Exercise 8

ANALYSIS OF CARBOHYDRATES
Name:
Group &
Sec:

Bridgette M.
Juarez
Group 3 Sec EF

Date
Performed:
Date
Submitted:

October 24,
2016
November 16,
2016

I.Objective:
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 dark 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
Pineapple juice
Cotton

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
Extremely dark green solution, almost black
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
Benedicts Test
2%
glucose

(3rd) brick red

solution; lighter
than galactose

2%
fructose

(1st) brick red

soln; brightest
colored solution

2%
arabinose

(5th) dull reddish

brown solution,
darker than
maltose
(4th) dull reddish

2%

Observations
Barfoeds Test
Seliwanoff
Test
th
(4 ) deep blue
Orange
solution; brick
solution
red ppt.

Orcinol test

(1st) deep blue

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

Cherry red
solution
Greenish
yellow ppt

Clear light
yellow soln;
no sign of
change
Soln turned
brownish
yellow with
ppt
Light blue
solution

(-) light blue

Orange

Soln turned

maltose

brown soln;
brighter than
arabinose
(-) light blue
solution; no
significant
change

soln; no ppt

solution

dark violet
with ppt

(-) light blue

soln; no ppt

Cherry red
solution

5%
galactose

(2nd) brick red

soln; darker
than galactose

Red-orange
soln with ppt.

5%
sucrose

(-) light blue

solution; no
significant
changes

(2nd) deepest
blue soln
among
samples; brick
red ppt
(-) light blue
soln; no ppt.

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

2%
sucrose

Cherry red
soln

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 yelloworange soln
Clear yelloworange soln
Dark yelloworange soln
Cherry bright red
soln
Blood red soln
Clear yelloworange soln

Table C4. Hydrolysates C (2)

Samples
2% glucose
2% fructose
2% arabinose
2% maltose
2% sucrose
5% galactose

Benedicts Test
Clear soln; no ppt
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

Observations
Barfoeds Test
Soln with red ppt
Light yellow soln

Orcinol Test
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

5% sucrose
Starch
Dextrin

Ligt yellow to light

brown ppt at top
Yellow soln

Yellow green soln

Cloudy light blue
soln
Cloudy light blue
solution
Yellow soln

Cotton

Blue green cloudy

season
Light-yellow soln

Inulin
Tulya

Yellow soln
Yellow soln

Green soln
Light blue green
soln

Liver

Light yellow soln

with tinge of blue
green

Yellow soln

Yellow soln with

light brown ppt
Metallic green
soln
Red violet soln
2 layers: upperclear
Bottom: brown
Brown ppt formed
Red ciolet soln
with dark violet
ppt
Light yellow soln

III. Discussions:
Carbohydrates originally refer to compounds with the general formula:

Cn (H 2 O)n . But not all sugars fit this formula, only the monosaccharides that
consists of a single polyhydroxy aldehyde or ketone unit. Other sugars such as
oligosaccharides and polysaccharides that are based on the monosaccharide
units have slightly different general formulas. The difference in the general
formulas is caused by the loss of a water molecule for each newly formed link in
non-monosaccharide sugars. Carbohydrates can either be an aldose, the
carbonyl group at the end of its chain is an aldehyde, or a ketose which has a
ketone at the end of its chain. The common number of carbons in each
monosaccharide can range from three to six.
The tests conducted for carbohydrates can be classified into two: general
and specific. The general tests include Molisch test and Anthrone test, while the
specific tests include iodine test, Benedicts test, Barfoed test, Seliwanoff test,
Orcinol test, Osazone test and Mucic acid test. The general tests are used to
detect the presence of sugar in a qualitative or quantitative manner. The specific
tests are used to detect certain sugars or group of sugars according to their
structure or reaction.
The samples for this experiment consist of monosaccharides (glucose,
galactose, fructose & arabinose), oligosaccharides (maltose, dextrin, sucrose &
gum arabic), polysaccharides (cotton, starch), extracts (tulya, liver, saliva and
pineapple juice)
MOLISCH TEST:
This is a qualitative test for presence of carbohydrates. The sample is first
treated with a strong acid (conc.

