PRACTICAL 1: DETERMINATION OF REDUCING SUGAR USING THE

DINITROSALICYLIC (DNS) COLOURIMETRIC METHOD

INTRODUCTION

UV-Vis Spectroscopy is used to determine analyte concentration either at one time or often over
a desired time period. The technique measures the absorption of light across the ultraviolet and
visible light wavelengths through a liquid sample. Samples are dispensed into cuvette and
placed in the path between a UV-Vis light and a detector. According to Beer-Lambert’s law,
with a constant light path length and known absorption coefficient (dependent upon
wavelength), the concentration of a compound in question can be determined from the light
absorbed by the sample at that wavelength. The spectroscopy used for quantitation of analytes,
quality assurance and control and maximum wavelength determination. Advantages using the
spectroscopy are fast sample analysis, suitable for a wide variety of analytes, much simpler than
chromatographic techniques, user-friendly interface and little maintenance required.

OBJECTIVES

To determine the reducing sugar using the dinitrosalicylic (DNS) colourimetric method

MATERIALS AND APPARATUS

Double-beam spectrophotometer, Analytical balance, plastic cuvettes, beakers, vortex mirror,

pipettes, volumetric flasks, test tubes rack, water bath(boiling), spatulas, wash bottles, ice water,
glucose standard, 3,5- dinitrosalicylic acid (DNS), 2 molar NaOH, sodium potassium tartrate
tetrahydrate, distilled water, jam, juice

METHOD

1. Preparation of DNS reagent

For solution A, firstly 10g of DNS was dissolved in 200 ml 2M NaOH with warming and
vigorous stirring. Meanwhile for solution B, 300g sodium potassium tartrate tetrahydrate
was dissolved in 500 ml of distilled water. Lastly, both solution A and solution B was
mixed and make up to 1 L with distilled water
2. Preparation for sample

a) For solid sample

Firstly, 3 g of the solid sample was weighed accurately into a beaker and 50 ml of
distilled water was added. The beaker was put on the hot plate for warmed it and
stirred for 5 minutes or until it dissolved. Next, the solution was filtered into 100 ml
volumetric flask and the residue was washed into the volumetric flask with a small
amount of distilled water then make up to volume 100 ml and mixed them well with
repeated inversion of the flask. Lastly, 10 ml of the sample was pipette and diluted to
250 ml of volumetric flask with distilled water and mixed them well by inversion of
the flask.

b) For liquid sample

Firstly, 5 ml of the liquid sample was pipette and filtered into a 100 ml volumetric
flask. Then, the residue was washed into the volumetric flask with a small amount of
distilled water and make up to volume 100 ml followed by mixed them well with
repeated inversion of the flask. Lastly, 10 ml of the sample was pipette and diluted to
100 ml of volumetric flask with distilled water and mixed them well by inversion of
the flask.

3. Preparation of glucose standard solution

For prepared glucose stock solution of 100 mg/ml firstly, 10 g of glucose standard
was dissolved in 50 ml distilled water in a beaker. Next, the glucose solution was
transferred into a 100 ml volumetric flask. Then, the residue was washed into the
volumetric flask with a small amount of distilled water and make up to volume 100
ml followed by mixed them well with repeated inversion of the flask. Then, a series
of glucose standard solution of 1 mg/ml, 2 mg/ml, 3 mg/ml, 4 mg/ml, 6 mg/ml and 8
mg/ml was prepared by dilution of the glucose stock solution using distilled water in
100 ml volumetric flask. Lastly, mixed them well by repeated inversion of the flask
 4. Absorbance measurement
Firstly, 1 ml of distilled water was pipette into a test tube and labeled as ‘Blank’ and
each of concentration of glucose standard solution 1 ml was pipette in other labelled
test tubes. Secondly, 1 ml of the samples solution was pipette into test tubes labelled
as ‘sample’. Blank, standard and sample was prepared in triplicate. Then, 1 ml of
DNS reagent and 3 ml of distilled water was added into each test tubes prepared
above and mixed well using vortex mirror. Next, all test tubes was arranged in a test
tube rack and the rack was placed in the boiling water bath for exactly 5 minutes.
After 5 minutes, the test tubes were transferred immediately into a container of iced
water. After that, 10 ml of distilled water was added to each test tube and mixed well
using vortex mirror. Next, the maximum wavelength were determine using double-
beam spectrophotometer by measuring the absorbance spectrum of 3 mg/ml glucose
standard solution and the spectrum within the wavelength range of 400 nm to 600 nm
was measured. Then, the spectrophotometer wavelength was set using the highest
absorbance obtained. Lastly, the absorbance for each standard solution and the
sample solutions were measured.

RESULTS

Table 1.0: Absorbance data for DNS colorimetric method

Standard / Sample concentration Absorbance reading at λ = nm

Abs 1 Abs 2 Abs 3 Abs. average ±
SD
Blank 0.000 0.000 0.000 0.000
1 mg/ml 0.672 0.716 0.730 0.705 ± 0.030
2 mg/ml 1.409 1.429 1.486 1.441 ± 0.040
3 mg/ml 1.725 1.830 1.858 1.804 ± 0.070
4 mg/ml 1.935 1.855 2.015 1.935 ± 0.080
6 mg/ml 1.919 1.972 1.945 1.945 ± 0.027
8 mg/ml 1.910 1.968 2.072 1.983 ± 0.082
Apple juice 0.420 0.458 0.470 0.449 ± 0.026
Jam 0.354 0.370 0.343 0.356 ± 0.014
Calculation:

Example for prepare concentration of glucose solution from the stock solution given.

