The determination of amino acids by spectral reflectance

Abstract

The extensive literature dealing with the direct estimation of amino acids of paper chromatograms reveals that there is a reproducible relationship between the color density of the ninhydrin complexes of the acids and their concentration on the chromatograms. Furthermore, a number of investigators have shown that reflection measurements can be carried out on powders with an acceptable degree of precision if certain experimental conditions are met. In view of these data, the application of spectral reflectance to the identification and determination of substances, such as amino acids, resolved on thin-layer plates seemed feasible. For the purpose at hand probably the most appropriate theory treating diffuse reflection and the transmission of 1ight scattering layers also happens to be the most general theory developed by Kubelka and Munk (63, 64). When applied to an infinitely thick opaque layer, the Kubelka-Munk equation may be written as (1-R ͚ˡ)^2/2Rˡ͚ = k/s Where R ͚ˡ is the absolute reflectance of the layer, k is its molar absorption coefficient, and s is the scattering coefficient (for the derivation of the equation See appendix, page 111). Instead of determining R ͚ˡ, however, it is customary in practice to work with the more convenient relative diffuse reflectance, R ͚ which is measured against a standard such as MgO or BaSO4. In these cases it is assumed that the k values for the standards are zero and that their absolute reflectance is one. Since the absolute reflectance of the standards exhibiting the highest R ͚ˡ values never exceeds 0.98 to 0.99, however, one is actually dealing in such instances with the relationship R ͚ˡ sample/ R ͚ˡ standard = R ͚ which indicates that a 1inear relationship should be observed between F(R ͚) and the absorption coefficient, k, provided s remains constant. S is rendered independent of wave length by employing scattering particles whose size is large relative to the wave length being used. During the research described herein, the constancy of s was ensured by making use of powders consisting of particles having an approximate diameter of 5µ. A straight-line relationship between F(R ͚) and k is observed, however, only when dealing with weakly absorbing substances and only when the grain size of the powders employed is less than 1 p in diameter. Furthermore, any significant departure from the state of infinite thickness of the adsorbent layer assumed in the derivation of the Kubelka-Munk equation results in background interference which, in turn, is responsible for non-ideal diffuse reflectance. When absorbents having a large grain size or when large concentrations of the absorbing species are used, plots of F(R ͚) versus k or concentration deviate from straight lines in that there is a decrease in slope at higher concentrations. In his explanation of this phenomenon, G. Kortüm (65, 69, 70, 71) postulates that the reflected radiation is the result of both regular and diffuse reflectance. The first can be described as a mirror reflection whereas the second occurs when impinging radiation is partly absorbed and partly scattered by a system so that it is reflected in a diffuse manner, that is to say, with no defined angle of emergence. Regular reflectance for cases involving normal incidence is described by the Fresnel equation Rreg = I(refl/I˳) = ((n-1)^2 + n^2 k^2) / ((n+1)^2 + n^2 k^2 where k is the adsorption coefficient and n is the reflective index. Diffuse reflectance is described by the Kubelka-Munk function given earlier. Since regular reflectance is superimposed on diffuse reflectance, a distortion of the diffuse reflectance spectrum results which is responsible for the anomalous relationship observed between F(R ͚) and k at high concentrations of the absorbing species. It is essential, therefore, to eliminate as far as possible the interference caused by regular reflectance, Rreg This can be accomplished by selecting appropriate experimental conditions. Especially effective are the use of powders having a small grain size and the dilution of the light absorbing species with suitable diluents. Although Kortüm et al (109) suggest the grinding of samples for twelve to fourteen hours in a ball mill as a means of diminishing the interference caused by regular reflectance. it was felt that there was no need to resort to such a procedure in this instance. Commercial grade adsorbents for thin-layer chromatography. since they consist of particles having an average diameter of 5µ. seemed to be suited for reflectance measurements as received from the manufacturers. In addition, the grinding operation recommended by Kortüm is not only too inefficient and tedious for a routine analytical procedure, but also could result in the contamination of the samples.