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Group 3 Sec EF
Exercise 8
ANALYSIS OF CARBOHYDRATES
I.Objective/s
1. To identify carbohydrates based on their reactions on different tests
II. Results
A.General Test for Carbohydrates
Table A1. Molisch Test
Samples
1% Glucose
1% Sucrose
1% Arabinose
1% Starch
Saliva
Pineapple juice
Cotton
Liver filtrate
Tulya Filtrate
Observations
Purple layer in the middle; cloudy layer on top; green layer
under purple layer; transparent layer at the bottom
Purple layer in the middle; cloudy layer on top; brown layer
under purple layer; transparent layer at the bottom
Purple layer in the middle (less than sucrose); cloudy layer on
top; brown layer under purple layer transparent layer at the
bottom
Purple layer in the middle; cloudy layer on top (more cloudy
than other samples); green layer under purple layer; pale green
on the bottom
No purple layer; green layer in the middle; transparent layer
with bubbles on top; transparent layer on the on the bottom
Dark color layer in the middle; yellow in top; brown layer
below violet layer; transparent on the bottom
2 layers: upper is dark violet and the lower is transparent
Purple layer in the middle; cloudy on top; transparent layer on
the bottom
Dark brownish-violet layer in the middle; murky green layer
on top; brown layer below violet layer; transparent layer on the
bottom
Observations
Extremely dark green solution, almost black
Extremely dark green solution, almost black
Extremely dark green solution, almost black
Extremely dark green solution, almost black
Dark green solution, least dark color solution among all test
samples
Pineapple juice
Extremely dark green solution, almost black
Cotton
Yellowish solution with brown particles at the bottom
Liver filtrate
Extremely dark green solution, almost black
Tulya Filtrate
Extremely dark green solution, almost black
Theoretical Result: deep green color
Experimental result: black coloration
Observations
Dull violet color; most precipitate observed among the other
test solutions
Brighter violet color; few precipitate
Yellow color; few precipitate
Light yellow color; no precipitate
Very light yellow; becomes colorless after 5 minutes
Colorless
Dark yellow; few precipitate
C. Hydrolysis of Polysaccharides
Table C1.
Samples
1% starch
Dextrin
Cotton
Inulin
Liver filtrate
Tulya filtrate
Observations
Colorless solution
Colorless solution
Heterogenous solution
Yellow solution; dark red particles floating on the entire
solution but more accumulated at the bottom of the test
tube
Faint yellow solution; clear solution; no precipitate
Muddy brown colored solution; turbid
2% fructose
Benedicts Test
(3rd) brick red
solution; lighter
than galactose
(1st) brick red
soln; brightest
Observations
Barfoeds Test
Seliwanoff Test
(4th) deep blue
Orange solution
solution; brick
red ppt.
st
(1 ) deep blue
Cherry red
solution; dark
solution
Orcinol test
Clear light
yellow soln; no
sign of change
Soln turned
brownish
colored solution
2%
arabinose
2% maltose
2% sucrose
5%
galactose
5% sucrose
Light blue
solution
Orange solution
Soln turned
dark violet with
ppt
Cherry red
solution
Soln become
more intense
yellow
compared to the
original ligter
yellow soln
Solution turned
violet
Soltion turned
brownish
yellow
Benedicts Test
Faint yellow color
Observations
Barfoeds Test
faint clear yellow
Dextrin
Cotton
Inulin
Tulya
liver
Seliwanoff Test
Clear yellow-orange
soln
Clear yellow-orange
soln
Dark yellow-orange
soln
Cherry bright red
soln
Blood red soln
Clear yellow-orange
soln
Observations
Barfoeds Test
Soln with red ppt
Orcinol Test
Yellow soln
Benedicts Test
Clear soln; no ppt
2% fructose
2% arabinose
2% maltose
2% sucrose
5% galactose
5% sucrose
Starch
Dextrin
Cotton
Inulin
Tulya
Yellow soln
Yellow soln
Liver
*The results for some of the positive test in Osazone test are put in the discussion.
*Mucic acid test positive result and some of the negative results are put in the discussion as
well.
III. Discussion
Carbohydrate, Cm(H2O) n, is an organic compound composed mainly of elements
carbon, hydrogen (H), and oxygen. Carbohydrates make up most of organic substances and
play vital role in life processes. These are also called saccharides and are divided into
monosaccharides, disaccharides, oligosaccharides and polysaccharides depending on the
contained number of units of carbohydrates.
