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About Method Development

Creating a Method for Acquisition and Calibration

A method contains complete information for acquisition, integration, and calibration of a sample run. A method has several main sections. These are:

Instrument Setup

Integration Timed Events

Calibration

Custom reports

The method can also contain general data processing information such as data export options, valve programs, and user programs to run.

The Instrument Wizard can guide you through the essential steps for creating a method. To start the Instrument Wizard for creating a method, click Instrument
Wizard and then click Create or modify a method.

A method can be created from one of the Instrument windows, or when running an instrument offline. When you create a method from an Instrument window, you also
have access to starting runs and viewing data acquisition. When you create a method offline, you do not have access to instrument control (starting or stopping runs),
however you can set up a complete method, including instrument configuration, which can be opened at a later time and used for one of the Instruments on the system.

This section describes the steps required to prepare a method for data acquisition, optimize integration, and set up calibration. Later sections will detail report generation
and sequence operations.

Select a New Project

When instrument login and project management are turned ON, you must select a project when you log into an instrument. This project becomes the current project for
your instrument.

To change the project

1. On the File menu, click Project.

2. From the list of projects to which you have rights, click the project you want to use for this instrument, and then click OK.

Create an Acquisition Method

In order to acquire data and save it on the hard drive of your computer, you need to create a method that contains data acquisition information such as run time and
sampling rate. To create a new method or edit an existing method, use the Method Wizard and select the Create a New Method button. This will over-write current
method parameters with those of the current data system default method.

You can create a new method from Instrument Window whether or not the instrument is on line. However, you only have access to starting actual data acquisition when
you have an on line instrument connected.

Method Wizard

The Method Wizard is started from the Instrument Wizard by clicking the button labeled Create or modify a method.

When you click this button, the Method Wizard will appear, allowing you to select how you want to use the wizard.

Select Create a new method to start a new method with the system default method parameters.

Select Modify the current method to step through the current method and make changes.

Select Modify a method on disk to open a file saved on disk and step through it to make changes.

When you select one of these buttons, the Method Wizard sets up a bank of buttons in your application window that allow you to "step" through all dialogs of method
generation. A save button is also provided.

Save Method As
When you have completed the sections for creation of a data acquisition method, save the file on your hard disk before you use it for data acquisition. To save the
method, click the Save button, followed by Method, or select File followed by Method and then click Save As A dialog box will appear where you can give your
method a distinctive name and designate a disk folder (directory) where it will be saved. The dialog appearance and behavior will be slightly different for systems with
advanced file security enabled. Users will be limited to storing files within the current project folder and the Enterprise Common folder. A folder bar will be included in
the dialog to facilitate navigation.

Select the directory where you want to save the method. Enter the name of the new method in the File Name field. You can review and change the method description
using the Description box. To save the method, click the Save button.

You are now ready to use your method for data acquisition.

Baseline Check On Demand

To perform an on-demand baseline check, from the Control menu, click Baseline Check. A dialog will appear where you set the parameters for doing the baseline

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check.

To pass, the baseline must meet the test criteria for

This specifies the time during which the baseline must meet the criteria in order to pass the test.

Stop checking baseline if conditions are not met after

This specifies the maximum time that will be spent checking the baseline. If conditions are not satisfied in this time, then the baseline check fails.

Noise Test Method

Select the noise calculation method that should be applied to the acquired data.

Test Spreadsheet

This spreadsheet specifies the channels to be used for baseline calculations and the tests that should be performed on those channels. The number of rows of the
spreadsheet equals the number of acquisition channels based on instrument configuration plus the number of PDA Multichromatogram channels defined in the PDA
Options Window (if PDA is configured).
Channel

Each entry in this column contains the name of an acquisition channel specified by instrument configuration or the PDA multichromatogram definitions. The entries
cannot be edited.
Enable Noise Test

Checking this box indicates that detector noise should be calculated on the corresponding channel. If this box is unchecked, the Threshold edit field to its right is
disabled; otherwise it is enabled.
Noise Threshold

This is used to specify the maximum acceptable value for the calculated noise. If the calculated noise is greater than the Threshold, the baseline check is considered to
have failed.
Enable Drift Test

Checking this box indicates that detector drift should be calculated on the corresponding channel. If this box is unchecked, the Threshold edit field to its right is
disabled; otherwise it is enabled.
Drift Threshold

This is used to specify the maximum acceptable value for the calculated drift. If the calculated drift is greater than the Threshold, the baseline check is considered to
have failed.

Baseline Check Status

To check the status of the Baseline Check function, from the View menu click Baseline Check Status. A status box appears where you can view the status of the most
recent Baseline Check test. This box contains status information only. To change Baseline Check parameters or perform a Baseline Check test, use the Baseline Check
command from the Control menu.

About Pretreatment Files

An autosampler pretreatment (injector program) is comprised of a series of numbered lines, each of which defines an operation that the injector carries out sequentially.
If your autosampler supports a pretreatment program, the Pretreatment menu will appear on the menu bar. From this menu, you can create/edit an autosampler
pretreatment program, and set up properties such as a description and audit trail for the pretreatment program.

Open a Pretreatment File

To open an existing pretreatment file,

1. From the Instrument Window menu bar, click File and then Pretreatment followed by Open. A dialog will appear where you can select a pretreatment file to
open for the current instrument.

Select the file to open, or use the search boxes at the bottom to search for a file that matches criteria you select.

