
12
(9(17  &
7 ,0('
1&
12
(9(17  &
1&
7 ,0('
12
(9(17  &
1&

BCD

RS-232
7 ,0('
12
(9(17  &

610 LINK HS 40XL

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

HS 40 to AutoSystem GC Using a 600 Series LINK

7
 HS 40 System Connections

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 black
wire 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 Manager’s Guide.
6. Reconnect and plug in all power cords, and turn on all instruments.

8
 HS 40 Headspace Analysis
When you run an HS 40 analysis, you do the following:
• Create a TotalChrom method (<filename>.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 User’s Guide;
• Create an HS 40 method (<filename>.HSM) in the HS 40 Method Editor that
contains HS 40 instrument control parameters.
• Create a TotalChrom sequence that controls the HS 40, AutoSystem, data
acquisition, and analysis.
An HS 40 method must satisfy the following requirements for the TotalChrom
sequence to run:
• The AutoSystem GC and HS 40 methods must have the same 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 the
overlapping 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.

9
Creating an HS 40 Control Method

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.

10
HS 40 Headspace Analysis

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 the
thermostatting 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.

11
Creating an HS 40 Control Method

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.

12
HS 40 Headspace Analysis

For best reproducibility, set the needle and transfer temperatures to a value
5-10°C 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 the HS 40 Capillary Backflush Accessory Manual for more
information.
9. To activate the CryoFocusing accessory, enter the Pre- and Post-CryoFocus
times.
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.

13
Creating a Sequence in TotalChrom

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 User’s 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.

14
HS 40 Headspace Analysis

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.

15
Setting up a Sequence and Displaying HS 40 Status

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.

16
HS 40 Headspace Analysis

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.
External Device — 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.

17
Setting up a Sequence and Displaying HS 40 Status

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.

18
 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.
½ To create an HS 40 MHE Method:
Enter your MHE GC and HS 40 method and sequence parameters as you would
for a standard HS 40 application, with the following exceptions:
• TotalChrom Method — Use Calibration Factor is not a recommended
calibration 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 40
Method Editor.

19
Multiple Headspace Extraction (MHE)

• MHE sequence — An MHE sequence must contain at least two rows

and must have between 2 and 9 injections (extractions) from the same
vial.
• MHE Results Window — Develop calibration and sample lists of
TotalChrom result 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.

20
 Overview of HS 40 MHE Analysis

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.
½ 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.

21
Multiple Headspace Extraction (MHE)

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 MHE
application 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 <filename.mhe>.
½ 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 and
displays 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.

22
 Overview of HS 40 MHE Analysis

General Information
Component The name of the component that is displayed.
6WXG\1DPH The name of the study as entered in the Sequence Editor.
Sample Name — The name of the sample as entered in the Sequence Editor.
9LDO1XPEHU The number of the sample vial that contains the component
that is displayed.
6DPSOH$PRXQW The amount of sample in the sample displayed.
'LOXWLRQ)DFWRU 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 calibration
sample.
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

23
Multiple Headspace Extraction (MHE)

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.
½ 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.

24
 Managing MHE Files

½ 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 the
component 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
½ 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.

25
Multiple Headspace Extraction (MHE)

2. Enter or select the path(s) for the TotalChrom result files, and choose OK.
½ 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.
½ 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.

26
 MHE Theory and Calculations

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
= kC (1)
dt
where t is time, C is concentration, and k is a constant. Since the concentration at any
time, C, depends on the initial concentration C0 and the exponent k, the equation
becomes

C = C0 e − kt (2)

27
Multiple Headspace Extraction (MHE)

Since the extractions are performed in steps, the time t may be replaced by the
number of extraction steps, n. The initial concentration C0 is replaced by the peak area
from the first extraction step, A1, which occurs at time t = 0 or n = 1. The constant k,
which now includes instrument parameters, becomes k*:

An = A1e − k ( n −1)
*
(3)

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

ln An = − k * ( n − 1) + ln A1 (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 not
linear, 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 ΣAn becomes

∑A n = A1 1 + e − k * + e −2 k * + e −3k ... (5)

Written in a more general form, the equation for the sum of the areas is
A1
∑A n =
1 − e−k
* (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, the
amount 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, Theory
and Practice,” Wiley-VCH, 1997. PerkinElmer order number: N101-1210.

28
 Index

6 about 9
creating a method 11
600 series LINK, HS 40 connections 6 creating a sequence 14
displaying status 17
A printing audit trail 18
audit trail 18 printing method parameters 18
AutoSystem connections setting up a sequence 15
HS 40 4 starting 15
interfaces 4 HS 40 connections
ready/run signal 8 AutoSystem 4
interface 4, 6
C ready/run signal 8
shield wire 8
component information, MHE HS 40 Method Editor 10
about 23 HS 40 methods
viewing/printing 24 creating 11
constant thermostatting 12 printing audit trail 18
conventions, manual 1 printing parameters 18
cycle time 9, 12 requirements 9
HS 40 MHE analyses See MHE analyses
D HS 40 status 17
data processing, delaying 16
I
E instrument configuration, updating 12
integral LINK, HS 40 connections 4
equations, MHE 27
M
G
manual conventions 1
ground 8 messages, HS 40 status 17
methods
H GC 9
hardware connections 3 HS 40 11
HS 40 analyses HS 40 MHE 19

i
MHE shield wire 8
about 27 side-by-side plots 22
files, managing 25 starting a run 15
mode 12 status, HS 40 17
regression curves 21 stopping a run 16
MHE analyses suppress processing 16
about 19
creating a method 19 T
creating a result file 21
printing/viewing results 24 theory, MHE 27
regression curves thermostatting
displaying 22 extended 17
entering peak areas 22 mode 12
plotting 21 TotalChrom sequence See sequence
sequence 20
W
P Water Trap accessory 12
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 26
sampling modes 12
sequence
creating 14
HS 40 14
MHE 20
setting up 15

ii