Carbohydrates can be considered as reducing or non-reducing sugar. Any sugar that
has an aldehyde or ketone group is considered as a reducing sugar. Other sources base
reducing sugar on the free hydroxyl group in the anomeric carbon. Enolization of sugars is
really important especially in reduction tests in alkaline reagents, high pH and temperature.
The reduction ability of a sugar in alkaline test reagents is highly dependent on the aldehyde
or ketone group. These kind of sugars are potential substrates capable of reducing Cu 2+ to C+,
and Ag+ and Ag and so on. Thus, these are the ideal substrate for test on reducing sugars
including Benedicts test, Barfoeds test, and Osazone test. Thses tests involves test on sugars
such as 2% glucose, 2% fructose, 2% maltose, 2% arabinose, 2% sucrose, 5% galactose and
5% sucrose. Also, sugars contained in starch, dextrin, inulin, cotton, tulya and liver filtrates.
There are two ways of carbohydrate analysis, the qualitative and quantitative.
Quantitative test involves Anthrone test while qualitative test includes Molisch test with the
samples utilized including 1% glucose, 1% sucrose, 1% arabinose, 1% starch, saliva,
pineapple juice, cotton, and the filtrates of tulya and liver. Other qualitative tests for
carbohydrate includes Seliwanoff, Orcinol, and Mucic acid test.
Molisch test is a general qualitative test for carbohydrates, meaning further tests are
important to specifically characterize carbohydrate. Molisch test shows whether a
carbohydrate is present or not. Formation of purple ring indicates the presence of
saccharide/s. the reagent for this test is composed of 4% alpha-naphthol and concentrated
H2SO4. The reaction depends on the point that concentrated H2SO4 catalyzes the formation
of furfural (from pentoses) or hydroxymethyl furfural (from hexoses) by dehydration of
sugars. The visible result, which is a visible purple or violet colored product, is given off by
condensation of furfural derivatives with sulfonated alpha naphthol. In the case of
polysaccharides and glycoproteins, also give positive result, they are first broken down into
monosaccharide components by acid hydrolysis and followed by dehydration to form their
respective furfural derivatives. In this test, only saliva and pineapple juice did not give
positive result because they do not have carbohydrates in their composition or very little
amount only. In the case of human saliva, it is comprised of 99.5% mostly water, electrolytes,
mucus, white blood cells, glycoproteins, enzymes, and other antimicrobial agents.
OH
HO
O H
H
H
OH
C o n c . H 2S O
HO
OH
H D-glucose
OH
O
4
OH
a lp h a n a p h th o l
3H2O
Hydroxymethyl furfural
C o n d e n s a t io n p r o d u c t
( p u r p le o r v i o le t )
a.
b.
c.
d.
e.
a.
b.
positive. Probably there were abundant carbohydrates in the samples except that of the cotton
with yellowish solution and brown particles. These are probably due to random error. For the
ideal results, this test should have the same conclusion as the Molisch test where in saliva and
pineapple juice reacted negatively.
OH
O H
H
H
OH
C o n c . H 2S O
HO
OH
OH
H
A n th ro n e
B lu is h g r e e n c o m p le x
3H2O
OH
D-glucose
Hydroxymethyl furfural
a.
b.
f.
e.
g.
h.
c.
d.
I2 + I-
I3-
*cotton hydrolysis was not documented because the liquid evaporated after heating.
Benedicts test is a test for reducing sugar. A reducing sugar is referred to a
carbohydrate that contains a free aldehyde or a ketone group to be oxidized. Basically, all
monosaccharides react positively in this test but not all disaccharides. Some disaccharides are
not reducing sugar because their linkage is both on their respective anomeric carbon. Copper
sulphate is the main reagent in this test and is exposed to alkaline environment. The reason
why this test is for reducing sugars because the reaction takes in place mainly on the ketone
or aldehyde group. So, copper sulphate reacts with this free ketone or aldehyde group to from
cuprous oxide, a red brown precipitate. This precipitate is due to the reduction of Cu 2+ to Cu+
ions and oxidation of the carbohydrate. The more free carbonyl groups in a carbohydrates, the
more precipitate is formed. In this test, saccharides such as glucose, fructose, maltose,
arabinose, and galactose; give the positive results, while sucrose is not a reducing sugar, it
give a negative result. In the hydrolysates, since the substances are composed of
polysaccharides, their intensity of their color response depends on the carbohydrates present.
C u (O H )2
C u O + H 2O
OH
H
HO
H
OH H
H
OH
OH
OH
+ 2 C uO
HO
OH
H
OH H
H
OH
OH
OH
+ C u 2O
Positive test:
Negative test:
a.
b.