Launch Lab Monitor Software

To run the Lab Monitor program for supported instruments, from the Control menu, click Launch Lab Monitor Software.

Instrument Setup
The instrument setup portion of your method is where you define instrument control parameters and define acquisition parameters. If you are using a Generic
instrument, parameters appear that enable you to set up the acquisition. If you have installed one of the instrument control options and are using one of those configured
instruments, the instrument setup will contain parameters that are specific for the control of the selected instrument.

To open Instrument Setup,

1. From the Method menu or on the Navigation Pane, click Instrument Setup.

2. For analog instruments, click the Detector tab (or the tab with the detector name you designated during configuration). Complete the aquisition parameters
described below.

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3. Click other tabs for any additional parameters you wish to edit. These will become part of the method when you save it.

Detector 1…x

For each detector channel (x) configured on the instrument, define the following acquisition information.
Acquisition Channel On

Select this box to turn the acquisition for this channel ON. If this box is not selected, no data will be acquired for this channel.
Sampling

This is the rate at which data will be sampled by the system. You can choose how you want to specify the sampling rate. When you select a sampling rate, a prompt will
appear indicating the narrowest peak width for which this sampling rate will be adequate. It is recommended that you use the Graphical Events Programming to
determine the optimum sampling rate for your chromatography.
Frequency

This selection is in Hz (samples per second). This is the selection for most chromatography applications. Click on the down-arrow to get a list of the frequencies
available for the configuration of your system.
Period

When you select this type of sampling, you must select the number of seconds (or milliseconds) between data points. Enter the value, then select whether the period is in
milliseconds (mSec) or Seconds.
Run Time

Run Time determines the length of time data will be sampled.

Acquisition Delay

Acquisition Delay is the interval between the start of run (Trigger) and the time when sampling starts for this channel.

Import Instrument Setup

This command enables you to open just the instrument setup parameters from a saved method.

To import an instrument setup

On the File menu, click Method, and then click Import Instrument Setup. The Method Open dialog box will appear where you can select the method from which you
wish to import the instrument setup parameters.

Trigger
To select the type of trigger to use for a method, from the Method menu, click Instrument Setup and then click Trigger.
Trigger Type

Determines how the data sampling is started.

Trigger selection Trigger effect

Sampling starts immediately after clicking on Start. Sequence acquisitions do not
None
pause between runs.
Operator has to press ENTER to start the run. Sequence acquisitions pause for
Manual
confirmation between runs.
If the data sampling is started from an external trigger, select this option. The type
External
of trigger is designated when the instrument is configured.

When you have completed the acquisition parameters, click the X box in the upper right corner of the dialog box to exit the dialog.

External Events

Once you have configured external events for your instrument, you can program the events for your method using the External Events tab in Method > Instrument
Setup. Note that this tab will not appear on your menu unless you have configured external events for your instrument. When you select this tab, a spreadsheet appears
where you can select the events and designate when and how they activate during the run.

To program an event, select it from the drop-down list of configured events by clicking the Event field. Enter the time at which the event is to actuate, the length of time
you wish the event to remain in the desired state (Duration), and the State of the event during the event (Close or Open).

You may configure up to 25 events for the run.

Baseline Check

When Baseline Check in the configuration options for an instrument is enabled, the Baseline Check Tab will appear in Instrument Setup, where you can set the
parameters to use when doing a baseline check while acquiring data using this method.

When a method containing baseline check parameters is used for data acquisition, a baseline check will occur when either:

The Perform Baseline Check checkbox is checked in the Single Run dialog, or

The Run Type of the current Sequence line includes Baseline Check.

When a baseline check is to occur as part of data acquisition, the software will first download the initial conditions from the method to the instrument. These initial
conditions are then used to acquire the baseline check data before the normal data acquisition. If the baseline check data fails to meet the threshold for any channel, then

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the data acquisition is aborted.

If the baseline check results indicate a failure of the test, then the current run is aborted and the baseline check data and results are stored in the data file without any
other acquisition data. If the run is aborted (by the user or because of a hardware error) prior to the completion of the baseline check data acquisition, then no data is
saved.

To pass, the baseline must meet the test criteria for

This specifies the time during which the baseline must meet the criteria in order to pass the test.

Stop checking baseline if conditions are not met after

This specifies the maximum time that will be spent checking the baseline. If conditions are not satisfied in this time, then the baseline check fails.

Noise Test Method

This is used to specify the noise calculation method that should be applied to the acquired data.

Test Spreadsheet

This spreadsheet specifies the channels to be used for baseline calculations and the tests that should be performed on those channels. The number of rows of the
spreadsheet equals the number of acquisition channels based on instrument configuration plus the number of PDA Multichromatogram channels defined in the PDA
Options Window (if PDA is configured).
Channel

Each entry in this column contains the name of an acquisition channel specified by instrument configuration or the PDA multichromatogram definitions. The entries
cannot be edited.
Enable Noise Test

Checking this box indicates that detector noise should be calculated on the corresponding channel.

If this box is unchecked, the Threshold edit field to its right is disabled; otherwise it is enabled.
Noise Threshold

This is used to specify the maximum acceptable value for the calculated noise. If the calculated noise is greater than the Threshold, the baseline check is considered to
have failed.
Enable Drift Test

Checking this box indicates that detector drift should be calculated on the corresponding channel.

