Anthrone in the Estimation of Hexose Sunars With Special Reference to Pentose Interference RONOULD JOHANSON Plant Industry, Commonwealth Scientific and Industrial Research Organization, Canberra, Aurtralia
A method has been developed whereby errors in the estimation of hexose sugars resulting from the premature and/or irregular mixing of the reagent and test solution are eliminated. A spinning disk is used to produce a centrifugal force which keeps reactants in separate layers until the moment of mixing. With controlled anthronation the study of interference from pentoses was greatly facilitated. Increase in pentosehexose ratio and/or concentration of sulfuric acid in the reagent produces a shift from 625 to 680 m p in maximum absorption. With less concentrated acid it remains unchanged at 625 mrrand the total interference of pentoses is greatly reduced.
TIfF
uide use of the anthrone reagent introduced by Dreywood '' (4), in the estimation of sugars, oligosaccharides, and derivatives of polyhexose units ( 1 , S, 6, 6 , 8-12) confirms the usefulness of this reagent. Numerous modifications in procedure, adapted to obtain reproducible results, are evidence of the difficulty in controlling the anthronation reaction. It appears that the concentration of acid in the reagent, and the manner in which the reagent and test solution are brought together determine the nature and intensity of the color developed, and so affect the reproducibility of results. The method dcscrihed here affords some degree of precision. During investigations of the anthrone reaction there was evidence t h a t the presence of pentose sugars could cause serious interfercnce with the determination of hexoses (7). When pentose alone is anthronated under conditions similar to those used for hexose a tramient blue-green color develops and rapidly deteriomttos to yellow. Under special conditions, this blue-green color cltn be made to persist long enough for it to form the basis of a method of pentose estimat,ion (5). The more stable yellow complex is equivalent a t 625 mp t o a 6% equimolar hexose. However, an antbronated pentoso-hexose mixture may give B complor which differs vastly from that obtained on sepnrately mixing anthronated hexose and pentose. The data indicate that, pentose interference in hexose determinations depends largely on the composition of the anthrone reagent. The methods described greatly reduced interference hut with some materials, care is necessasy for the interpretation of resu1t.s. M'
The anthrone reagent norn . 92 to 95 volume % mlfurio acid. When- this reagent is mixed with the aqueous solution of test sample, considerable heat is evolved. Slight variations in the mixing technique have been found to give marked differencesin the intensity of color development, Morse ( I O ) and Barnett and Miller ( f )added the reagent so that a separate layer wa6 formed below the test solution, and then rapidly mixed the two layers. V i l a and Silverman ( f a ) added the reagent rapidly with continuous mixing. Koehler ( 8 ) used this method, but cooled the test tubes during mixing. Black ( 3 ) and Fairbairn ( 6 ) avoided heating through hydration by using a lower concentration of acid, and then heated the mixture on a water bath t o obtain full development of color. Difficulties have been experienced in this laboratory in mproduring results when these techniques wem used. The use of
weaker acid holps to minimize the eriect of irregular mixing, but is accompanied by loss of sensitivity and slower color development (Table I). Below a cerlsin concentration of acid (approximately 56 volume %) separation of anthrone and decomposition of the complex occurs. The use of more concentrated acid appears to be essential if the method is applied to certain complex polyhexoses, and it is desirable for general routine purposes where there is no interference from pentoses. The problem was to devise a means of getting the reagent into proximity with the test solution so that instantaneous mixing could he attained. This can be done with the aid of a, small conical flask centrally mounted on a spinning disk (Figure 1). The roagent is added down the side of the rotating flask, and eontrifugal force collects it around the wide bottom of the flask. This allows ample time for delivery of the viscous reagent without the risk of premature reaction. When addition is complete, the flask is removed from the disk, shaken quickly, and then allowed t o stand in a. water bath for optimum color development. Using this method, the standard error of a single reading on an absorptiometer, graded from 0 to 1, is 0.004,
Figure 1.
Spinning Disk XJ,sed in Anthronation Procedutr
..."..."". ....
