Quantitative Analysis of Carbohydrates Using Nelsons Assay Marcus Natividad, Barbara Ngo, Lexley Ong and Jane Jenelle

Quilaneta Group 8 2C Pharmacy Biochemistry Laboratory ABSTRACT

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INTRODUCTION This experiment aims to make the students able to determine the amount of reducing sugars specifically glucose using Nelsons Test. Carbohydrates are the most abundant class of organic compounds found in living organisms. They originate as products of photosynthesis, an endothermic reductive condensation of carbon dioxide requiring light energy and the pigment chlorophyll. n CO2 + n H2O + energy - CnH2nOn + n O2 As noted here, the formulas of many carbohydrates can be written as carbon hydrates, Cn(H2O)n, hence their name. The carbohydrates also known as saccharides, are a major source of metabolic energy, both for plants and for animals that depend on plants for food. Aside from the sugars and starches that meet this vital nutritional role, carbohydrates also serve as a structural material (cellulose), a component of the energy transport compound ATP, recognition sites on cell surfaces, and one of three essential components of DNA and RNA[1]. Carbohydrates are divided into three general classes depending on the number of carbohydrate molecules they contain. Monosaccarides are simple sugars that cannot be hydrolyzed. Oligosaccharides are those that contain 2-10 monosaccharide units. Polysaccharides contains more than 10 monosaccharide unit[2] Carbohydrates can be defined as compounds that have reactive aldehyde or ketone functional group and multiple hydroxyl groups. The most common carbohydrate is glucose (C6H12O6)[5].

Glucose, or D-glucose, is classified as an aldohexose, a monosaccharide with six carbon atoms with an aldehyde group at the end and a reducing sugar. It occurs widely in plants and in the blood of man and other animals. Glucose is an important source of energy for the body since it can be utilized directly without any intervening digestive process[3]. One of the processes used to determine glucose is the Nelsons method. The Nelsons method, more oftenly called the Nelson-Somogyi method, is a widely-used classical method for quantitative determination of reducing sugars. It demonstrates how much glucose is liberated from glycogen during enzymatic and acid hydrolysis [4]. EXPERIMENTAL A. Sample Used Sample Used: Nelsons reagent A, Nelsons reagent B, Arsenomolybdate reagent, Glucose standard, distilled water B. Procedures Qualitative Analysis Using Nelsons Assay. In Nelsons Assay, Nelsons reagent was made by mixing 12.5 mL Nelsons A with 0.5 mL Nelsons B. Eight test tubes were labeled and the measured amounts of standard glucose were transferred into the test tubes. The following amount of standard glucose was shown in the table below: 1.

Fig1. Structure of D-Glucose Table 1. Protocol for glucose standard curve

Tube No. 1 2 3 4 5 6 7 8

Glucose Standard (mL) 0 0.1 0.2 0.4 0.6 0.8 1.0 0

Distilled Water (mL) 1.0 0.90 0.80 0.70 0.60 0.50 0.40 0.60

Unknown sample (mL) 0 0 0 0 0 0 0 0.4

Wherein the volume of the standard glucose was computed using ratio and proportion which is shown in Table 2. Table 2. Computed volume of standard glucose Test tube no 1 2 3 4 5 Volume of standard glucose 0mg = __x___ mL 0.10mL 0.1mg = __x___ mL 0.10mL 0.2mg = __x___ mL 0.10mL 0.4mg = __x___ mL 0.10mL 0.6mg = __x___ mL 0.10mL 0.8mg = __x___ mL 0.10mL 1.0mg = __x___ mL 0.10mL

