Page 274 - Tandem Techniques

Page 258

in a number of unique ways to aid in chromatographic analysis. The most common uses of the tandem
system are as follows.

1. To monitor the separation at an optimum wavelength and thus provide the maximum
chromatographic sensitivity.

2. To test the purity of a solute peak by calculating the absorption ratios across the peak.

3. To extract the spectrum of a peak and compare it to a library standard to confirm the identity of a
substance.

An example of the use of the tandem instrument to enhance the sensitivity of detection has already been
given, and other examples will be provided later. The method for testing the purity of a peak using the
tandem combination is particularly interesting. If the composition of the peak is chemically
homogeneous throughout the whole elution curve, then the ratio of the absorption at any particular
wavelength to that taken at another significantly different wavelength will be constant throughout the
entire peak. This constant ratio will be independent of the nature of the spectra and whether there is any
fine structure present or not. It follows that if this ratio is plotted against time, across the breadth of a
pure peak, a perfectly rectangular trace will be obtained. In contrast, if another substance is co-eluted,
under the peak, then the rectangular shape will be significantly distorted. An example of this method of
purity-testing is given in Figure 7.2.

The chromatogram, which was monitored at 274 nm, is shown in the lower part of Figure 7.2. As a
diode array detector was employed, it was possible to ratio the output from the detector at different
wavelengths and plot the ratio simultaneously with the chromatogram monitored at 274 nm. Now, as
already explained, if the peak was pure and homogeneous, the ratio of the adsorption at the two
wavelengths (those selected being 225 and 245 nm) would remain constant throughout the elution of
the entire peak. The upper diagram in Figure 7.2, shows this ratio plotted on the same time scale, and it
is seen that a clean rectangular peak is