Lab 1: Carbohydrate Qualitative tests

1. Molisch’s Test:
It is the general test for all carbohydrates. Monosaccharides give a rapid positive test. Disaccharides and
polysaccharides react slower. WHY?
Principle:
The reaction is due to the formation of Furfural and it’s derivatives by the dehydrating action of acid on
sugar in the event of Sugar being (poly saccharide or disaccharide) the acid first hydrolyzes it into
monosaccharides and then acts as a dehydrating agent.

The Molisch reagent dehydrates pentoses to form furfural. It dehydrates hexoses to form 5-hydroxymethyl
furfural. The furfurals further react with α-naphthol present in the test reagent to produce a purple product.

■ Method:
• 1ml test solution + 2 drops of α-naphthol
• mix well
• Add conc. H2SO4 down the side of the tube to form the ring at the interface of the two layers.

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-ve +ve

Lab results: Glucose, maltose, arabinose and starch will all display the purple ring compound at the interface of
the acid and solution.
Limitations:
-Not specific to carbohydrates
-Generalized test that cannot distinguish carbohydrates and further testing must be undertaken to identify the
carbohydrate.

2. Benedict’s Test:
All reducing sugars give positive Benedict's Test
Principle: reducing sugars have free aldehyde or keton group, it under go converting in to enediol forms
under hot alkaline condition. The enediol are strong reducing agents converting cupric ions (Cu+2) of
the Benedict's solution into Cuprose ion which altimately oxide as a red precipitate red copper(I) oxide
by aldehydes.
• Benedict's reagent contains blue copper(II) sulfate (CuSO4) · 5H2O which is reduced to red
copper(I) oxide by aldehydes, thus oxidizing the aldehydes to carboxylic acids.
• The copper oxide is insoluble in water and so precipitates. The color of the final solution ranges from
green to brick red depending on how many of the copper (II) ions are present.
•

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Hot alkaline
medium Hot alkaline
medium

■ Method:

• 1ml test solution + 1ml Benedict's reagent

• heat the mixture in Boilingf Water Bath for
(5min)
• Reddish brown ppt.

3. Barfoid’s Test:
The test is used to distinguish between monosaccharides and reduced disaccharides

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• This reaction will detect reducing monosaccharides in the presence of disaccharides. Reagent uses
copper ions to detect reducing sugars in an acidic solution. Barfoed's reagent is copper acetate in dilute
acetic acid (pH 4.6)

• Reducing monosaccharides are oxidized by the copper ions in a weak acidic medium to form a
carboxylic acid and a reddish ppt of Cu2O (cuprous oxide).

• Reducing disaccharides (lactose but not sucrose) undergo the same reaction but at slower rate.
Therefore, boiling time is critical and positive test within ( 2min.) indicate the monosaccharides in case
of disaccharides they are first converted into monosaccharide and then colored precipitate are formed.

• So the porolonged boiling may hydrolyses disaccharides to give a false positive test.

• Method:
• 1 ml of the solution to be tested + 2 ml of freshly prepared Barfoed's reagent.

• Place test tubes into a boiling water bath and heat for 2 minutes.
• Remove the tubes from the bath and allow to cool.
• Formation of a green, red, or yellow precipitate is a positive test for reducing monosaccharides.
• Do not heat the tubes longer than 3 minutes, as a positive test can be obtained with disaccharides if they
are heated long enough.

-ve
+ve

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4. Seliwanoff’s Test:
The test reagent dehydrates ketohexoses to form 5-hydroxymethylfurfural

HCl
-3H2O

5-hydroxymethylfurfural

5-hydroxymethylfurfural further reacts with resorcinol present in the test reagent to produce a red product
within two minutes.

Resorcinol red product

Note:
1. Aldohexoses react to form the same product, but do so more slowly.
2. In case of sucrose, avoid over-boiling because sucrose may be hydrolyzed to its component (glucose
and fructose) and gives false positive result.
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■ Method:
• 1/2 ml of a sample + 2ml of Seliwanoff's reagent (a solution of resorcinol and HCl) is added.
• The solution is then heated in a boiling water bath for two minutes.

• A positive test is indicated by the formation of a red product.