If this box is unchecked, the Threshold edit field to its right is disabled; otherwise it is enabled.
Drift Threshold

This is used to specify the maximum acceptable value for the calculated drift. If the calculated drift is greater than the Threshold, the baseline check is considered to
have failed.

Aux Traces

To turn on acquisition and display of auxiliary traces for an instrument,

1. From the Method menu click Instrument Setup and then click Aux Traces (if available). This tab is used to acquire traces of instrument status parameters
during normal data acquisition. Many instruments have the ability to report continuous monitoring data on status parameters such as flow rate and oven
temperature. When this option is enabled, this tab appears, allowing you to designate which status parameters you wish to monitor during the run. The actual
entries in this list will vary depending on the instrument configuration. Changes to the aux. traces for the Agilent 1100 Column Comp, RI detector, Isocratic
pump, Binary pump and Quaternary pump are logged in the audit trail.

To have status information logged for a parameter listed in this dialog, click on the check box for that row and select the appropriate units for that parameter. When data
acquisition is performed, status information will also be acquired. The resultant data will be presented as an additional data channel whenever the data file is viewed.

Method Properties
Before you use a method to acquire data, be sure that the options such as automatic post-run analysis of the data (if desired) are turned on. To check these parameters,
from the Method menu, click Properties. A tab box will appear where you set the properties for the method.

Method Properties Description

To enter a description for your method,

1. From the Method menu, click Properties.

2. Select the Description tab. A dialog box will appear where you can enter text information about your method. You can enter any information you wish. The
description can be viewed from the Open File dialog, and therefore can be useful in sorting quickly through methods to find the right one.

Method Properties Options

To set the analysis options for your method,

1. From the Method menu, click Properties and then select Options.

Analyze during acquisition

If you select this box, the chromatogram will be automatically analyzed during the run at the interval you specify in the box.

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Analyze after acquisition

If this box is selected, the chromatogram will be analyzed automatically after every acquisition. This is the default condition. If this box is not selected, you must either
manually analyze the chromatogram by clicking the Analyze button, or analyze the sample as part of a sequence reprocessing after the run has been completed.

Enable compression for data file

When this box is checked, the data files acquired using the data system will automatically be compressed before saving. (Compressed data files are smaller but take
longer to load.) Once a file has been saved using compression, the only way to decompress it is to save it as another data file with compression turned off.

Method Properties Calibration

To set calibration defaults for your method,

1. From the Method menu, click Properties and then select Calibration.

Automatically average consecutive replicates of the same level

When this box is checked, calibration replicates (consecutive injections of a single calibration level) will always be averaged. If this box is not checked, calibration
replicates will not be averaged unless you so specify at the time of calibration.

Number of replicates in rolling average

If you wish to use a rolling average, enter the number of replicates per average here.

Response Factor Definition

Select how the response factors are to be calculated and displayed - Area/Amount or Amount/Area.

Method Properties Audit Trail

Select the Audit Trail tab. The Enable Audit Trail checkbox is an important box because, if checked, subsequent changes to the method will be logged in the method.
To enable the method audit trail,

1. From the Method menu, click Properties and then select Audit Trail.

Note: Once the Enable Audit Trail box is selected, it cannot be de-selected. The method will continue to have audit trail enabled unless you save the file under a new
filename.

If you select the Enable Audit Trail box, the following warning will appear.

If you select No, you will be returned to the Audit Trail tab and you can continue. If you select Yes, the Enable Audit Trail will be permanently checked for this method.

Once the Enable Audit Trail box is checked, the following options are available for documenting changes.

Prompt for reason at every change

This option requires the user to enter a reason for every subsequent change to the method at the time the change is made.

Prompt for reason when saving Method

This option requires the user to enter a reason for each change when the Method is saved.

Do not prompt for reason

When this option is selected, changes are documented, but the user is not required to enter a reason for the changes.

Audit Trail Reasons

When you have selected the Audit Trail Option with reasons, you will be prompted for the reason for changes, either at the time of the change, or when the
method/sequence/data/system is saved.

If you have selected the option to enter a reason at every change, the dialog will appear each time you make a change to the item being audit trailed. A description of the
change appears in the Source/Activity box. Select the reason for the change in the Reason box or type in your own reason, then click OK.

If you selected the option to enter a reason upon saving the method/sequence/data, the dialog will appear when you save the method/sequence/data and you must enter a
reason for all changes to the method/sequence/data. Use the Back and Next buttons to view the descriptions of the changes. A change item number is displayed on the
lower right of the window, along with the total number of changes. If you want to enter one reason for all changes, type the reason, then click the Apply to All button.
After you have entered changes for all reasons, click OK.

Data Properties
Data Properties presents tabs where you can view and change the description for the current data file, and turn on audit trail for the current data file. To view the
properties for a data file,

1. From the Data menu, click Properties.

Data File Description

The information shown in the Description box is the description currently stored in the current data file. Because the description can be viewed from the Open File

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dialog, the description can be useful in sorting through the data files on your disk. If you want to change the description for the current data file, you can enter or edit
from this box.

Data file descriptions can be entered in the sequence at time of data acquisition, or can be entered from the Single Run dialog when running a single sample.

Data File Audit Trail

Data File Audit Trail

Data files always have an associated audit trail. The Data File Properties>Audit Trail tab allows you to select whether or not you want the system to prompt you for
reasons whenever a change is made, by selecting the appropriate button.

