Appendix UV detection using ion-pair reagents

Significant effects are achieved if the aqueous mobile phase contains both hydro-phobic, UV-absorbing ions and hydrophilic ions, e.g. salts. The distribution equilibrium of the UV-absorbing ions between the mobile and stationary phases is specihcally altered following sample injection. The sample components in the UV detector then produce a signal, despite being non-UV-absorbing themselves. Hence, 

To obtain good results the following recommendations for the design of a mobile phase have been given  

Chromatograms for such systems show positive and negative peaks depending on the charge and k values of the sample components, and also one or more system peaks (those that cannot be ascribed to a particular component) (Fig. 13.3). These phenomena have been described in detail and substantiated in the original papers cited.  

1 mM tetrapropylammonium bromide in 50 mM phosphate buffer-acetonitrile, gradient from 5 to 60% acetonitrile temperature, 50°C fluorescence detector, 350/410 nm. Peaks 1 =a-ketoglutaric acid  

3 = a-ketoisovaleric acid 4 = a-ketoisoca-proic acid 5 = a-keto-j5-methylvaleric acid IS = a-ketocaprylic acid (internal standard). 

---