Practical Biochemistry I

Prepared by:
Dr. Arwa Yahya
Dr. Faiza Ghlab
Dr.Najlaa Amer
Practical Biochemistry I

Carbohydrates identification tests

# Introduction:-
Carbohydrates are white ,crystalline substance with sweet taste (poly saccharides
are taste less), they are soluble in water.

# Classification :-
1- Whether the CHO can be breakdown to small unit or not by hydrolysis.

→ Monosaccharaides ( simple sugar)

→ Disaccharides
→Oligosaccharides
→ Polysaccharides

2- No. of carbon
if 3 -----→ Triose
4 -----→Tetrose
5 ----→ pentose and so on ……

3-Functional group
- Aldoses
- Ketoses

Monosaccharaides

1- Glucose: -
- The most abundant hexose in our diet.
- The major metabolic fuel of mammals.
- The precursor for synthesis of all other CHO in the body.

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2- Galactose: -
- Found in lactose.
- Found in cell membrane of CNS.

3- Fructose :-
-Sweet CHO .
- Ketosugar
- Found in sucrose .

Disaccharides

1- Maltose (malt sugar)

2- Lactose (milk sugar)
3- Sucrose ( table sugar)

polysaccharides

1- Glycogen 2- Starch 3- Cellulose 4- Dextrin.

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CHO identification tests

1-Molisch’s test: (general for all CHO)

Principle

Concentrated sulphuric acid (H2SO4) hydrolyses glycosidic bonds to give the

monosaccharides which are then dehydrated to furfural derivatives (Pentoses are
then dehydrated to furfural, while hexoses are dehydrated to 5
hydroxymethylfurfural). These products then combine with sulphonated -
naphthol to give a purple complex.

Caution:
Molisch’s reagent contains concentrated sulfuric acid, which is toxic and corrosive. It can
cause severe burns. Prevent eye, skin, clothing, and combustible material contact. Avoid
ingesting the substance. If you spill any reagent or acid, immediately notify your laboratory
instructor.

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Procedure Observation Result

1- Add two drops of the
-naphthol solution to 1 ml • Reddish-violet ring • presence of CHO
of unknown solution between two layers (Pentose, hexose
form).
2-Then carefully pour
about 2 ml conc. H2SO4
down the side of the tube
so as to form two layers.
• No reddish-violet • Absence of CHO
3-Then, observe any color ring between two
change at the junction of layers
the two liquids.

2-Iodine test: (Specific for Polysaccharides (Starch or glycogen)

Principle
The amylose component of starch has helical structure, when treated with iodine
solution, Iodine trapped inside the coil and the complex has intense blue color.

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Procedure Observation Result

Add 3 drops of iodine • Dark Blue/Black • Presence of Starch

solution to 1 ml of color solution
unknown solution
• Reddish brown • Presence of
color solution glycogen

• Yellow color • Not polysaccharides

solution

3-Barfoed’s test: (specific for monosaccharaides)

Principle
By controlling PH & time of heating, the aldoses and ketoses can reduce the cupric
ion to cuprous in acidic medium.

Procedure Observation Result

1- Add 1 ml of • Brick red • Presence of
Barfod’s reagent to1 ml precipitation at bottom monosaccharide
o of the unknown solution of tube before 5min.

22- boil for 5 min, and • Non change • Presence of

allow to stand before 5min disaccharide
Caution:
Barfoed's reagent is corrosive and an irritant. If you spill any of the solution on yourself or on the
bench, immediately notify your laboratory instructor.

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4- Benedict’s Test (specific for reducing sugar)

Principle
The free aldehyde or ketone group in the CHO sol. Can reduce cupric ions to
cuprous in alkaline condition. The cuprous ion combines with OH- ion to form
yellow cuprous hydroxide, which up on heating is converted to red cuprous oxide.