Data File Electronic Signatures

To view the electronic signatures for a data file,

1. Open the data file.

2. From the Data menu, click Properties and then click E-Signatures.

Electronic Signatures

Users whose privileges include data results sign-off can electronically "sign-off" results. To access the data results sign-off, from the Data menu, click Apply electronic
signature… .

Enter your name, password, and domain. You must also enter a Reason by selecting from the list displayed. You can also enter a comment if desired. Your "electronic
signature " will be logged into the data file audit trail. Once you have clicked the Signoff button, the data is locked to further analysis. Each data file can have multiple
electronic signatures (no maximum).

Revoke Electronic Signatures

Users with appropriate rights can Revoke an electronic signature using the Data/Revoke electronic signatures… command. No reason is required to revoke electronic
signatures, although you can add a reason if desired. Once you have revoked the electronic signatures, all electronic signatures will be revoked for the data file and it
can once-again be analyzed. When the electronic signature is revoked, an entry is made in the audit trail to that effect.

Electronic signatures are available for printing on custom reports. See the Custom Report section.

Revoke Electronic Signature

Users with appropriate rights can Revoke an electronic signature. To revoke an electronic signature,

1. From the Data menu, click Revoke electronic signatures… . (Note: If there are no electronic signatures for this data file, this command will not be available.)

2. Enter your user name and password, and type a reason to revoke an electronic signature.

3. Click Signoff.

4. Once you have revoked the electronic signatures, all electronic signatures will be revoked for the data file and it can once-again be analyzed. When the electronic
signature is revoked, an entry is made in the audit trail to that effect. A user may only revoke the electronic signatures of the data file if no-one with a higher
signing authority has signed the data file.

When a data file is opened in the context of a project other than that in which it was signed, only a user with System Administration privileges can revoke the signatures
on the data file.

Apply Electronic Signatures to Multiple Files

To apply your electronic signature to a batch of files simultaneously,

1. From the Data menu, click Apply Signatures to multiple files… .

2. In the Open data file dialog select the files from the list box at the top, and then click the Add button to add them to the list of files to be signed. When a file is
selected for signature, it will appear at the bottom of the dialog box.

To delete a file selected for signature, select the file in the list at the bottom, and click the Delete button.

When you are ready to sign, click the Open button, and the Electronic Signature dialog box will appear where you enter your electronic signature and password, and
select a reason. The same reason will be applied to all files signed in this manner.

Multiple File Signature Processing

When signing multiple files in a batch process, a dialog will appear when you click the Signoff button in the Electronic Signature dialog that will show the status of the
files you are signing or for which you are revoking the signatures.

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While the signatures are being processed, the Abort button will be active and you can choose to abort the process. If you select this, any files not signed will not be
signed. Any files already signed (success) will not be affected.

The Print button will print the list of signed files.

This dialog will appear during Multiple File Signatures and during Revoking Multiple File Signatures.

Revoke Signatures of Multiple Files

To revoke your electronic signature to a batch of files simultaneously,

1. From the Data menu, click Revoke signatures of multiple files....

2. In the Open data file dialog box, select the files from the list box at the top, and then click the Add button to add them to the list of files to be revoked.

3. When you have added all of the files to revoke signatures, click Open.

4. Enter your User name and Password, and a reason for revoking the signatures in the Revoke Electronic Signatures dialog box, and then click Signoff.

To delete a file selected for revoking, select the file in the list at the bottom, and click the Delete button.

When you are ready to revoke the signatures, click the Open button, and the Revoke Electronic Signature dialog box will appear where you enter your electronic
signature and password, and select a reason. The same reason will be applied to all files revoked in this manner.

View the Method Audit Trail

To view the audit trail log for the current method,

1. From the File menu, select Method followed by Audit Trail. A listing of the audit trail will appear that includes the User responsible for the change, Time
Logged, method location (Source) of the change, what was changed (Activity), and Reason for the change.

To see full information for a logged change, print one of the entries, or print all the entries, first select a row by clicking on it with the mouse to highlight it, then do a
right mouse click.

Show Detail

Select this option to view the full information on the selected activity.

Print All

Select this option to print all of the audit trail entries.

Print Selection

Select this to print only the highlighted entry.

View the Data File Audit Trail

To view the audit trail for the current data file,

1. From the File menu, select Data followed by Audit Trail and a listing of data audit trail events will appear. These include the name of the logged user and time
activity was logged, the source of the change, the change or activity for the data file, along with the method used to analyze the data, MIF table changes, and
changes to the data file description, along with reasons, if any.

To see full information for a logged change, print one of the entries, or print all the entries, first select a row by clicking on it with the mouse to highlight it, then do a
right mouse click.

Show Detail

Select this option to view the full information on the selected activity.

Print All

Select this option to print all of the audit trail entries.

Print Selection

Select this to print only the highlighted entry.

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View the Instrument Activity Log

As you use a method to acquire data, a log of activity from the current instrument is kept. To view this log, from the File menu, click Instrument Activity Log and then
click Display Log.. A window with the Instrument Activity Log appears. The window displays the User who used the instrument, the time the activity was logged into
the Instrument Activity Log, and a description of the activity.

To view details of any line in the instrument activity log, click on the line to highlight it, then do a right mouse click within the spreadsheet. From this pop-up menu you
can view details of the highlighted line, print it, or print the entire activity log. In addition, you can export, archive, or purge the activity log from this menu.

Note: Over time, the instrument activity log file may become large, so periodically you should archive the file to a floppy or another location and then purge it.

