Enhancement of UV Absorption Detection Sensitivity

Monitoring an isotachophoresis separation by UV absorption can be very helpful but, unfortunately, many substances to not exhibit UV absorption and so alternative procedures must be adopted. The UV response of a non-absorbing ion can be evoked in isotachophoresis by employing a UV absorbing counter ion. The counter ions have different concentrations and different pH values in those zones and, thus, show different UV absorbance and as consequence can be used to detect the eluting ion band. An example of this system is shown in figure 23.

The separations depicted were the analysis of a mixture of chlorate (0.01M), acetate, (0.01M), formate (0.01 and glutamate (0.01M) employing a sample volume of 1 ml for the isotachophoretic analysis. The terminating electrolyte, morpholinoethanesulphonic acid was employed in both separations and the current was maintained at constant value of 30 micro-amps. The separation was monitored by both electrical conductivity and by UV absorption (256 nm) and the isotachoferograms also include the first derivative of the measured resistance with time; the migration period was about 15 minutes. 

In the isotachopherogram depicted on the right hand side of Figure 23 the counter ion buffer was e-aminocaproic acid and it is seen that no absorption steps appear on the isotachopherogram. The peaks that are seen (as opposed to steps) result largely from concentration differences at the band boundaries causing refractive index changes and, thus, some light scattering. In contrast, the isotachopherogram on the left, which was buffered with creatine, clearly shows UV absorption steps corresponding to those obtained from resistance monitoring. It is seen that though the ions do not themselves absorb in the UV the UV trace obtained with the UV absorbing counter ion could be used equally well for quantitative measurements as the trace obtained by resistance measurement≥.