Procedure Observation Result

1-Add 1 ml of Benedict's • Red, brown, • Reducing sugar

reagent to 1ml of the yellow or green
unknown solution color solution

2- put it in a water bath • Non reducing sugar

for 3 min • No change

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5- Seliwanoff ’s Test: (specific for ketohexose sugar)

Principle: -
Ketohexoses on treatment with HCL form 5- hydroxy methyl furfural, which then
condensed by resorcinol to give a cherry-red colored complex.

Procedure Observation Result

1-Add 2 ml of • Cherry – red color • This solution is

Seliwanoff's reagent to solution ketohexose (fructose)
1ml of unknown solution

2- warm in a boiling
water bath for 5 • Non cherry – red • This solution is not
min. color solution ketohexose

Caution:
Seliwanoff's reagent is toxic and corrosive. If you spill any of the solution on yourself or on
the bench, immediately notify your laboratory instructor.

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Scheme for unknown carbohydrate solution

Molish test

Violet ring (CHO) No violet ring ( No CHO)

Protein or lipid

Iodine test

Blue black color ( Starch) No blue black color

( Not polysaccharide )

Barfoed’s test

No red ppt (Diasaccharide) Brick red ppt.

(monosaccharide)

Benedict's test
Seliwanoff's test

No yellow – Yellow ,brown,

red-green
red, green Ketohexose ( fructose)
No reducing (reducing sugar No red color
sugar (sucrose) disaccharide No ketohexose
(lactose or
maltose)

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Proteins identification tests

1-BiuretTest: (general test for all protein by detect the presence of peptide bond)
Principle
Cupric ion in an alkaline medium react with compound containing 2 peptide
bond or more to form violet color complex. The reaction is not absolutely
specific for peptide bonds, since any compound containing two carbonyl
groups linked through a nitrogen or carbon atom will give a positive result.

Procedure Observation Result

1 ml of the unknown
solution followed by 1 ml • Violet color • This solution is protein
of NaOH and 1ml of solution
cuso4; mix then note the
colors produced. • No violet color • Not protein

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2-Xanthoproteic reaction: Detect presence of amino acid which contain an

aromatic nucleus  their salt [glycine, tyrosine, tryptophan and phenylalanine]

Principle
Amino acids which contain an aromatic nucleus form yellow nitro derivatives on
heating with concentration nitric acid. The salts of these derivatives are orange.

Procedure Observation Result

Add an equal volume of
conc HNO3 to about 0.5
ml of the amino acid • Yellow color
solution, cool, and
observe the color change.

Add sufficient NaOH to This solution contain a

make the solution an aromatic nucleus
strongly alkaline.
A yellow color in acid
solution which turns • orange color
bright orange with
alkali constitutes positive
result. Repeat the test
with the Phenol

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Practical Biochemistry I

3- Isoelectric point: (for identification of casein)

Principle
At isoelectric point the protein is become less soluble, it will precipitate when
reached to its isoelectric point by adding acid or alkaline.

Procedure Observation Result

1 ml of the unknown
solution followed by 3 • green color • Casein is present
drops of BCG then add a precipitate
few drops of acetic acid
• No green color • Not Casein
precipitate

4- Glyoxylic reaction (specific for identification of Peptone)

Principle
Glyoxylic acid react with Indole group of tryptophan or protein containing
tryptophan in the presence of sulfuric acid formed violet ring.

Procedure Observation Result

Add 2 ml of glyoxylic
acid to 1 ml of the test • Violet-ring at • This solution is Peptone
solution, then pour about junction of two
2 ml of conc. H2SO4 layers
carefully down the sides
of a sloping test tube so
as to form two layers. • No violet-ring at
junction of two • This solution is Gelatin
Observe any color change layers
at the liquid junction.

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Practical Biochemistry I

Lipids identification tests

lipid

Fat (animal derive) oil (plant derive)

• Consist of glycerol + • Consist of glycerol +
high number of saturated FA. high number of unsaturated
FA.
• Solid. • Liquid.

- Properties: -
• Soluble in organic solvent as ether, chloroform.
• Insoluble in water.

