Diffuse Reflectance Spectroscopy; Applications, Standards, and Calibration (With Special Reference to Chromatography)

The multitude of areas in which diffuse reflectance spectroscopy can be applied has been described in several books and reviews and ranges from color measurements of textiles, pharmaceuticals, building materials, paper and pulp materials etc., to adsorption studies and other basic investigations in physical, inorganic and organic chemistry. The major area of application is still the measurement of color which has become indispensible in the quality control of colored products, dyes and pigments. Color matching practices and techniques with sophisticated instrumentation which can be fully computerized as well as the use of simpler filter instruments for quality control are mentioned. Transferability of reflectance data i.e., color coordinates, depends on the quality of standards particularly when absolute measurements are desired. The difficulty of finding suitable “white standards” with good reflection properties at low UV and with a good long term stability is discussed. Similar arguments hold for sphere coating materials. For the measurement of fluorescing surfaces suitable standards are lacking which renders transfer of such data almost impossible. The usefulness of diffuse reflectance techniques to study adsorption phenomena on small particle adsorbents is demonstrated with a malachite green-o-carboxylic acid lactone system studied by Kortüm. This or similar systems could be adopted to the measurement of relative surface areas on certain chromatographic adsorbents yielding more realistic values than the BET-method. The most recent area of application has been in the field of chromatography for the in situ evaluation of chromatographic zones in flat-bed chromatography, electrophoresis and isoelectric focusing. In chromatography, standardization is less problematic since usually relative measurements are sufficient. On the other hand one has to find suitable calibration procedures. The use of the Kubelka-Munk function is often questionable since we are usually not dealing with layers of infinite thickness and below 300 nm the conventional adsorbents such as silica gel, alumina or cellulose are strongly absorbing. Experiences with a new function combining the laws of Kubelka-Munk and Lambert-Beer are therefore presented. The problem is also to find calibration techniques which account for chromatographic parameters. Until recently it was believed that a quantitative evaluation of chromatograms required a number of reference zones to be developed on the same chromatogram. In our experience this is no longer true. A novel calibration technique which utilizes the concept of transferable calibration factors is discussed. With this approach a quantitative evaluation of a chromatogram with only one reference spot is possible. Here again scanning and data acquisition can be fully automated. The application of proper calibration procedures to differential reflectance techniques and the measurement of multi-component systems is briefly mentioned. Finally it is demonstrated that it is possible to carry out in situ quantitative measurements on low UV absorbing compounds (down to 190 nm) separated on silica gel surfaces, provided suitable techniques and instrumentation are used.