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R.A. van Iterson Drenthe College Emmen the Netherlands

Quantitative Methods in chromatography

This document is written to describe various methods for obtaining quantitativeinformation from chromatograms such as normalising peak areas, internalstandards, external standards and standard addition methods.

1. Introduction2. Normalising Peak Areas Method3. Internal Standard Method4. External Standard Method5. Standard Addition Method

1. IntroductionData from chromatograms may be used to obtain the relative

concentrations of components in a mixture, providing good resolution isachieved. The Peak area, from integration of the detector signal duringelution of a component, is proportional to the amount of that component inthe sample. However, the response of a detector varies from onecompound to another; for example, the HPLC ultraviolet detector dependson absorption of electromagnetic radiation, a GC flame ionization detectordepends on the formation of ions and a GC electron capture detectordepends on electron affinities. Thus, a set of detector response factorsneeds to be determined for a particular analysis. Although manyintegrators include area % in the printout this is not the true ratio of thecomponents. Area % is simply the area of an individual peak calculated as

a percentage of the total areas recorded for all peaks in thechromatogram. It can be useful for a quick check of replicate analyses.There are four principal methods for obtaining quantitative information:

normalising peak areas, internal standards, external standards andstandard addition methods.

2. Normalising Peak Areas MethodThe area of each peak is obtained from a series of replicate injections of amixture containing equal (or known) amounts of all the components.Acceptable precision is essential to obtain satisfactory data. One

component is chosen as the referenceand the relative responses of theother components are determined by dividing the peak areas by that of thereference component. The detector response factors(DRF) may thenbe used to calculate corrected peak areas(A correct) for other analysesinvolving these components and hence their percentage ratios in themixture may be determined.

3. Internal Standard Method

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R.A. van Iterson Drenthe College Emmen the Netherlands

The internal standard methodis a variation on the above, and isrecommended for accurate quantitative work. It eliminates the need foraccurate injections since a reference standard is included in each sampleanalysed. An internal standard is selected which has a retention time suchthat it is eluted in a suitable 'gap' in the chromatogram. The procedure

involves analysing a test sample containing known amounts of eachcomponent plus a predetermined amount of the internal standard (I.S.).Since peak area is proportional to the amount of an eluted component andthe detector response factor (DRF) for an individual component x:

Ax = DRFx x Cxand for the internal standard: AIS = DRF,IS x CISwhere C is the amount of component x or internal standard, IS.The relative response of a component (DRF'x) to the internal standard isthereforeDRF'x = ( DRFx / DRF, IS ) = ( Ax / Cx ) / ( AIS / CIS ) = ( Ax / AIS ) x ( CIS

/ Cx )

Response factors for all components are calculated in the same way.Analysis of an unknown mixture is achieved by adding an accuratelyknown amount of internal standard and then carrying out thechromatography. The concentration of each component is calculated usingthe equation above, rearranged to give

Cx = ( Ax / AIS ) x ( CIS / DRFx )

The precision of the analysis is not dependent on injection of an accuratelyknown amount of sample, but does depend on accurate measurement ofpeak areas. This is not a problem with electronic integrators and an overallprecision or covariance of < 4 % should be readily obtained.

4. External Standard MethodAutomated sample injection systems and multiport injection valves (HPLC)have good reproducibility so that a series of injections can be made with avariation in sample volume of < 1 %. A set of standard mixtures containingknown concentrations of the analytes is analysed and their peak areasrecorded. A calibration graph of area versus concentration can be drawnfor each analyte to confirm a linear detector response and from which theamount of the analyte in a mixture can be determined. Alternatively for anestablished method a replicate series of one standard mixture is injectedand the area/unit amount of analyte calculated. ASTANDARD = xmg/litreThe mixture is then analysed and the amount of the components in thesample calculated using the peak area data for the standard mixture.Therefore, if the recorded peak area for the component in a samplemixture is AMIX then the amount of component x is

Amount x = ( x AMIX ) / ( ASTANDARD ) mg/litre

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R.A. van Iterson Drenthe College Emmen the Netherlands

5. Standard Addition MethodStandard addition methodis used in many techniques in analyticalchemistry. It is of limited use in chromatography because of the difficulty ofinjecting accurately known amounts of sample. A sample mixture isanalysed for the analyte of interest by adding a specified amount of this

analyte to the sample, thus increasing its concentration. The analysis isthen repeated and the resulting increase in peak area due to addition ofthe standard amount is noted. Hence, the concentration of the analyte inthe original sample may be calculated. If the peak area for the firstanalysis is A1 and with the standard addition of x mg is A2, then the peakarea corresponding to x mg (or x mg/litre) is (A2 - A1). Thus, the originalamount of the analyte x in the sample corresponding to A1, is given byAmount x = ( x A1 ) / ( A2 - A1 ) mg/litreAn allowance for dilution due to addition of the standard amount has to bemade. The main difficulty with this method concerns the reproducibility ofthe sample injection. A precision of better than 1 % should be achieved if

valid quantitative results are to be obtained.An alternative approach is first to analyse the sample, noting the area, A1,for the analyte. Successive standard amounts of the analyte are thenadded, each sample standard mixture being analysed and the areasrecorded. A graph of peak area versus concentration is drawn and theamount of analyte in the sample obtained by extending the calibration lineto intersect the abscissa as shown in Figure 1.

(Figure 1. Standard addition method)