H 2 SO 4 ) to hydrolyze the sugars into its

monosaccharide components. Upon the reaction of the acid and the

monosaccharide, 3 moles of water is expelled and it gives a furfural or furfural
derivative (5-hydroxymethylfurfural). Pentoses yield furfural and hexoses yield

furfural derivative. Furfural is then condensed with 2 moles of -naphthol to give

the purple ring or complex. (Fig. 1)
Fig. 1

In this test, as expected, all carbohydrates (glucose, sucrose, arabinose,

starch and cotton) gave a positive result showing a violet ring at the interface of
the two layers. The liver and tulya extracts gave positive results as well because
they contain 0.02% and 5%-20% carbohydrates respectively. Pineapple also
showed a dark colored complex because fresh pineapple juices contain 10%-13%
carbohydrates. Saliva gave a negative result because of the absence of
carbohydrates.
Fig. 1.1
Positive tests:

Arabinose

Glucose

Sucrose

Cotton

Starch

Tulya

Pineapple

Liver

Negative test:

Saliva
ANTHRONE TEST:
The same with Molisch test, Anthrone test is also used to detect the
presence of sugars. The difference with this test from Molisch is that it is more of
a quantitative test that bases the amount of sugar present on the intensity of the
color given in the result. The carbohydrates are first hydrolyzed with sulfuric acid
to form furfurals and/or hydroxy-methly furfural. These furfural are then
condensed by anthrone reagent to form a blue green color complex. (Fig 2)

Fig. 2

All carbohydrates and samples (except for saliva) tested positive in

Anthrone same as in Molisch test. The positive result is an extremely dark green
to black solution. The faintness of the green color in saliva implies the lack of
carbohydrates. (Fig. 2.1)
Fig. 2.1
Positive test ex.:

Tulya

Negative test:

IODINE TEST
The amylose constituent of starch has helical structure and when it reacts
with iodine solution, the iodine gets trapped inside the helical structure giving
the solution blue color, which indicates the presence of starch. (Fig. 3)
Fig. 3
Starch-iodine complex

I 3

I 2+ I

Only starch and dextrin gave positive results. Dextrin is a derivative of

starch so its expected t give a blue color. The yellow color found in the other
samples that tested negative is a result of the dilution of the iodine solution
used. (Fig 3.1)
Fig. 3.1
Before addition of straw yellow iodine

5 mins. after addition of straw yellow

iodine

BENEDICTS TEST
This test is for the presence of reducing sugars that uses an alkaline
reagent. The reagent is composed of copper sulfate that provides cupric ions,

Sodium carbonate which causes the alkalinity of the solution and Sodium citrate
that prevents cupric ions from precipitating. The mixture is then heated in a
water bath. A positive result for the presence of reducing sugars is indicated by
the formation of a precipitate and a change in color. Benedicts reagent contains
blue copper (II) ions, which are reduced to copper (I). These are precipitated as
brick-red copper (I) oxide, which is not soluble in water (Fig. 4). The color of the
solution depends on the concentration of the sugar present (Fig. 4.1)
Fig. 4

Fig. 4.1

All sugars except for sucrose gave a positive result (Fig. 4.2 & Fig. 4.3) since
maltose along with the other monosaccharides are reducing sugars. The
concentration of sucrose did not matter sice its not a reducing sugar. Most of the
monosaccharides reacted fast giving a brick-red precipitate while maltose was in
second to the last. Arabinose was the last to form precipitate probably due to the
fact that the dropping of the Benedicts reagent to the samples were not
simultaneous.
Fig. 4.2 Positive results

Maltose

Arabinose

Fig. 4.3 Negative results

Glucose

Galactose

Fructose

Sucrose
BARFOEDS TEST
Similar to Benedicts test except for the use of an acidic reagent, Barfoeds
test is also a test for reducing sugars. Monosaccharides are oxidized by the
copper ion in solution to form a carboxylic acid and a reddish precipitate of
copper (I) oxide (Fig. 5). Reducing disaccharides undergo the same reaction, but
do so at a slower rate.
Fig. 5

All the monosaccharides gave a positive result for this test while maltose
and sucrose did not form red precipitates (Fig. 5.1). Maltose is a disaccharide
reducing sugar so it should have tested positive but should be allotted longer
time.

