Direct spectrophotometric quantitation of phenylthiohydantoin derivatives of amino acids from thin layers of silica gel

The main deterrent to researchers for use of spectrophotometric techniques for quantitation of compounds separated on silica gel thin layers has been the inability to obtain pure spectra from eluates of the compounds (10). This problem, as regards amino acid phenylthiohydantoins, has also been encountered using paper chromatography (16, 17).A method has been presented which demonstrates quantitation of phenylthiohydantoin derivatives of amino acids with effective complete recovery of these compounds. Pretreatment of thin layers of silica gel with methanol seems to be the key to attaining a surface from which the compounds may be eluted with methanol to yield pure ultraviolet spectra. The method has proved usable in analysis of 10⁻⁸ mole of those compounds. This corresponds to about 3 × 10⁻³ gm of a protein such as fibrinogen (MW = 330,000) or to about 5 × 10⁻⁵ gm of a protein such as insulin. By use of special spectroscopic techniques, e.g., longer path cells and scale expansion, the limits could be lowered.