Quantification of a given analyte is based on the construction of a calibration curve
obtained using a pure, authentic sample of the analyte. The construction of the
calibration curve is carried out using the general principles discussed in Section
1.4.6. Most commonly the calibration curve is based on the use of relative peak areas
obtained using an internal standard that has been subject to any preliminary extrac-
tion procedures adopted for the test samples. The standard must be carefully chosen to
have similar physical and structural characteristics to those of the test analyte, and in
practice is frequently an isomer or structural analogue of the analyte. Ideally, it should
have a retention time close to that of the analyte but such that the resolution is greater
than 99.5%.11.2.6 Sample clean-up
Whilst chromatographic techniques are designed to separate mixtures of analytes, this
does not mean that attention need not be paid to the preliminary purification (clean-
up) of the test sample before it is applied to the column. On the contrary, it is clear that,
for quantitative work using HPLC techniques in particular, such preliminary action is
essential, particularly if the test analyte(s) is in a complex matrix such as plasma, urine,
cell homogenate or microbiological culture medium. The extraction and purification of
the components from a cell homogenate is often a complex multi-stage process. For
some forms of analysis, for example the analysis of low-molecular-weight organic drugs
in biological fluids, sample preparation is relatively easy.Solvent extractionis based
on the extraction of the analytes from aqueous mixtures using a low-boiling-point
water-immiscible solvent such as diethyl ether or dichloromethane. The extract is
dried to remove traces of water before it is evaporated to dryness (often under nitrogen
orin vacuo), the residue dissolved in the minimum volume of an appropriate solvent
such as methanol or acetonitrile, and a sample applied to the column. This solvent
extraction procedure tends to lack selectivity and is unsatisfactory for protein clean-up
and for the HPLC analysis of analytes in the ng cm^3 or less range.
The alternative to solvent extraction issolid phase extraction, which unlike solvent
extraction can be applied to the pre-purification of proteins. The test solution is
passed through a small (few millimetres in length) disposable column (cartridge)
packed with an appropriate stationary phase similar to those used for HPLC (Section
11.3). These selectively adsorb the analyte(s) under investigation and ideally allow
interfering compounds to pass through. For example, for the purification of a mixture
of proteins a reversed phase, affinity or ion exchange chromatography cartridge
would be selected as these are ideal for desalting or concentrating protein mixtures
containing in the range of femto- to picomoles of protein. Once the test solution has
been passed through the column, either by simple gravity feed or by the application of
a slight vacuum to the receiver vessel, the column is washed to remove traces of
contaminants and the adsorbed analyte(s) recovered by elution with a suitable eluent.
Complex protein and peptide mixtures can be partially fractionated at this stage by
gradient elution. Several commercial forms of this solid phase extraction technique
are available, for example Millipore Ziptips™, and the termpipette tip chromatog-
raphyhas been coined to describe it.444 Chromatographic techniques