1- The solubility of lipids (Detect the solubility of lipids in the following solvents
[water, chloroform, ether and alcohol]

Principle
Solubility, the main groups of lipids have different solubility characteristics and
this property is used in their extraction and isolation from biological materials.
Emulsion. Most lipids are soluble in 95% v/v ethanol but form an emulsion of fine
droplets on addition of water. This gives the solution a characteristic milky
appearance and is a very sensitive test for fats.

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Procedure Observation Result

1-with water
Fat and water remain separate Fat is insoluble in water
Take a small amount of fat and is lighter in weight.
(butter could be used) in test
tube and add a few drops of
water

2- with Chloroform
Fat completely dissolves Fat is soluble in
Take a small amount of fat chloroform chloroform (organic
in a test tube and add some solvent)
chloroform – cover
completely, shake

3- with Ether Fat completely dissolves in Fat is soluble in ether

Ether (organic solvent)
Take a small amount of fat
In test tube and add some
ether to cover the same,
completely shake.

4- with Alcohol
Fat dissolves in Alcohol Fat is soluble in alcohol
Take a small amount of (organic solvent)
fat in a test tube and add
some alcohol (absolute to
cover the same), shake

5- Emulsion test
Milky solution Fat for emulsion
0.5ml of water+ o.5 ml of
oil+ 0.5 ml organic solvent,
shake vigorously for 2 min.

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Other reactions of lipid identification

Procedure Observation Result

Sudan III

1ml of oil+1ml of Red – orange ring in upper Lipid is present

water+ 3 drops of layer
Sudan III

Cupper acetate test • Blue color in lower layer and • Un saturated fatty
green color in upper layer acid
2 drops oil+2ml ether
shake then add 2ml • Colorless in upper layer and • saturated fatty acid
cupper acetate solution. green in lower layer

Cholesterol test

2ml of cholesterol +10 Deep green color Cholesterol is present

drops acetic
anhydride+2ml conc
H2SO4

Glycerol test
2 drops glycerol+1ml Black color Glycerol is present
conc H2SO4, then heat
by water bath for 3min.

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 Practical Biochemistry I

Identification of carbohydrates
Name …………………………………………………………………………… Group ………………………….

Test Observation Result

Molisch’s test

Iodine test

Benedict’s test

Barafoed’s test

Seliwanoff’s test

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 Practical Biochemistry I

Identification of carbohydrates
Name …………………………………………………………………………… Group ………………………….

Test Observation Result

Molisch’s test

Iodine test

Benedict’s test

Barafoed’s test

Seliwanoff’s test

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 Practical Biochemistry I

Identification of carbohydrates
Name …………………………………………………………………………… Group ………………………….

Test Observation Result

Molisch’s test

Iodine test

Benedict’s test

Barafoed’s test

Seliwanoff’s test

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 Practical Biochemistry I

Identification of carbohydrates
Name …………………………………………………………………………… Group ………………………….

Test Observation Result

Molisch’s test

Iodine test

Benedict’s test

Barafoed’s test

Seliwanoff’s test

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 Practical Biochemistry I

Identification of carbohydrates
Name …………………………………………………………………………… Group ………………………….

Test Observation Result

Molisch’s test

Iodine test

Benedict’s test

Barafoed’s test

Seliwanoff’s test

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Practical Biochemistry I

Protein identification test

Name: ……………………………………… ………. Group…….

Test Observation Result

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Practical Biochemistry I

Protein identification test

Name: ……………………………………… ………. Group…….

Test Observation Result

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Practical Biochemistry I

Protein identification test

Name: ……………………………………… ………. Group…….

Test Observation Result

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Practical Biochemistry I

Protein identification test

Name: ……………………………………… ………. Group…….

Test Observation Result

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Practical Biochemistry I

Lipid identification test

Name: ……………………………………… ………. Group…….

Test Observation Result

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Practical Biochemistry I

Lipid identification test

Name: ……………………………………… ………. Group…….

Test Observation Result

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