Pharmaceutical Biochemistry Laboratory – PH BIOCHEM (2012 – 2013) 2B-Ph Group 3 Experiment 3

Isolation, Hydrolysis and Color Reactions of Starch from Potato

Colleen Ann Caragay, Rizzalaine Caringal, Bett Shannen Carpio*,

Ancell Julienn Cruz, John Matthew Cruz
Department of Pharmacy, Faculty of Pharmacy
University of Santo Tomas, Espana Street, Manila 1008

Date Submitted: February 13, 2013

Abstract

In this experiment, potato was used as a sample to extract starch. Starch

solution was successfully extracted from the potato. Several tests were done to help
prove the extraction of starch solution from the potato. Molisch's test, iodine reaction
and Benedict's test gave a positive result that indicates the presence of starch. On the
other hand, general tests for carbohydrates including Benedict's test, Barfoed's,
Seliwanoff's and Bial's - Orcinol tests confirmed that glucose and galactose is an
aldohexose, xylose is an aldepentose, fructose is a ketohexose and lactose and sucrose
are disaccharides.
______________________________________________________________________

INTRODUCTION

Carbohydrates are organic Starch, which was isolated in this

molecules with the general formula of experiment, is a polymer of glucose,
CHO in 1:2:1 ratio. They provide the raw found in roots, rhizomes, seeds, stems,
fuel for cellular energy production. tubers and corms of plants, as
Carbohydrates are classified according microscopic granules having
to the number of molecules they characteristic shapes and sizes. Most
contain. animals, including humans, depend on
these plant starches for nourishment.
Monosaccharides are simple The structure of starch is more complex
sugars which cannot be hydrolyzed. On than that of cellulose.
the other hand, oligosaccharides contain
2-10 monosaccharide units, while This experiment aims to isolate
polysaccharides contain more than 10. polysaccharides from the plant source,
perform general tests for carbohydrates
Monosaccharides are further and explain the principles involved in the
classified into the number of carbons processes.
that they contain: pentoses (5 carbons)
and hexoses (6 carbons). Examples of
monosaccharides are glucose, fructose, METHODOLOGY
galactose, deoxyribose and ribose.
A. Extraction of Starch from Potato
Oligosaccharides are subdivided The potato was comminuted and
into disaccharides (2 –monosaccharide grinded using a mortar and pestle.
subunits), trisaccharides (3 – The mixture was transferred in a
monosaccharide subunits), and so on. small beaker, added with 100 mL of
Examples of disaccharides are sucrose water, and mixed. It was then
maltose and lactose. strained using a cheesecloth. The
starch was allowed to settle. After the
Polysaccharides can be extraction, the general tests and
composed of hundreds of thousands of hydrolysis of polysaccharides were
monosaccharide units connected in performed on the isolate.
various patterns. Common examples of
polysaccharides are starch, cellulose B. General Tests for Polysaccharides
amd glycogen. 1. Molisch’s Test
 Molisch’s reagent is composed of small flame until almost dry.
5% α-naphthol in 95% ethanol. A (Use an alcohol lamp and do
few drops of it was added into a 1 not scorch.) It was cooled at
mL starch solution. Down the room temperature. The crystals
side of the tube, 2 mL of were examined under the
concentrated H2SO4 was microscope. If no crystals
carefully poured to form a layer. appeared, let the glass slide
The color at the junction of the stand until the next period.
two liquids was then observed. 3. Phenylhydrazone Test
2. Iodine Reaction The phenylhydrazine reagent
A few drops of 0.01M iodine were was prepared by mixing 2 grams
added into a 1 mL starch solution. of phenylhydrazine hydrochloride,
Then the mixture was warmed in 3 grams sodium acetate and 10
a water bath. The change of color mL distilled water. The reagent
was observed. It was cooled and was placed in a warm water bath
the result was noted. and stirred until it was clear. In
different test tubes, 2 drops of the
C. Hydrolysis of Polysaccharides carbohydrate samples were
1. Acid Hydrolysis mixed with 4 drops of freshly
Five drops of concentrated HCl prepared phenylhydrazine. It was
was added to 5 mL of the isolate. mixed well and then covered with
It was covered with a marble and a cotton. The test tubes were later
boiled in a water bath for 30 heated in a boiling water bath for
minutes. The hydrolysate was 30 minutes. The time when yellow
kept for Benedict’s test. The crystals first appeared were
hydrolysate must be stored in the recorded. The test tubes were
ref if the test cannot be performed cooled and the crystals were
on the same date. observed.
D. Qualitative Tests for
Carbohydrates
1. Benedict’s, Barfoed's,
Seliwanoff's and Bial's - RESULTS AND DISCUSSION
Orcinol Test.
In separate test tubes, five The isolated starch was a light
drops of each of the
yellow, turbid solution. The successful
carbohydrate sample solutions
– 0.1M glucose, xylose, extraction of starch was proved by
fructose, lactose,sucrose and acquiring positive results from the
1% starch – were mixed with 1 following tests which tested the
mL of Benedict's reagent. presence of starch in the extracted
(One test on the different solution – Molisch’s test, iodine reaction,
carbohydrate solutions must be and Benedict’s test.
performed at the same time.)
The test tubes were then
placed into a boiling water bath Table 1. Results of the General Tests
and were removed whenever for Polysaccharides
the solutions for one test gave TEST RESULT
a visible result. The result and
Molisch’s Test purple ring (+)
the time it took for the visible
result to form in each test was Iodine Reaction red color (+)
noted. The same procedure
was applied with Benedict’s, Molisch’s test is a general test
Barfoed's, Seliwanoff's and for carbohydrates. The positive result is
Bial's - Orcinol test. a violet interphase which is due to the
2. Mucic Acid Test. dehydration of carbohydrate by sulfuric
In the mucic acid test of acid to produce an aldehyde, which then
galactose and lactose, 3 drops condenses with two molecules of phenol
of the carbohydrate samples (α-naphtol) to form a purple colored
were mixed with 3 drops of product. The extracted starch from the
concentrated HNO3. The potato produced this positive result.
mixture was placed on a glass
slide which was passed over a
 A positive iodine test indicates Barfoed’s is more specific than
the presence of starch in a solution. A Benedict’s test because it shows visible
positive result of iodine test for starch is results. Barfoed’s test is used to distinguish
a violet solution. This is due to the whether a carbohydrate is a
monosaccharide or a disaccharide. Its
coiling of starch which will trap the
reagent consists of (Cupric acetate)
iodine ion and iodide to form starch- Cu(CH3COO)2 and (acetic acid) CH3COOH.
iodine complex (triiodide). All monosaccharides are reducing sugars,
+2
they reduce Cu ions to a brick-red Cu2O
The starch isolate underwent when heated. A positive result for Barfoed’s
test is indicated by the formation of this
acid hydrolysis and produced a turbid, brick-red precipitate within 5 minutes time.
viscous solution. Acid hydrolysis is a All Monosaccharides were tested positive.
chemical process in which acid is used Ketoses here do not isomerize.
to convert cellulose or starch to sugar. It
Table 3: Seliwanoff’s Test Results
implies a chemical mechanism Sugar Visible result
of hydrolysis catalyzed by a Brønsted- Fructose Cherry red product
Lowry or Arrhenius acid. By contrast, it Lactose Pale yellow solution
does not usually imply hydrolysis by Glucose Pale yellow solution
Xylose Pale yellow solution
direct electrophilic attack—as may
Sucrose Cherry red product
originate from a Lewis acid. Starch Pale yellow solution

