BY M. V. TRACEY Rothamsted Experimental Station, Harpenden, Hertfordshire
(Received 20 January 1950)
A method for the determination of pentoses in the presence of large amounts of hexoses and uronic acids has been developed for use in the examination of extracts of leaf fibre after enzymetreatments. Methods previously described are not satisfactory, owing to interference by hexoses or uronic acids or to lack of sensitivity. The survey by White & Green (1932) of colour tests for sugars and their derivatives in urine indicated that their qualitative 'aniline' test for pentoses might be developed. In this test, the sugar solution with an equal volume of glacial acetic acid and a fewdrops of aniline is heated to boiling, allowed to stand for 2 min., cooled and the colour extractedwith chloroform. A quantitative modification of this test is now described, in which the interference by glucose and other sugars is reduced by allowing the reaction to take place at room temperature. It has the advantage that little manipulation or attention is required.
Biochem. 1950, 47EXPERIMENTAL
100 ml. glacial acetic acid, 10 ml. 5 % (w/v) aqueous oxalic acid, 24 ml. water and 16 ml. colourless aniline are mixed and stored in a dark-glass bottle. The reagent should be stored out of direct light and not be used more than a week after preparation. Reagent (6 ml.) is added from a burette to each of a series of test tubes (75 x 15 mm.) matched for use in a photoelectricabsorptiometer (the Evans Electroselenium Portable model was used). The solution to be tested (containing 10-80,sg. pentose) is added, together with enough water to bring the final volume to 8 ml. and the contents of the tube well mixed. At the same time a blank (2 ml. water + 6 ml. reagent) and two standards of xylose (10 and 50,ug.) are set up. If it is known that the pentose in the solution isnot xylose, standards of that pentose are employed. Standard solutions of pentose containing 50 ,ug./ ml. are conveniently made up in saturated benzoic acid solution. These standards do not deteriorate at room tem28
M. V. TRACEY
long periods. The tubes are then kept for 20-24 hr. in -the dark at room temperature. The colour developed is measured with theabsorptiometer using Ilford filter 622, the reagent-water blank being used for setting the instrument. The amount of pentose present is then calculated from the readings obtained on the standards by direct proportion, for the relation between instrument reading and the amount of pentose present is linear over the range used.
Factors influencing colour development
In examining the effect of variedconditions on the specificity and sensitivity of the test, glucose and galactose were tested in addition to pentoses, as preliminary tests had shown that these two sugars were the most important interfering substances. Sensitivity was determined by calculating the scale reflexion/mg. sugar produced on the absorptiometer using filter 622, and interference by calculating the deflexion/mg. as apercentage of that produced by xylose under the same conditions.
furfural (see below, p. 435), the effects of NaCl, Na2HPO4 and oxalic acid were investigated, all of which Adams & Castagne found to stabilize and deepen the colour given by furfural. All three substances were added to the original acetic acid-aniline mixture individually and in all combinations. The results showed that only oxalicacid had any effect on colour development. The absorptions given by glucose and galactose using filter 622 were reduced by about 50 and 20 % respectively after 24 hr. at room temperature, and the xylose colour was increased by about 50%. Since oxalic acid effected an increase both in specificity and sensitivity it was incorporated in the reagent. Oxalic acid had the further effect of reducing the...