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Report Reducing Sugar

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INTRODUCTION

In this experiment, the reducing sugar compound in the carbohydrate of


the sample was be determined. A sugar is only a reducing sugar if it has an open
chain with an aldehyde or a ketone group. Monosaccharides which contain an
aldehyde group are known as aldoses, and those with a ketone group are known
as ketoses. Reducing monosaccharides include glucose, fructose, glyceraldehyde
and galactose. Many disaccharides like lactose and maltose also do have a
reducing form, as one of the two units may have an open-chain with an aldehyde
group. However, sucrose and trehalose, in which the anomeri carbons of the two
units are linked together, are non-reducing disaccharides.
Nowadays threre are several methods were established in order to
determine the reducing sugar such as Dinitrosalicylic Acid method, Benedict's
reagent, Fehlings solution and Nelson-Somogyi Method. The Dinitrosalicylic Acid
method was used because it is simple, sensitive and adoptable during handling
of a large number of samples at a time. Benedict's reagent and Fehling's solution
are used to test for the presence of a reducing sugar. The reducing sugar reduces
copper(II) ions in these test solutions to copper(I), which then forms a brick red
copper(I) oxide precipitate. While The Nelson-Somogyi method is one of the
classical and widely used methods for the quantitative determination of reducing
sugars. Different reducing sugars give blue colored products of varying
intensities when estimated this method, thereby implying that a mixture of
reducing sugars should not be estimated using this protocol. Proteins present in
the reaction medium should be eliminated by zinx hydroxide precipitation as
they can interfere with this assay.
In this experiment the Nelson-Somogyi method was applied. The principle
of this method is The reducing sugars when heated with alkaline copper tartrate
reduce the copper from the cupric to cuprous state and thus cuprous oxide is
formed. When cuprous oxide is treated with arsenomolybdic acid, the reduction
of molybdic acid to molybdenum blue takes place. The blue color developed is
compared with a set of standards in a colorimeter at 620nm.

APPARATUS
Material :

a) 200mL beaker
b) Pipette
c) Volumetric flask.
d) cuvvete
e) spectrophotometer.
Chemicals :
a) Low-alkalinity copper reagent sodium potassium tartrate (40g) and
anhydrous (28g) were dissolved in about 700 mL of water. A solution
of 10% cupric acid sulphate pentahydrate in water (80mL) was
added in small amounts with stirring. 180 g anhydrous was added in
small amounts and was dilute to 1 litre. After 1 day of standing, the
clear supernatant solution was used.
b) Arsenomolybdate reagent- 25g of ammonium molybdate in 450 mL
water was added 21mL of 95% sulphuric acid, followed by 3.0g of
disodium hydrogen arsenate heptahydrate dissolved in 25mL water.
The mixed solution was incubated 25 hours at 37OC and stored in a
glass- stoppered brown bottle.
c) Standard glucose solution stock solution was diluted containing
1mg/mL glucose in sturated benzoic acid solution to give a standard
solution of 100 mg/mL and 300 mg/mL
Sample : carbonated drinks.

PROCEDURES
A : sample preparation

1) The bottle containining the carbonated drinl=ks was shaken.


2) 100mL of the drink was poured into 250Mlbeaker.
3) The drink were transfered from ane beaker to another. This steps was
repeated until nogas remains in the dark.
4) The degassed sample was pipette out 5,0 mL into 250 volumetric flask
and the volume was diluted. This is calle dsolution is A.
5) 10mL of solution A was pipette out into a 100mL beaker and 50mL of
distilled water. The solution was neutralized. The solution was transfer
into 100ml volumetric flask and the volume was diluted. This is solution is
B.
B : determination of reducing sugar
1) A blank and standar solutios containing between 0 and 450 ug glucose
were prepared as similar to the manual.
2) A sample solution of containing not more than 2mL of solution B was
made. A series of dilution was made and added distilled water until 2mL.
3) Samples, blanks and standard sugar solution were added with 2mL copper
reagent and the content was mix properly.
4) Samples, blanks and standard sugar solution was heated 10min in
avigorously boiling water bath and then was cooled under running water
for 5 min
5) 1 mL of arsenomolybdate reagent was added to altest tubes and mix well.
6) When all the cuprous oxide was dissolved after mixing, the solution was
diluted to the 10 mL using volumertric flask and then allowed to stand and
at least 15 minute, but not more than 40min.
7) Read absorbance was read at 520nm.

