The External Standard Method

In the external standard method, the solute chosen as the reference is chromatographed separately from the sample. However, results from two chromatograms will be compared so chromatographic conditions must be maintained extremely constant.

To reduce the effect of any changes in the operating conditions the sample and reference solutions can be chromatographed alternately. The data from the reference chromatograms run before and after the sample are then used for calculating results of each assay.

With the external standard method, the reference standard (or standards) can be chosen to be the same as the solute (or solutes) in the sample. This eliminates the need for response factors. In addition, the external standard(s) can be made up to have concentration(s) closely similar to the component(s) of the sample, thus, errors that might arise slight detector non-linearity significantly reduced.

Assuming, the response index of the detector is unity, if the (p)th solute in the mixture is at a concentration of (cp(s)) in the sample and (cp(st)) in the standard

solution, then

(29)

where (ap(s)) is the area of the peak for solute (p) in the sample

chromatogram,

(ap(st)) is the area of the peak for solute (p) in the reference

chromatogram,

and (cp(st)) is the concentration of the standard in the reference

solution.

If peak heights are employed, then

(30)

where (hp(s)) is the height of the peak for solute (p) in the sample

chromatogram,

(hp(st)) is the height of the peak for solute (p) in the reference

chromatogram,

and (cp(st)) is the concentration of the standard in the reference

solution.

If the value or the response index, (r), is not unity, then the corrected area and height must be used. Thus again assuming

then equation (29) becomes

(31)

and equation (30) becomes

(32)

Theoretically, if chromatographic conditions are kept constant, the reference chromatogram need only be run once a day. However, in practice, the reference chromatogram should be at least every two hours and, many analysts run a reference chromatogram immediately before and after each sample.