; x= 0 ; x=0.01 ; x=0.02 ; x=0.04 ; x=0.06 ; x=0.08 ; x=0.1

After preparing the test tubes, 1.0 mL of Nelsons reagent was added to each test tube. The test tubes were heated simultaneously in a boiling water bath for 20 minutes. It was then removed and placed in a beaker of water to cool down. Then, 1.0 mL of arsenomolybdate reagent was added to each test tube and was shaken occasionally for 5 minutes until the Cu2O precipitate dissolved. Then the absorbance of the standards and unknown was measured against the blank reagent at 480 nm. Finally, the concentration of the unknown was determined by constructing a glucose standard curve by plotting absorbance readings against concentration of standard solutions. RESULTS AND DISCUSSION Nelson's Assay is used to establish a standard curve for glucose. Samples containing accurately known concentrations of glucose are subjected to this assay, absorbance readings recorded, and the data plotted as a standard curve. It should be borne in mind that this method is a general test for reducing sugars and does not distinguish between reducing monosaccharides (such as glucose) and reducing disaccharides (such as maltose) To find out the concentration of the standard glucose which is used in the Nelsons Assay. The equation below was used:

6 7

The obtained volume of standard glucose is used for the computation of concentration of the standard glucose solution in the different test tubes. Table 3. Concentration of Glucose Standard Test Tube No. 1 2 3 4 5 6 7 Glucose Standard (mg/mL) 0 = 0 3mL 0.01 = 3.33x10-3 3mL 0.02 = 6.67x10-3 3mL 0.04 = 0.013 3mL 0.06 = 0.02 3mL 0.08 = 0.0267 3mL 0.1 = 0.033 3mL

  

 

Table 3 shows the computed value of concentration of the standard glucose The total volume of the solution is determined by adding the volume of the standard glucose, volume of the distilled water, volume of the Nelsons reagent and the volume of the arsenomolybdate reagent. The total volume for each test tube in this experiment is 3 mL.

The amount of carbohydrates present in a given sample is measured by Nelsons Method which is based on the capacity of the free reducing groups of sugars in a carbohydrate sample to reduce Cu+2 in an alkaline solution. In this determination, the amount of free reducing sugars in the sample is directly related to the molybdenum blue formed via series of oxidation/ reductions, and is measured colorimetrically.

In table 4, we were able to measure the amount of light transmitted at 480 nm of the standard solutions and of the unknown sample. The absorbance is directly proportional to the concentration of the standard glucose solution. This means that as the concentration increases, the absorbance also increases.

2.5 2

Glucose Standard Curve

Absorbance

1.5 1

y= 18.306x R2= 0.4731

0.5 0 0 0.01 0.02 0.03 0.04

Glucose standard (mg/mL)

Figure 3. Glucose Standard Curve This shows the glucose standard curve that was constructed by plotting the absorbance reading against the concentrations of the standard solutions. The line or the best fit line represents the ideal absorbance readings relative to the concentration. It was done using the linear regression equation: Table 4. Absorbance at 480 nm Test Tube no 1 2 3 4 5 6 7 8 Absorbance 0.997 1.307 1.38  1.794 1.810 1.87 1.893 1.817   Wherein y is equal to the absorbance, x represents the concentration of the glucose standard and m is the slope of the line The linear equation for glucose standard curve is y=18.306x

Figure 2. Samples used in Nelsons Assay Figure 2 shows that Cu+2 is reduced to Cu+1 by the reducing activity of the sugar. It results in blue (reduced) arsenomolybdous acid.

References [1]http://www2.chemistry.msu.edu/faculty/reusc h/virttxtjml/carbhyd.htm [2]Crisostomo A. et.al.(2010).Laboratory Manual in General Biochemistry.Quezon City: C&E Publishing Inc.do.edu/hndbksupport/ochemlabtech.html 2003 [3]http://staff.science.nus.edu.sg/~dbsyhh/lab3. htm [4]http://www.esu.edu/~jfreeman/317/chem317 l/Lab%20folders/317lcarbpro/317lcarbpro.htm [5]Boyer, Rodney. Concepts in Biochemistry. Third. Hoboken, New Jersey: John Wiley and Sons Pte Ltd, 2006. Print.