Fig. 5.1
Positive test ex.:

Fructose

Negative test:

Sucrose

Maltose

Sucrose

SELIWANOFFS TEST
This test is used to differentiate ketoses from aldoses. The reagent
dehydrates ketohexoses to form 5-hydroxymethylfurfural. 5hydroxymethylfurfural further reacts with resorcinol present in the test reagent
to produce a red product. Aldohexoses react to form the same product, but do so
more slowly (Fig. 6)

Fig. 6

Since in all the sugars used only fructose is a ketose and there is fructose in
sucrose, theyre the only ones that should give the positive result of having
cherry red solution (Fig. 6.1). Other sugars are more yellow in color than red.
Fig. 6.1 Positive results

Sucrose

Fructose

ORCINOL TEST
This test is specific for pentoses. The components of the reagent include
orcinol, hydrochloric acid, and ferric chloride. When pentose is present, it will be
dehydrated to form furfural which then reacts with the orcinol to generate a
colored substance. The solution will turn bluish and a precipitate may form (Fig.
7).
Fig. 7

Since arabinose is the only pentose sugar in the samples it is the only one
that gave a light blue solution upon its reaction (Fig. 7.1). The other sugars
produced a yellow solution.
Fig. 7.1 Positive test

Arabinose

OSAZONE TEST
Reducing sugars which have either a free aldehyde or keto group are
reacted with phenylhydrazine to form osazone crystals. Three molecules of
phenylhydrazine reacts with the first two carbons in the carbohydrate chain. This
affects the -carbon oxidation with the formation of bis-phenylhydrazone or
osazone (Fig. 8). The crystals formed are either ball-shaped crystals by lactose,
broomstick/needle-shaped crystals from glucose and fructose or sunflowershaped crystals from maltose.
Fig. 8

The only non-reducing sugar used in this experiment is sucrose so ideally it

will not create an osazone. When viewed under the microscope it was clear that
the crystals formed by sucrose were needle shaped and the crystals formed by
maltose were vaguely sunflower-shaped (Fig. 8.1).
Fig 8.1 Positive results ex.:

Fructose (needle-shaped crystals)

Maltose (sunflower-shaped crystals)

MUCIC ACID TEST

When most monosaccharides are oxidized by a strong acid (nitric acid) and
subjected to heat they form a soluble dicarboxylic acid. This is a specific test for
galactose due to its structure; when it is oxidized it produces an insoluble
dicarboxylic acid known as mucic acid (Fig. 9).
Fig. 9

Only galactose formed rhombic-plate crystals (Fig. 9.1). Other sugars

showed one or two strands of needle-shaped crystals but they were insignificant
compared to the amount of crystals produced by galactose.
Fig. 9.1 Positive result

Galactose
IV. Conclusion
Carbohydrates can be detected by using general tests that can either be
qualitative or quantitative. They can also be identified by using specific tests
that react with sugars with certain characteristics or structures. Usually
monosaccharides are more reactive than oligosaccharides or polysaccharides
and reducing sugars are more reactive than the non-reducing sugars because
they are easily oxidized.
V. References
https://allmedicalstuff.com/anthrone-test-carbohydrates/
http://www.permahealthcare.com/glme-composition.htm
https://books.google.com.ph/books?
id=Ws570Ql8krAC&pg=PA33&lpg=PA33&dq=lab+manual+in+biochemistry+a
nthrone+test&source=bl&ots=ubuZqvTxtG&sig=1g2Dj3ejB6olDV0FWPblEpfTeT
s&hl=en&sa=X&ved=0ahUKEwjwPCigKvQAhXCJ5QKHbHmCvMQ6AEIIDAB#v=onepage&q=lab%20manual%20in

%20biochemistry%20anthrone%20test&f=false
http://eprints.utm.my/6174/1/MasnizaSairi2004_ChemicalCompositionAndSens
oryAnalysis.pdf
https://www.google.com.ph/url?
sa=t&rct=j&q=&esrc=s&source=web&cd=5&cad=rja&uact=8&ved=0ahUKEw
jl0PPp-KrQAhVFs48KHR__A48QFggxMAQ&url=http%3A%2F%2Fwww.umich.edu
%2F~bmsteach%2Flopatin%2Fsalivarygland%2Flectures%2Fdownload
%2FChem_Comp_%26_Funct.ppt&usg=AFQjCNF6o7cWICGhCj1V1rwhEZY_eWBng
http://chemistry.elmhurst.edu/vchembook/548starchiodine.html
https://www.reference.com/science/benedict-test-function-a2b30da3be1056be
http://usmle.biochemistryformedics.com/osazone-test/
https://www.scribd.com/doc/60136819/Osazone-Test
http://generalchemistrylab.blogspot.com/2011/12/mucic-acid-test-forgalactose.html