SACAY, JEMINA ANDREA JULY 27,2019

TAN, PATRICIA MAE LOCKER NO. 46

Experiment 5: Analysis of Carbohydrates (Post-lab report)

V. Observations

GENERAL TESTS FOR CARBOHYDRATES

Sample Tests Molisch Test Anthrone Test
1.) Glucose Purple ring Blue Green

2.) Sucrose Purple ring Blue Green

3.) Arabinose Purple ring Blue Green

4.) Starch Purple ring Blue Green

5.) Cotton Purple ring Light Blue

6.) Chicken Liver Purple ring Blue Green

7.) Shell Mostly dark green, but presence Blue Green

of purple color is visible

IODINE TEST FOR Color Response to Iodine

POLYSACCHARIDES
1.) Starch Blue-black

2.) Dextrin Yellow/Brown

3.) Gum-arabic Yellow/Brown

4.) Agar-agar Yellow/Brown

5.) Chicken Liver Dark Brown

6.) Shell Yellow/Brown

 Test Solution Benedict’s Barfoed’s Seliwanoff’s Orcinol Osazone Mucic acid
(time of
formation)
1.) Glucose Redish brown brick red (+) Yellow (-) Bluish green Clear (-)
(+) (+)
2.) Galactose Orange (+) brick red (+) Yellow (-) Peachy/orange Clear sol.
(-) w/ crystals
(+)
3.) Fructose Brown (+) brick red (+) Red (+) Bluish green Clear (-)
(+)
4.) Arabinose Brown (+) brick red (+) Yellow (-) Bluish green Clear (-)
(+)
5.) Maltose Orange (+) Light Blue (-) Yellow (-) Bluish green Clear (-)
(+)
6.) Lactose Orange (+) Light Blue (-) Yellow (-) Bluish green Clear sol.
(+) w/ crystals
(+)
7.) Sucrose Green (+) Light Blue (-) Red (+) Bluish green Clear (-)
(+)
8. Hydrolysate
from
a.) Starch Brick red (+) Light Blue (-) Yellow (-) NA NA
b.) Dextrin Brown (+) Brick Red (+) Yellow (-) NA NA
c.) Cotton Yellow (+) Light Blue (-) Yellow (-) NA NA
d.) Chicken Light Blue (-) Yellow (-) NA NA
Liver
e.) Shell Green (+) Light Blue (-) Yellow (-) NA NA
f.) Gum arabic NA Bluish green Clear (-)
(+)
g.) Agar-agar Brown (+) NA Bluish green Clear (-)
(+)
 Sketch for Mucic Acid Crystals
Test Solution Sketch Test Solution Sketch
(Hydrolysate)

1.) Glucose a.) Starch

2.) Galactose b.) Dextrin

3.) Fructose c.) Cotton

4.) Arabinose d.) Gum-arabic

5.) Maltose e.) Agar-agar

6.) Lactose

7.) Sucrose
VI. Analysis

1.) Correlate the results of the iodine test on the polysaccharides with their
structure.
- A solution of iodine (I2) and potassium iodide (KI) in water has a light
orange-brown color. If it is added to a sample that contains starch, the color
changes to a deep blue. Starch is a carbohydrate found in plants. It consists of
two different types of polysaccharides that are made up of glucose units which
are connected in two different ways. One is the linear amylose and the other is
the branched amylopectin. Amylose is the compound that is responsible for the
blue color. Its chain forms a helix shape, and iodine can be bound inside this helix
The colors are caused by so-called charge transfer (CT) complexes. Molecular
iodine (I2) is not easily soluble in water, which is why potassium iodide is added.
Together, they form polyiodide ions of the type In–. The negatively charged
iodide in these compounds acts as charge donor, the neutral iodine as a charge
acceptor. Electrons in such charge-transfer complexes are easy to excite to a
higher energy level by light. The light is absorbed in the process and its
complementary color is observed by the human eye

2.) Correlate the results of the tests on cotton hydrolysate with the structure of
cellulose.

3.) Why does fructose, a ketone, reduce Benedict’s reagent?

