PHL 224 Biochemistry II

Kingdom of Saudi Arabia

Ministry of Higher Education
Salman Bin Abdulaziz University
College of Pharmacy- Girls’ branch

PHL 224 Biochemistry II

Lab. Manual

Academic Year
1435/1436 - 2014/2015

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PHL 224 Biochemistry II

Attendance Sheet

Week Date Exp. Marks

1st 25/01/2015 05/04/1436 _ _ _ _
22d 01/02/2015 12/04/1436 Glucose, fructose
3rd 08/02/2015 19/04/1436 Lactose, Sucrose, starch, potato
4th 15/02/2015 26/04/1436 Cont. Sucrose, maltose
5th 22/02/2015 03/05/1436 Lipid & buffer
6th 01/03/2015 10/05/1436 Amino acid & protein
7th 09/03/2015 17/05/1436 Amino acid & protein
8th 15/03/2015 24/05/1436 Amino acid Revision
9th 22/03/2015 02/06/1436
10th 29/03/2015 09/06/1436
11th 05/04/2015 16/06/1436
12th 12/04/2015 23/06/1436
13th 19/04/2015 20/06/1436
14th 26/04/2015 07/07/1436
Total marks

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 PHL 224 Biochemistry II

One of the crossover fields of chemistry, sometimes called "biological chemistry", is the study of chemical
processes within and relating to living organisms.
We like biochemistry because we learn about things that are inside of us. We can relate to what happens when
we eat and how our bodies are constructed, you will need to understand the information in both biochemistry
and organic chemistry. Why? Because the movement of atoms in the bio-chem world follows the same rules you
will learn in o-chem.

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Are aldehyde or free ketone derivatives of polyhydric alcohols.

Classification of Carbohydrate
Monosaccharaides: can't hydrolyzed into simpler. They may classified as:
 trioses, tetroses, pentoses, hexoses or heptoses depending on the number of carbon atoms.
 And aldoses (e.g glucose, galactose and mannose) or ketoses(e.g fructose) depending upon whether they have an
aldehyde or ketone group.

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1. Disaccharides: are condensation products of two monosaccharaide units. ex. Maltose, lactose and sucrose.
2. Oligosaccharides: are condensation products of three to ten monosaccharaide units.
3. Polysaccharides: are condensation products of more than ten monosaccharaide units ex. Starch, glycogen and dextrin
Reducing sugar is a carbohydrate possessing either a free aldehyde or free ketone functional group.

Glucose: is the most important biological carbohydrates, it is produced by plants during photosynthesis. Glucose is reducing sugar.

Its stored as Starch in plant and as Glycogen by animals. Disaccharide Sucrose is Not reducing sugar.

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No. Carbohydrate Classified as Found in

Glucose hexa – aldoses- Monosaccharide Plant
1.
*(Blood sugar or grape sugar)
Galactose hexa –aldoses- Monosaccharide not normally found in nature, but is mostly
2. hydrolyzed from the disaccharide lactose

Mannose hexa - aldoses -Monosaccharide Fruits, vegetables

3.
4. Fructose*(fruit sugar) hexa - ketoses - Monosaccharide Fruit , honey
Maltose (glucose + glucose) Reducing Disaccharide Barley, germinating grain
5. *(malt sugar)
Lactose (glucose + galactose) Reducing Disaccharide Milk
6.
Sucrose(glucose + fructose) Non-Reducing Disaccharide Cane sugar,Beat sugar
7.
*(Table Sugar)
Starch -(C6H10O5)-n Homo- Polysaccharide Potatoes, Wheat, corn, rice
8.
*known as

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Digestion:
1- Salivary alpha- amylase(stop in stomach because high acidity)
2- Pancreatic amylase in small intestine( pH 7.1)
3- Intestinal enzymes

Monosaccharide

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Disaccharide

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No. Test Observation Inference

Solubility: Soluble in cold water Mono and/or disaccharides
1. Compound + water Insoluble in cold water but form colloid in Polysaccharides present
hot water
Molisch test:
Principle: carbohydrates when treated with con.
Violet/purple ring at the junction Sugar present.
Sulphuric acid undergo dehydration to give furfural
of two liquids Its sensitive but a non-specific test and is
derivatives which condense with alpha naphthol to
form colored hexoses yield 5-hydroxy methyl given by all types of carbohydrates, if the
2. furfurals Oligosaccharides or Polysaccharides are

Procedure: Mix 2ml of sugar sample with 5 drops of present they are first hydrolysed to

Molisch's Reagent in a test tube. Add gently through monosaccharaides which are then

the side by tilting the tube, about 2 ml of dehydrated to give the test positive.

concentrated H2SO4 so as to form a bottom layer.

Benedict’s test: Reducing sugar

Principle: carbohydrates with free aldehyde or
ketone groups have the ability to reduce cupric ions
3. Cu2+ to cuprous which combine with OH ions to form
yellow cuprous hydroxide upon heating converted to
Green, yellow, orange or brick
red precipitate is observed
Present
Benedict's test is a semi-quantitative test.
The color of the precipitate gives a rough
estimate of reducing sugar present in the

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red cuprous oxide(Cu2O). sample.

