Quantitative Quantitative Analysis Analysis of Carbohydrates Arienne Astorga, Jermaine Balbanida, Ma.Fatima Louise Barreiro, Jenina Marie Caabay, Sarleen Castro, *Shelley Lauren Cham 2D-MT
ABSTRACT Carbohydrates are polyhydroxyaldehydes (aldoses) and polyhydroxyketones (ketoses) with the general formula (CH 2O)n where n equals 3 or more. Carbohydrates are the most important sources of energy and are essential components in many foods. They are widely distributed in plants and are even found in certain animal tissues such as the liver and muscles. The experiment was performed in order to determine the components present in a given sample and to correlate those standard sugars presented with that of the acid hydrolysate using the Nelson’s test. Glucose standard curve was pl otted using the absorbance reading against the concentrations of the standard solutions. A zig zig line graph was obtained in the experiment. There were errors made in the experiment due to some technical problems.
INTRODUCTION
Carbohydrates are organic compounds consisting of carbon, hydrogen, oxygen in which the last two is in the 2:1 ration. It can be defined as compounds that have reactive aldehyde and ketone functional group and multiple hydroxyl groups. Carbohydrates are also known as saccharides and are divided into four chemical groupings: monosaccharides, disaccharides, oligosaccharides, and polysaccharides. In general, monosaccharides, and disaccharides, which are smaller (lower in molecular weight) carbohydrates, are commonly referred to as sugars. Basically, its primary function is to provide energy for the body, especially in the brain and the nervous system. (1)
Fig. 1 Chemical structures of Saccharides
Glucose, or D-glucose, is classified as an aldohexose, a monosaccharide with six carbon atoms with an aldehyde group at the end. It is found 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. One of the processes used is the Nelson’s method. (2) The Nelson’s method is often called Nelson-Somogyi method. It is a widely used classical method for quantitative determination of reducing sugars. Determination of reducing sugar using Nelson method is based on the absorbance 480 nm of a colored complex formed between a copperoxidized sugar and arsenomolybdate. The amount of carbohydrate present is determined by comparison with a calibration curve using a spectrophotometer. Under the proper conditions, the Somogyi-Nelson method is accurate to ± 0.01 mg for D-glucose, D-galactose, and maltose. (3)
The primary objective of the experiment is to determine the amount of reducing sugars using Nelson’s test and explain the principle involve.
METHODOLOGY
RESULTS
In the experiment, Nelson’s reagent was prepared by mixing 12.5 ml of Nelson’s A with 0.5 ml of Nelson’s B . The seven test tubes were labelled and were filled with measure amounts of standard glucose solution presented in the table below.
The following table is the tabulated data for the quantitative analysis of carbohydrates and absorbance reading.
Table 1: Dilution of Samples
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 0.9 0.8 0.6 0.4 0.2 0 0.6
Unknown Sample (ml) 0 0 0 0 0 0 0 0.4
Then, 1.0 ml Nelson’s reagent was added into each test tube, and was shaken well. The test tubes were heated in water bath for about 20 minutes. The test tubes were removed simultaneously and were cooled in a beaker of water afterwards. Then, 1.0 ml arsenomolybdate reagent was added into each test tube. The test tubes were shaken occasionally for five minutes or until the Cu 2O precipitate dissolves. The solutions were diluted with 13 ml more water so that the absorbance can be read easily. The absorbance of the standards and the unknown was taken against a reagent blank at 480 nm. Standard curve was constructed by plotting absorbance reading against concentrations of standard solutions. Finally, concentration of the unknown in mg/ml and mg/tube was determined.
Table 2: Concentrations of Glucose and Absorbance Readings
Test Glucose tube Std. No. (mg/tube) 0 0.01 0.02 0.04 0.06 0.08 0.1 0
1 2 3 4 5 6 7 8
Glucose Std. (mg/ml)
Absorbance 480
0 0.00063 0.00125 0.00250 0.00375 0.00500 0.00625 0
0.000 0.571 0.450 0.375 0.551 0.819 0.601 0.328
Below is a graphical representation of the data
1 y = 136.41x R² = -0.247
0.8
e e c n 0.6 a b r o 0.4 s b A
0.2 0 0
0.002
0.004
0.006
0.008
Concentration (mg/ml)
Fig. 2 Glucose Standard Curve
The following table is the tabulated data for the unknown in (mg/tube) and (mg/ml).
Table 3: Concentration of Unknown
Unknown (mg/tube) Unknown (mg/ml)
0.0006 0.0018
DISCUSSION The
table 2 above showed the different computed concentrations of glucose solution per test tube and ml. It also showed the absorbance readings of each. The test tube contains different volumes of the standard glucose solution. The concentration (mg/ml) was obtained using the formula: Concentration= Volume of glucose standard/ Total volume of solution in tube The total volume of the solution is determined by adding the volume of standard glucose, volume of the distilled water, volume of the Nelson’s r eagent, volume of the dilution water, and volume of the arsenomolybdate reagent. The total volume for the each test tube in this experiment is 16 ml. Spectrophotometer was used to get the absorbance reading of the standard solutions and of the unknown sample. The absorbance at 480 nm is directly proportional to the concentration of the standard glucose solution. This means that as the concentration increases, the absorbance also increases. Unfortunately, the absorbance read in the performed experiment did not exhibit a direct proportionality with the concentration of the glucose standard (shown in Fig 2 above). This may be due to contamination of the solution. Contamination may be due to dirty apparatus like the test tubes or cuvette. (4)
REFERENCES
1.) Flitsch, SL & Ulijn, RV (2003). Sugars tied to the Spot. Nature 421: 219-220 2.) Lehninger, Albert L. Biochemistry. Second. New York, New York: Worth Publisher, Inc., 1976. 3.) “Determination of Reducing Sugars by Nelson-Somogyi method.” ePlant Science. com. N.p., n.d. web. 24 Feb 2011. 4.) Campbell, M.K., and Farrell, S.O. (2006). Biochemistry, Fifth Edition. California, USA: Thomson Brooks/Cole.