Instrument Activity Log Manual Entry

To enter a manual entry in the instrument activity log,

1. From the File menu, click Instrument Activity Log and then click Manual Entry.

2. Type in the information you wish to enter in the instrument activity log, then click OK.

Instrument Activity Log Export

To export the instrument activity log,

1. From the File menu, click Instrument Activity Log and then click Export… . A dialog appears.

File name

Enter the name to be used to store the instrument activity export file.

Save as type

Select the type of file you wish to save from the choices displayed.

Select record ranges

Select the radio button next to the range desired.

Click the Save button to save the instrument activity log for the range selected in the file specified.

Instrument Activity Log Archive

To archive the instrument activity log for an instrument, from the File menu, click Instrument Activity Log and then click Archive. A dialog will appear where you
can select the location for the archive file. A default name is assigned, with the .logarc extension. To view the archived file, use the Archived Log Viewer that can be
run from the Chromatography program group in Windows.

Instrument Activity Log Purge

To purge the current instrument activity log,

1. From the File menu, click Instrument Activity Log and then click Purge. The following message will appear.

If you have set the enterprise options such that a purge is only allowed after archive, you will be presented with the Instrument Activity Archive dialog if the log has not
been archived when you click either the Yes or the No button.

If you have not set the enterprise options such that a purge is only allowed after archive, you will be presented the Instrument Activity Archive dialog if you click Yes.
If you click No, the instrument activity log will be purged.

Archived Log Viewer

To view an archived log file, run the Archived Log Viewer from the Chromatography program group in Windows. You can also print the archived log from this
program.

Integrating the Chromatogram

The data system comes with a default method that is adequate for data acquisition and analysis of simple chromatograms. However, your chromatography may require
more elaborate treatment of the data, or special integration of specific peaks. Integration events are normally entered in the Integration Timed Events Table. This section
describes how to optimize a method for proper integration of your data files.

Integration Events Table

When you add an integration event to the Integration Events Table, the integration changes will be used on all subsequent samples analyzed using this method.

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(Assuming the method is saved.) This is the default selection for events that are normally used to modify integration of peaks that always occur in the sample.

To view the method Integration Events table, from the Method menu, click Integration Events.

The Integration Events Table contains all current Integration Timed Events for the current method channel.

To add an event manually,

1. In a new line of the spreadsheet, click the Event field, and select the event you wish to add.

2. Type an appropriate Start and Stop time and a Value for the event (if required).

3. To remove an event’s effect from an analysis, yet keep the event in the table, click on the check-mark next to the event. Only events with a red check-mark will be
used in subsequent analyses.

To remove an event entirely from the table,

1. Click on the row number of the event, followed by the DELETE key on your keyboard.

A right-hand mouse click anywhere in the table produces a menu of commands for manipulating cells and rows in the spreadsheet.

The Cut, Copy, and Paste commands enable you to cut, copy, and paste the contents of a cell.

The Insert Paste command will insert a line and at the same time do a paste to the location.

The Insert Line command simply inserts a blank line where your cursor is located.

The Clear command clears the contents of the current cell or selected row.

The Clear All command clears the contents of the entire spreadsheet.

Required Integration Events

Two Integration events are required for each run: Width, and Threshold. These events are used to detect peak start, stop, and apex, and to distinguish true peaks from
noise. The system uses default values of Width = 0.2 minute and Threshold = 50. For details on setting Width and Threshold, as well as other integration timed events,
see Integration.

Sampling Rate

The sampling rate used to acquire your data determines how much information the integration algorithm has for drawing and integrating the chromatogram. The
sampling frequency is set in the Instrument Setup part of your method. To make sure you have the proper sampling rate, use the Suggest Sampling Frequency
command in graphical programming. Slight over-sampling of data is corrected with the Width integration parameter and is not a problem. Try to avoid gross over-
sampling of data, however, as it does not give better integration and it wastes space on the hard drive of your computer. More important, however, is not to under-
sample, as there is no way to correct for data points that are not sufficient to define and integrate your peaks.

Graphical Integration Optimization

Optimizing integration using Graphical Programming is easy to do. With your chromatogram displayed in the chromatogram window, click anywhere in the window
with the right mouse button. From the pop-up menu that appears, move the cursor to Graphical Programming. A list of available graphic events will appear. These
events are also available from the Integration Toolbar if displayed.

Note: Before attempting to add integration timed events, make sure your chromatogram has been analyzed using the current method. To make sure, look for baselines
and start/stop tic marks on your chromatogram. Or, click the Analyze button on the command ribbon.

Graphically Setting Width

From the graphical programming list, click Width. (Or click the Width button on the Toolbar.) Follow the instructions in the status bar at the bottom of the window to
optimize the width integration parameter. Click once at the beginning of your narrowest peak, then once again at the end of the narrowest peak. EZChrom Elite will
suggest a width value. Select Analyze Now to add it to your method and reintegrate using the new width value. Click Add to Table if you want to simply add the event
to your integration timed events table and go on without integrating the chromatogram at this point.

The Manual Integration Fixes table is used for special baseline integration and is described below. Do not select this option now unless you want this timed event to
apply only to this sample and not become a regular part of the method integration.

Graphically Setting Threshold

Set the threshold using Graphical Programming next. This time, instead of using the pop-up menu, click the Threshold button on the integration toolbar. As instructed
in the status bar at the bottom of the window, click the mouse once at the beginning of a section of your chromatogram where no peaks elute, then once again at the end
of that section of chromatogram. The software will suggest a threshold value. Select Analyze Now to add the value to your method and reintegrate the chromatogram.