Copper(II) Copper(I) oxide (Cu2O)
Green precipitate 0.5 gm%
Procedure: Take 5 ml of Benedict's qualitative
reagent, add 8 drops of sugar solution. Boil over a Yellow precipitate 1 gm%
flame for 2 minutes or place in boiling water bath for
Orange precipitate 1.5 gm%
3 minutes. Allow to cool.

Brick red precipitate  2 gm%

Fehling’s test: Yellow or brick red precipitate

Principle: Reducing sugar reacts with Fehling's Reducing sugar
reagent in alkaline medium where reduction of deep
Present
blue solution of cupric(II) to red ppt of cuprous oxide Used to differentiate between
4.
(Cu2O). Copper(II) Copper(I) oxide (Cu2O) reducing and non-reducing sugar
Procedure: 2 ml of Fehling's solution A + 2ml of
Fehling's solution B + 2 ml of Sugar solution Boil.

Barfoed’s test Red Precipitate Monosaccharide present

Principle: aldoses and ketoses can reduce cupric ions
Positive test indicates presence of
5. even in acidic conditions. reducing monosaccharide
On prolong heating disaccharides can
Copper(II) Copper(I) oxide (Cu2O)
also give test positive so solution should
This test is used to distinguish reducing be boiled for 3 minutes only

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monosaccharides from disaccharides by controlling

pH and time of heating, monosaccharides react very
fast whereas disaccharides react very slowly.
Procedure: Sugar solution + Barfoed’s reagent (Cu
Acetate/Acetic acid), heat for 3 min. on boiling W.B
Scanty brick red precipitate
Seliwanoff's Test
Principle: keto hexoses on treatment with Rose Red Color Keto - hexoses Sugar present
hydrochloric acid form 5-hydroxy methyl furfural
Positive test given by Keto-hexoses.
which on condensation with resorcinol gives a cherry
6. On prolong heating aldoses
red colored complex.
converted to Keto-hexoses and give
Procedure:Sugar solution+ few crystals of Resorcinol
positive reaction
+ Equal volume of conc. HCl and warm on W.B.

Bial's test:

Principle:the test reagent dehydrates pentoses to

Specific for pentose
form furfural.
7- Hexoses generally react to form green,
red or brown products.
Furfural further react with orcinol and the iron
bluish product.
present in the test reagent to produce a bluish
All other colours indicate –Ve

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product.

Procedure: 2 ml of sample solution is placed in a test

tube and 2 ml of Bial's reagent is added, solution is
heated gently in hot W.B

Osazone test

Principle: A solution of reducing sugar when heated Glucose, fructose and Maltos and
with phenyl hydrazine, charactreristic yellow As under microscope: lactose.
crystalline compounds called osazone are formed.
Glucosazone: Needle shaped crystals
These crystals have definite crystalline structure,
galactosazone: Rhombic shaped crystals.
8. preciptation time and melting point for different
Maltosazone: Sun flower shaped crystals
Glucose, fructose and mannose
reducing sugars. produce the same osazone
lactose: Powder puff/hedgehog shaped
Galactosazone crystals are formed
Procedure: crystals.
in 7 min. Maltosazone crystals are
1- Add 10 drops of glacial acetic acid to 5 ml of
formed in 10-15 min
sugar solution in test tube.
2- Then add a knife point of phenyl hydrazine

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hydrochloride and double the amount of sodium

acetate crystals.
3- Mix and warm a little to see that the solids are
dissolved.
4- Filter the solution, and keep the filtrate in a
boiling W.B for 20 min.
5- Cool slowly in water bath not under the tap to
have better crystals.
6- Examine the crystal under microscope.

Hydrolysis of sucrose (Inversion test) Perform Benedict's and Seliwanoff's tests.

Principle: sucrose on hydrolysis with HCl is converted

Boiled portion gives +ve Benedict's
to glucose and fructose.
and Seliwanoff's
9.
the prescence of these two monoscharides can be
confirmed by Benedict's and Seliwanoff's tests.

Procedure: add 2 drops of HCl and 1 drop of thymol

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blue to 5 ml of sucrose solution.

The development of pink color indicates that the

solution is acidic.

Divide it in to two equal parts

Boil one portion for about one min., cool under tape
water. Neutralize both portions by adding 2% sodium
carbonate drop by drop. Formatison of blue color
indicates neutralization .

Iodine test: potato turns black

Principle: If iodine is added to an peeled potato then
it will turn black.
10. Procedure: iodine is added to an peeled potato
Starch

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Iodine/Potassium iodide test: Blue-black complex

Starch confirmed
Principle: iodine forms a coordinate complex
between the helically coiled polysaccharide chain and The color obtained depends on the length

iodine. of the unbranched or linear chain.

11.
Procedure: Two ml of a sample solution is placed in a Amylose linear chain deep blue

test tube. Two drops of iodine / potassium iodide

a) Lugol's Iodine b) Starch solution Amylopectin branched chain purple
solution and one ml of water are added. colour
b) Starch solution with iodine

Results: On the basis of above observations the given sample was found to be sugar.

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