- The presence of hydrogen atoms in the aldehyde group, give the aldehyde
molecule its’ reduction properties. Aldehydes are easily oxidized via mild
oxidizing agents like Benedict’s reagent (Cu2+ complexed with citrate). Ketones
are not oxidized under the same conditions. This allows for Benedict’s reagent to
be used in distinguishing aldehydes from ketones. Fructose reduces such
reagents even though it contains no aldehyde group. Reduction occurs because
the reagents are basic solutions and fructose is readily isomerized to a mixture of
aldoses (Glucose and mannose) under basic conditions.

4.) Which sugars gave positive result upon prolonged heating with Seliwanoff’s
reagent? Why?
- The sugars that gave positive result for the Seliwanoff’s reagent are fructose and
 sucrose. When it is mixed with seliwanoff’s reagent, ketopentoses and
ketohexoses react within 2 minutes to from a cherry red ppt. Fructose gave a
positive result because it is a ketohexose. Other carbohydrates and aldohexoses,
like glucose upon prolonged heating will give a faint reddish color due to the
conversion of glucose to fructose by HCL or hydrolysis of a disaccharide or
polysaccharide to yield fructose which will result to a false positive result.

5.) What is the use of NaOAc in the osazone reagent?

- Some sugars react with Phenylhydrazine to give crystalline compounds known as
osazones. Phenylglucosazone, which is formed from glucose and also fructose, is
in the form of sheaves of needle-like crystals. Phenyllactosazone crystals, on the
other hand, are smaller and more feathery than those obtained from glucose.
The osazone test will give some indication whether a sugar is either glucose or
lactose, but the solubility of the crystals formed should be determined to verify
the microscopical appearance, as so many factors influence the crystalline
structure.

6.) Which sugars form the same osazone? How can you differentiate both in the
actual osazone test?
- N/A. We were not able to perform the osazone test due to the lack of
Phenylhydrazine reagent.

7.) Give the Chemistry/Principle behind the tests involved vis-à-vis results. Give
pertinent equations.
- Molisch’s Test Principle: Carbohydrates when treated with concentrated Sulfuric
acid undergo dehydration to give furfural derivatives. These compounds
condense with Alpha naphthol to form colored products. Pentoses yield furfural
while Heroxses yield 5-Hydroxy methyl furfurals.
Anthrone Test Principle: Starches are dried out with concentrated H2SO4 to frame
“Furfural”, which gathers with anthrone to shape a green shading complex which
can be measured by utilizing colorimetrically at 620nm (or) by utilizing a red
channel. Anthrone reacts with dextrins, monosaccharide, disaccharides,
polysaccharides, starch, gums and glycosides. If this happens, the yield of shading
is where is to frame sugar to starch.

Iodine Test Principle: Iodine forms a coordination complex between the helically
coiled polysaccharide chain and iodine centrally located within the helix due to
absorption. The color obtained depends upon the length of the unbranched or
linear chain available for complex formation.

Benedict’s Test Principle: Carbohydrates with free aldehyde or ketone groups can
reduce solutions of various metallic ions. Reducing sugars under alkaline
conditions tautomerize and form enediols. Enediols are powerful reducing agents
and can reduce cupric ions to cuprous form and are themselves converted to
sugar acids. The cuprous ions combine with OH- ions to form yellow cuprous
hydroxide which upon heating is converted to red cuprous oxide.

Barfoed’s Test Principle: Aldoses and ketoses can reduce cupric ions even in
acidic conditions. This test is used to distinguish reducing monosaccharides from
disaccharides by controlling pH and time of heating. Monosaccharides react very
fast whereas disaccharides react very slowly.
 RCHO + 2Cu2+ + 2H2O → RCOOH + Cu2O↓ + 4H+

Seliwanoff’s Test Principle: Keto hexoses on treatment with hydrochloric acid

from 5-hydroxy methyl furfural which on condensation with resorcinol gives a
cherry red colored complex.

Bial’s/Orcinol Test: The test reagent dehydrates pentoses to form furfural.