Suggest Sampling Frequency

Optimization of your sampling frequency can also be assigned graphically. Click on the Sample Frequency button, or the (right mouse click) Graphical
Programming>Suggest Sampling Frequency command. Following the instructions on the status bar, click once at the beginning of your narrowest peak of interest,
then once at the end of that peak. The software will suggest a sampling rate or period for acquisition of the sample. You can enter this value for your sampling rate using

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the Instrument Setup button.

Adding and deleting integration timed events

All of the integration timed events can be added to your method using the Graphical Programming technique. Each time you add an event, it will be stored in the
Integration Timed Events Table or Manual Integration Fixes Table. To view the Integration Events Table, click the Integration Events button, or select
Method>Integration Events from the menu.

Each row of the Integration Events spreadsheet represents an integration event in the method. To manually add an event, click on an empty row Event, and click the
down-arrow. Select the event to enter, then enter a Start Time, Stop Time, and Value (if applicable) for the event.

You can temporarily remove an event from the table by de-selecting the check mark adjacent to the event. When the check mark is not displayed, the event will not be
applied to the integration. To permanently delete an integration event, click the row of the event to select it. Click the right hand mouse button.

Select the Cut command. This will cut the event from the list. If you change your mind, you can reenter it using the Paste command. You can also use the right mouse
click commands to insert a line, clear a selected cell or row, clear all events, or select all events.

For a complete list of integration events, along with examples of how they work, see the Examples of Integration Events section.

Manual Integration Fixes

A Manual Integration Fix is an adjustment to integration required only for a particular chromatogram. Manual integration corrections that are created using graphical
integration are logged into the Manual Integration Fixes Table. These manual changes can also be entered in the Manual Integration Fixes Table by selecting the
desired integration change from the drop-down list, then entering a Start and Stop time, and a Value if appropriate. These integration changes are applied only to an
individual chromatogram rather than becoming part of the method, and are saved in the data file only. The Manual Integration Fixes are saved in the data file so that
the integration of the chromatogram can be re-created at a later date.

When the Data>Manual Integration Fixes command or the Manual Integration Fixes Table button is selected, the Manual Integration Fixes table will appear, where
you can view or edit the current Manual Integration events.

About Advanced Method Options

Advanced Method Options are available for users who have a need to go beyond the simple data acquisition and analysis provided by customizing results or linking to
external data systems or networks. When you select Method followed by Advanced option tabs will appear where you can set up various advanced options for your
method.

Option Lets you

Data Export Turn on data export and set export parameters.
Graphics Export Set up export for chromatograms and traces.
Custom Parameters Define custom parameters
Column/Performance Set up automatic calculation of system performance values.
Files Designate user programs and baseline file subtraction.
Advanced Reports Turn on printing and export of advanced reports.

Export Data

The Data Export function of a method allows you to save results automatically in an ASCII file after each analysis is completed. You select the type of data to export:
Peaks, Groups, Standard Reports, or Chromatogram. For Peaks and Groups, you may choose the parameters you want to export. When you export Standard Reports,
only the contents of the report will be exported.

Note: In order to export advanced reports, a printer must be configured on the computer doing the exporting.

To set up data export,

1. From the Method menu, select Advanced... followed by Export Data.

2. Click the Export Enabled box to turn data export On for the method. While this option is enabled, data export will occur after each Analysis of the data. Since
the export of data will occur whether the analysis occurs automatically at the end of a run, or when executed manually, you should turn this option off while you
are developing methods.

3. Choose the type of information to export from the drop-down list. For each type of export chosen, you can select parameters for export. If you have defined any
Custom Parameters, they appear in the appropriate list of items you can choose to export.

Exporting Peaks

When you select Peaks to be exported, a list of available peak export parameters will be displayed. Select a parameter for export by double-clicking on it, or clicking on
it to highlight it, then click the Green (Top) arrow button to move it to the list of export items in the right-hand box. To remove an item from the export box, highlight it,
then click the Red (bottom) arrow button.

Exporting Groups

If you select Groups for export, a list of group parameters will be presented in the left-hand box. To select an item for export, double-click it, or click on it to highlight
it, then click the Green (Top) arrow. To remove an item from the export list, highlight it, then click the Red (Bottom) arrow.

Exporting Standard Reports

When you choose to export Standard Reports, a list of available standard reports will be displayed. Choose a report to export by double-clicking it, or highlighting it
with the mouse, then clicking the Green (Top) arrow button.

Exporting Chromatograms

If you select Chromatograms to be exported, you will be given the option to export in either/or AIA (*.CDF) file format, or in ASCII format.

Selecting the AIA option causes Elite to create a *.CDF (Chromatograph Data File) in the standard format specified by the Analytical Instrument Association (AIA).
AIA Level 2 file export is supported. This includes the raw chromatogram, and integration results. This is also called ANDI file format (Analytical Data Exchange).

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This allows the results to be read by other chromatography data systems.

Note: AIA Level 2 support is for export only. Import of AIA files is supported only at Level 1 (raw data).