Sucrose is not a reducing sugar because its glycosidic linkage is on both anomeric
carbons of glucose and fructose.
Barfoeds test has the same principle with Benedicts test, both are test for reducing
sugars only that this test is subjected to acidic environment. So basically, this test is for
reducing sugars and differentiating monosaccharides from disaccharides. Monosaccharides
react faster in this test compared to disaccharides. Barfoeds reagent is composed of cupric
acetate in acetic acid solution. Reduction of Cu2+ to Cu+ takes place in between the reaction
of monosaccharide and the reagent forming Cu2O, the brick red precipitate. The reason
behind is that monosaccharides are oxidized readily in weak acid solutions. Disaccharides
also can have the positive result as long as they are reducing sugars, but the reaction is much
slower. The monosaccharides then the phosphomolybdic acid in the phosphomolybdate
colour reagent to form phosphomolybdenum blue. Same as with disaccharides only that the
reaction is slower as mentioned above. So for this test, sucrose should not react positively
while monosaccharides such as glucose and fructose for example, should have a positive
visible result.
(C H 3C O O )2C u + 2 H 2O
Cupric acetate
Cupric hydroxide
2 C H 3C O O H + C u (O H )2
C u (O H )2
C u O + H 2O
OH
H
HO
H
OH H
H
OH
OH
+ 2 C uO
OH
HO
OH
H
OH H
H
OH
OH
+ C u 2O
OH
HO
OH
OH
OH
C onc. H C l
O
3 H 2O
OH
re s o rc in o l
OH
c o n d e n s a tio n p r o d u c t
(re d )
D-fructose
hydroxymethyl furfural
Reaction 6. Seliwanoff test reaction
a.
b.
Figure 10. Positive result but slow reaction of 2% arabinose (left) and 2% glucose
(right)
Orcinol test is also called Bials test, named after a German physician. This test is
purposed to distinguish pentose sugars from other types of sugars. The test reactions are also
almost the same with Seliwanoff test. Here, reagent contains HCl, orcinol, and ferric choride.
Hydrolysis of pentose sugar occurs in order to form a furfural derivative. The furfural
derivative is the one that reacts with orcinol through condensation reaction to form the
colored complex, green-yellow for this test. Disaccharides and polysaccharides composed of
pentose units are broken down first by hydrolysis in their glycosidic bonds. Hexoses are also
hydrolysed and react with the orcinol but form a red to brown complex rather than a greenyellow coloration. Arabinose is the only one to react positively in this test based on the
samples used, because it is the only pentose among samples.
OH
OH
H H OH
HO
OH
OH H
H
H3C
OH
o r c in o l
b lu e - g r e e n c o m p le x
F e C l3
D-arabinose
furfural
Reaction 7. Orcinol Test reaction
Osazone test is mainly for reducing sugars and differectiate them based on the crystals
formed . Osazone reagent is composed of phenylhydrazine. Reaction of the carbonyl carbon
of sugar and phenylhydrazine leads to the formation of osazone. The positive test for osazone
is the appearance of the crystals which differ among sugars. Osazone crystals are yellow in
color and differs in shape, melting point, and time of formation of crystals depending on the
types of sugar.
2% Sucrose
Liver
5% sucrose
Inulin
Figure 12. Some of the positive tests in osazone test showing different shapes of crystals;
Fibrous needle-like shape for sucrose, flower like in liver and irregular shape for inulin
Mucic acid test is highly specific for galactose, either as free sugar or derived from
hydrolysis of lactose. Heating galactose with HNO3, a strong oxidizing agent, forms
saccharic acid. Mucic acid or galactaric acid is formed from galactose via oxidation reaction
of the aldehyde group and alcoholic group in Carbon 1 ad 6. The positive result for this test is
the presence of crystals. These crystals came from saccharic acid being insoluble in cold
water, thus it remains and helps in the identification of galactose. In the conduction of this
test, the group was able to encounter crystal like images of the samples other than galactose.
Probably it was only fibrous material happened to be in the slide or the cover slip just like in
Figure 13b below.
Positive result:
crystal
V. References
http://chemistry.elmhurst.edu/vchembook/548starchiodine.html
https://biochemistryisagoodthing.wordpress.com/2013/02/17/lab-review-1/
https://mynotesarchive.wordpress.com/2012/02/18/chem-lab-notes/
http://vlab.amrita.edu/?sub=3&brch=63&sim=631&cnt=2
http://www.slideshare.net/katealyssacaton/mucic-and-barfoeds-test