Page 272 - Tandem Techniques

Page 256

An example of the use of the diode array detector to select a specific wavelength, to give a high
response to specific compound types and consequently allow the separation to be monitored at the
maximum sensitivity, is shown in Figure 7.1. The separation is that of a series of common fatty acids
and was carried out on a reversed phase column, using water buffered with phosphoric acid as the
mobile phase. In order to achieve adequate sensitivity, the total separation was monitored by the diode
array detector, the UV absorption at 210 nm recalled, and the chromatogram obtained by plotting the
absorption at this wavelength against time.

Column: Spherisorb® Octyl, 25 cm x 4.6 mm
I.D., 5 µm particles. Mobile phase: 0.2 M phosphoric
acid. Flow rate 0.8 ml/min. monitored at 210 nm. 1.
tartaric acid, 2. lactic acid, 3. malic acid, 4. formic acid,
5. acetic acid, 6. citric acid, 7. succinic acid, 8. fumaric acid.
Figure 7.1
The Separation of Some Carboxylic Acids
Monitored by UV Absorption at 210 nm
Courtesy of Supelco Inc.

Due to the relatively low absorption of carboxylic acids at higher wavelengths, (e.g. at 254 nm, which is
the wavelength of most fixed wavelength detectors), the acids are usually separated by ion exchange
chromatography, and the separation monitored by the electrical