Notes on Rounding for CDF Export

A multiplier is used to convert the floating point numbers to integers. The code tries to determine the precision of the numbers by the Y axis label. It maps as follows:
microvolt, uvolts, uv, uau to a multiplier of 1. It maps millivolt, mvolts, mv, mau to a multiplier of 1000. If it does not find the y axis label specified in the CDF file in
either of these lists, then it determines the multiplier by picking the largest number that will not overflow a 22-bit floating point number. It does this by taking the largest
value in the data stream and multiplying it by 1000000, 1000, 1 and picking the largest multiplier that does not overflow the 22-bit float.

Files that have a y-axis label of "ua" do not map to any known label. In this case, 1000 will be used for the multiplier.

The converter does allow for the user to specify multipliers and axis labels in an .ini file called aia.ini located in installation directory. The format of the file is as
follows:

[YLabelSection]

YLabelCount=<number of labels>

YLabelString1=<y axis label name 1>

YLabelValue1=<multiplier 1>

YLabelString2=<y axis label name 2>

YLabelValue2=<multiplier 2>

Note: Microsoft Data Access Components (MDAC) 2.5 or higher is required for ODBC export. A version of this is on the software CD under \Updates.

Once this tab dialog is completed, it becomes part of the method.

Export Graphics
The Graphics Export function of the method will cause designated graphics to be exported, in .WMF format, each time an analysis is performed using the method. To
enable graphics export for a method,

1. From the Instrument Window, click Method followed by Advanced and click the Graphics Export tab.

2. In the spreadsheet, click Export and then type the filename to be used for the export.

3. The graphics window displays the current graphic for export. To change the graphic, do a right mouse click in the window. All the standard graphics parameters
are available.

4. Select the path to be used for export of the graphics file. The graphs exported will be saved with file name <Data file name> + “ _ “ + <Export Name> + “.wmf”

Custom Parameters

A custom parameter is a user-defined value that can be defined in one of two ways:

1. As a calculation whose result is calculated by a Windows executable program or VB script.

2. As a parameter defined as part of a sequence.

Custom parameters can be reported in a custom report, exported as a result, and annotated on chromatograms (per-peak parameters only). In addition, the custom
parameter can be read by name from a VB script or from Advanced Reports. The user supplies the custom parameter program or VB script. To set up custom
parameters for the method, click the Custom Parameters tab of the Advanced Method Options tab dialog box.

1. To define a Custom Parameter, enter the title you want to use to report the result in the Parameter Name field.

2. Next, move the cursor to the Type field and click your mouse. You will be given a choice of System-wide or Per-peak.

A System-wide parameter is one that is calculated once for the whole chromatogram. System-wide parameters can be displayed in a custom report, and will be printed
at the bottom of the report. An example of a System-wide parameter would be a BTU calculation, where the program multiplies each peak by its BTU content, then
sums the calculated BTU values providing a single value equal to the BTU content of the entire mixture.

A Per-peak parameter is one that is calculated once for each peak in the chromatogram. Per-peak parameters can be displayed in a custom report column similar to the
way the peak number or concentration would be displayed. Per-peak parameters can also be annotated on the chromatogram. An example of a Per-peak parameter
would be HETP (height equivalent to a theoretical plate). For HETP, the Custom Parameter program would take the length of the column (in centimeters) and divide it
by "n", the theoretical plates for the component which is already calculated by the system.

Note: If the custom parameter is designated as a sequence table parameter, any change to the Type (per-peak or system-wide) will not be updated until you reopen the
sequence custom parameter dialog from the sequence.

3. Select Number or String from the Returns drop-down list to select what type of value is returned by the parameter or calculation.

4. Click the Source field. From the Parameter Source dialog box, select from the two options:

From sequence table - Select this if you want to define the custom parameter as part of the sequence.

Calculate using - Select this if you want to use a custom parameter program or VB script to define the custom parameter. If you are using a program, it must be a
Windows .DLL that satisfies all the requirements for a custom parameter program given in the User Program section. If you select this, you must then enter or select the
name of the program or VB script that is to be used to calculate the custom parameter. The parameters available are listed in the table below.

5. Use the Additional Parameters field as necessary for your custom parameter application.

Available Custom Parameters

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European Pharmacopoeia Signal to

6-Sigma Noise Test Baseline Check Calculations Noise test (rms noise) Savitsky-Golay Smoothing
Noise
6-Sigma Signal-to-Noise Calib Weight External Standard Calibration Curves Normalization Report Calculation Scaling
Area % Report Calculation Calibration Averages External Standard Report Calculation Peak Centroid Signal Conversion
Area/Amount Response Factor USP (United States Pharmacopia)
Calibration Curve Calculations Internal Standard Amounts Peak-to-Valley Ratio
Definition calculation method
USP (United States Pharmacopia)
Area/Height calculation method Calibration Curves Internal Standard Report Calculation Performance Calculations
Signal to Noise
ASTM LOD (Limit of Detection) Internal Standard vs External
Capacity Factor (k') Plates/Meter Weighting and Scaling
Calculation Standard and Normalization
ASTM LOQ (Limit of Quantitation) Cubic Fit Internal Standards Point-to-Point Fit Weighting Method (LSQ Weight)
DAB (German Pharmacopia) Japanese Pharmacopoeia (JP)
ASTM Noise Calculations Quadratic Fit
calculation method calculation
Determining Concentrations for
ASTM Signal to Noise Linear Fit Relative Retention (Selectivity)
Uncalibrated Peaks

Reporting "Zero" for Internal

Automatic Averaging Drift Test for System Suitability Matrix Operations
Standard Concentrations
Average RF
EMG (Exponential Modified
Modified Least Squares Calculation Response Factor Definition
Gaussian) calculation method

Once you have a custom parameter defined in your method, the software will attempt to run the Custom Parameter Program(s) designated whenever you analyze your
chromatogram.

