Page 256 - Vogel's TEXTBOOK OF QUANTITATIVE CHEMICAL ANALYSIS

8 COLUMN AN0 THlN-lAVER LlîlUlO CHROMATOCRAPHV

conditions are often difficult to apply to techniques other than reverse phase
and ion exchange chromatography.
The amperometric detector is currently the most widely used electrochemical
detector, having the advantages of high sensitivity and very small interna1 ce11
volume. Three electrodes are used:
1. the working electrode, commonly made of glassy carbon, is the electrode at
which the electroactive solute species is monitored;
2. the reference electrode, usually a silver-silver chloride electrode, gives a stable,
reproducible voltage to which the potential of the working electrode is
referred; and
3. the auxiliary electrode is the current-carrying electrode and usually made of
stainless steel.
Despite their higher sensitivity and relative cheapness compared with ultraviolet
detectors, amperometric detectors have a more limited range of applications,
being often used for trace analyses where the ultraviolet detector does not have
sufficient sensitivity.

In liquid chromatography, in contrast to gas chromatography [see Section 9.2(2)],
derivatives are almost invariably prepared to enhance the response of a particular
detector to the substance of analytical interest. For example, with compounds
lacking an ultraviolet chromophore in the 254 nm region but having a reactive
functional group, derivatisation provides a means of introducing into the
molecule a chromophore suitable for its detection. Derivative preparation can
be carried out either prior to the separation (pre-column derivatisation) or
afterwards (post-column derivatisation). The most commonly used techniques
are pre-column off-line and post-column on-line derivatisation.
Pre-column off-line derivatisation requires no modification to the instrument
and, compared with the post-column techniques, imposes fewer limitations on
the reaction conditions. Disadvantages are that the presence of excess reagent
and by-products may interfere with the separation, whilst the group introduced
into the molecules may change the chromatographic properties of the sample.
Post-column on-line derivatisation is carried out in a special reactor situated
between the column and detector. A feature of this technique is that the
derivatisation reaction need not go to completion provided it can be made
reproducible. The reaction, however, needs to be fairly rapid at moderate
temperatures and there should be no detector response to any excess reagent
present. Clearly an advantage of post-column derivatisation is that ideally the
separation and detection processes can be optimised separately. A problem
which may arise, however, is that the most suitable eluant for the chromatographic
separation rarely provides an ideal reaction medium for derivatisation; this is
particularly true for electrochemical detectors which operate correctly only
within a limited range of pH, ionic strength and aqueous solvent composition.
Reagents which form a derivative that strongly absorbs UV/visible radiation
are called chromatags; an example is the reagent ninhydrin, commonly used
to obtain derivatives of amino acids which show absorption at about 570 nm.
Derivatisation for fluorescence detectors is based on the reaction of non-
fluorescent reagent molecules (fluorotags) with solutes to form fluorescent