-ve +ve

Lab result: Fructose is a ketose sugar that will react with the reagent to form the cherry-red complex after 1
minute. The Glucose solution will also form a pink to red complex but not after 1 minute. It will form the
complex after a longer period.

Limitations:

Although this test is able to adequately distinguish a ketose sugar from an aldose sugar, it is not very specific
since an aldose may also form the complex. The difference is the time taken to do so.

This test also is a generalized test that does not differentiate the specific ketose present but rather illustrates that
a ketose sugar is present. Specific ketose sugar identification must be performed by further testing.

5. Iodine’s Test:
(Test for Polysaccharides)
Poly Saccharides adsorb I2
And form colour complex Starch gives blue colour while
glycogen gives reddish-brown colour.

Note: On heating (poly saccharide-Iodine) complexes

losses blue colour because of dissociating the complex

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while on cooling the blue colour again appears due to re-association of complex
a- Starch:
• 1/2 mL of the fresh starch solution + 1 drop of the iodine solution.
• A dark blue color indicates a positive test for starch.
• If the yellow color of the iodine reagent simply becomes diluted, no starch is present.
• Record the observation as positive (blue) or negative (yellow).
b- Dextrin:
• 1/2 mL of the fresh dextrin solution + 1 drop of the iodine solution.
• A violet color indicates a positive test for dextrin.
• If the yellow color of the iodine reagent simply becomes diluted, no dextrin is present.
• Record the observation as positive (violet) or negative (yellow).
6. Bial’s Test:
This test is used to distinguish pentose sugars.
The reaction: Bial reagent contains HCl, orcinol and ferric chloride. The pentose sugars are hydrolysed by the
HCl to form a furfural derivative. This derivative then reacts with the orcinol to form a green -yellow complex
in the presence of ferric ions via a condensation reaction. Polysaccharides made up of pentose units are
hydrolysed to break the glycosidic bonds and then undergo the same reaction to form the complex. Hexoses are
also hydrolysed and react with the orcinol but form a red to brown complex rather than a green-yellow
colouration.

Procedure: right=+ve
Left:-ve
1. Take 1 ml of Standard and Test sugar solutions in four test tubes
2. Add 2.5 ml of Bial’s reagent to each tube and heat in a boiling water bath for 1 minute and allow the tubes
to cool down to room temperature. Look for the formation of blue-green colour.

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Lab results: Arabinose is a pentose sugar that will yield the green yellow colour change. Gum Arabic is a
polymer of arabinose, rhamnose and galactose and therefore when hydrolysed, will react similar to arabinose
and give the green-yellow colour change. However, glucose is a hexose sugar and therefore will not react
similar to arabinose and will therefore not yield a green colour change. Rather it may produce a red to brown
colour change or no change at all.
Limitations:
It is not specific to which pentose is present and further tests must be conducted to identify specific pentose
sugar.

7. Phenylhydrazene’s Test (Osazone Test)

Osazones are formed when the sugars (monosaccharides) react with a compound known as
phenylhydrazine. These sugars are reducing ones which have either a free aldehyde or a ketone group to
react with the phenylhydrazine. This reaction is complete in 3 step and consume 3 moles of
phenylhydrazine

During reaction with monosaccharides, additional phenyl hydrazine is consumed in oxidizing the
adjacent OH-group to carbonyl group which then forms a second phenyl hydrazone.

Such bisphenyl hydrazones are called osazones.

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H H
C O C NNHPh
H OH C NNHPh
H HO H
HO H
H OH + xs H2NN H OH
H OH H OH
CH2OH
CH OH
2 phenylosazone

D-glucose
phenylhydrazine of D-glucose

Mannose, a pentahydroxyaldehyde that differs in configuration from glucose only at C-2, gives an osazone with
exactly the same structure as that of the osazone of glucose

How can these reactions help us determine whether we have glucose or mannose as an unknown? The answer is
by timing the reactions. The

osazone from glucose forms in approximately five minutes; whereas, the osazone from mannose forms in less
than one minute. Thus, if we allow our unknown glucose or mannose to start forming an osazone at exactly the
same time we start osazone formation with authentic samples of glucose and mannose, our unknown will form
its osazone at the same time as one of the known compounds but not the other.

Osazone is a crystalline compound with a sharp melting point will be obtained

Since only C1 & C2 of a saccharide are involved in osazones, sugars with the same configuration at the
remaining carbon atom gives the same osazone.

