Post Lab Conference on Carbohydrates

CARBOHYDRATES:
Organic compounds containing hydrogen and oxygen with the ratio the same as in water,
thus called hydrates of carbons.
The definition is not perfect because there are compound that comply with the
definitions but are not carbohydrate. Example acetic acid (CH3COOH).
Carbohydrates are aldehyde or ketone derivatives of polyhydricalcohol.
COLOR REACTIONS OF CARBOHYDRATES:
A. General Test for Carbohydrates:
* The presence of carbohydrates in a preparation can be detected by either the
Molisch Test or Anthrone Test
* Both tests are based on the hydrolyzing and dehydrating action of
concentrated
mineral acids.
* In these tests the concentrated mineral acid catalyzes the hydrolysis of any
glycosidic bonds present in the carbohydrate sample and the dehydration of
furfurals (pentoses[ or hydroxymethyl furfurals [hexoses].
* These furfurals then condense with the organicphenols to form
colored products.
1. Molisch's Test:
* Reagents: Alcoholic alpha naphthol andconcentrated sulfuric acid is allowed to
flow along the side of the test tubes containing the carbohydrate sample
* Result:A violet or purple colored ring at the point of junction between the
carbohydrate solution and the acid.
2. Anthrone's Test:
Isa very sensitive test.
It willgive a positive reaction with filter paper [cellulose].
Anthrone reaction had been adopted for the quantitative determination of
glycogen, inulin and blood sugar in the laboratory.
* Reagents: Anthrone and concentrated sulfuric acid.
* Result: Ablue green colored complex.
* All carbohydrates and glycoprotein give positive reaction with Molisch and
Anthrone's Tests, except triose and tetrose sugars.
B. Test for Ketoses:
1. Seliwanoff's Test:
*A timed color reaction specific test for ketose sugars.
* With concentrated hydrochloric acid. Ketoses undergo dehydration to yield
furfural derivatives more rapidly than aldoses.
* The furfural derivatives condense with resorcinol to form colored products.
* Will differential aldose from ketose sugar.
* Aldose sugar willalso give a positive reaction with Seliwanoffs Test when the
concentration is high or on prolonged heating time.
* For comparative purposes, tubes containing different sugar solution should be
immersed simultaneously in the same water bath.
*A positive result is a cherry red colored complex.
C. Test for Reducing Property of Sugars:
The reducing property of sugars is dependent on the presence of an actual or
potentially free aldehyde or ketone group in the molecule.
* When solution of such sugars is heated in the presence of certain metallic ions, the
active group is oxidized and the metallic ions are reduced to a lower valence state.
*The reagent used are solution of metallic saltsS, usually in an alkaline medium mixed
with an organic compound containing alcoholic groups.
*The salt of copper, mercury, bismuth, and silver had been used.
* Sodium potassium tartrate (Rochelle salt], sodium citrate or glycerol containing one
or more hydroxyl are additional reagents.
Sugars containing free aldehyde or ketone group or potentially free reducing
group [that is. hemiacetal or hemiketal carbons] are subject to oxidation by a
 variety of general
*
oxidizinsuchg agents.
Whereas, sugars lacking
* glycosidic linkage, as in groups (for example, those containing only
The initial attack of sucrose] are resistant to these agents.
silver, the general oxidizing
ferricyanide,
of the sugar
* Thus,
agents (for example, cupric, bismuth,
and aromatic nitro Compounds]
[hence, the term reducing groups. occurs at the reducing group
sugars
contaitheningadded the
Since, they reduce these easily oxidized groups are called "reducing sugars,"
Aiter the initial attack.. subseguent oxidizing agents.
oxidation and isomerization may condinue dom
ne carbon chain yielding a large numberof products with ow molecular weight
All monosaccharides and disaccharides are reducing
sugars, except sucrose.
Polysaccharide with four or more monosaccharides units are non- reducing.
1. Benedict's Test:

Benedict'stest solution contains sodium citrate and anhydrous sodium carbonate

and copper sulfate. The cuprous hydroxide Cu[OHJ2 forms a soluble complex with
the alcoholic group of sodium citrate.
Benedict's solution is not readily reducible by non-carbohydrate reducing substances
such as uric acid, creatine, conjugated glucuronate, and other productsoccurring
normally in the urine.
* Positive result varies depending on the concentration of the reducing sugars.
Apple green [traces], yellow [plus 1], orange [plus 2] or red [plus 3].
2. Barfoed's Test:

* This is a copper reducing test but different from Benedict's test in that the reduction
of metallic ions occurs in an acidic medium.
* Barfoed's reagent contains copper sulfate and acetic acid.
* In making use of the Barfoed's reagent advantage is taken of the fact that sugars are
less reactive in acidic than in alkaline medium and also disaccharides are less reactive
than monosaccharides.
*The test as performed is used to differentiate monosaccharides from disaccharides.
* Prolonged heating, however, may give positive reaction with disaccharides.
*To ensure correct reading of the test, the bottom of the test tube be carefully
inspected for the presence of a scanty brick red precipitate in a positive result.

