ISOLATION, HYDROLYSIS AND CHARATERIZATION OF STARCH FROM POTATO

Mark Kenneth Ong, Leah Kristine Reyes, Renz Mervin Rivera, Nadine Sabado, Joannah Marie Sahagon
Group 7, 2B Medical Technology

Abstract
Carbohydrates are the most abundant organic compounds in the plant world. They act as storehouses
of chemical energy (glucose, starch, glycogen); are components of supportive structures in plants
(cellulose),crustacean shells (chitin), and connective tissues in animals (glucosaminoglycans); and are
essential components of nucleic acids (D-ribose and 2-deoxy-D-ribose). Starch is a carbohydrate
consisting of a large number of glucose units joined by glycosidic bonds. Starch was extracted from the
potato using the Molischs Test and I
2
Reaction. Acid hydrolysis and enzymatic hydrolysis was also
performed on the sample. The acquired hydrolysate was then used for characterization and
chromatography. Different qualitative tests were executed to characterize the sample. The test were as
follows; Benedicts Test, Barfoeds Test Seliwanoffs Test Bials-Orcinol Test, Mucic Acid Test and
Phenylhydrazone Test. It was established that glucose, fructose and galactose were the monosaccharaides
present within the sample. The reducing sugars were also gglucos fructose and galactose. The
ketohexoses discovered were fructose and sucrose. There was only one pentose revealed, which was
xylose. Thin-layer chromatography was performed to verify the components of the hydrolysate. Nelsons
Method was accomplished in order to determine the quantity of the reducing sugars.
INTRODUCTION
Carbohydrates are a major source of metabolic
energy, both for plants and for animals that
depend on plants for food. Aside from the sugars
and starches that meet this vital nutritional role,
carbohydrates also serve as a structural material
(cellulose), a component of the energy transport
compound ATP, recognition sites on cell surfaces,
and one of three essential components of DNA
and RNA. Formulas of many carbohydrates can
be written as carbon hydrates, C
n
(H
2
O)
n
, hence
their name.[1]
Starch is manufactured in the green leaves of
plants from excess glucose produced during
photosynthesis and serves the plant as a reserve
food supply. Starch is a soft, white, tasteless
powder that is insoluble in cold water, alcohol, or
other solvents. The basic chemical formula of the
starch molecule is (C
6
H
10
O
5
)
n
. Starch is
a polysaccharide comprising glucose monomers
joined in 1,4 linkages. The simplest form of
starch is the linear polymer amylose;
amylopectin is the branched form.[2]

Fig 1 (Wheat starch
granules stained with
iodine)

Starch can be separated into two fractions--
amylose and amylopectin. Natural starches are
mixtures of amylose (10-20%) and amylopectin
(80-90%).
Amylose forms a colloidal dispersion in hot water
whereas amylopectin is completely insoluble. The
structure of amylose consists of long polymer
chains of glucose units connected by an alpha
acetal linkage. All of the monomer units are alpha
-D-glucose, and all the alpha acetal links connect
C#1 of one glucose to C#4 of the next
glucose.[3]

Fig 2 (Structure of Amylose)
The acetal linkages of Amylopectin are alpha
connecting C#1 of one glucose to C#4 of the
next glucose.
The branches are formed by linking C#1 to a C#6
through acetal linkages. Amylopectin has 12-20
glucose units between the branches. Natural
starches are mixtures of amylose and
amylopectin.[3]

Fig 3 (Structure of Amylopectin)
This experiment aimed to:
Perform the general tests for
carbohydrates and explain the principle
involved.
Compare the products of acid and
enzymatic hydrolyses of the isolated
carbohydrate.
Illustrate the specificity of alpha-amylase
on the hydrolysis of the isolated
carbohydrate.

EXPERIMENTAL
A. Isolation, General Test and Hydrolysis
1. Extraction of Starch from Potato
a) The sample was grinded.
b) The sample was transferred into a small
beaker.
c) 100 mL of water was added.
d) Cheesecloth was used to strain.
e) The starch was allowed to settle.
f) General tests for polysaccharides were
performed.

2. General Tests for polysaccharides
a) Molischs Test
i) Few drops of Molischs reagent were
added into 1 mL glycogen solution.
ii) 2 mL conc. H
2
SO
4
was carefully poured
down the side of the tube to form a
layer.
iii) The junction of the two liquids was
observed
b) I
2
Reaction
i) Few drops of 0.01 M I
2
into 1 mL
sample solution were added.
ii) The mixture was warmed in a water
bath.

