Robert Binman Queens College of CUNY Flushing, New York 11367
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Analysis of Reducing Sugars
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A general chemistry experimenf
in ~reakfastCereal and Other Foods
There has heen a trend to incorporate "relevant" experiments into the chemical curriculum. Many descriptions of experiments have appeared recently in this Joumol and in new laboratory manuals, illustrating the attempts heine made to arouse and maintain student interest in the laho;atory (I). The experiment descrihed here has been introduced in the freshman chemistw laboratow and has been found t o be of interest to the &dent and &structive as well. Alkaline 3,5-dinitrosalicyclic acid (DNS) forms a redbrown reduction product, 3-amino-5-nitrosalicylate, when heated in the presence of reducing sugars (2); it is recognized, however, that the occurrence of side reactions may make the chemistry of the test complicated (2-4). The alkaline DNS test for reducing sugars is simple, fast, and reliable, and has therefore been widely used in carbohydrate research and medical diagnosis. Since its introduction in 1921 a s a reagent for the determination of reducing substances in urine (3, DNS has been used for the determination of blood sugar (6),measurement of reducing end-groups produced in the hydrolysis of starch (7, 81, and assay of carhohydrase activities (9, 10). In this experiment, the intensity of the color developed a t 540 nm on reduction of DNS by reducing sugars is used to determine the reducing carbohydrate content of food samples. DGlucose solutions are used to construct a standard curve. Because the molar reducing capacities of different carbohydrates are not identical (2, 6, 11-13) results are reported in terms of the percent glucose by weight in each food sample. Each student is expected t o analyze a t least two foods and five standard glucose solutions. The students are given a brief introduction to the importance of carbohydrates for storage of energy by plant and animal cells; hydrolysis of polysaccharides to monosaccharides by acids or enzymes is also discussed. Apparatus
Colorimeter equipped with an adapter for 15 X 1.8 cm test tubes or culture tubes Test tubes or culture tubes, 15 X 1.8 em, preferably matched for colorimetry One 2-02 mortar and pestle per student Three 1.0-ml transfer pipets or two 1.0-ml and one 2.0-ml transfer pipets per student Reagents
DNS Reagent. Dissolve, with warming and vigorous stirring, 10 g of 3.5-dinitrosalicyclic acid in 200 ml of 2 N NaOH. Dissolve 300
Procedure 1) of Powdered Food. Place more than 50 me of -. Reoaration . the food m a mortar. Use the pestle to grnd it to a fine powder. Weigh out a sample of about 50.0 mg ai the powdered fuod Kecord the weight to the tenth of mrlligram. Place the sample in a labeled 15 x 1 . 8 cm test tuhe. Repeat the procedure using a different food. 2) Hydrolysis of Polysaccharides. To eaeh test tuhe add 10 ml of 1.5 M H~SOI.Heat in a boiling water bath for 20 min. Stir the contents occasionally. Cool the tubes under the tap. Cautiously add 12 ml of 10%NaOH. Mix. 3) Filtration of the Hydmlysates. Warm the test tubes containing the hydmlysates and filter by gravity while warm. (The filtration will proceed slawly if the samples are not warm. If wheat germ is used, it will be necessary to filter by suction instead of by gravity; requisition a Large sidearm test tube; after the fdtration, transfer the filtrate from the side-arm test tube to a 20 x 2.5 cm teat tube.) The filtrates are to be collected in 20 X 2.5 em test tubes. In order to transfer each hydrolysate quantitatively from the 15 x 1.8 em tube to the funnel where filtration is occurring, wash eaeh hydrolysate tube with warm distilled water and add the washing to the filter paper. Bring the final volume of each 20 X 2.5 em tube to 60 ml. (This may be done by making a calibration mark corresponding to60 ml onane tube.) Mix well. 4) Addition of DNS to Fdtered Hydrolysotes. Withdraw 1.0 ml of each hydmlysate. Place each 1.0-ml aliquot in a labeled 15 x 1.8 em test tube or culture tuhe. To each tube, add 1.0 ml of DNS reagent. Add 2.0 ml of water to each tube, making an effort to wash the hydmlysate and DNS toward the bottom. 5) Addition of DNS to Standard Glucose Solutions. Glucose solutions of 250, 500, I W , 1250, and 1500 fig per ml are pmvided. Take six clean, dry 15 X 1.8 cm test tubes or culture tubes. Label them 250, 500, 1000, 1250, 1500, and "blank." Add 1.0 ml of distilled water to the blank, and 1.0 ml of the various glucose solutions to the appropriate tubes. The same 1.0-ml pipet may be used if you start with the most dilute solution and rinse the pipet eaeh time with the solution you are about to pipet. To each tube add 1.0 ml of DNS reagent and 2.0ml ofwater. 6) All eight tubes from steps 4 and 5 are heated in a boiling water bath for 5 min to allow the reaction between glucose and DNS to occur. 7) Cool all tubes promptly in cold running water to ensure complete stoppage of the reaction. Adjust the volume of each tube to 25 ml with distilled water. Mix well. 8) Read the absorbance at 540 nm (alternatively, a colorimeter with a green filter may be used) of each sample after the blank has been adjusted to read 100% transmittance (zero absorbance). 9) Plot the absorbance at 540 nm (ordinate) against the amount of glucose in micrograms (abscissa). 10) Calculate the percentage of reducing sugars (in terms of glucose) by weight in the sample of each food. ~
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Results
'Sodium potassium tartrate is added for stabilization of the color developed in thereaction ( 4 ) .