After performing the qualitative Seliwanoff’s test is used for

ketohexoses. It consists of resorcinol
tests, namely, Benedict’s, Barfoed’s,
(C6H4(OH)2) in 6M HCl. The concentreated
Seliwanoff’s, and Bial’s orcinol tests, on hydrochloric acid allows ketoses to undergo
glucose, xylose, fructose, lactose, and dehydration than aldoses which further
sucrose, the following results were condenses forming a cherry red product.
obtained: Ketose containing compounds can also yield
a positive result Frustose and sucrose are
ketoses while lactose, glucose, xylose, and
Table 1: Benedict’s Test Results galactose are aldoses.
Sugar Visible result
Fructose Brick red precipitate Table 4: Bial’s Orcinol Test Results
Lactose Blue solution Sugar Visible result
Glucose Brick red precipitate Fructose Turbid yellow solution
Xylose Brick red precipitate Lactose Clear yello solution
Sucrose Blue solution Glucose Clear yellow solution
Starch Blue solution Xylose Blue-green solution
Sucrose Brown solution
Benedict’s test is used to distinguish Starch Clear yellow solution
non-reducing and reducing ones in
carbohydrates.. The Benedict’s reagent
uses the reagents (cupric sulfate) CuSO 4, Bial’s orcinol test shows a positive
NaOH, and tartaric acid. All result for pentoses and is used to
monosaccharides can reduce the blue differentiate pentoses from hexoses. Bial’s
+2
cupric ion Cu ions to brick-red Cu2O. The reagent consists of orcinol, conc.
color brick red precipitate is the positive (Hydrochloric acid)HCl, and (ferric
result for Benedict’s test. When heated by chloride)FeCl3. Pentoses are allowed to
the mixture in a boiling water, it gives the dehydrate yielding to furfural that condenses
reaction a higher rate. orcinol and ferric ion forming blue green
solution.

Mucic acid test is used for

galactose. Oxidation of monosaccharides by
Table 2: Barfoed’s Test Results conc. Nitric acid HNO3 yields soluble
Sugar Visible result dicarboxylic acids. Mucic acids are insoluble
Frustose Brick red precipitate with the said oxidizing agent. A positive
Lactose Blue solution result for mucic acid test is the formation of
Glucose Brick red precipitate an insoluble crystalline precipitate and would
Xylose Brick red precipitate indicate the presence of galactose. Lactose
Sucrose Blue solution and galactose were tested positive with
Starch Blue solution mucic acid test.

The phenylhydrazone test detects

reducing sugars like monosaccharides and
disaccharides. Phenylhydrazone reagent
consists of phenylhydrazine hydrochloride
and NaCH3COO. Aldoses and ketoses when
they react to phenyl hydrazine, they form
crystalline product which is known as the
(osazones).

SUGAR DESCRIPTION
Phenylhydrazone yellow needle
Crystals of Fructose shaped
Phenylhydrazone badminton ball
Crystals of Lactose shaped
Phenylhydrazone yellow needle
Crystals of Glucose shaped

REFERENCES

[1] Bayquen, A.V., Cruz, C.T., de

Guia,R. M., Lampa, F.F., Pena, G.T.,
Sarile,A.S., T orres, P.C. (2009).
LaboratoryManual in Organic Chemistry.
C&Epublishing, Inc

Online sources
[2]http://himedialabs.com/TD/HTBC002.
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[4]http://www.wisc-
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[5]http://www2.chemistry.msu.edu/facult
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