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 bodys 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-5-
nitrosalicylic 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 =CL
Where A = absorbance
= 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 Beers
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 didnt 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
http://www.bio.davidson.edu/courses/bio111/bio111labman/preface%20d.html
http://www.jenway.com/adminimages/A09_008A_Determination_of_glucose_in_drin
ks.pdf
http://employees.csbsju.edu/mcampos/bio114/labmaterials/lab.2.writeup.03.pdf
http://biochem.ncsu.edu/faculty/brown/Lab%20Exercise%200ne%202008.ppt
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