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Colorimetric Method for Determination of Sugars and Related Substances

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Colorimetric Method for Determination of Sugars and Related Substances Colorimetric Method for Determination of Sugars and Related Substances MICHEL DUBOIS, K. A. GILLES, J. K. HAMILTON, P. A. REBERS, and FRED SMITH Division of Biochemistry, University of Minnesota, St. Paul, Minn. Simple sugars, oligosaccharides, polysaccharides, and their glycosides, it is of limited use for met,hylated sugars and their derivatives, including the methyl ethers with free t.he pentoses. Although butanol-propionic acid-water is an ex- or potentially free reducing groups, give an orange- cellent solvent for separating the disaccharides (4),the residual yellow color w-hen treated with phenol and concentrated propionic acid interferes xith the 1-napht,holsulfonate method. sulfuric acid. The reaction is sensitive and the color Aniline phthalate (S8)and aniline trichloroacetate (17)have been is stable. By use of this phenol-sulfuric acid reaction, utilized for the colorimetric determination of sugars and their a method has been developed to determine submicro derivatives (2, 3); these reagents, however, are unsatisfactory for amounts of sugars and related substances. In conjunc- ketoses. tion with paper partition chromatography the method Phenol in the presence of sulfuric acid can be used for the is useful for the determination of the composition of quantitative colorimetric microdetermination of sugars and polysaccharides and their methyl derivatives. their methyl derivatives, oligosaccharides, and polysaccharides (15). This method is particularly useful for the determination of small quantities of sugars separated by paper partibion chro- OLORIMETRIC tests for reducing sugars and polysaccha- matography with the phenol-wat,er solvent and also for those rides have been known for a considerable time. The rea- sugars separated with solvents vihich are volatile-e.g., but'anol- gents such as 1-naphthol (33) for carbohydrates in general; ethanol-water (39)) ethyl acetate-acetic acid-water (26), or benzidine for pentoses and uronic acids (27, 49, 50); naphtho- methyl et,hyl ketone-lT-ater (4, 39). The method is simple, resorcinol for uronic acids (61); and resorcinol (43),naphthore- rapid, and sensitive, and gives reproducible results. The rea- sorcinol (.%), and resorcinol disulfonic acid (31) for ketoses are gent is inexpensive and st,able, and a given solution requires only well-knon-n examples of colorimetric tests that may be carried one st,andard curve for each sugar. The color produced is out in acid solution. Such tests as these and modifications of permanent and it is unnecessary to pay special attention to the them using aromatic amines and phenols (4,22, 38) have re- caontrol of the conditions. cently gained added importance since the ext,ensive development, of partition chromat'ography for the separation and character- DETERMIhATION OF COYCENTR4TION OF PURE SCG4R izat,ion of minute amounts of sugars and their derivatives (1, 4, SOLUTIOYS 8, 11, 12, 17, 18, 21-23, 26, S6, 39, 47). Polyols and carbohy- Reagents and Apparatus. Sulfuric acid, reagent grade 95.5%, drates with a reducing group may be detected by the Tollens conforming to -4CS specifications, specific gravity 1.84. silver reagent (39, 52), perhaps one of the best reagents in the Phenol, 80% by weight, prepared by adding 20 grams of glass- distilled water to 80 giams of redistilled reagent grade phenol. art, of chromatography. Reducing sugars are also detectable This mixture forms a v-ater-n-hite liquid that is readily pipetted. by picric acid (7, l7), 3,4-dinitrobenzoic acid (5), 3,5-dinitro- Certain preparations have been known to remain water-white salicylic acid (6, 32, 48),o-dinitrobenzene (17, 401, and methylene after a years' storage, while others turn a pale yellow in 3 or 4 blue (54), Tvhile diazouracil is said to be specific for sucrose as months. The pale yellon- color that sometimes develops does not interfere in the determination, inasmuch as a blank is included. well as oligosaccharides and polysaccharides containing the Coleman Junior, Evelyn, Klett-Summerson, or Beckman sucrose residue (42). Model DU spectrophotometers. All \vere used with satisfactory Volunietric procedures involving the use of potassium ferri- results in this inwstigation. cyanide (19), ceric sulfate (is), copper sulfate (16, 44),and sodium hypoiodite (20) are ap- plicable to the determinat,ion of small amounts 120- of reducing sugars after separation hy parti- t,iori chromatography. However, experience 1.00- - show that t,hese methods require considerable skill and are time-consuming and sensit,ive to slight variation in the condit,ions. The anthrone (13, 14, 28, 3dJ 35, 53) and the 1-naphtholsulfonat,e (10) reagents are ex- cellent for st,andard sugar solut,ions (3$)>but, when applied to the anal)-sis of sugars sepa- rated by partition chromatography, the pres- ence of only traces of residual solvent dr- veloper may render them useless. 