Figure 4.37
'Wall-jet' amperometric detector.retention time, tR, (p. 85) – compare gas chromatography (p. 109). As in GC, members of a homologous
series show a linear relation between log tR and the number of carbon atoms. The trapping of HPLC
peaks for examination by other techniques such as infrared, nuclear magnetic resonance and mass
spectrometry is straightforward, providing that the time taken for the peak to reach a sample vial via a
transfer line from the detector can be accurately measured. However, direct interfacing is now the
preferred technique. Complete UV/visible spectra are recorded by diode array detectors or the flow can
be stopped with the peak in the cell of a conventional dispersive spectrophotometric detector while the
complete spectrum is scanned.
Quantitative analysis using valve injection, has a relative precision of 0.2% or better. Precision is
highest where 50– 200 μl sample loops are used. Isocratic elution is preferable to gradient elution
because conditions are more reproducible. Internal standards are not always required, the concentration
of samples being determined from previously prepared calibration curves or by using a factor if the
curve is linear. For routine analyses, automatic sampling systems are better because they eliminate
operator error. Either peak height or peak area can be measured, the former being suitable for sharp,
early-eluting peaks, where peaks are not fully resolved, and for trace analysis. Peak area measurements
give better precision and are more reliable if peaks are asymmetrical; they are less susceptible to
changes in chromatographic conditions.