3. Enzymatic Hydrolysis
a) 10 mL of the isolated carbohydrate was
placed in a beaker.
b) Saliva was added.
c) It was allowed to stand at room
temperature for 30 minutes.
d) The solution was then introduced into a
collodion bag and was suspended
overnight in a small flask with 50mL
distilled water.
e) The dialyzing bag was removed.
f) The solution inside the flask was
concentrated to the volume of 10 mL
using an open flame.
g) Benedicts test was performed to test for
the presence of reducing sugar in the
hydrolysate.

B. Qualitative Tests for Carbohydrates
1. Benedicts, Barfoeds, Seliwanoffs and Bials
Orcinol Tests.
a) 5 drops of the carbohydrate solution and 1
mL of the required reagent were mexed in
separate test tubes.
b) Tests on the different carbohydrate
solutions were performed at the same
time.
c) All the test tubes were placed into a
boiling water bath at the same time.
d) All test tubes from the water bath were
removed after one test gave visible result.
e) Results were noted.
f) The same tests for the unknown were
repeated.

2. Mucic Acid Test
a) 3 drops of the carbohydrate solution and 3
drops conc. HNO
3
were mixed on a glass
slide.
b) The mixture was passed over a small
flame until almost dry.
c) It was cooled at room temperature.
d) The crystals were examined under the
microscope.

3. Phenylhydrazone Test
a) 2 g of phenylhydrazine hydrochloride, 3 g
Ch
3
CHOONa and 10 mL distilled water
were mixed to prepare the
phenylhydrazine reagent.
b) The reagent was placed in a warm water
bath.
c) 2 drops of carbohydrate solution was
mixed with 4 drops of freshly prepared
phenylhydrazine reagent in different test
tubes.
d) The tubes were covered with cotton.
e) The mixture was heated in a boiling water
bath for 30 minutes.
f) The tubes were cooled and the crystals
were observed under the microscope.

C. Thin-Layer Chromatography
a) 40 mL of the solvent system was placed in
the developing chamber.
b) It was covered with an inverted watch
glass.
c) A pencil line across one end of the TLC
plate were drawn (2cm from the bottom)
d) Equidistant points along the origin of the
TLC standards, and the acid and
enzymatic hydrolysates were marked.
e) The standards were applied five times and
the hydrolysates were applied ten times
using capillary tubes.
f) The TLC plate was placed inside the
developing chamber. It was covered and
developed until the solvent is about 1 cm
from the top of the TLC plate.
g) After the development, the chromatoplate
was removed and the solvent front was
marked with a pencil.
h) The chromatoplate was air-dried and
sprayed with p-anisaldehyde visualizing
agent.
i) The chromatoplate was heated for 10
minutes. The sugars were evident because
of the appearance of colored spots.
j) The spots were encircled by a pencil.
k) The Rf values of each spot were
calculated.
l) The components of acid and enzyme
hydrolysates were identified.

D. Quantitative Analysis
a) 12.5 mL Nelsons A was mixed with 0.5
mL Nelsons B to prepare the Nelsons
reagent.
b) The test tubes were labeled and amounts
of standard glucose solution was
transferred according to the protocol
provided.
c) 1.0 mL Nelsons reagent was added into
each test tube.
d) All the test tubes were placed
simultaneously in a boiling water bath for
20 minutes.
e) , 1.0mL arsenomolybdate reagent was
added to each tube and was shook
occasionally until the Cu
2
O precipitate
dissolves.
f) The absorbance of the standards were
read at 450nm.


RESULTS AND DISCUSSIONS

A. Isolation, General Tests for Starch
The isolated starch appeared to be creamy
and white. A purple interface was observed after
performing the Molischs Test which indicates a
positive result. A Bluish Black solution was
perceived after executing I
2
reaction. If the
sample was to be observed under the
microscope, it would appear as eccentric.[4]
These results also indicated a positive result for
the I
2
Reaction.
The galactose appeared to be a clear solution.
It gave a positive result with Molischs Test but
did not respond with the I
2
reaction.



