1.

TESTING SUGAR AND DETERMINATION OF STARCH IN PLANT

TISSUE

1.1.

OBJECTIVE

To determine qualitatively presence or absence of sugar in given

sample.
To identify the type of sugar present in unknown sample.
To determine quantitatively of starch content in plant tissue.

1.2.

INTRODUCTION

Carbohydrates are important constituents of all living cells. These are

defined as aldehydes or ketones of polyhydric alcohols and also include
those biopolymers which yield these compounds on hydrolysis. Sugar
such as glucose are amongst the major sources of energy whereas
starch and glycogen function as storage polysaccharides in plants and
animals, respectively.
Iodine forms coloured adsorption complexes with polysaccharides.
Starch gives blue color with iodine, while glycogen react to form
reddish brown complex. Hence it is useful, convenient and rapid test
for detection of amylose, amylopectin and glycogen.
Picric acid is test for detecting of reducing sugar. The reducing sugar
react with picric acid to form a red coloured picrimic acid. Benedicts
test is more convenient and this reagent is more stable.
Non reducing sugar that cannot donate electron to other molecule
and therefore cannot acts as a reducing agent. For example sucrose.
Reducing sugar is any sugar that is capable of acting reducing agent
because it has a free aldehyde group or free ketone group.

Starch is a carbohydrates consisting of a large number of glucose

unit joined by glycosidic bond.

1.3.

MATERIALS AND EQUIPMENT

1.4.

Iodine

1.5.

Picric acid

1.6.

Benedicts reagent

1.7.

Samples (1% test solutiion of glucose, sucrose, starch,

fructose, lactose, maltose and galactose)

1.8.

Boiling water bath

1.9.

Tabletop centrifuge

1.10.

Refrigerated centrifuge

1.11.

Spectrophotometer

1.12.

Anthrone - sulphuric acid reagent

1.13.

80% (v/v) ethyl alcohol

1.14.

52% perchloric acid

1.15.

1 mg/mL glucose standard

1.16.

1.17.
PROCEDURE
1.18.
1.18.1.
TESTING SUGAR
1.19. By using nine samples including three unknown samples will
be use to run the experiment test.
1.19.1.1.Iodine test
1 mL of test solution have put in test tube.
4 5 drops of iodine solution has been added and mixed
gently the content in test tube.
The colour of the product have observed and have noted
1.20.
1.20.1.1.Picric acid test

1 mL of saturated picric acid has been added to 1 mL of

sample solution followed by 0.5 mL 10% Na2CO3.

The test tube heated in a boiling water bath.

1.21.
1.21.1.1.Benedicts test

0.5 1 mL of the test solution has been added to 2mL

of Benedicts reagent.
The test tube was placed in vigorously water bath.
The formation of red precipitates otherwise no change
observed.
1.22.

1.22.1.

STARCH DETERMINATION
1 g of finely corn flour was taken in a 50 mL centrifuge
tube and has been added 10 mL of 80% alcohol. The
tubes was vortex for 2 min and centrifuged at 3000 rpm
for 10 min to decant the supernatant. This extraction

repeated for once.

The residue was cooled in ice water and was added 6.5
mL of 52% perchloric acid while stirring and then

centrifuged at 4C.
The supernatant was collected and make up the volume

to 100 mL with water.

5 mL aliquot of diluted extract was taken and placed in
cold water with the tubes for 5 min. 10 mL anthrone
reagent has been added, mixed properly and have
transferred the tubes to boiling water bath for 7.5 min.
After cooling the tubes was running under tap water and

the absorbance of these solution at 630nm have noted.

By using 0 to 100 g glucose, a standard curve have
prepared and the amount of glucose have calculated.

1.23.
RESULTS
1.23.1.
1.23.2.
TESTING SUGAR
1.23.3.

1.23.4.

SU

GAR
1.23.10.

Gl

ucose solution
1.23.14.

Su

1.23.5.
OBSERVATION
1.23.8.
PI 1.23.9.
BE
1.23.7.
IO
CRIC ACID
NEDICTS
DINE TEST
TEST
TEST
1.23.11.
1.23.12.
+
1.23.13.
+

+++++
+++++
+
1.23.15.

1.23.16.
+
1.23.17.
+

crose solution
1.23.18.
St

1.23.19.
+

+++++
1.23.20.

+++++
1.23.21.

arch solution
1.23.22.
Fr

+++++

uctose
solution
1.23.26.

1.23.23.

La

1.23.27.

1.23.24.

+++
1.23.28.

1.23.25.

+++++
1.23.29.

ctose solution
1.23.30.
M

1.23.31.

+++++
1.23.32.
+

+++++
1.23.33.
+

altose solution
1.23.34.
Un

+
1.23.35.

+++
1.23.36.

+++++
1.23.37.
+

known 1
1.23.38.

Un

1.23.39.
+

+++++
1.23.40.

+++++
1.23.41.

Un

++++
1.23.43.

+
1.23.44.
+

+
1.23.45.
+

known 2
1.23.42.
known 3
1.23.46.

+++++
+++++
Table 1 : Present and absence of sugar in various
sample

1.23.47.
1.23.48.

Based on the result obtained, Unknown 1 is

assumed as fructose solution, Unknown 2 is lactose solution and

Unknown 3 is maltose solution.

1.23.49.
STARCH DETERMINATION
1.23.50.
1.23.51.

Glucose
1.23.53.
1.23.55.
1.23.57.
1.23.59.
1.23.61.
1.23.63.

concentration
0
0.2
0.4
0.6
0.8
1.0

1.23.52.

