commonly used form of constant displacement pump. Such pumps produce small
pulses of flow and pulse dampeners are usually incorporated into the system to
minimise this pulsing effect. All constant displacement pumps have inbuilt safety
cut-out mechanisms so that if the pressure within the column changes from pre-set
limits the pump is inactivated automatically.
11.3.3 Detectors
Since the quantity of material applied to an HPLC column is normally very small, it is
imperative that the sensitivity of the detector system is sufficiently high and stable to
respond to the low concentrations of each analyte in the eluate. The most commonly
used detectors are:
- Variable wavelength detectors: These are based upon ultraviolet–visible
spectrophotometry. These types of detector are capable of measuring absorbances
down to 190 nm and can give full-scale deflection (AUFS) for as little as 0.001
absorbance units. They have a detection sensitivity of the order of 5 10 –10gcm–3
and a linear range of 10^5. All spectrophotometric detectors use continuous flow cells
with a small internal volume (typically 8 mm^3 ) and optical path length of 10 mm
which allow the continuous monitoring of the column eluate. - Scanning wavelength detectors: These have the facility to record the complete
absorption spectrum of each analyte, thus aiding identification. Such opportunities
are possible either by temporarily stopping the eluent flow or by the use of
diode arraytechniques, which allow a scan of the complete spectrum of the
eluate within 0.01 s and its display as a 3D plot on a VDU screen in real time
(Fig. 11.6). - Fluorescence detectors: These are extremely valuable for HPLC because of their greater
sensitivity (10–12gcm–3) than UV detectors but they have a slightly reduced linear
range (10^4 ). However, the technique is limited by the fact that relatively few analytes
fluoresce. Pre-derivatisation of the test sample can broaden the applications of the
technique. - Electrochemical detectors: These are selective for electroactive analytes and are
potentially highly sensitive. Two types are available,amperometricandcoulometric,
the principles of which are similar. A flow cell is fitted with two electrodes, a stable
counter electrode and a working electrode. A constant potential is applied to the
working electrode at such a value that, as an analyte flows through the flow cell,
molecules of the analyte at the electrode surface undergo either an oxidation or a
reduction, resulting in a current flow between the two electrodes. The size of the
current is recorded to give the chromatogram. The potential applied to the counter
electrode is sufficient to ensure that the current detected gives a full-scale deflection
on the recorder within the working analyte range. The two types of detector differ
in the extent of conversion of the analyte at the detector surface and on balance
amperometric detectors are preferred since they have a higher sensitivity (10–12gcm–3
as opposed to 10–8gcm–3) and greater linear range (10^5 as opposed to 10^4 ).
449 11.3 High-performance liquid chromatography