D-fructose and D-mannose give the same osazone as D-glucose

seldom used for identification; we now use HPLC or mass spectrometry

Depending on the time required to form the insoluble yellow osazone, different sugars can be
classified into the following:

Mannose: 1-5 min

Fructose: 2 min

Glucose:5 min

Xylose: 7 min

Arabinose: 10 min

Galactose: 20 min
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Maltose osazone soluble in hot water

Reagents:

1% solutions of glucose, fructose, maltose, mannose, and xylose

Phenyl hydrazine mixture (2 parts phenyl hydrazine hydrochloride are mixed with 3 parts
sodium acetate).

To 300 mg of phenyl hydrazine mixture add 5 ml of the tested solution,

Shake well, and heat on a boiling water bath for 30 – 45 min.

 Allow the tubes to cool slowly (not under tap) and examine the crystals microscopically, draw the
shapes of the crystals

Glucosazone fructosazone

Xylosazone Galactosazone

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8. Hydrolysis of Starch using Saliva or mineral acids:

Outline. The experiment illustrates the conversion of starch to a reducing sugar by the action of
hydrochloric acid at boiling point. The longer the starch is exposed to the acid the further hydrolysis
proceeds. The experiment is intended to show the contrast with enzymes, which do not need high
temperatures and prolonged exposure to reagents and give a quick reaction.

Objective
It demonstrates the hydrolysis of starch into glucose.

 Polymers are broken down by hydrolysis which is essentially the reverse of condensation.
 An –OH group from water attaches to one monometer and a H attaches to the other.
 This is a hydrolysis reaction because water (hydro) is used to break (lyse) a bond.
 When a bond is broken, energy is released.
 Polysaccharides such as starch, dextrin and glycogen, give positive iodine test.
 Starch is a non reducing polysaccharide therefore it does not give positive result with Benedict’s,
Fehling’s and Barfoed’s reagents, nor does it form any Osazone.
 However after hydrolysis into monosaccharide by the actions of strong acid , its components (glucose
molecules) give all the test positive.
PROCEDURE
 Take test tubes and label them as ‘T’ (for test) ,Take 15ml of starch sol in test tube
 Add 10 drops of conc. HCl in test tube ‘T’
 Put test tube in boiling water bath and start performing iodine test after every min in an indicator dish by
taking 1 drop of iodine and 1 drop of starch sol till the iodine test becomes –ve in test tube ‘T’
 Heat test tubes for 2 min more
 Take out test tube from boiling water bath, then cool them at room temperature
 Add very small quantity of solid sodium carbonate in test tube

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 Continue to add sodium carbonate till the effervescence stops in test tube ‘T’
 Now from test tube take small quantities in separate test tubes to perform Benedict’s ,Selivanoff’s and
Osazone tests.
INTERPRETATION
In test tube ‘C’ Benedict’s, Selivanoff’s, Osazone test remain –ve because due to absence of conc. HCl in this
test tube, the starch is not hydrolyzed. In this test tube iodine test will also remain +ve.

In test tube ‘T’ Benedict’s, Selivanoff’s , Osazone test become +ve because due to presence of conc. HCl in this
test tube the starch is hydrolyzed into glucose, which is a strongly reducing monosaccharide.

Although acid completely hydrolyze starch to give glucose but this process occurs through various
stages.Before complete hydrolysis it gives various products which react with iodine and produce different
colors.

Stage of hydrolysis Color

Starch(insoluble) Blue

Starch(soluble) Blue

Amylodextrin Blue purple

Erythrodextrin Red

Achrodextrin No color

Maltose No color

Glucose No color

PRINCIPLE
 Heating of starch in the presence of conc. HCl causes its hydrolysis into glucose. because glucose have free
Aldehyde group, therefore it is a strongly reducing monosaccharide, and hence Benedict’s, Selivanoff’s and
Osazone tests become positive.
 Sodium carbonate is added to neutralize excessive HCl, because the reducing ability of reducing sugars is
high in alkaline medium, and hence gives good results of Benedicts ,Selivanoff’s and Osazone tests.
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 Erythrodextrin give red colour.its further hydrolysis produces achrodextrins, which gives negative iodine,
test. When the iodine test becomes negative, we heat test tubes for two minutes more.The reason being is to
provide time to complete hydrolysis of achrodextrin into maltose and maltose into glucose.

