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Quantitative Analysis of Carbohydrates; Nelsons Method

Riczen Vila, Kathyrene Visco, Rafael Vivar, Jeninah Zafra, Jeselle Ziganay Group 8, 2-G Pharmacy.

I.

Abstract

This experiment aims to determine the amount of sugar in the carbohydrate obtained from hydrolysis using Nelsons method. Nelsons method makes use of what we call the Nelsons regeant, and is made by mixing 12.5 ml of Nelsons A with 0.5 ml of Nelsons B. We prepared seven test tubes with different glucose concentration and an eighth one as an unknown. After the addition of Nelsons regeant we were asked to observe the samples under a regeantblank at 480 nm. Through this method we were able to determine the concentration of the unknown.

II.

Introduction
sugars and Carbohydrates mono, di, are tri, are classified poly The into and smallest whereas as starch,

Carbohydrates: Mainly

starches, together constituting one of the three principal types of nutrients used as energy sources (calories) by the body. Carbohydrates can also be defined oxygen. Carbohydrates come in simple forms such as sugars and in complex forms such as starches and fiber. The body breaks down most sugars and starches into glucose, a simple sugar that the body can use to feed its cells. Complex carbohydrates are derived from plants. Dietary intake of complex carbohydrates for saturated fat. can lower blood cholesterol when they are substituted chemically as neutral compounds of carbon, hydrogen and

heterosaccharides. carbohydrates such as polysaccharides

monosaccharides

glucose such

cellulose and glycogen can be large and even indeterminate in length. The energy produced by

carbohydrates is 4 calories per gram. Proteins also provide 4 calories per gram. Fats are high-cal; they provide 9 calories per gram. Nelson's method for the quantitative determination liberated layer of reducing glycogen is sugars during used the demonstrates how much glucose is from enzymatic and acid hydrolysis. Thin chromatography to qualitatively demonstrate

difference

between

the

reaction

IV. Results and Discussions


Quantitative assay for reducing sugars used the Nelson's Test. The standard curve generated by measurement of known concentrations solutions provides a relationship between the concentration of substance (glucose) present and the intensity of absorbance (color) formed in the reaction. A plot of the data provides a standard curve. The graph can then be used to determine the concentration of reducing sugar in an unknown solution which has been assayed in an identical manner. Indirect colorimetric assays require that the color forming reagents be present in excess concentration.

products of the acid hydrolysis of glycogen and the enzymatic hydrolysis of glycogen.

III. Methodology
1. Eight test tubes were prepared by mixing 0.5 mL of Nelsons Reagent A and 0.5 mL Nelsons Reagent B. The test tubes were labeled and were prepared in corresponding with their glucose standard solution in mL (0, 0.1, 0.2, 0.4, 0.6, 0.8, 1.0 and 0) and amount of distilled water (1.0, 0.9, 0.8, 0.6, 0.4, 0.2, 0 and 0.6) and on the 8th test tube 0.4 of the unknown sample. 2. Prepared test tubes were heated simultaneously in boiling water for 20 minutes. 3. The test tubes were removed and cooled in a beaker of water.4. 1.0 mL of arsenomolybdate were added to each test tube and shake for five minutes until the Cu2O precipitates were dissolved 5. The absorbance of the standard was measured at 480 nm. The standard curved for glucose was constructed.

Figure 1. Graph of Glucose Standard Curve

Glucose Standard Curve

1.800

absorbance (480 nm)

1.600 1.400 1.200 1.000 0.800 0.600 0.400 0.200 0.000 0.000 0.010 0.020 0.030 0.040 0.050 0.060

y = 10.763x + 0.2006 R2 = 0.1488

concentration (mg/mL)

The graph shows that there is no line of best fit that we cannot use it to know the concentration of the reducing sugar of the unknown sample. In other words the light absorbed by a sample is proportional to the number of molecules of absorbing substance through which the light passes. Insufficient concentrations of reagent results in nonlinear data at high concentrations of the unknown. These is obtained by absorbance against concentration and this is probably due to one or other of the following conditions not fulfilled such as: 1)Light must be of narrow range and preferably monochromatic ,2)The wavelength of light used should at the maximum absorption of the solution: this also gives great sensitivity, 3) There must be no ionization, association, dissociation or solvation

of the solute with concentration or time, 4)The solution is too concentrated, giving an intense color and 5) The law only holds up to a threshold maximum concentration for a given substance. Several types of errors may occur in using the Beer-Lambert Law for spectrophotometric analysis. These includes the errors in reading the absorbance or transmittance values; errors from dirty cuvettes. The principle of the Nelsons method for reducing sugar assay is based on the formation of enediols by reducing sugar in alkaline solutions. Enediols are readily oxidized to sugar acids by a variety of agents including ferricyanide, 3-52+ dinitrosalicylate and Cu . In the Nelson test the sugar is heated with an alkaline copper (Cu2+) reagent resulting in the formation of cuprous

oxide (Cu2O). The Cu2+ is then oxidized by arsenomolybdate, which is deep blue in its reduced form. The amount of reduced arsenomolybdate is proportional to the amount of sugar. This test is sensitive and reproducible.

V. Sources
*http://www.visionlearning.com/librar y/module_viewer.php?mid=61 *http://www.medterms.com/script/m ain/art.asp?articlekey=15381 *http://en.wikipedia.org/wiki/Carbohy drate