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Quantification Concepts and Integration

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In This Section, We Will Discuss:

Quantification calculations.

When to use peak area or peak height.

The difference between precision and accuracy.

How to properly integrate a chromatogram.

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Beers Law and Quantification

Absorbance Detectors

Fixed Wavelength

Light

Flow

Source

Cell

Lenses

Slits

Detector

Elements

I

Io

log _____ = A = abc

I

Io

I = Incident Radiation Intensity

I = Transmitted Radiation Intensity

A = Absorbance

a = molar absorptivity

b = path length

c = solute concentration

o

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Equation:

Absolute Amt of x =[(Responsexx RF) - yintercept]

x M x D

Responsex = peak area orheight

RFx = Amountx

Responsex

M = Multiplier

D= Dilution Factor

External Standard Calibration

AMT (ng/uL)

600

500

400

300

200

100

0

0 100 200 300 400 500

ESTD

Response = 1 .18 (Amt) - 0.317

r = 1.0002

A

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Increase the Precision with an Internal Standard

ISTD

Salicylamide

AMT Ratio

Re

sponseRatio

0 1 2 3 4 5

2.0

1.6

1.2

0.8

0.4

0.0

Response Ratio = 0.371

r = 1.000

(Amt Ratio) + 0.000535

2

Permits compensation for any variations inpeak area; i.e. sample size

Permits compensation for variation due to

sample preparation

Equation:

Actual Amt of x =[(RFx x ResponseRatiox x AmountISTD) -

yintercept] x M xD

Response Ratio = ResponsexResponseistd

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Choice of Internal Standard

Pure and easily obtainable.

Similar chromatographic and detector propertieswith the sample.

Elute in the chromatogram during a gap.

ISTD

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Peak Height or Peak Area for Calculations

0

5

10

15

20

-3 -6 -9 -12 -15

Flow Rate Change (%)

C

hangeinPeakBehavior(%)

Area

Peak Height

Peak heights unaffected by flow

variations.

Peak area should be used for

badly tailing peaks.

Both are imprecise with

irreproducible injection volume.

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Precision and Accuracy

Precision

Dependent on the ability to define and

control the instrument conditions ofanalysis

Accuracy

Dependent on calibration of the system

Reliable standards

AccuratePrecise

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Building a Calibration Table

Inject standards with concentrations bracketing your

unknowns.

Set-up appropriate integration events for the low standard.

Integrate to obtain the peak height or area. Save the

integration events to the method. Begin a new calibration table with level one, the lowest

level.

Integrate each additional standard level and add to thecalibration

table. Examine the calibration curve and save it to the method.

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When a Signal is Loaded, Integration May Occur

Automatically

Integrate by:

Selecting Integrate

after load in Load

Signaldialog box or

Preferences dialog

box.

Selecting Integrate or

Auto Integrate from

the menu.

Selecting the

Integration or Auto

Integration Tool.

Running a method

where the Run

Time Checklist

includes Data Analysis.

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Initial Settings - Events Peak Width controls the ability ofthe integrator to distinguishpeaks from baseline noise. In

general, increasing the peak width

will result in broader peaks.

Area reject- All peaks whose

areas are below this value willnot be reported.

Height reject- All peaks whose

heights are below this value will

not be reported.

0.05 0.1

Slope sensitivity decreasing slopesensitivity will result in detecting

smaller and broader peaks.

Shoulder Detection Mode shoulders

detected using the second derivative of

peak

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Initial Settings For All Signals

Tangent Skim Mode only applies when

conditions for the following settings are

met: Tail Peak Skim Height Ratio, or

Front Peak Skim Height Ratio, and

Skim Valley Ratio

Peak To Valley Ratio

Baseline Correction must beAdvanced

Some Events in this table are dependent

on other events in this table.

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Tangent Skim Mode

Tangent Skim Modes

New Exponential

ExponentialStraight

Standard

The Tail Peak Skim Height RatioandSkim Valley Ratio will be used

to determine whether a tangent skim will be applied to calculate the

area of a child peak on the trailing edge of a parent peak.

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Tail Peak Skim Height Ratio

H

p

Hc

Hp

Hc

Ratio must begreater than set

value to be

skimmed

Setting the value to zero

disables tangent skimming

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Skim Valley Ratio

Hc

Hv

Height Child Peak

Height Valley

When the ratio is less than

the set value the child will

be skimmed.

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Tail Peak Skim Height Ratio

No Tangent Skimming

Tangent Skimmed

Tail Peak Skim Height Ratio = 3

Skim Valley Ratio = 20

Tail Peak Skim Height Ratio = 0

Skim Valley Ratio = 20

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Front Peak Skim Height Ratio

176.9880 483.1385

New Exponential

Area = 209.3318

449.7947

Hc

Hv

Hp

Straight

Very similar to Tail Peak SkimmingHc

H

p Set Front Skim Height Ratio

Hc

Hv Set Valley Height Ratio

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Advanced Baseline

Advanced Baseline

tracks the curvatureof baselines better

Classical

Advanced

Integrator tries to improve

the start and end points

of a peak, re-establish the

baseline for a cluster ofpeaks and remove

baseline penetrations.

Uses Peak to Valley

Ratio

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Integration Example - Default Parameters

Default integration

parameters may not

always be the best

for your analysis

Possible Problems

Noise selected as peaks

Baseline tracking difficulties

Drop lines inappropriate

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Practical Integration Advice Starting Point

1. Set the slope sensitivity to 50.

2. Estimate the peak width from

the initial integration. Use the

smallest peak width from a real

chromatographic peak, not noise.

Set initial height and area reject

to zero.

3. Set the Tail Peak Skim Height Ratio

to 3, the Front Peak Skim Height Ratio

to 6, and the Skim Valley Ratio to 20.

4. Baseline correction is Advanced with

Tangent Skim Mode New Exponential.

5. Integrate and view the results.

6. If all peaks of interest were not

integrated, lower the slope sensitivity

until all real peaks are integrated.

7. If there are still peaks that cannot be

integrated, lower the peak width setting.

8. Use timed events if necessary.

9. Remove undesired peaks with the height

orarea reject.

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Example Initial

Set initial values,

then re-integrate

Current Integration from

DEF_LC.M default

integration values

Pick narrowest real

chromatographic

peak for peak width.

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First Integration Results

Better, but stillneeds work.

Desired peaks not

Integrated

Baseline problems

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Adjust Initial Parameters

Lower the slope sensitivity

to detect smaller, broader peaks.

Here the slope sensitivity was

changed from 50 to 20.

The smaller, broader peaks

were detected.

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Insert Timed Events

Use drop box to

select desired event.

Click on

chromatogram

at the desired time.

Or, use the inserttimed event tool.

Use the delete timed

events tool to

remove an event.

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Insert Timed Events

After timed event inserted

and integration

Before

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Finish Integration

Use area and height reject to

ignore unwanted peaks

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Save and Close

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Save Integration Events as Part of a Method

When finished creating the integration events, save them to the method.

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Auto Integrate

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 as 90% area of most symmetrical peak.

Autointegrates based on your

settings in For All Signals.

Can be a good starting point

for integration events.

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Draw Peak Baseline

Negative PeakTangent Skim Peak

Split peak

Delete Peak(s)

Manual Integration