GLUCOSE ASSAY BY DINITROSALICYLIC COLORIMETRIC METHOD

DEPARTMENT OF BIOTECHNOLOGY ENGINEERING INTERNATIONAL ISLAMIC UNIVERSITY MALAYSIA BIOTECHNOLOGY ENGINEERING LAB BTE 2221

EXPERIMENT 1 (BIOCHEMISTRY) GLUCOSE ASSAY BY DINITROSALICYLIC COLORIMETRIC METHOD DATE OF EXPERIMENT : 16/4/2014

NAME

: MOHAMAD ABDULAH BIN MD NOORDIN

MATRIC NO.

: 1221663

INSTRUCTOR

: BR SAHEED

INTRODUCTION Objectives : Measure the concentration of glucose in a given unknown sample by DNS method.

Background : Glucose, a monosaccharide (or simple sugar), is the most important carbohydrate in biology. Transported via the blood stream, it is the primary source of energy for the body’s cell. Glucose levels are tightly regulated in the human body. Failure to maintain blood glucose in the normal range leads to conditions of persistently high (hyperglycemia) or low (hypoglycemia) blood sugar. Diabetes mellitus, characterized by persistent hyperglycemia, is the most prominent disease related to improper blood sugar regulation.

The determination of glucose levels in blood is critical in the control of diabetes. A dinitrosalicylic acid (DNS) assay has been available since 1955 but more recently, several enzymatic assay using either hexokinase-glucose-6-phosphate dehydrogenase or glucose oxidase-peroxidase for glucose quantification have been developed. The nanoenzymatic assay quantitates all reducing sugars whereas the enzymatic assay is specific for glucose, allowing for more accurate quantification.

In this experiment we determine the amount of sugar or carbohydrates in a given unknown sample by a spectrophotometric (colorimetric) method. The method is based upon the color which forms when sugars reduce 3,5-dinitrosalicylic acid (DNS) to 3-amino-5nitrosalicylic acid, as shown in the equation.

Several reagents have been employed which assay sugars by using their reducing properties. This method tests for the presence of free carbonyl group (C=O), the so-called reducing sugars. This involves the oxidation of the aldehyde functional group present in, for example, glucose and the ketone functional group in fructose. Simultaneously, 3,5-dinitrosalicylic acid (DNS) is reduced to 3-amino-5-nitrosalicylic acid under alkaline conditions, as illustrated in the equation above.

The spectrophotometer measures absorbance. Absorbance values, by themselves, do not describe the concentration of a substance. However, we can determine the concentration of a substance in a solution using a standard curve. A standard curve translates absorbance values into concentration. We can construct a standard curve by making solutions with a known concentration of the substance we are measuring and then measuring their absorbance. Graphing the concentration on the x-axis and the absorbance on the y-axis, we can see that there is a linear relationship between concentration and absorbance as seen in Fig 2-1 below. Thus a standard curve is not really a curve, but a straight line. Beers Law describes this linear relationship:

A =εCL Where A = absorbance ε

= molar absorptivity of the substance

C = concentration (for clinical measurements, the concentration often used is mg/dl) L = pathlength (the length the light travels through the cuvette)

We assume that the y-intercept is zero. In other words we are assuming that the absorbance of a solution is zero when none of the substance we are measuring is present. We do this by using a blank. A blank solution contains all the substances present in the solution except the substance to be measured. Before making measurements with the Spec 20, the blank is measured, and its absorbance value is set to zero. By using a blank, if water or sugar absorbs light, that absorption will be set to zero, and thus the y-intercept on our linear Beer’s Law equation would indeed be zero.

RESULT AND CALCULATIONS From the spectrophotometer readings, we have :

0.069 | 0.058 | 0.123

We take the average by:

(0.069 + 0.058 + 0.123) = 0.0833 3 Therefore y = 0.0833

From the given standard curve, substituting y to 0.0833 : Y = 3.5329 x

x = 0.0833 3.5329 x = 0.0235

DISCUSSION

We pour three identical solutions that we have prepared into three cuvettes to measure the absorbance and take the average. From there we got 0.0833 absorbance of unknown solution (average). From the given standard curve equation y = 3.5329 x, we divide the absorbance by the slope of the standard curve. We calculated our concentration (x) to be 0.0235 mg/mL.

From here, we know that on average, only 0.0235 mg/mL of glucose present in the 0.0833 mg/mL of our unknown solution. In other words, glucose appears to be 28.2% of the actual unknown concentration. Our result might differ from other groups based on our pipetting process. If our pipetting has been accurate and precise, everyone should have the same result.

Heating is necessary to accelerate the chemical reaction. We heat our mixture of DNS solution and unknown solution until it develops to red-brown color. It is aware that the heating process should only be between 5 to 15 minutes. Overheating will cause the chemical reaction to stop. Therefore, we use a timer to make sure we didn’t stop the reaction.

Some safety precautions that we need to observe includes using test tube holders to handle hot tubes to avoid hand injuries. Since we are dealing with DNS solution which is toxic, we need to wear goggles at all times to avoid any contact with the skin. If any is spilled, wash thoroughly with soap and water. At the end of the session, we need to clean up before we go for others convenience.

CONCLUSION

Alhamdulillah we have successfully calculate the amount of glucose present in the given unknown solution which is only 0.0235 mg/mL. We use method of DNS by which we mix our unknown solution with a DNS agent. The concentration of our glucose was measured using the absorbance in the spectrophotometer.

REFERENCES 

Dr. Mohamed E. S. Mirghani, Lab Manual for Biotechnology Engineering Lab 1 BTE 2221

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http://www.bio.davidson.edu/courses/bio111/bio111labman/preface%20d.html

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http://www.jenway.com/adminimages/A09_008A_Determination_of_glucose_in_drin ks.pdf

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http://employees.csbsju.edu/mcampos/bio114/labmaterials/lab.2.writeup.03.pdf

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http://biochem.ncsu.edu/faculty/brown/Lab%20Exercise%200ne%202008.ppt

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http://bric.postech.ac.kr/myboard/down.php?Board=exp_qna&filename=Determinatio n%20of%20sugar%20as%20glucose.pdf&id=42071&fidx=1



http://www.kau.edu.sa/GetFile.aspx?id=156654&fn=bioc_211(lab1).doc