Glucose P
AMall:oger (moniosacchario e)

3. Nylander's Test
* Nylander's reagent: bismuth nitrate, potassium sodium tartrate and ammonium
 hydroxide
Positive result, black precipitate of metallic bismuth
4. Tollen's Test
* Reagent include ammoniacal solution of silver nitrate
*Carbohydrates react with Tollen's reagent and form asilver mirror on the inner walis
of the test tube.
Silver ions are reduced to metallic silver

5. Picric Acid Test:

carbonate
* Reagent include saturated picric acid solution and sodium
* Picric acid is also called trinitrophenol
* Reaction involves reduction of nitrophenol to aminophenol, resulting to picramic acid
which is colored red.
* Creatine will also give positive reaction to the test.
6. Moore's Test:
* Reagent: sodium hydroxide
*Test is based on the ability of reducing sugars to undergo oxidation in alkaline
solution.
Inthe presence of an alkali (sodium hydroxide], reducing sugars undergo
tautomerization toform a resinous substance called "caramel."
* Positive result varies in yellow, orange and brown color.
D. OXIDATION OF CARBOHYDRATES:
1. Mucic Acid Test:
*Concentrated nitricacid has a unique property that it oxidizes two terminal
carbons of aldohexoses, forming the corresponding dicarboxylic acids [saccharic
acidJ.
* In general,saccharic acid are soluble in dilute nitric acid, but mucic acid, the
particular saccharic acid formed by galactose (either free or as released by
hydrolysis] is insoluble in dilute acids.
* Thus, the formation of an insoluble saccharic acid when concentrated nitric acid
 is heated with a carbohydrate and subsequently diluted signifies the presence of

galactose and hydrolyzed lactose

2. Phenylhydrazine Reaction:
phenylhydrazine
* Most sugars have a free carbonyl group which can react with therefore
oluble in water, and
to form phenylhydrazones, which however, are
has little use in the identification of sugars.
adjacent to the carbonyl
*In the presence of excess phenylhydrazine, the carbon
which can condense with another
group is oxidized to another canbonyl group
Compounds known as osazones.
molecule of phenylhydrazine to form yellow CHO
CH20H
CHO
HÌ -C-H
H-C-OH C=0
HO -C-H
HO -C-H
HO - C-H
H-C-0H
H-C-OH
H-C-OH
H-C- OH
H-C-OH
H-C-OH

CH20H
CH20H
CH20H Mannose
Fructose
Glucose
Phenylhydrazine Reaction: following
compounds for the identification of sugars for the
Osazones are valuable
reasons:
each osazone is guite detinite.
1. The time formation of
relatively insoluble.
2. Some osazones are
specific forms.
3. Osazone crystallizes in dried, has definite
osazone, if carefully purified by recrystallization and
4. Each
melting time. CH.NHC6H5
CH.NHC6H5
CH.NHC6H5

CH.NHC6H5
CH.NHC6H5 CH.NHC6H5

HO-C-H HO -C-H
HÌ -C-H

H-C-OH H-C-OH
H -C-OH

H- C-0H H-C-OH
H-C-OH

CH20H CH20H
CH20H
Fructosazone Mannosazone
Glucosazone
Osazone Crystals:
* Glucosazone - needle shaped or broomstick crystals,
* Galactosazone - rhombic plates
* Lactose - powder puff or hedged hog crystals
* Maltosazone - sun flower shaped
* Glucose, fructose and mannose will yield similar osazone.
Time Formation for Osazone:
* Fructosazone crystals form in 2 minutes,
* Glucosazone in 4 to 5 minutes,
* Galactosazone in 7 minutes,
* Maltosazone in 10 to 15 minutes.
*Sucrose in 30 to 35 minutes [after being hydrolyzed]
 solubility of Osazone:
# Both lactosazone and
maltosazone are soluble in hot water.
E. INTERCONVERSION OF SUGARS:
* Aldoses in the presence of a weak alkalimavy be converted into
ketoses, or otner
aldoses or vice versa
*Hence, starting with either glucose, mannose or fructose, after interconversIon
at equilibrium, there will be presence of equal amounts of the three sugars.
Seliwanoff's Test
Before Inversion After Inversion
Glucose +Ba•OH]2 Yellow Orange
Glucose +Water Yellow Yellow
Fructose +Ba[OH]2 Red Orange
Fructose + Water Red Red
F. INVERSION OF SUCROSE:
fructose
*Invert Sugar - isa solution of eguimolar mixture of glucOse and
by the enzyme invertase or by
resulting from the hydrolysis of sucrose catalyzed
an acid and heat
After Inversion
Before Inversion
Positive
Benedict' Test Negative
Positive
Barfoed's Test Negative
Positive
Seliwanoff's Test Negative
POLYSACCHARUDES:
1. Starch- the storage form of carbohydrate in plants.
A. Reactions of Starch:
* Results: lodine test: Blue color.
Benedict's test: negative
B. Hydrolysis of Starch:
lodine Test Benedict Test
Deep blue Negative
Starch
Deep blue Negative
Amylodextrin
Purple Negative
Amylose
Erythrodextrin Red Negative
Achrodextrin Colorless Negative
Colorless Positive
Maltose
Colorless Positive
Glucose
C. After Hydrolysis:
*lodine test; Colorless. Benedict' test: positive
*Phenylhydrazine test:Glucosazone
2. Glycogen:
*the storage form of carbohydrate in animals
*stored mainly in the liver and the skeletal muscles.
A. Preparation of Glycogen from animal liver
B. Reactions of Glycogen:
*Results: lodine test: Reddish brown. Benedict's test: negative
C. Hydrolysis of Glycogen:
* Attained after boiling
* After hydrolysis, perform lodine test and Benedict's Test.
* Results: lodine test: Colorless.
Benedict's test: positive
Phenylhydrazine test: Glucosazone crystals.