Absorbance good operating range

Refractive index detectors are not as sensitive as uv absorbance detectors. The best noise levels obtainable are about 1CT7 riu (refractive index units), which corresponds to a noise equivalent concentration of about 10-6 g cmT3 for most solutes. The linear range of most ri detectors is about 104. If you want to operate them at their highest sensitivity you have to have very good control of the temperature of the instrument and of the composition of the mobile phase. Because of their sensitivity to mobile phase composition it is very difficult to do gradient elution work, and they are generally held to be unsuitable for this purpose. 

Advantages of the technique are its relatively low cost combined with quickness and ease of operation. One can work with dilute solutions (indeed they are usually a prerequisite) and water is a good solvent as it does not absorb in the UVMsible range. Quartz cells are required for observation of absorptions in the UV range, otherwise glass or plastic (for aqueous solutions) can be used for visible frequencies. Flow cells are available for coupling to separation equipment and absorption can either be monitored at a fixed wavelength for detection of a specific compound or class of compounds, or, if a diode array is available, a complete spectrum can be recorded for each fraction on-line. 

The photodiode-array based UV absorbance detector, usually abbreviated as either the PDA or DAD, but in this chapter as DAD, has been used in a wide range of applications since it was introduced commercially almost two decades ago. It allows collection of the complete UV absorbance spectrum of the HPLC column eluent The general principles and operation of the DAD have been reviewed by Huber and George in a book on DADs and their application [2]. This book is also a good primer on the use of the DAD in fields such as clinical, pharmaceutical, environmental, polymer, and biotechnology analyses. 

The solutes separated in Figure 2 are carbohydrates which do not absorb in a normal UV range and would be difficult to detect by other detectors. The column was a type of reversed phase and the mobile phase an acetonitrile/water mixture. The above example is a good illustration of the advantage of having a refractive index detector available for special applications. The only practical alternative methods of detection for these components would be to use derivatives or employ a UV detector and operate at very low wavelengths (190-200 nm). Unfortunately, at these wavelengths, baseline stability is difficult to maintain. 

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