Note how the elution order of the six components in the mixture alters with
the mobile phase composition.Methods of identifying unknown solutes separated by chromatographic
techniques are described in Topic D2. In the case of HPLC, there are four
alternatives:● Comparisons of retention factors (k) orretention times(tR) with those of
known solutes under identical conditions, preferably on two columns of
differing selectivity to reduce the chances of ambiguous identifications.
● Comparisons of chromatogramsof samples spikedwith known solutes with
the chromatogram of the unspiked sample.
● Comparisons of UV-visible spectra recorded by a diode-array detector with
those of known solutes. This is of limited value because most spectra have
only two or three broad peaks so many solutes have very similar spectral
features.
● Interfacinga high-performance liquid chromatograph with a mass spectro-
meter. This enables spectral information for an unknown solute to be
recorded and interpreted. Identifications are facilitated by searching libraries
of computerized spectra (Topics F3 and F4).Methods used in quantitative chromatography are decribed in Topic D2, and
alternative calibration procedures are described in Topic A5. Peak areasare
more reliable than peak heights as they are directly proportional to the quantity
of a solute injected when working within the linear range of the detector. Most
HPLCdetectors have a wide linear dynamic range (Topic D6, Table 4), but
response factors must be established for each analyte as these can vary consider-
ably. Calibration is normally with external standardschromatographed sepa-
rately from the samples, or by standard addition.Constant volume loops for
sample injection give very good reproducibility (about 0.5% relative precision),
making internal standards unnecessary, cfgas chromatography (Topic D5), and
auto-injectors are frequently employed for routine work. An overall relative
precision of between 1 and 3% can be expected.Quantitative
analysis
Qualitative
analysis
D7 – HPLC: modes, procedures and applications 173