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ISOLATION AND CHARACTERIZATION OF

CARBOHYDRATES
G.A.N Obligado

Department of Mathematics and Natural Sciences, College of Arts, Sciences and Education
Aquinas, University of Legazpi, Rawis, Legazpi City, Philippines
DATE PERFORMED: December , 2014
ABSTRACT
Starch from potato and glycogen from chicken liver were isolated for the
general test for polysaccharides, which are the molischs test and the iodine
reaction test, hydrolysis of polysaccharides (acidic and enzymatic) and for the tests
such as Benedicts, Barfoeds, Seliwanoffs, and Bials Orcinol Tests. Quantitative
analysis was performed with glucose solution employing absorption spectrometry
which determined the concentration of the given unknown sample using the
equation of the line obtained from the graph. The absorbance of the unknown
solution was 0.620 resulting to the quantifying its concentration which was
0.000896 M.
INTRODUCTION second class utilizes solutions with
copper (II) ions. The carbohydrate
Carbohydrates play essential reduces the ions to copper (I) oxide.
part in almost all the biological Reducing sugars are all aldoses
processes. They are one of the four containing either a free aldehyde
macromolecules vital to natural group or a cyclic hemiacetal.
processes. They serve as a major
energy source for living organisms. For METHODOLOGY
example, glucose which is the main
energy source in plants and animals. Extraction of Glycogen from
They also transport energy, serve as a Chicken Liver
structural material and an originator
for vast molecules. Three grams of chicken liver
A carbohydrate with the were weighed and placed in the petri
functional group aldehyde is classified dish. The liver was cut with scissors
as aldose and ketose when it has and 12 ml boiling water was added.
ketone functional group. The Then, the mixture was put into a
subgroups under carbohydrates are beaker and set to boil for 2 minutes.
monosaccharides, oligosaccharides, The mixture was grinded with mortar
polysaccharides and and pestle. 2ml of distilled water was
glyconoconjugates. Glycogen is an added and transferred into the beaker
example of a polysaccharide. again. The mixture was heated for 30
The first type of carbohydrate minutes. Glycogen solution was
test reagents is the two-step analysis obtained by filtering. The solution was
with the use of dehydrating acids then divided into four test tubes.
the condensation reagents.
Dehydrating acids convert pentoses Extraction of Starch from Cassava
into furfural, and hexoses into 5- and Potato
hydroxymethyl furfural which then
react with the phenolic compounds to The potato was pelled, chopped,
yield highly colored products. The grinded and placed in small beaker
with 100 ml water. The starch was Nelsons reagent was prepared
filtered using cheesecloth. by mixing 12.5 ml and 0.5 ml of
Nelsons A and Nelsons B, accordingly.
Molischs Test Glucose and distilled water were
mixed in seven test tubes following
Few drops of molischs reagent the volumes indicated in the
was added into the 1 ml starch laboratory manual. Then, 1 ml of
solution. 2 ml concentrated H2SO4 was Nelsons reagent was added. The test
poured. tubes were heated for 20 minutes then
cooled. 1 ml of arsenomolybdate
I2 Reaction reagent was dropped then the
absorbance of the solutions was
Iodine, 0.01 M, was dropped measured using a spectrophotometer.
into a 1 ml sample solution. The The unknown sample was also
mixture was then warmed in a water subjected to the spectrophotometer
bath. Finally, the mixture was cooled for the determination absorbance that
for observation. was used to calculate for its
concentration.
Acid Hydrolysis

Concentrated HCl, 5 drops, was RESULTS AND DISCUSSION


added to the 5 ml isolate in a test tube
and was covered with a marble then Isolation and General Tests for
boiled in a water bath for 30 minutes. Polysaccharides
Afterwards, it was used for the
Benedicts test. Descriptio Remar
Test Result
Enzymatic Hydrolysis n k
Molisch Turbid Negativ
In a beaker, 10 ml of the isolate Fail
s white e
and 2.3 ml of saliva were mixed. After Brownish
30 minutes, it was placed inside the I2 with
prepared dialyzing bag and stored Negativ
Reactio floating Fail
overnight. The next day, the mixture e
n precipitat
without the dialyzing bag was heated e
then used for Benedicts test. Table 1. Results for Molischs and
I2 Reaction Test for Starch
Benedicts, Barfoeds,
Seliwanoffs, and Bials Orcinol
Tests

In each test, 5 drops of glucose,


fructose, maltose, lactose, sucrose and
starch (one test tube per specific
carbohydrate solution) were all mixed
with 1 ml of the reagent. Afterwards,
the test tubes were placed into a
boiling bath all at the same time.