3Iost sugars can be separated on filter paper hy a phenol- O0 ' lb ' 210 ' 310 ' 40 ' 20 ' $0 ' '710 ' do ' 9'0 ' ,bo ' Ilb ' ,io s-ater solvent (,E?),but the!- cannot then be MICROGRAMS OF SUGAR determined by the anthrone reagent because Figure 1, Standard curves residual phenol, held tenaciouslJ- in the paper, 1. D-Xylose, Coleman Jr., 480 mp, 17 mg.of phenol interferes TT-ith the green color produced by the 2. D-Mannose Beckman Model DU 490 mp 40 mg. of phenol anthrone reagent. Moreover, the anthrone 3. D-Mannose' Evelyn filter No. 496, 40 mg.'of phenol 4. o-Galactose Coleman Jr 490 mp 33 mg. of phenol reagent is expensive and solutions of it in sul- 5, L-Arabinose', Coleman Jr:', 480 mp: 17 mg. of phenol 6. o-Galacturonic acid, Coleman Jr., 485 ma. 17 mg. of phenol furir acid are not stable (30,34). The anthrone 7. L-Fucose Coleman Jr. 480 mp 40 mg. of phenol 8. D-Glucurbne, Coleman'Jr., 485'nw 17 mg. of phenol method also suffers from the disadvantage that, 9. 2 3.4,6-Tetra-o-methyl-~-glucoseColeman Jr 485 mp, 17 mg. of phenol while it is satisfactory for frw sugars and 10. ;-Glucose, Beckman Model DU,'4!?0 mp, 100 mg. of phenol 350 VOLUME 28, NO. 3, MARCH 1956 351 order to obtain good mixing. The tubes are allowed to stand 10 minutes, then they are shaken and placed for 10 to 20 minutes in a xvater bath at 25" to 30" C. before readings I20 are taken. The color is stable for several hours /" and readings may be made later if necessary. The absorbance of the characteristic yellow- 1.00 orange color is measured at 490 nip for hexoses and 480 nip for pentoses and uronic acids. Blanks are prepared by substituting distilled water for the sugar solution. The amount of sugar may then be determined bj. reference to a standard curve previously constructed for the particular sugar under esamination. All soliitions are prepared in triplicate to mininiizc errors resulting from accidental con- tamination \vith cellulose lint. If it is desired to avoid the use of niicaro- pipets, the phenol may be added as a 5% solu- tion in v;ater. The amounts of reactants are I 0 IO 20 30 40 50 60 70 80 90 100 110 I20 then: 1 or 2 nil. of sugar solution, 1 ml. of MICROGRAMS OF SUGAR 5% phrnol in n-ater, and 5 ml. of concentrated sulfuric acid. -All other steps are the same as Figure 2. Standard curves above. 1. Sucrose, Beckman Model DV, 490 mr, 100 mg. of phenol 2. Potatostarch, Beckman Model DU, 490 mp, 100 mg. of phenol Standard Curves. *A series of t)yic.al ,*tmd- :. Dextran from Leuconostoc mesenteroides strain NRRL 515 1 Beckman Model DU. 490 mu. 103 mn. of Dhenol ard curws is shown in Figures and 2. In- D-G~u~os~,Evelyn, filter No. 490, 80 mgof p'henol .eluded in these figures are esamples of some 4: L-Rhamnose. Coleman Jr., 480 mfi, 40 mg. of phenol 6. Raffinose, Beckman RIodel DE. 490 mp, 100 me. of phenol of the sugars usually encountered in carbohy- 7. D-Fructose, Beckman Model Dr. 490 mp3200 mg. of phenol drate studiep-namely, pentose, deorypentow, 8. 2-Deoxy-D-ribose, Coleman Jr., 490 mp, 80 mg. of phenol mcthylpentose, aldohesoee, ketohesose, ht.w- ronic arid, disaccharide, trisaccharide, :nid 1.00 I,,,,,,, certain methylated derivatives. In order to test t,he method, the experiments were repeated on different days and by different D-MANNOSE I80 q) operators. In all cases the variations between esperiment,s and .90- between operators vere no more than 0.01 t,o 0.02 unit in all- HYOROXYMETHYLFURFURAL sorbance, which n-as t,he same order of magnitude as the vwia- .80 - tion between the triplicate samples. The experimental data for the various carbohydrates, esrept, .70- 2-deoxyribose, given in Figures 1 and 2 may be t,abulated by 0-GALACTOSE (80 CI) calculating the value of a,, the absorbance index, in the equation 9,= a,bc (Table I). The absorbance, A,, is a dimensionless .60 - Tao~ver, ratio equal to log,, __6 where I' is per cent transmittance, w-0 Tsolutiou' - b is the length of light. pat'h, expressed in centimeters, and c is m4.50 the concentration, in micrograms of sugar per milliliter of final w-m volume. 4.40 - Discussion of Results. ABSORPTIOSCCRVES. The rurves obtained by plotting ahsorixinw t3.s. wave length (Beckman >lode1 .30- 20- D-XYLOSE (80 71 .IO- 01 ' ' I,,, WAVE LENGTH-MILLIMICRONS Figure 3. -4bsorption curves Fast-delivery 5-ml pipet, to deliver 5 ml. of concentrated sul- furic acid in 10 to 20 seconds. This is easily prepared by cutting a portion of the tip of a standard 5-ml. pipet. Series of matched colorimetric tubes, internal diameter between 16 and 20 mm.
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