RESULT

Glucose
solution
(100g/ml)
Glucose
solution
(300g/ml)
Distilled
water (ml)
Absorbance
reading

Tube 1
0.0

Tube 2
0.5

Tube 3
1.0

Tube 4
1.5

Tube 5
2.0

Tube 6
-

Tube 7
-

1.0

1.5

2.0

1.5

1.0

0.5

0.0

1.0

0.5

0.055

0.297

0.507

0.779

1.029

1.641

1.962

Graph of glucose standard curve

Glucose Standard Curve


2.5
2

f(x) = 0x + 0.04
R = 0.98

1.5
Absorbance 520nm

1
0.5
0
0

50 100 150 200 250 300 350 400 450 500


Amount og glucose g

Carbonated drinks results:


Sample

Amount (ml)

Absorbance

A1
A2
B1
B2

0.5
0.5
1.0
1.0

From standard curve,


y = 0.004x + 0.044
The amount of glucose in the sample,
Sample A1
X

= (0.244 0.044) / 0.004


= 50g

Sample A2
X

= (0.246 0.044) / 0.004


= 50.5g

Sample B1
X

= (0.428 0.044) / 0.004


= 96g

Sample B2
X

= (0.406 0.044) / 0.004


= 90.5g

Calculation of reducing sugar concentration

0.244
0.246
0.428
0.406

For 0.5ml sample


A1

= 50 x 10-6g x 50 x 10 x 4
= 0.100 g/ml

A2

= 50.5 x 10-6g x 50 x 10 x 4
= 0.101 g/ml

Average
= (0.100 g/ml + 0.101g/ml) / 2
= 0.1005g/ml

For 1.0ml sample,


B1

= 96 x 10-6g x 50 x 10 x 2
= 0.096 g/ml

B2

= 90.5 x 10-6g x 50 x 10 x 2
= 0.0905 g/ml

Average
= (0.096 g/ml + 0.0905 g/ml) / 2
= 0.09325 g/ml

Final concentration of carbonated drinks reducing sugar is ;= 0.1005 g/ml + 0.09325 g/ml) / 2
= 0.097 g/ml

DISCUSSION

For this experiment, the Somogyi Nelson method have been used in
determining the concentration of reducing sugar in the sample which is
carbonated drink. This method is commonly used in determining the reducing
sugar rather than the DNS method because of its effectiveness for all types of
sample. That is because, it does not matter about the color of the sample, it can
be easily applied in determination of reducing sugar.
For this experiment, the sample that we used is Coke drink which is a kind
of carbonated drink. There several steps in doing this method and it started with
removing the gas of that carbonated drink. Then, the sample solution is diluted
with the distilled water for neutralization. After the several dilution of the sample,
the sample is added with copper reagent and arsenomolybdate reagent. After
mixing and incubated at room temperature for several minutes, the absorbance
of the sample can be read by spectrophotometer at 520nm wavelength.
In principle, during the first step, glucose (or a reducing sugar) is oxidised
using a solution of Cu(II) ion which in the process is reduced to Cu(I). In the
second step the Cu(I) ions are then oxidised back to Cu(II) using a colourless
hetero-polymolybdate complex, which is, in the process, reduced to give the
characteristic blue colour. Finally the absorption of the hetero-poly molybdenum
blue is measured using a colorimeter and compared to standards prepared from
reacting sugar solutions of known concentration, to determine the amount of
reducing-sugar present.
When the reading of absorbance is got, the concentrations of reducing
sugar can be determined by refer to the reducing sugar standard curve that has
been produced before. In order to achieve the exact concentration of the
reducing sugar, we need to multiply the result with the dilution factor. And after
all, the concentration of reducing sugar of coke drink that we got is only 0.097
g/ml. From the result we got, the concentration of reducing sugar that we
determined is not really high.

CONCLUSION
The concentration of reducing sugar of the carbonated drink is not really
high which is only about 0.097 g/ml. However, if compared to the other type of
drinks, the amount of reducing sugar of carbonated drink is much higher.
REFFERENCES
1) http://en.wikipedia.org/wiki/Reducing_sugar
2) http://www.eplantscience.com/index_files/plant
%20protocols/Carbohydrates/reducing_sugars_by_NelsonSomogyi_method.php

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