Furfural further reacts with orcinol and the iron ion present in the test reagent to
produce a bluish product.

Mucic Acid Principle: Galactose is converted to Saccharic acid on heating with

HNO3(a strong oxidizing agent). Saccharic acid which is insoluble in cold water
and thus helps in the identification of galactose.

VII. Conclusion

A carbohydrate is an organic compound with the general formula Cm(H2O)n, that

is, consists only of carbon, hydrogen and oxygen, with the last two in the 2:1 atom
ratio. Carbohydrates make up the bulk of organic substances on earth and perform
numerous roles in living things. The carbohydrates (saccharides) are divided into four
chemical groups: monosaccharides, disaccharides, oligosaccharides and
polysaccharides. Polysaccharides serve for the storage of energy (e.g., starch in
plants and glycogen in animals) and as structural components (e.g., cellulose in
plants and chitin in arthropods). Structural polysaccharides are frequently found in
combination with proteins (glycoproteins or mucoproteins) or lipids
(lipopolysaccharides). The 5-carbon monosaccharide ribose is an important
component of coenzymes (e.g., ATP, FAD and NAD) and the backbone of the genetic
molecule known as RNA. For the hydrolysis of Polysaccharide, five samples (starch,
dextrin, cotton, chicken liver, and shell filtrate) were added with three drops of
concentrated HCl , the samples were then covered with marbles and were boiled for
an hour. The same procedure with the hydrolyzation of gum-arabic and agar-agar it
was added with 2mL of conc. HCl.
For the general test for carbohydrates, two tests were performed, Molisch Test
and Anthrone Test. In Molisch’s Test all samples gave a positive result. Molisch’s Test
is a sensitive chemical test for all carbohydrates, and some compounds containing
carbohydrates in a combined form, based on the dehydration of the carbohydrate by
sulfuric acid to produce an aldehyde (either furfural or a derivative), which then
condenses with the phenolic structure resulting in a red or purple-colored compound.
Anthrone test is also another general test for all carbohydrates. In this test also,
carbohydrate gets dehydrated when react with conc. H2SO4 to form furfural. This
furfural reacts with anthrone to give bluish green colored complex. Iodine test is an
indicator for the presence of starch. Iodine solution (iodine dissolved in an aqueous
solution of potassium iodide) reacts with starch producing a blue-black color. Starch
gave blue-black color which is an indication for positive test. In Benedict’s Test, it is a
test to determine whether the carbohydrate contains a free aldehyde or ketone
group. In Benedict's reaction, copper is reduced, and the product forms a red
precipitate. (Benedicts reagent contains sodium bicarbonate, sodium citrate and
copper sulfate). Almost all the samples gave positive results for Benedict’s Test
except for the hydrolyzed cotton since it gave a yellow precipitate.

Barfoed’s Test is a test that can distinguish monosaccharides from di- and
polysaccharides because with the conditions of lower pH and shorter incubation time,
only monosaccharides can react fast enough to reduce copper ions. The reagent is
similar to Benedict's except that the pH is lower (around pH 4.5). The samples that
gave a positive result are glucose, galactose, fructose, arabinose, and dextrin.
Seliwanoff’s Test distinguishes between aldose and ketose sugars. Ketoses are
distinguished from aldoses via their ketone/aldehyde functionality. If the sugar
contains a ketone group, it is a ketose and if it contains an aldehyde group, it is an
aldose. This test is based on the fact that, when heated, ketoses are more rapidly
dehydrated than aldoses. This test gave a positive result to fructose and sucrose with
the presence of red color. Bial’s/Orcinol test is used to distinguish between pentoses
and hexoses. They react with Bial’s reagent and are converted to furfural. Orcinol and
furfural condense in the presence of ferric ion to form a colored product. Appearance
of green colour or precipitate indicates the presence of pentoses, and formation of
muddy brown precipitate shows the presence of hexoses. Lastly, the Mucic acid test
is highly specific for galactose which is either independently present in solutions or
obtained by the hydrolysis of lactose.