Note: In order to enter a sequence custom parameter in a sequence spreadsheet, the method must have a custom parameter defined as sequence-based, and that method
must be saved.

Column/Performance Parameters

The Column/Performance tab allows you to set up automatic calculation of system performance values such as Theoretical Plates, Capacity Factor, Resolution, or Peak
Asymmetry. To set up column performance calculations,

1. From the Method menu, click Advanced... followed by Column/Performance.

2. Click the Calculate performance parameters for this channel box. Then fill in the information for the given fields required for the calculations.
Unretained Peak Time (min)

Enter the retention time of an unretained peak for this column.

Column Length (meters)

Enter the length of the column, in meters or cm.

Particle Diameter (microns)

Enter the particle diameter, in microns, for the column you are using (if applicable).
Column Serial Number

Enter the serial number for the column (if applicable). This information will be saved with the data and available for reporting.
Column Installation Date

Enter the date the column was installed in the instrument (if desired). This information will be saved with the data and available for reporting.
Column Description

Enter column description if desired. This information will be saved with the data and available for reporting.
Calculate performance parameters for this channel

Select one or more calculation methods. The choices include USP, EMG (Exponential Modified Gaussian), DAB (German Pharmacopeia)/ BP (British
Pharmacopeia)/EP (European Pharmacopeia)/ ASTM, AOH (Area/Height), and JP (Japanese Pharmacopeia), calculation methods.

Once you have completed the Column/Performance tab, the system will calculate performance parameters after each analysis. These parameters can be exported,
annotated on the chromatogram, and printed in a custom report.

Select User Programs and Baseline Files

To designate program files to associate with the method,

1. From the Method menu, click Advanced... and then click Files.

User Programs

In this section you can designate user programs to be run Before each run, Before Analysis of the chromatogram, After Analysis of the chromatogram, or After
export. Enter the user program name, or select it from a list by clicking the File button. For details on writing User Programs, see the User Programs appendix at the end
of this manual. (Note: Include the full UNC path name for the file.)

Baseline File

If you want the data system to automatically subtract a stored baseline file after each run, select the Baseline File box. Designate a baseline file name, and then check
each channel from which the baseline file is to be subtracted. The chromatogram will be analyzed after the subtraction is performed. (Note: Include the full UNC path
name for the file.)

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Advanced Method Reports

This tab is used to specify one or more reports to be printed and/or exported when the method is used to analyze data.

Print

Select this box if you want to print the designated report when data is analyzed with this method.

Export

Select this box if you want to export the designated report when data is analyzed with this method. This results in tab-delimited export only.

Report Template Name

Enter or select the name of the report template file to be used to generate this report.

Export Path

If the report is to be exported, enter or select the path for the export file to be saved.

Export File Name

Select the file naming from the types presented in the list. The export file name will be created using the identification selected.

Save a Default Method

A set of default values are loaded whenever you create a new method. You can create a default method file for each instrument to include parameters that are frequently
used in your laboratory. To save a default method, from the File menu, click Method followed by Save as Default. The default method will be called Instrument xxx
Default Method.met (where x = instrument ID). For example, the default method for Instrument 1005 would be Instrument 1005 Default Method.met. To view an
instrument's ID number, do a right mouse click on the instrument icon in the Main screen, and click Properties. The assigned instrument ID is displayed. The default
method files are stored in the project method folder, so you can have different default methods for each project.

A non instrument specific default method can also be created and saved in the program folder. This will be loaded whenever an instrument-specific default method
cannot be found. If you want to create such a default method, name it Default.met, and save it in the program folder.

Note For SS420X instruments, the number assigned for the default method will be the SS420X ID +1 (for example, SS420X 10006 will be Instrument 10007 Default
Method.met).

Create Multi-channel Methods

You can create a single method that will acquire and process data independently from multiple channels simultaneously. For each channel in a method, you can set
totally independent data acquisition, integration, reporting, calibration, and advanced options.

To create a multi-channel method, you must have the instrument configured for multiple detectors (channels). For details on how to configure an instrument, see the
Installation chapter of this manual.

Designate a Channel

If your instrument is configured for more than one detector, you can select the current channel by using the drop-down list on the menu bar. Once you select a channel,
the method parameters you select will apply to that channel, and will be saved separately in both the method file and in all data files acquired using this method.

Offline Instruments
Starting an Instrument Offline allows you to create and edit methods and sequences, and do post-run data analysis, while data is being acquired on the instrument. All
functions described in Method Development, Sequence Operations, and Custom Reporting are available when you are running an instrument offline. The only
commands not available in Offline Processing are the Control commands that directly affect start and stop of data acquisition.

To open an instrument Offline,

1. From the Main Menu do a right mouse click on the instrument icon and then select Open Offline.

The offline instrument window is exactly the same as using one of the Instrument windows "online" with respect to method development, sequence development, and
reprocessing of data. The Offline Processing window looks just like the Instrument window, except the prompt "Offline" appears at the top of the window, and control
functions that relate to data acquisition are not available (appear greyed-out).

Once you have created and saved a method in an offline instrument, it can be used to acquire data by opening the method file in an online Instrument window and
proceeding as usual.

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