The number of micrograms of glucose present in the 1.0-ml aliquot of each hydrolysate is determined from the standard curve. The number of micrograms of glucose in the 60-ml hydmlysate is obtained by multiplying the number read from the graph by 60. Beer's law was oheved in the concentration ranee used and the color prbducei in the reaction was stable. Commercial breakfast cereals having a range of carbo-
of sodium ootassium tartrate tetrahvdratel in 500 ml of distilled water. Mix the two ~ l u t i u n sand adlust the final volume to 1.0 I with drstrllcd water. Stondord Gluroae Sdur!ons Prepare solurionn of 250. 500. 1 W . 1230,and 1500 rg per ml m distilled water. Slow in the cold.
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Journal of Chemical Education
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hydrate.contents were chosen. The student results were as follows: Rice Chex (Ralston Purina Co.), 74% (manufacturer's listed value, 87.5% total carbohydrates); BeechNut Oatmeal Cereal, 64% (manufacturer's listed value, 66.4%). It should be noted that the method measures storage polysaccharides, such as starch, not total carbohydrates. Other foods have been used, e.g., Apollo Wheat Germ, 50% (manufacturer's listed value, 44.5%). Listings of protein, fat, and total carbohydrate contentof various foods are available (14). The variety of possible samples that may be used makes this a versatile experiment. Literature Cited (I1 Soe for example. B m m , 0. B., and R i d m a n , L. B., J. CHEM EDUC.. M. 211
(21 ~ m t t kF ,. . B o ~ ~ E., I , and B U ~ I ,H., H ~ Uc .h i m . ~ ~ t a34.2132(1%1). . (31 Gilman. H.. "(hganie Chemistry: An Advanced ~reatim:' ~ o h nwiley and sons. New York. 1938. Volume2. p. 1523. (41 ~ i ~ ~0.eL r. , A ~ ~them., I. 31.426 ( I % ~ I . ( 5 ) sumner, J. B.. a n d ~ r a h a m v.A.. . J. B ~ Ocham.. I 4?.5(1921). (6) Sumner. J.B., J. B i d Chem., 62.267 (1924-5). (7) ~ d t i xG., , and ~ ~ ~ P., f ~H ~i Ddc h. ,m . A C ~81, ~ 286 , (19481. (81 smith, ~ . ~ . , a n d s t o e k ~ r , c . , ~ r e h . 21.%(1%9). ~i~~hr~., (9) Bcmfeld. P., "Methods in Enzymology: (Editors: Coloanck, S. P., and Kaplan. N. 0.1, Academic Pres, New York. 1955, Volume 1, p. 149. (101 Fiaeher. E. H., and Stein, E. A,, Biorhem. Fw..8.27 (1961). (111 Behi. D.J..Manners. D.J., andPslmer,A..J Chom. Soc., 3760(1%21. (121 M e w . K. H.. van der Wyk, A. J.A., and Few. C-P., Hdu. Chim. A m , 37. 1619 (I%~I. (131 Bruner, B. L., in " ~ e t h o d ain c m b o h ~ & ~ tchemidw," e ( ~ d i t o cwhistler, R.), A c a d e m i c h . New York. I S 4 . Volume4, p. 61. (I41 See for crampie, Wmsfer. H. A., Jr., "Nutritional Data: 2nd Ed., H. J. Hein. Co., Pittsburgh. Pa.. 1954.
(19731. and rohrmcsaeited therein.
Volume 51, Number 1, January 1974
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