REGOROSA, NICOLE C.

Date Performed: October 24, 2016

Group 3 Sec EF

Date Submitted: November 16, 2016

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

Table A2. Anthrone Test

Samples
1% Glucose
1% Sucrose
1% Arabinose
1% Starch
Saliva

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

B. Iodine Test for polysaccharides

Samples
1% Starch
Dextrin
Inulin
Gum Arabic
Agar-agar
Liver filtrate
Tulya filtrate
Table B.

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

Table C2. All sugars

Samples
2% glucose

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

(5th) dull reddish

brown solution,
darker than
maltose
(4th) dull reddish
brown soln;
brighter than
arabinose
(-) light blue
solution; no
significant change

5%
galactose

(2nd) brick red

soln; darker than
galactose

5% sucrose

(-) light blue

solution; no
significant changes

ppt (most ppt.

observed)
rd
(3 ) deep blue
soln; dark ppt
(small or few)

yellow with ppt

Greenish yellow
ppt

Light blue
solution

(-) light blue; no

ppt

Orange solution

Soln turned
dark violet with
ppt

Light blue soln;

no ppt

Cherry red
solution

Soln become
more intense
yellow
compared to the
original ligter
yellow soln
Solution turned
violet

(2nd) deepest blue

Red-orange
soln among
soln with ppt.
samples; brick
red ppt
(-) light blue
Cherry red soln
soln; no ppt.

Soltion turned
brownish
yellow

Table C3. Hydrolysates C (1)

Samples
Starch

Benedicts Test
Faint yellow color

Observations
Barfoeds Test
faint clear yellow

Dextrin

Murky light green

Faint clear yellow

Cotton

Faint yellow soln

Inulin

Faint light solution

Faint yellow; almost

colorless
Murky faint yellow

Tulya
liver

Bright yellow soln

Light yellow soln

Light yellow soln

Murky light yellow;
almost colorless

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

Table C4. Hydrolysates C (2)

Samples
2% glucose

Benedicts Test
Clear soln; no ppt

2% fructose
2% arabinose
2% maltose
2% sucrose
5% galactose
5% sucrose
Starch
Dextrin

Yellow to red pp.

formed
Red ppt at the
bottom
Red ppt at the
bottom
Yellowish-light
brown soln with ppt
Brwn red ppt formed
Ligt yellow to light
brown ppt at top
Yellow soln

Cotton

Blue green cloudy

season
Light-yellow soln

Inulin
Tulya

Yellow soln
Yellow soln

Liver

Light yellow soln

with tinge of blue
green

Light yellow soln

Yellow soln with

brown ppt
Light brown soln

Red orange ppt

Dark violet ppt

Blue green soln

Yellow ppt. formed

Light orange soln

Red violet ppt

formed
Yellow soln with
light brown ppt
Metallic green soln

Yellow green soln

Cloudy light blue
soln
Cloudy light blue
solution
Yellow soln
Green soln
Light blue green
soln
Yellow soln

Red violet soln

2 layers: upper-clear
Bottom: brown
Brown ppt formed
Red ciolet soln with
dark violet ppt
Light yellow soln

*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

Reaction 1. D-glucose with Molisch test

Some of the positive tests:

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.

Figure 1. (a-f) 1% arabinose, 1% glucose, 1% sucrose, cotton, and tulya.

Negative results:

a.

b.

Figure 2. (a) saliva, (b) pineapple juice

Anthrone test is a quantitative test for carbohydrates. Anthrone reagent is composed

of 0.2% (w/v) anthrone in solution in concentrated H2SO4. So basically, this test has the
same principle as molisch test only that this one accounts for the concentration of total sugars
in a sample. Sugars react with the anthrone reagent under acidic conditions to yield a bluegreen or green color. Ideally in the samples we had, only saliva and pineapple juice would
have a very small visible result or none at all because these two substance contain very least
amount amount carbohydrates or none at all. But, what happened in our conduction is that
almost all test solutions went very dark. It was hard to distinguish whether it was a positive
test or really and error probably. But the group still considered the very dark green result as

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

Reaction 2. D-glucose with Anthrone test.

a.

b.
f.

e.

g.
h.

c.

d.