C 1 V 1=C 2 V 2

100 mg 1 mg
C 1= ,V 1=? , C2= , V 2=100 ml
ml ml

(100)¿) = (1)(100)

V 1=1 mg/ml

Determine sample concentration from the equation derived from the linear standard curve.

x = concentration of glucose in diluted sample

y = average absorbance of sample

Apple juice:

Equation, y = 0.642x and average absorbance of apple juice, y = 0.449

y = 0.642x

0.449 = 0.642x

x = 0.6994 mg/ml

Concentration of glucose in original apple juice:

0.6994 mg/ml 100 ml 100 ml

= 1 ml
×
5 ml
×
10 ml

= 139.88 mg/ml

= 0.1399 g glucose in 1 ml apple juice

= 13.99 g glucose in 100 ml apple juice

= 13.99 % glucose in apple juice

1st dilution factor = 100 ml / 5 ml

2nd dilution factor = 100 ml / 10 ml

Jam:

Equation, y = 0.642x and average absorbance of jam, y = 0.356

y = 0.642x

0.356 = 0.642x

x = 0.5545 mg/ml

Concentration of glucose in original jam:

0.5545 mg/ml 100 ml 250 ml

= 1 ml
×
3g
×
10 ml

= 462.08 mg/g

= 0.46 g glucose in 1 g jam

= 46 g glucose in 100 g jam

= 46 % glucose in jam

1st dilution factor = 100 ml / 3 g

2nd dilution factor = 250 ml / 10 ml

DISCUSSIONS (BWH NI INRO)

The principle of uv-vis spectroscopy is when a photon hits a molecule and is absorbed, the
molecule is promoted from its ground state into a higher energy state. The energy difference
between the two is the band gap. The energy of the photon must exactly match the band gap in
order for the photon to be absorbed. The chemical structure determines the band gap, therefore
molecules each have unique absorbance spectra. Absorbance follows Beer's Law, which states
absorbance equals the molar attenuation coefficient times the path length and concentration.
Beer's law can be used to calculate sample concentration, if the absorptivity is known, or a
calibration curve can be used. Most UV-Vis spectrophotometers use a deuterium lamp for the
UV range, which produces light from 170–375 nm, and a tungsten filament lamp for the visible
range, which produces light from 350–2500 nm. The light source used usually is lamp, so it has
broad wavelength ranges. It needs a monochromator in order to select the specific absorbance
wavelength.

In this experiment, samples need to be prepare first in order to do the measurement of the
absorbance. The technique for prepare the samples have to be correct for obtained the colour
changes for sample to be measure. If the color changes does not occur, it can effect the reading
of spectroscopy. So it will be an error for the measurement. Before the measurement of the
absorbance for the samples begin, allows the lamp to warm up by turn on the spectrophotometer
for a period of time to stabilize them. The cuvette need to be place in the correct position for
making the light can easily pass through the sample. The maximum wavelength have to be
determine by measure the absorbance spectrum of 3 mg/ml glucose standard solution. The
spectrum need to be measure within the wavelength of 400 nm to 600 nm. The maximum
wavelength was used to set the spectrophotometer using the highest absorbance obtained for
measure the samples. Based on this experiment, the highest absorbance reading obtained at
wavelength 508 nm. The absorbance for each standard solution and sample solution was
measured using the wavelength obtained. From the Table 1.0, it is shows the absorbance data for
DNS colorimetric method and by using the data standard curve graph can be plotted. Based on
the Figure 1.1, it is shows that the graph of absorbance versus concentration of the glucose for
non-linear curve meanwhile in Figure 1.2, it is shows that the graph of absorbance versus
concentration of the glucose for linear curve. The equation derived from the linear curve is y =
0.642x and the linear regression obtained from the graph is R2= 0.977. From the linear curve
obtained, the range for concentration of glucose is between 0 to 3 mg/ml.

CONCLUSION

As a conclusion, the objective was determined and the experiment was successfully conducted.
A linear and non-linear graph can be plotted using the data obtained from the experiment. The
concentration of glucose in the sample can be calculated using the equation derived from the
linear graph.
REFERENCES

1. Howell,J.A.(n.d).Ultraviolet and visible molecular Absorption Spectrometry.In

Handbook of Instrumental Techniques for Analytical Chemistry,Settle,FA.ed.,New
Jersey:Prentice Hall

2. Unknown.(n.d.).Visible and Ultraviolet Spectroscopy. Retrieved from

https://www2.chemistry.msu.edu/faculty/reusch/virttxtjml/spectrpy/uv-vis/spectrum.htm

3. Weerakkody, A. (2012, September 11). Ultraviolet-Visible (UV-Vis) Spectroscopy |

Analytical Chemistry. Retrieved from https://pharmaxchange.info/2011/12/ultraviolet-
visible-uv-vis-spectroscopy-principle/
Figure 9.1: The samples BEFORE colour change occurs

Figure 9.2: The samples AFTER colour change occurs