R.A.

van Iterson Drenthe College Emmen the Netherlands

Quantitative Methods in chromatography

This document is written to describe various methods for obtaining quantitative information from chromatograms such as normalising peak areas, internal standards, external standards and standard addition methods. 1. Introduction 2. Normalising Peak Areas Method 3. Internal Standard Method 4. External Standard Method 5. Standard Addition Method

1. Introduction Data from chromatograms may be used to obtain the relative concentrations of components in a mixture, providing good resolution is achieved. The Peak area, from integration of the detector signal during elution of a component, is proportional to the amount of that component in the sample. However, the response of a detector varies from one compound to another; for example, the HPLC ultraviolet detector depends on absorption of electromagnetic radiation, a GC flame ionization detector depends on the formation of ions and a GC electron capture detector depends on electron affinities. Thus, a set of detector response factors needs to be determined for a particular analysis. Although many integrators include area % in the printout this is not the true ratio of the components. Area % is simply the area of an individual peak calculated as a percentage of the total areas recorded for all peaks in the chromatogram. 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 and standard addition methods. 2. Normalising Peak Areas Method The area of each peak is obtained from a series of replicate injections of a mixture containing equal (or known) amounts of all the components. Acceptable precision is essential to obtain satisfactory data. One component is chosen as the reference and the relative responses of the other components are determined by dividing the peak areas by that of the reference component. The detector response factors (DRF) may then be used to calculate corrected peak areas (A correct) for other analyses involving these components and hence their percentage ratios in the mixture may be determined. 3. Internal Standard Method

R.A. van Iterson Drenthe College Emmen the Netherlands

The internal standard method is a variation on the above, and is recommended for accurate quantitative work. It eliminates the need for accurate injections since a reference standard is included in each sample analysed. An internal standard is selected which has a retention time such that it is eluted in a suitable 'gap' in the chromatogram. The procedure involves analysing a test sample containing known amounts of each component plus a predetermined amount of the internal standard (I.S.). Since peak area is proportional to the amount of an eluted component and the detector response factor (DRF) for an individual component x: Ax = DRFx x Cx and for the internal standard: AIS = DRF,IS x CIS where C is the amount of component x or internal standard, IS. The relative response of a component (DRF'x) to the internal standard is therefore DRF'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 accurately known amount of internal standard and then carrying out the chromatography. The concentration of each component is calculated using the equation above, rearranged to give Cx = ( Ax / AIS ) x ( CIS / DRFx ) The precision of the analysis is not dependent on injection of an accurately known amount of sample, but does depend on accurate measurement of peak areas. This is not a problem with electronic integrators and an overall precision or covariance of < 4 % should be readily obtained. 4. External Standard Method Automated sample injection systems and multiport injection valves (HPLC) have good reproducibility so that a series of injections can be made with a variation in sample volume of < 1 %. A set of standard mixtures containing known concentrations of the analytes is analysed and their peak areas recorded. A calibration graph of area versus concentration can be drawn for each analyte to confirm a linear detector response and from which the amount of the analyte in a mixture can be determined. Alternatively for an established method a replicate series of one standard mixture is injected and the area/unit amount of analyte calculated. ASTANDARD = x mg/litre The mixture is then analysed and the amount of the components in the sample calculated using the peak area data for the standard mixture. Therefore, if the recorded peak area for the component in a sample mixture is AMIX then the amount of component x is Amount x = ( x AMIX ) / ( ASTANDARD ) mg/litre

R.A. van Iterson Drenthe College Emmen the Netherlands

5. Standard Addition Method Standard addition method is used in many techniques in analytical chemistry. It is of limited use in chromatography because of the difficulty of injecting accurately known amounts of sample. A sample mixture is analysed for the analyte of interest by adding a specified amount of this analyte to the sample, thus increasing its concentration. The analysis is then repeated and the resulting increase in peak area due to addition of the standard amount is noted. Hence, the concentration of the analyte in the original sample may be calculated. If the peak area for the first analysis is A1 and with the standard addition of x mg is A2, then the peak area corresponding to x mg (or x mg/litre) is (A2 - A1). Thus, the original amount of the analyte x in the sample corresponding to A1, is given by Amount x = ( x A1 ) / ( A2 - A1 ) mg/litre An allowance for dilution due to addition of the standard amount has to be made. The main difficulty with this method concerns the reproducibility of the 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 then added, each sample standard mixture being analysed and the areas recorded. A graph of peak area versus concentration is drawn and the amount of analyte in the sample obtained by extending the calibration line to intersect the abscissa as shown in Figure 1.

(Figure 1. Standard addition method)