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Lab2: Carbohydrate quantitative tests
Test: Determination of Glucose using Benedict’s Solutions

1. Estimation of Glucose by Benedict’s Method:

During qualitative analysis of sugars we have already learnt that glucose reduces copper sulphate in Benedicts
reagent under alkaline conditions and a red precipitate is formed. This qualitative method has been exploited for
its use in quantitative analysis.

The Benedicts quantitative reagent contains the following ingredients, copper sulphate, sodium carbonate,
sodium or potassium citrate, potassium thiocyanate and potassium ferrocyanide. Of these, copper sulphate has
to be very accurately measured as the amount of copper sulphate reduced will correspond to the amount of
glucose present in solution.

Due to presence of potassium thiocyanate in Benedict’s reagent a white precipitate of cuprous thiocyanate
instead of red precipitate of cuprous oxide will be formed when copper sulphate is reduced. As the precipitate
formed is white it is very easy to determine the end point. Blue tint of Benedict’s reagent disappears completely
at this point.

The small amount of potassium ferrocyanide added helps to prevent the oxidation of cuprous oxide. Sodium or
potassium citrate added does not allow the formation of copper carbonate. The alkaline condition is produced
by sodium carbonate which is a mild alkali in comparison with NaOH and is, therefore, less destructive for the
sugar. The Bennedict’s reagent prepared as follows is stable for long periods of time.

To prepare quantitative Benedict’s reagent 18.0 gm. of crystalline copper sulphate is dissolved in 100 ml of
water (solution A). Further, 100 gm. of sodium carbonate, 200 gm. of anhydrous sodium citrate and 125 gm. of
potassium thiocyanate are dissolved in 800 ml of water with heating (solution B). If solution B is not clear it
should be filtered. Solution A is added slowly to solution B with stirring. Then 5 ml of potassium ferrocyanide
solution is added and the volume is finally made up to 1 litre after cooling.

The reaction of CuSO4 with glucose is quite complicated and a number of molecules of CuS0 4 are reduced by
one molecule of glucose. Therefore, it is not possible to write the stoichiometric equation for reaction between

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CuSO4 and glucose. But it has been found that 25 ml of the above mentioned quantitative reagent corresponds
to 50 mg glucose. Determination of the unknown amount of glucose will be based on this.
Procedure:
Pipette out in a conical flask 25 ml of the Benedict’s quantitative reagent. Add about 5 to 10 gm. of Na 2CO3
and a few porcelain chips to the flask to prevent bumping. Heat the contents of conical flask to boiling and then
run in the glucose solution from a burette at first rapidly and then slowly until the blue colour becomes fade.
Allow it to boil for 2-3 minutes more and add glucose solution drop by drop till the solution becomes
colourless. Note down the volume of the glucose solution used and calculate the percentage of glucose in
solution as described below. Sometimes the solution in the flask becomes too much concentrated due to
evaporation of water. To avoid it more water may be added.

Suppose 20 ml of the glucose solution is required to titrate 25 ml of Benedict’s quantitative reagent. As 25 ml of

the Benedict’s quantitative regent is equivalent to 50 mg of glucose, hence 20 ml of the solution contains 50 mg
of glucose. Therefore, 100 ml of the glucose contains 50×100/ 20 =250 mg of glucose and the strength of the
solution 250 mg per cent.

Estimation of Lactose by Benedict’s Quantitative Regent:

Principle is same as for glucose, only difference being 25 ml of Benedict’s quantitative regent is equivalent
to 67 mg of lactose.

Even sucrose after acid hydrolysis can be estimated by this method.

2. Glucose Oxidase Method for Estimation of Glucose:

In this method, the aldehyde group of β-D-Glucose is oxidized by glucose oxidase to give gluconic acid and
hydrogen peroxide.

β-D-Glucose + H2O + O2 → gluconic acid + H2O2

The hydrogen peroxide may be broken down to water and oxygen by a peroxidase and if an oxygen acceptor is
present, it will convert to a coloured compound which can be measured. The reagent usually used is oxidation
product of phenol condensed with 4-aminophenazone to give a coloured product as in determination of alkaline
phosphatase.