Most detectors and chemical assay systems give a linear response with
increasing amounts of the test substance over a given 'working range'.
Alternative ways of converting the measured response to an amount of
- External standardization: this is applicable where the sample volume is
sufficiently precise to give reproducible results (e.g. HPLC). You measure
the peak areas (or heights) of known amounts of the substance to give a
calibration factor or calibration curve which can be used to calculate the
amount of test substance in the sample.
- Internal standardization: where you add a known amount of a reference
substance (not originally present in the sample) to the sample, to give an
additional peak in the elution profile. You determine the response of the
detector to the test and reference substances by analysing a standard
containing known amounts of both substances, to provide a response
factor (r), where:
|⇒ Equation [32.5]
||peak area (or height) of test substance
|peak area (or height) of reference substance
Use this response factor to quantify the amount of test substance (Qt) in
a sample containing a known amount of the reference substance (Qr),
from the relationship:
|⇒ Equation [32.6]
||[peak area (or height) of test substance]
|[peak area (or height) of reference substance]
Internal standardization should be the method of choice wherever
possible, since it is unaffected by small variations in sample volume (e.g.
for GC microsyringe injection). The internal standard should be
chemically similar to the test substance(s) and must give a peak that is
distinct from all other substances in the sample. An additional advantage
of an internal standard which is chemically related to the test substance is
that it may show up problems due to changes in detector response,
incomplete derivatization, etc. A disadvantage is that it may be difficult
to fit an internal standard peak into a complex chromatogram.