B. Enzymatic Hydrolysis of Starch
The hydrolysate seemed to be very gelatinous
after the hydrolysis. Benedicts Test was
performed in order to determine the presence of
reducing sugars. It was concluded that a positive
result was acquired. The starch reduced the
cupric ions of the Benedicts reagent, and formed
cuprous oxide, Cu2O, which formed the yellow-
red precipitate indicating a positive result.[5]

C. Color Reactions

Carbohy
-drate
Solution
Visible Results
Benedict
s
Barfoed
s
Seliwanoff
s
Bial
s
Glucose
Brick Red
ppt (+)
Brick Red
ppt (+)
Orange (-) Yellow
(-)
Fructose
Brick Red
ppt (+)
Brick Red
ppt (+)
Cherry red
(+)
4 min
Yellow
(-)
Xylose
(-) Brick Red
ppt (+)
Orange (-) Blue
green
soln
(+)
Lactose
(-) (-) Orange (-) Yellow
(-)
Sucrose
(-) (-) Cherry red
(+)
5 min
Yellow
(-)
Starch
(-) (-) Orange (-) Yellow
(-)
Galactose
Brick red
ppt. (+) 40
secs
Brick red
ppt (+)

Table 1 (Results of Color Reaction)

The Benedicts test is a Qualitative or
quantitative test for reducing sugars. Benedicts
solution reacts with reducing sugars on heating
and reduces the Cu(II) ion to Cu(I) producing a
precipitate of red copper oxide. The resulting
colour change depends on the type and
concentration of sugar, so this test can be used
semi-quantitatively to indicate approximate
concentrations.[6] Glucose, Fructose and
Galactose responded to the test and each of
them formed brick red ppt. With that fact, the
group clearly stated that glucose, fructose and
galactose were indeed positive to Benedicts Test.

Barfoeds Test is a test for
monosaccharides conducted in an acid solution.
Cupric acetate is reduced to cuprous oxide, a red
precipitate. Reducing monosaccharides are
oxidized by the copper ion in solution to form a
carboxylic acid and a reddish precipitate of
copper (I) oxide within three minutes. Reducing
disaccharides undergo the same reaction, but do
so at a slower rate.[7] A positive result is
indicated by the formation of a reddish
precipitate which was formed within 3 minutes.
The fact that glucose, fructose, xylose and
galactose are monosaccharides, gave the group a
clear and positive conclusion.






Fig 4 ( Positive and negative result for Barfoeds
Test)
On the other hand, Seliwanoffs Test
simply shows positive results with ketoses. The
test reagent dehydrates ketohexoses to form 5-
hydroxymethylfurfural. 5-hydroxymethylfurfural
further reacts with resorcinol present in the test
reagent to produce a red product within two
minutes. Aldohexoses react to form the same
product, but do so more slowly.[8] A positive
result is indicated by the formation of a red
product. Fructose and Sucrose gave a positive
result.

Fig 5 (Positive and negative result of Seliwanoffs
Test)
Lastly, the Bials Test displays positive
results for pentoses. Xylose displayed a positive
result which would mean that it is a pentose. The
test reagent dehydrates pentoses to form
furfural. Furfural further reacts with orcinol and
the iron ion present in the test reagent to
produce a bluish product.[9]
D. Thin-layer Chromatography

Fig 6 (Thin-Layer Chromatography)




The figure (Figure 6) shows the result of
the thin layer chromatography performed. Visible
spots appeared on the plate as seen on the
figure. Distances travelled by the samples were
also illustrated on the sample.

The table (Table 2) points out that the
enzymatic hydrolysate travelled farther than that
of the acid hydrolysate.

Tryptophan was the least polar because it
has the greatest affinity on the mobile phase.
Glycine was the most polar since it has the
greatest affinity on the stationary phase. It also
states that it has the lowest Rf value. the Rf
value of the unknown component with that of the
standard amino acids determine the amino acid
present. The enzyme hydrolysates Rf value is
proximate to that of Prolines and Tyrosines.
Thus verifies the result for the Paulys test and
Xanthoproteic test.

E. Quantitative Analysis
Tube
No.
1 2 3 4 5 6 7 8
Glucos
e Std
(mg/tu
be)
0 0.08 0.02 0.04 0.06 0.08 0.10
Glucos
e std.
(mg/m
L)
0 0.008 0.002 0.00
4
0.00
6
0.00
8
0.01
0

Absorb
ance
0 0.262 0.254 0.32
9
0.24
3
0.39
5
0.55
7
0.04
2
Table 3 (Quantitative analysis)

The Nelsons Method was used in the
quantitative analysis. Nelson's test for reducing
sugar is a pretty old test and is quite generic in
its scope. It basically uses the reduction of some
dye compound and then relies on
spectrophotometry to determine the level of
chemical dye remaining at a specific wavelength.
It is important to have a good control sample to
compare with when you do this test.

According to the table (Table 3), Test tube
number 7 amassed the highest reducing sugar.
Test tube no. 7 had the most amount of the
glucose standard and the least amount of the
distilled water. With these results, the amount of
free sugars in the sample is proportional to the
molybdenum formed by the series of
oxidation/reduction reactions.



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