Annexure 2 - Glucose Standard Curve

Experiment No.
: 01
PREPARATION OF GLUCOSE STANDARD CURVE
AIM:
To prepare a standard curve of glucose through DNSA method.
THEORY/PRINCIPLE:
The relationship between a substance's concentration (in this case, glucose concentration) and
absorbance at a specific wavelength is shown graphically by the standard curve. To measure
the absorbance of any unknown material and estimate its glucose concentration, the created
standard curve must be used. One notable colorimetric test for determining reducing sugars is
the Dinitro-salicylic Acid (DNSA) technique. Since glucose is a reducing sugar, its presence
in any sample can be ascertained using the DNSA technique. The orange-red 3-amino,5-nitro
salicylic acid is produced when glucose breaks down the pale yellow-colored alkaline 3,5-
Dinitro salicylic acid (DNS).
Fig. 1. Showing chemistry behind estimation of reducing sugar through DNSA method
According to Beer Lambert's law, the color's intensity is correlated with the amount of
glucose in the solution. A colorimeter or spectrophotometer is used to measure the intensity
of colour change by measuring the absorbance at 540 nm wavelength. Since this is the
wavelength where orange-red colour absorbs the most, it is set at 540 nm. Test tubes are filled
with a series of liquids with various known glucose concentrations, and each solution has a
known amount of DNS added to it. A colorimeter/spectrophotometer is used to measure the
optical densities of these test tubes after they have been heated for a few minutes in a water
bath. A graph is then plotted with amount of glucose on X axis and the observed optical
density at Y axis. The plot thus obtained is called a standard curve of glucose.
REQUIREMENTS:
A. Reagents
1. Glucose/Dextrose standard solution (1 mg/mL)
2. Phosphate Buffer (0.1 M, pH-7.0)
3. DNSA Reagent
B. Instruments and Glassware:
4. Colorimeter/Spectrophotometer
5. Glass/Micro Pipettes and pipette tips
6. Colorimeter cuvettes
7. Test tubes
8. Test tube stand
9. Water bath
PROCEDURE:
1. A series of glucose standard solutions with concentrations ranging from low to high (10
μg/mL – 200 μg/mL) was prepared
2. Phosphate buffer was used for dilution to make up the volume till 1 ml.
3. 2 ml DNSA reagent was added in standard solution of glucose in each test tube.
4. The tubes were incubated in boiling water bath (100 °C) for 10 minutes.
5. The colorimeter was set at wavelength 540 nm and the absorbance of the developed
colour was measured. The absorbance values for all the solutions were recorded.
8. A graph with concentration of glucose on the x-axis and absorbance on the y-axis was
plotted.
OBSERVATIONS TABLE:
Sr. No. Volume of Volume of Volume of Concentratio Absorbance

glucose buffer (μl) DNSA n of glucose at 540 nm
standard reagent (μg/ml)
solution (μl) added (ml)
Incubate in water bath at 100 °C for 15 mins.

1. (Blank) 0 1000 2 0
2. 20 900 2 100 0.01

3. 40 800 2 200 0.04
4. 60 700 2 300 0.06
5. 80 600 2 400 0.16
6. 100 500 2 500 0.18
7. 120 400 2 600 0.14
8 140 300 2 700
0.16
9 160 200 2 800
0.18
10 180 100 2 900
0.2
11 200 0 2 1000
0.26
Standard Curve
Glucose standard curve

0.3
0.25
Absorption at 540 nm
0.2
0.15
0.1
0.05
0
0 20 40 60 80 100 120 140 160 180 200
Maltose concentration (ug/ml)
RESULT:
A glucose standard curve was prepared successfully.
PRECAUTIONS:
1. DNSA reagent is highly sensitive to light and must be stored in amber-colored bottles or
glass jars.
2. Do the pipetting carefully to avoid the handling errors.
3. Role of individual component of DNSA reagent:
Dinitro-salicylic Acid: The DNS reagent reacts with reducing sugars present in the sample.
Reducing sugars are sugars that have a free aldehyde or ketone functional group that can
undergo oxidation. During the reaction, DNSA in the reagent is reduced by the reducing
sugars, resulting in the formation of a coloured compound.
Sodium potassium tartrate: Sodium potassium tartrate, also known as Rochelle salt, is often
used in the Dinitro-salicylic Acid (DNSA) method as a stabilizer and catalyst. It reduces the
tendency to dissolve O2 by increasing the ion concentration in the solution.
Crystalline Phenol: Crystalline phenol enhances the colour development in the DNSA
reaction. When reducing sugars react with Dinitro-salicylic acid (DNSA), they form coloured
compounds. It makes it easier to measure the absorbance and quantify the amount of reducing
sugars present in the sample.
Sodium hydroxide: NaOH is added to the reaction mixture to create an alkaline

environment. The alkaline conditions help break down complex sugars and facilitate their
conversion into simpler compounds that can react with DNSA.
Sodium Sulphite: Na2SO3 is sometimes used as an additive in the Dinitro-salicylic Acid

(DNSA) method to improve the accuracy and reliability of the assay. It serves as a reducing
agent and antioxidant.

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Experiment No.

PREPARATION OF GLUCOSE STANDARD CURVE

To prepare a standard curve of glucose through DNSA method.

B. Instruments and Glassware:

Sr. No. Volume of Volume of Volume of Concentratio Absorbance

Incubate in water bath at 100 °C for 15 mins.

2. 20 900 2 100 0.01

Glucose standard curve

A glucose standard curve was prepared successfully.

Sodium hydroxide: NaOH is added to the reaction mixture to create an alkaline

Sodium Sulphite: Na2SO3 is sometimes used as an additive in the Dinitro-salicylic Acid

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