Quantitative Analysis
Picture 1. Results for Molischs
and I2 Reaction Test for Starch

Molischs test and iodine


reaction test are assessments for the
presence of carbohydrates and
polysaccharides. Starch is a
carbohydrate and a polysaccharide so
it must be positive for these tests.
Confirmation that a sample is positive
for Molischs test is that there must be
dark violet region between the
junctions of two layers due to the
formation of an unstable condensation Picture 2. Result for Acid
product of beta-naphthol with furfural Hydrolysis
(produced by the dehydration of the
carbohydrate). For the iodine reaction
test, there mixture should have been
turned blue for the presence of
polysaccharide because iodine forms
coloured adsorption complexes with
polysaccharides. Therefore, the
performed experiment for this part
was unsuccessful.

Hydrolysis of Polysaccharides

Descripti Rema
Test Result
on rk
Acid Picture3. Results for Acid and
Turbid Negati
hydrolysi Fail Enzymatic Hydrolysis
white ve
s
Enzymati According to references, the
c Negati solutions for this part must turn red so
Yellow Fail the experiment for hydrolysis of
hydrolysi ve
s polysaccharides was unsuccessful.
Table2. Results For Acid
hydrolysis and Enzymatic Qualitative Tests for
Hydrolysis Carbohydrates

Legend: + (positive), - (negative)

Carbo
Benedi Barf Seliw
hydrat Bial
ct oed anof
e
Glucos Red Blue Yello Brow
e Precipi with w n
tate red gold -
+ + - Barfoed
Red Blue
Cher s
Brow
Fructo Precipi with
ry
n
se tate red
red
-
+ + +
Red Yello
Brow
Maltos PrecipiBlue w
n
e tate - gold
-
+ -
Light Brow
Sucros White Blue
red n
e - -
+ -
Yello
Brow
White Blue w
Starch n
- - gold Seliwano
-
- f
Red Yello
Brow
Lactos Precipi Blue w
n
e tate - gold
-
+ -
Table 3. Results for the
Qualitative Tests

Benedict
s

Bials

Table4. Photos for the results for


each test.

The basis for deciding whether


the tests resulted positive and
negative outcomes is shown in the
Table 5. If the observation in each Table6. Values for Concentration
carbohydrate from the experiment did and Absorbance for Quantitative
not match the stated standard Analysis
appearance in the table for positive
results, therefore, those carbohydrates 0.6
are non-reducing sugars,
oligosaccharides or polysaccharides, 0.4 f(x) = 378.19x + 0.28
aldoses and hexoses for Benedicts, R = 0.22
Absorbance
Barfoeds, Seliwanofs and Bials test, 0.2
respectively.
0
Appearanc 0 0 0 0 0 0 0
e Concentration
Test Test for?
For positive
result
There must Figure1. Graph of Concentration
be red vs. Absorbance for the
precipitate Quantitative Analysis
Reducing
Benedict due to
sugars
production Poor linearity is shown by the
of plot and by the square of the
Cu(1) oxide correlation coefficient. Having known
With red in the absorbance of the unknown
blue sample, which is 0.620, its
solution concentration was determined using
Monosacc
Barfoed due to the equation of the line obtained from
arides
production the graph. The concentration of the
of unknown sample was 0.000896 M.
Cu(1) oxide
Cherry red CONCLUSION AND
Seliwanof solution Ketoses RECOMMENDATION
must occur
Blue-green The general tests and hydrolysis
Bial Pentoses
product for polysaccharides were unsuccessful.
Table5. Basis for the conformation The experiment should have been
for positive and negative results performed very carefully and following
in the Table 3. the procedures systematically.
Reagents for each test should have
Quantitative Analysis been assured to be uncontaminated.
The mucic acid test and the
Concentration Absorba phenylhydrazone test were not done
(M) nce due to lack of time. The group should
0 0 have been more aware of the time so
0.0000555 0.42 that distribution and exhibition of
0.000111 0.48 tasks were equal and fast.
0.000222 0.47
0.000333 0.39 REFERENCES
0.000444 0.42
0.000555 0.44
1) http://amrita.vlab.co.in/? ( 0.000222 M ) (0.0001 L)
sub=3&brch=63&sim=631&cnt 3) 0.001 L
=2
2) http://www.ausetute.com.au/red ( 0.000333 M ) (0.0001 L)
sugar.html 4) 0.001 L
APPENDICES ( 0.000 444 M ) (0.0001 L)
( 0.0000555 M ) (0.0001 L) 5) 0.001 L
1) 0.001 L
( 0.000555 M )( 0.0001 L )
( 0.000111 M ) (0.0001 L) 6) 0.001 L
2) 0.001 L
7) y = 378.19x + 0.2813
0.620= 378.19x + 0.2813

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