_ll e Apparatus. A disk (11-em. d.-.&.- yl.,, for holding a 30-ml. conical flask in 8. recem a t the center, is mounted on a vertical shaft (10 em. high), The disk also acts as a pulley for a belt driven by a stirrer motor. The spinning movement of the disk is started and stopped by moving the stand away from or toward the motor, thus tightening or loosening the tension in the belt. The spectral measurements were made on a Beckman Model DU spectrophotometer with 1-em. Carex-glass cells and anthronated blanks. Mahmum intensities of color developed were measured on Hilger ahsorptiometer H 760. Chemicals. One hundred milligrama of anthrone (British Drug Houses laboratory reagent) is dissolved in 100 ml. of sulfuric mid diluted to the required concentration using analytical grade [specific gravity 1.838 a t 20" C.), shaken to ensure homogeneity,
ANALYTICAL CHEMISTRY
1332 Table I. Absorptiometer Readings for Glucose Anthronated under Varying Conditions Aci; VOl. 96.4 94.5 81.0 75.2 69.4
T:mp.,
C 80 80 90 90 90
(Means of duplicate estimations) Time, Glucose. y hIin. 30 60 90 5 0.882 0 . 7 5 0 0.634 6 0.867 0.752 0.640 7 0.907 0.815 0.714 20 0.923 0.835 0.742 1.5 0.913 0.826 0.757
120 0,536 0.542 0.642 0.668 0.670
180 0.342 0.342 0.493 0.510 0.560
Ratio of reagent to test solution is 3 to 1 except for 69.4 acid vol % which was 5 to 1. a
Table 11. Wave Lengths for )laximum Absorption for Complexes Produced by Anthronation
Molecular ratio, arabinose toglucose Maximum absorption, mp
0.75:l 1 : l 635
645
1.5:l 1.7:l 650
665
2:l
2.4:l 3:l
670
675
680
of pentoses and hexoses under a range of conditions. The interference appears to be due to an interaction between the tJvo types of sugar molecules and/or their products; it reaches R maximum value when the pentose-hexose molecular ratio is 2.7 ( h 0 . 3 ) to 1. Moreover, the form of the absorption curve is changed. Effect of Pentose-Hexose Ratio. Solutions containing 60 y of glucose, and varying amounts of arabinose were anthronated with concentrated (96.4 volume 70)reagent. The absorption curves were determined and are given in Table 11. The maximum absorption wave length was found to shift from 625 mp for pure hexose to 680 m r for the 3 to 1 ratio of arabinose to glucose. Absorption measurements a t 625 mp therefore do not measure the full interfcrcnre by arabinose.
3.5:l 4:l 676
r
670 0.5
and allowed to stand for a t least 12 hours. Anthrone reagent keeps well when stored away from light and a t temperatures of 0' to 3" C. Before using, the reagent is warmed to 20" C. The following sugars were used: glucose (B.D.H. analytical grade), L-arabinose, D-xylose, D-ribose and fructose (both B.D.H. and Light's, laboratory grades), and D-levulose (c.P., Pfanstiehl Chemical Co., Kaukegan, Ill.). Anthronation Technique. The reaction is carried out in 30ml. Erlenmeyer flasks, selected for uniformity, x-ith 3 f 0.003 ml. of test solution in each flask. Each flask in turn is placed on the disk and allowed to revolve a t about 400 r.p.m. From a pipet with rapid delivery 9 f 0.05 ml. of reagent is added on the side in such a way that i t "slides" along the walls and forms a separate layer in the flask. 4s the flask is removed from the disk the two layers are immediately mixed x5ith a sharp shake. Then the flask is placed on a wire platform, a t a depth of 1.5 cm., in a water bath a t a selected temperature of 80" or 90" C. for 5 to 30 minutes, depending on the acid concentration of the reagent. At the correct time each flask is cooled to room temperature in a secsond water bath and allowed to stand for 30 minutes. Intensity of color developed is measured in 1-cm. cuvettes on a photoelectric absorptiometer using filters for maximum transmittance a t 625 mp wave length. The readings are interpreted as sugar values from graphs prepared of standard glucose solutions included in the same group of determinations.
h I \
0.4
Y
f 0.3 m
8 0.2
-5 0.1
-4 -3 -2
DISCUSSION
,-I
Factors Influencing Color Development. Absorptiometer 450 550 650 750 readings for a range of conditions, summarized in Table I, illusWAVE LENGTH, Mr trate the general effects of acid concentration, time, and temFigure 2. Absorption Spectra for Sugar-.%nthrone perature on color formation. As color increases, so deterioraComplexes at 22' C. tion occurs; thus, a clear solution becomes turbid with decomFor all curves final concentration of anthronated sugars wa6 150 y position products. Experience soon indicates the degree of of arabinose and/or 75 y of glucose in 12 ml. of solution. color, N hich can be developed safely without i n t e r f e r e n c e from this source. I n general, Table 111. Pentose Interference the lower the concentration Pentose-Glucose Ratio, y of acid, the greater the time Acid, 25:30 25:60 50 :60 100:60 100:120 and/or temperature required VoLa Temp., Time, As Hexose, y % C. hlin. Diff.b Dev.C Diff. Dev. Diff. Dev. Diff. Dev. Diff. Dev. to develop the optimum color. Arabinose When optimum color is de
