Experiment 7: Quantitative determination of sugar in honey and milk

Part B: Determination of reducing sugar in honey by the Arsenomolybdic

AcidReagent (Nelson method)

Results:

Figure 1: The color of test tubes starting from left to right with order of blank
sample, glucose standard tubes 1, 2, 3, 4, 5, 6, and honey sample.

Test tube Concentration of Absorbance(600nm)

reducing sugar (mg/mL)
Blank 0 0
1 0.00625 0.012
2 0.0125 0.033
3 0.01875 0.102
4 0.0250 0.228
5 0.0375 0.232
6 0.0500 0.533
Honey 0.0603 0.587
Table 1.0: Result from our group
Test tube Concentration of Absorbance(600nm)
reducing sugar (mg/mL)
Blank 0
0
1 0.00625
0.007
2 0.0125
0.029
3 0.01875
0.125
4 0.0250
0.213
5 0.0375
0.255
6 0.0500
0.389
Honey 0.0735
0.595
Table 1.2: Results from other group

Calculation:

Concentration of reducing sugar

M 1 V 1=M 2 V 2

Test tube 1

M2
(50 g/mL)(0.25ml) = ( )(2ml)

M2 =6.25 g/mL

=0.00625 mg/mL
Test tube 2

M2
(0.05mg/mL)(0.5ml) = ( )(2ml)

M2 =0.0125 mg/mL

Test tube 3

M2
(0.05mg/mL)(0.75ml) = ( )(2ml)

M2 =0.01875 mg/mL

Test tube 4

M2
(0.05mg/mL)(1.0ml) = ( )(2ml)

M2 =0.025 mg/mL

Test tube 5

M2
(0.05mg/mL)(1.5ml) = ( )(2ml)

M2 =0.0375 mg/mL

Test tube 6

M2
(0.05mg/mL)(2.0ml) = ( )(2ml)
 M2 =0.05mg/mL

Standardization of glucose
0.6

0.5
f(x) = 11.26x - 0.09
0.4
R = 0.91
0.3
Absorbance
0.2

0.1

0
0 0.01 0.02 0.03 0.04 0.05 0.06

Concentration (mg/mL)

Figure 2.0 The standard curve of absorbance against concentration of glucose in solution

Chart equation:

From the standard curve we obtained,

y = 11.256x - 0.0914

0.587 = 11.256x-0.0914

x = 6.03 x 10-2 mg/mL

Concentration of reducing sugars in 1mL of honey sample is 6.03 x 10-2 mg/mL

Mass of reducing sugars = 6.03 x 10-2 mg /mL x 1mL

=0.0603mg
 40 mg
The mass of reducing sugars of honey in isotonic = 2mL x 1000 mL

= 0.08mg honey

In 0.08 mg honey conduct 0.603mg of reducing sugar.

Concentration of reducing sugars in honey (g):

0.0603mg
Mass of reducing sugars/100g of honey = 0.08 mg 100 g = 75.38g

Concentration = 75.38 / 100 g of honey

( 6.03 x 102 ) + ( 7.35 x 102 ) +(6.47 x 102 ) 2

Mean = 3 = 6.617 x 10

Standard deviation, s=

s=

(6.03 x 1026.617 x 102 )2+(7.35 x 1026.617 x 102)2 +(6.47 x 1026.617 x 102)2
31

s= 9.0347 x 105
2 s = 6.721 x 10-3

Answer: 6.617 x 10
2
6.721 x 10-3

Discussion
In this experiment, amount of reducing sugars in the honey was determined by using
Nelson method. That is the concentration of reducing sugar will be determined based on
their absorbance measured in honey. The amount of glucose in honey is measured using
glucose oxidase method when the glucose will react and turbidity increases in the
solution. The amount of reducing sugar is determined by Nelson method when blue color
appears as the reducing sugar react with acid reagent. The result will be further used with
other groups' results to calculate mean and standard deviation to get more accurate
results.

In fact, the Nelson method determines the concentration of reducing sugar using
arsenomolybdic acid reagent (Nielsen 2003). When reducing sugar is heated with isotonic
sodium sulphate copper sulphate solution, cupric ion in the solution is reduced to cuprous
and thus forming cuprous oxide. When cuprous oxide is treated with the arsenomolybdic
acid reagent, arsenomolybdic acid is reduced to molybdenum blue. The blue color
developed is compared by measuring the absorbance.

RCHO +2Cu(OH)2 + NaOH >RCOO-Na+ + Cu2O + 3H2O (Nielsen 2003)

Based on the result from Nelson method, the color of the test tubes changed after adding
with arsenomolybdic acid. The blue color was becoming darker from blank sample,
glucose standard tubes 1, 2, 3, 4, 5, 6 to honey sample. Hence, the absorbance measured
was increasing. As the concentration of reducing sugar increased in test tubes from 1 to 6,
the absorbance measured became higher.

By using Nelson method, the absorbance of honey sample is 0.587. Standard curve is
prepared from six standard tubes. And from the standard curve in the graph (Figure 1.1),
absorbance of honey at 600nm which is 0.587 was plotted, cross link the standard curve
to the point at which concentration is determined as 6.03 x 10-2 mg/mL.

The absorbance of honey sample measured was high that the concentration of reducing
sugar in it was calculated as 75.38/100g honey. According to Buba, Gidao and Shugaba,
the reducing sugar contents in honey varied between 65.53 and 91.05 g/100 g with an
average of 72.40 6.65 g/100 g (Buba et al. 2013). This shows that resulting
concentration of reducing sugar was reliable. Besides, the mean concentration of
 2
reducing sugar in honey sample was 6.617 x 10 with standard deviation of 6.721 x

10-3. As the standard deviation value was more than 10% of the mean, the result had
dispersion, thus it is not reliable.

Nevertheless, process of nelson method is more complex compare than other method in
determination of reducing sugar such as DNS method (Haigler & Weimer 1991). For
example, the sample need to be deproteinized before heated with alkaline cupric ion
(Haigler & Weimer 1991). Yet, the alkaline cupric working solution which is the isotonic
solution is unstable (Haigler & Weimer 1991).The other disadvantage of carry out Nelson
method is toxic problem of arsenic of arsenomolybdic acid reagent. The particular
chemical groups of arsenic are toxic to environment (Hatanaka, Kobara 1980).

There are some precaution steps when doing Nelson method. Test tube tong is used to
carry the test tube into and out of the boiling water when handling test tubes in boiling
water bath. Pipette used to measure arsenomolybdic acid reagent and dropper should be
rinsed with distilled water after each use to prevent cross contamination of different
solutions. Moreover, after each using cuvette, it should be rinsed with distilled water to
prevent contamination.

Conclusion

Honey consists of glucose and fructose which are both reducing sugars. The mean of the
2
concentration of reducing sugar in honey is 6.617 x 10 with a standard deviation of

4.52 x10-5.

Reference

Buba, F., Gidado, A., Shugaba, A., 2013. Analysis of Biochemical Composition of Honey
Samples from North-East Nigeria. Biochem Anal Biochem 2: 139. doi:
10.4172/2161-1009.1000139
Haigler, C. H., Weimer, P. J., 1991. Biosynthesis and Biodegradation of Cellulose. Marcel
Dekker. Inc.

Hatanaka, C., Kobara, Y., 1980. Determination of Glucose by a Modification of

Somogyi-Nelson Method . Agriculture and Biological Chemicstry, 44 (12), 2943-
2949.

Nielsen, S. S., 2003. Food Analysis, 3rd ed. New York: Plenum Publisher