Absorbance at 630nm, A630 nm

1.23.54.
0
1.23.56.
1.508
1.23.58.
2.899
1.23.60.
3.687
1.23.62.
3.825
1.23.64.
4.000

1.23.65.

Standard Curve of Glucose

4.5
4

f(x) = 4.88x
R = 0.95

3.5
3
2.5
Absorbance ar A630nm

Absorbance at 630nm

Linear (Absorbance at 630

2
1.5
1
0.5
0
0 0.2 0.4 0.6 0.8 1 1.2

Glucose concentration (mg/mL)

Table 2 : Glucose concentration and absorbance at 630nm of

glucose
1.24. Group
1.26. 1
1.28. 2
1.30. 3

1.25. Absorbance at 630nm,

1.27. 0.035 (not valid in calculation)

1.29. 0.394
1.31. 0.394

1.32. 4
1.34. 5
1.36.

1.33. 0.047
1.35. 0.018 (not valid in calculation)
y

1.37. Substitute the value of absorbance at 630nm, by using the

x
equation of glucose standard curve to get the value of :
1.1.

Glucose concentration
(mg/mL)

1.2.

Amount of starch
(g/mL)

1.3.

8.0758

1.4.

1.5.
1.9.

8.0758
Mean

1.6.

1.7.

0.9634

1.10.
1.11.

0.00727

0.00727

1.8. n 0.000867
=
= 0.005136

(x )

1.12.

Standard Deviation

1.13.

s=

1.14.

(0.015407 0.005136)

3
1.15.

= 0.00593

y
4.8788 y 4.8788 x

x
1.38.
1.39.
1.40.

( x) 100mL

mg / mL
1.41.

Glucose concentration(

)=

g / mL
1.43.
1.44.

1.42.

Amount of starch(

) = Glucose concentration 0.9

1.16.
1.18.

1.22.

GROUP
1

1.34.

1.46.

1.17.
CALCULATION
1.19.
Absorbance at 630nm,
A (g) = 0.035
1.20.
(not
valid
in
calculation)
1.21.
1.23.
Absorbance at 630nm,
y = 0.394
1.24.
1.25.
X = 0.080758
1.26.
1.27.
Glucose concentration
= 0.08076 x 100 mL
1.28.
= 80.76 mg/mL
1.29.
1.30.
Amount of starch =
80.76 x 0.9
1.31.
=
72.684 mg/mL
1.32.
=
72.684 x 10-3 g/mL
1.33.
=
0.072684 g/mL
1.35.
Absorbance at 630nm,
y = 0.394
1.36.
1.37.
X = 0.080758
1.38.
1.39.
Glucose concentration
= 0.08076 x 100 mL
1.40.
= 80.76 mg/mL
1.41.
1.42.
Amount of starch =
80.76 x 0.9
1.43.
=
72.684 mg/mL
1.44.
=
72.684 x 10-3 g/mL
1.45.
=
0.072684 g/mL
1.47.
Absorbance at 630nm,
y = 0.047
1.48.
1.49.
X = 0.00963
1.50.
1.51.
Glucose concentration
= 0.00963 x 100 mL
1.52.
= 0.963 mg/mL
1.53.
1.54.
Amount of starch =
0.963 x 0.9
1.55.
= 0.8667
mg/mL
1.56.
= 0.8667 x
10-3 g/mL
1.57.
= 0.000867
g/mL

1.45.
A
P
P
E
N
D
I
X
1.46.

1.47.

DISCUSSION

1.48.

Experimental of testing sugar had practically use three

method to test nine sample include three unknown sample of presence

or absence of sugar. The three method are, iodine test, picric acid test
and benedicts test. Those sample give reaction by changing the
colour of solution after the reagent and sample mix well.
1.49.

Some example of sample that show the presence of

sugar is starch solution when added a drop of iodine have change

colour from yellowish to blue. Lactose solution, after added picric acid
and sodium carbonate was heated in water bath until the colour
change from light yellow to light orange. Benedicts reagent change
sample solution from blue colour to red precipitates show presence of
reducing sugar in sample. Such as sucrose.
1.50.

Three unknown sugar have been tested. Unknown 1

show positive reaction when tested with benedicts reagent, so

unknown 1 is fructose solution. Unknown 2 show positive reaction
when tested with iodine shows that, it was lactose solution. Unknown 3
give positve feedback on picric acid test shows it was maltose solution.
1.51.

To determine the content of starch in plant tissue, many

step have been applied to get a solution for absorbance at 630nm.

Among them, separate solvent and supernatant to get sample aliquot
and after get the value of absorbance, we can calculate for amount of
glucose and starch. The amount of starch in 1 g of powdered sample is
0.00593.
1.52.
1.53.
CONCLUSION
1.54.
1.55. All the experiment was successful. The sample solution give
positive reaction to either one of the out of three test which showing
them reducing agent or non reducing agent. The three unknown

sample successfully identified. The amount of starch in plant tissue

also obtained.

1.56.
REFERENCES
1.57.
1.58.
McKee, T and Mckee, J.M. (2003). Carbohydrates in Biochemistry The
molecular basis of life, Third Edition. McGraw-Hill. New York. pp. 201-231.
1.59.
1.60.
Jane B. Reece, Lisa A. Urry, Michael L.Cain, Steven A. Wasserman, Peter V.
Minorsky and Robert B. Jackson. (2011). Campbell Biology Ninth Edition. Pearson.
United States of America. pg 115-122.
1.61.
1.62.
Sawhney, S.K. and Randhir, S. (2005). Carbohydrates in Introdcutory practical
Biochemistry. Alpha Science International Ltd.,Harrow. U.K. pp.15-32.
1.63.