Standard Data Analysis -Integration

ChemStation - Level 1 - Training

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Integration locates the peaks in a signal and calculates their

size

What is Integration

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The Integration Process consists of the following:

• construct initial baseline

• peak recognition – Start and end Time

• finds the apex of the peak

• baseline allocation (construction)

• peak area measurement

Integration Process

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1 Slope and Curvature within limit

2 Slope and curvature above limit: peak?

3 Slope remains above limit: peak recognized

4 Curvature becomes negative: front inflection

5 Negative slope: Apex

6 Curvature becomes positive: rear inflection point

7 Slope and curvature within limit: end of peak?

8 Slope and curvature within limit: end of peak

5

1

2

3

4 6

7

8

Start, End and Apex of a Peak

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Fit highest data point and those on each sideto a quadratic equation, solve for highest point

Determining Peak Apex

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BB

BV

VB

BB

BV

VB

Baseline Tick Marks

The default baseline is constructed as a series of straight line segments between: the start of the baseline, the tick marks, and the end of the peak.

Default Baseline Construction

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Standard or Enhanced Integrator The ChemStation includes two integrators:

the Enhanced Integrator (default) and the Standard Integrator (Genie)

New methods by default use the Enhanced Integrator and can not be down graded to the Standard Integrator

Old methods can be

updated to use the Enhanced Integrator ormaintained using the Standard Integrator so customers

can continue using validated methods.

The default method “defoldlc.m” has the Standard Integrator

The default method “def_lc.m” has the Enhanced Integrator

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Both integrators co-exist so you may continueto use validated methods

The Enhanced Integrator offers better: baseline allocation on wandering baselines rejection of noise peaks through the use of an initial peak

height parameter control of integration start and stop marks peak allocation on noisy signals peak shoulder allocation through second derivative

calculations

Standard or Enhanced Integrator

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Loading Signals

Select path, data file name and channel

Select

or ‘Load Signal’ from File Menu

Show/Hide the Signal Information

Choose to load all available or selected signals only

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You may also integrate by:

•Selecting Integrate or Auto Integrate• from the Integration menu.•Selecting the Integration Tool.•Running a method where the Run• Time Checklist includes Data Analysis

When a Signal is Loaded, Integration May Occur Automatically

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Initial Values

ThresholdPeak WidthArea RejectShoulders

Standard Integrator - Initial Values

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What timed events could you use to fix the

integration below?

Standard Integrator - Timed Events

What timed events could you use for this integration?

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Auto Integrate determines the optimum values for the initial threshold, peak width, and area reject by integrating the chromatogram twice.

Threshold - based on signal range and averaged baseline noise.Peak Width - proportional average of the peak widths of the first five peaks.Area Reject - 1/1000st of the greatest area found.

Auto Integration - Standard Integrator

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Select Integration Tasks Integrate CurrentChromatogram

Auto Integrate

Integration Events

Manual IntegrationDraw Peak Baseline

Manual IntegrationNegative Peaks

Manual IntegrationTangent Skim

Manual IntegrationSplit Peaks

Manual IntegrationRemove Peaks

Integration Tools - Standard Integrator

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The Enhanced Integrator offers better: baseline allocation on wandering baselines rejection of noise peaks through the use of an initial peak

height parameter control of integration start and stop marks peak allocation on noisy signals peak shoulder allocation through second derivative

calculations

Enhanced Integrator

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You may update methods developed on previous revisions of the ChemStation to the new Enhanced Integrator. Once you have updated the new method, you cannot revert back to the old integrator.

Enhanced Integrator

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Enhanced Integrator UI Define integration parameters just by selecting and clicking on the chromatogramThere are two sets of

integration events :

- intial events

- time-based

Use: Slope Sensitivity to define peak sensitivityPeakwidth to set an initial sampling interval for the

integrator to distinguish peaks from baseline noise

Area Reject to filter small peaksHeight Reject to set noise rejection

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Advanced Baseline

12 13 14 15 16 17 18 19 20

Abso

rbance

Time [min.]

Options OffAdvanced Integration

Options OnAdvanced Integration

Peak

Baseline

Peak

AreaUnassigned

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Using Initial Parameters

Peak Width

HeightReject

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Add Timed Events

Delete a Timed Event

Enhanced Integrator - Timed Events

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Solvent peak (Skim peak)

Tangent Peak

Straight Line Calculation

Exponential Calculation

Tangent Skim

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Shoulders occur when two peaks are so close together that no valleyexists between them

Peak with shoulders

Tangent Shoulders Drop Line Shoulders

Shoulder Detection

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Enhanced Integrator - Auto Integration

Examines beginning and end regions to estimate noise.

Assigns initial Slope Sensitivity and Height Reject.

Assigns temporary Peak Width value for first pass integration.

Sets Area Reject to zero.

Performs trial integration, may be repeated several times.

Calculates Peak Width based on early eluting peaks.

Refines Slope Sensitivity and Height Reject.

Computes Area Reject from initial Peak Width and noise level.

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Integration TasksIntegrate CurrentChromatogram

Auto IntegrateIntegration Events

Set Baseline Now

Set Baseline Hold

Set Tail Tangent Skim

Set IntegrationSet Slope Sensitivity

Set Area Reject

Set Fixed Peak Width

Set Detect Shoulders

Enhanced Integrator - Tool Bar

Use Auto Integrate to find suitable integration events

Only visible with

integration events open

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Save Events to Method

Save Integration Events as Part of a Method

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Manual Integration

Manual integration events

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Manual Integration

Use Cursor tools to perform manual integration by selecting the required action and dragging a line over or clicking on the chromatogram (depending on the task)

Zoom in using the zooming tools to better view your baseline

The new integration results are immediately visible in the user interface

If your chromatography is so demanding that you can’t use the the automatic integration process

Use the manual integration tool set

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Option 1: Save Manual Integration to Method

Select Integration, Copy Manual Events to Method

Save Manual Integration - To the method

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Step 1: Select View, Windows Functions, Save Window

Save Manual Integration - To the Raw Data File

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Note: manual changes are stored in the raw data file directory.

Step 2: Click OK to use default name or rename

Save Manual Integration - To the Raw Data File

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Select View, Windows Functions, Load Window

To Recall Manual Integration From Data File

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Lab Time !!!!! Lab 5 - Integration