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Contact your Alltech office or distributor for current or local
prices. Copyright 1998 Alltech Associates, Inc.
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
response
factor
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 g/ 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 that
allows us to identify and quantitate
individual components in a mixture.
Using individual standards and
reproducible conditions enables peak
identification by retention time. In most
cases this is absolute, that is unless there
are two peaks with exactly the same
retention time under the analysis
conditions. This same absolute property
cannot be applied to quantitation which
is affected by numerous variables.
Quantitation uses chromatographic data
to determine the amount of a given
component in a mixture. This data can
be in the form of either peak height or
peak area which is obtained from an
integrated chromatogram. It is veryimportant that this data is
gathered
accurately. It is best if the peak is totally
resolved from any neighboring peaks. A
co-elution or other anomalies such as
tailing or fronting will distort or obscure
the beginning and ending points of the
peak making it difficult to accurately
determine the size of the peak.
Quantitation MethodsThere are several types of
quantitationmethods commonly used. The five most
common 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 of
analytes 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.
PeakArea
Sample Amount
Linear
Calibration
Curve
PeakA
rea
Sample Amount
Non-Linear
CalibrationCurve
GCEducation
peak area
sample amount=
100,000
2,000
57,000
50= 1,140 gamount of
benzene=
amount of
analyte
peak area
response factor=
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Copyright 1998 Alltech Associates, Inc.
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 method
requires 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 internal
standard 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 the
concentration 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 internal
standard 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 the
multiple point internal standard method.
Each method varies with the degree of
accuracy and ease of performance. You
should pick the one which is right for you.
Prepare a sample containing 2,000g/mL of toluene (the internal
standard)and 1,000 g/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 1,000
2,000 x 67,000
= 0.8955
amount of specific compound =
amountIS x areaSC x IRFSC
areaIS
IS = Internal Standard
SC = Specific Compound of Interest
IRF = Internal Response Factor
EQUATION 3
Internal Response Factor =
areaIS x amountSC
amountIS x areaSC
IS = Internal Standard
SC = Specific Compound of Interest
Inject the sample containing 2,000g/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 using
Equation 4.
Amount of Benzene =
2,000 x 43,000 x 0.8955
122,000
= 631g
