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Quantitation Methods in Gas Chromatography

Single Point External Standard

Unlike the area percent method, the SinglePoint External Standard method requiresthe analysis of more than just the sampleof interest. Analyze a sample containinga known amount of analyte or analytesand record the peak area. Then calculatea response factor using Equation 1.

EQUATION 1

EQUATION 2

SINGLE PT. EXT. STD. EXAMPLE

responsefactor

Inject a sample with the unknown analyteconcentration and record the peak area.Then calculate the amount of analyteusing Equation 2.

Calculate an individual response factor foreach compound of interest.

An injection containing benzene at aconcentration of 2,000 µg/ ml is madeand results in a peak area of 100,000.Calculate the response factor forbenzene using Equation 1.

response factor = = 50

An injection of the sample with theunknown concentration of benzenehas a peak area of 57,000. Calculatethe amount of benzene present usingEquation 2.

Multiple Point External Standard

The Single Point External Standard methodassumes analyte response to be linear overa range of concentrations. (Figure 1). Usethe Multiple Point External Standardmethod when the concentration range islarge or if the single point external standardmethod is not linear (Figure 2). Thesamples used in this method cover theexpected analyte concentration range. Usea line fitting algorithm such as point topoint, linear least squares, or quadraticleast squares to produce a calibrationcurve. See Figure 2. Most modern datasystems include one or all of thesealgorithms.

FIGURE 2

FIGURE 1

Gas Chromatography is a useful tool thatallows us to identify and quantitateindividual components in a mixture.Using individual standards andreproducible conditions enables peakidentification by retention time. In mostcases this is absolute, that is unless thereare two peaks with exactly the sameretention time under the analysisconditions. This same “absolute” propertycannot be applied to quantitation whichis affected by numerous variables.

Quantitation uses chromatographic datato determine the amount of a givencomponent in a mixture. This data canbe in the form of either peak height orpeak area which is obtained from anintegrated chromatogram. It is veryimportant that this data is gatheredaccurately. It is best if the peak is totallyresolved from any neighboring peaks. Aco-elution or other anomalies such astailing or fronting will distort or obscurethe beginning and ending points of thepeak making it difficult to accuratelydetermine the size of the peak.

Quantitation MethodsThere are several types of quantitationmethods commonly used. The five mostcommon are area percent, single pointexternal standard, multiple pointexternal standard, single point internalstandard, and multiple point internalstandard.

Area Percent MethodArea percent is the simplest quantitationmethod. This method assumes that thedetector responds identically to allcompounds. This assumption, however,is not valid. This method provides arough estimate of the amounts ofanalytes present.

To calculate area percent take the areaof an analyte and divide it by the sum ofareas for all peaks. This value representsthe percentage of an analyte in thesample.

Peak

Are

a

Sample Amount

Linear Calibration Curve

Peak

Are

a

Sample Amount

Non-Linear Calibration Curve

GC Education

peak areasample amount

=

100,0002,000

57,00050

= 1,140 µgamount ofbenzene

=

amount ofanalyte

peak arearesponse factor

=

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Single Point Internal Standard

Unlike external standard methods, theinternal standard method accounts for anyvariances in gas chromatographperformance. The analyte chosen for theinternal standard has a predictableretention time and area, allowing it to beused to determine if abnormalities haveoccurred.

The Single Point Internal Standard methodrequires at least two analyses. The firstanalysis contains a known amount ofinternal standard and the compounds ofinterest. Calculate the response factorusing Equation 3.

Then add a known amount of the internalstandard to the sample containing analytesof unknown concentrations. Calculate theamount of the unknown analyte usingEquation 4.

EQUATION 4

SINGLE POINT INTERNAL STANDARD EXAMPLE

Multiple Point Internal Standard

This method, like the Multiple PointExternal Standard, uses several analyses.Each analysis contains the internalstandard whose concentration is keptconstant and the analyte of interestwhose concentration covers the range ofconcentrations expected. Plot the resultswith the ratio of the area of the analytesto the area of the internal standard onthe y-axis and the ratio of theconcentration of the analytes to theconcentration of internal standard on thex-axis. Fit this data to a curve usingmethods previously described.

Analyze the samples with unknownanalyte concentrations. Determine theratio of the analyte area to internalstandard area from the data. Thecorresponding ratio of analyteconcentration to internal standardconcentration is determined from thegraph. Multiply the concentration ofinternal standard in the sample by thisratio. This yields the concentration of theanalyte in the unknown sample.

Quantitation can be as easy as the areapercent method or as in depth as themultiple point internal standard method.Each method varies with the degree ofaccuracy and ease of performance. Youshould pick the one which is right for you.

Prepare a sample containing 2,000µg/mL of toluene (the internal standard)and 1,000 µg/mL benzene (the analyte).Then inject the sample. The resultingpeak areas are 120,000 for toluene and67,000 for benzene. Using Equation 3the response factor for benzene is:

Internal Response Factor =120,000 x 1,0002,000 x 67,000

= 0.8955

amount of specific compound =

amountIS x areaSC x IRFSC

areaIS

IS = Internal StandardSC = Specific Compound of InterestIRF = Internal Response Factor

EQUATION 3

Internal Response Factor =

areaIS x amountSC

amountIS x areaSC

IS = Internal StandardSC = Specific Compound of Interest

Description Part No. Price

CHROMATOGRAPHY SOFTWARE

PeakTrain Software* 12180

*Demo Disk Not Available

PeakTrain™

ChromatographyPeak IntegrationSoftwareExcellent Training Tool forChoosing the Best IntegrationMethod

• Requires Windows 3.1,Win95, or NT

All chromatography data systems giveyou many ways to integratechromatographic peaks. But how do youknow which method gives the mostaccurate answer? PeakTrain is the answerto this problem. This program lets youenter information on fourchromatographic peaks (retention time,width, and area) and then draws yourfour peaks on the screen.

Choose from several integration options:Perpendicular Drop (Baseline Forward),Valley-to-Valley, Shoulder, and even amanual integration option. The programreports the percent error in the area orheight when using a particularintegration option. You can even add abaseline shift, noise, and a hidden peak.This program is a valuable training toolfor any chromatography, illustrating theproper way to integrate peaks, andproduce the most accurate results.

View different integration methods andtheir results on screen.

Inject the sample containing 2,000µg/mL of toluene and an unknownamount of benzene using the samechromatography conditions. Theresulting areas are 122,000 for tolueneand 43,000 for benzene. Calculate theamount of benzene present usingEquation 4.

Amount of Benzene =2,000 x 43,000 x 0.8955

122,000

= 631µg

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