Figure 3. (a-f) starch, tulya, liver, 1% glucose, 1% sucrose, 1% arabinose

(g-h) pineapple juice-which supposedly be negative- and cotton.

In iodine test, it is used to distinguish polysaccharides from mono- and disaccharides.

This test actually is specific for substances like starch (polysaccharide). Starch is composed
of amylose, which is the one involved in this reaction. The principle in this test is mainly
adsorption. Iodine forms colored complex due it its adsorption on the polysaccharide chains.
The intensity of the color complex varies depending on the length of the chain that is readily
available for the complex formation. Blue black colored result is the positive indication for
this test. Iodine alone, as reagent, is insoluble in water. Therefore, it is dissolved in potassium
iodide solution resulting to potassium triiodide solution as the test reagent. For specific
reaction, potassium iodide dissociates, and then the Iodide ion reacts reversibly with the
Iodine to yield the triiodide ion.

I2 + I-

I3-

Reaction 3. Triiodide ion slips into the coil of

starch causing
a
n

intense blue black to violet solution

Thus, amylose, the unbranched helical
component of starch gives a deep blue or
violet color and amylopectin, the branched component gives red colour because the chains do
not coil effectively. Glycogen, which is also highly branched, gives red colour with iodine.
This test is conducted in acidic or neutral solutions.

Figure 4a. Before addition of

iodine. (left-right) starch,
dextrin, inulin, gum arabic,
agar-agar, liver filtrate, tulya
filtrate

Figure 4b. After addition of

iodine. (left-right) starch,
dextrin, inulin, gum arabic,
agar-agar, liver filtrate, tulya

Before proceeding to the remaining qualitative tests such as benedicts, barfoeds,

seliwanoff, orcinol, osazone, and mucic acid test, samples like dextrin, starch, cotton, inulin,
tulya, and liver were subjected to hydrolysis.
After Hydrolysis:

Figure 6. (left-right) Hydrolysates of dextrin, inuin, liver, 1 % starch, and tulya.

*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

Reaction 4.Benedicts test reaction

Positive test:

Figure 7a. (left-right) maltose, arabinose, glucose, galactose, fructose

Negative test:

a.

b.

Figure 7b. 2% sucrose (left), 5% sucrose (right)

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

Reaction 5. Barfoeds test reaction

Figure 8. Galactose showing the positive result

Seliwanoff test is to distinguish ketoses from aldoses. It is specific for ketoses since
the reaction is faster in ketoses. Although aldoses also react, but in a much slower rate.
Seliwanoffs reagent contains non-oxidizing acid such as HCl and resorcinol. Dehydration
takes place when HCl reacts with the ketose sugar which results to the formation of a furfural
derivative. Condensation reaction then follows with the reaction of the furfural derivative
with resorcinol forming the cherry red product/complex. In this test, only fructose, sucrose,
and inulin show the positive result because they are the only ones among the samples that
contain a ketose sugar.
OH
O
HO

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 9. Positive test for Seliwanoff. (a-c) 2% fructose, 5% sucrose, and

c.
2% sucrose

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

Figure 11a. Positive result for

arabinose

Figure 11b. Negative result hydrolysates (left)

and some sugars (right)

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.

Reaction 8. Reaction of sugar with phenylhydrazine

*Fructose was the first one to have yellow crystal precipitate.
Some of the osazone test results for sugars and hydrolysates:

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

Figure 13a. Positive test of galactose in Mucic acid test

Negative result:

Figure 13b. Liver, example of negative result.

IV. Conclusion
In conclusion, the group was able to identify carbohydrates based on their specific
reactions in different test with varying reagents. There are two ways to distinguish
carbohydrates, through a qualitative or quantitative test. Quantitative test involve solely
Anthrone test, which has almost the same mechanism as the Molisch test only that this one is
for determining the amount of sugar in a solution or a certain sample. Qualitative tests
include molisch test as general test for carbohydrates, iodine test for presence of amylose,
benedicts, barfoeds, and osazone tests for reducing sugars, seliwanoff test for ketoses,
orcinol test for pentoses, and lastly, mucic acid test specific for presence of galactose. These
are just the common and most accessible methods in analyzing carbohydrates.

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