The simultaneous determination of pentose and hexose in mixtures of sugars

Pentose and hexose can be estimated when present together in mixtures by heating them with orcinol and acid either alone or with ferric chloride. The colours produced are measured a t suitable wavelengths and the results calculated directly from the colorirneter readings by means of a nomogram. DURING investigations into the chemical composition of bacterial cell material it became necessary to determine pentoses and hexoses when present together. An approximate answer, at least, was needed even when the exact nature of the sugars present was not known, Colorimetric methods based on the orcinol reaction proved suitable and required only small amounts of material.When orcinol is heated with sugars in the presence of sulphuric or hydrochloric acid the product has an absorption band in the blue end of the spectrum. Fig. 1 shows the absorption spectra obtained when the reaction is carried out under the conditions prescribed on p. 81. Pentoses and hexoses both give a peak at 425 mp, but the absorption with the hexoses is the 560 600 Wavelength, rnp Fig. 1. Absorption spectra of material obtained on heating sugars with the orcinol-sulphuric acid reagent. Solutions containing 24 p g of the sugars per ml were treated with the reagent as described in the text. The absorption spectra of the solutions in a 1-cm cuvette were read on the Unicam spectrometer. 7w Fig. 2. Absorption spectra of material obtained on heating sugars with the orcinol-ferric chloride reagent. Solutions containing 24 p g of the two pentoses per ml or 72 p g of the hexoses per ml were treated with the orcinol-ferric chloride reagent as described in the text. The absorption spectra of the solutions in a l-cm cuvette were read on the Unicam spectrometer. Xylose , ribose # fructose 0 0 0 0 0 0 , galactose x-x-x-x-x glucose 0-0-0-0-0, lesser. If the colour is read on an " EEL " calorimeter* incorporating the appropriate filter, the colour ratio of pentose to glucose is about 1.3 to 1, or 1.1 to 1 if molar concentrations are considered (see Table I). This is the basis of the Tillmans-Phillippi method for assaying total carbohydrate.lP2 If, however, the reaction is carried out in the presence of ferric chloride,