UMM AL-QURA

UNIVERSITY FACULTY OF
MEDICINE DEPARTMENT OF
BIOCHEMISTRY

BASIC MEDICAL
BIOCHEMISTRY

Laboratory Manual
ABDULLATIF . T . BABAKR
(BSc., MSc., Biochemistry)

1435 1436

BASIC MEDICAL BIOCHEMISTRY- Practical Notes

Table of contents:
Week Practical
Introduction to Biochemistry Lab., Evaluation methods; Lab
1
conduct and safety precautions
Introduction to the commonly used instruments in the laboratory.
2
Containers: glassware, Plastics, metals, Disposables
3
Methods expressing concentration
Tutorial and E-Learning session
4

5
6
7
8

Mechanistic Principles of Qualitative Identification of

Carbohydrate I
Mechanistic Principles of Qualitative Identification of
Carbohydrate II
Quantitative Analysis of Carbohydrates
Tutorial and E-Learning

Page
03
12
19

25
26
27

9
10
11

Qualitative analysis of lipids-1

Qualitative analysis of lipids-2
Tutorial and E-Learning

31
34

12
13
14
15

Ionic properties of amino acids

Protein solubility and separation : Salting in & salting out
Protein size and separation (Gel Chromatography)
Tutorial and E-Learning

36
41
44

16

Revision

BASIC MEDICAL BIOCHEMISTRY- Practical Notes

I-

INTRODUCTION:

Biochemistry is one of the modern , most important , basic medical sciences. The
term define itself, however, a short rich definition is that : Biochemistry is the
study of life at the molecular level.
Biochemistry: From Atoms to Molecules to Cells (fig. 1):
Biochemical studies lead to a more fundamental understanding of life & impacts
the treatment of disease & solves environmental problems.
The Roots of Biochemistry:
All Living Matter Contains C, H, O, N, P & S:
Essential elements that make up the bulk of mass of any living organism. All
biological molecules (amino acids, proteins, glucose, polysaccharides, lipids,
nucleotides, DNA, RNA, etc.) are constructed from these elements.
Biological Macromolecules (Bio-molecules):
major classes are proteins, polysaccharides (carbohydrates), nucleic acids (DNA,
RNA); (lipids are a major class of biomolecules, but not polymeric).
Organelles, Cells & Organisms:
self-assembly of macromolecules into higher levels of order cell -cell structure &
organelles for prokaryotes & eukaryotes.
Biochemistry seeks to describe the structure, organization, and function of living
organisms in molecular terms. To understand life on the molecular level, you
must:
-know the chemical structures & function of biological molecules, to:
-understand the molecular processes in the expression of genetic information.
-understand bioenergetics (the study of energy flow in cells).

BASIC MEDICAL BIOCHEMISTRY- Practical Notes

So, there are Three Areas of Study:

1. Conformational:
structure and 3D arrangements of biomolecules.
2. Metabolism:
energy production and utilization.
3. Informational:
language for communication inside and between cells.

DNA

RNA

Protein

Cellular Processes.

FIG. -1- : BIOCHEMISTRY, FROM ATOMS TO ORGANISM

BASIC MEDICAL BIOCHEMISTRY- Practical Notes

Practical Biochemistry:
Biochemical studies are conducted in order to obtain information on chemical and
physico-chemical processes that take place in the cells and tissues of living
organisms in norm and in pathology.
Depending on the scope and target of intended investigation, special procedures
are applied to biological materials and adequate physico-chemical methods are
used that must be learned for conducting experiments in biochemistry.
Prior to getting down to practical work, it appears expedient to take a brief survey
of general principles and techniques employed in practical biochemistry.
Code of practice:
This code is a listing of the most essential laboratory procedures that are basic to
safe laboratory practice. In many laboratories, such a code may be given the status
of RULES for laboratory operation.
It is emphasized that good laboratory practice is fundamental to laboratory safety
and can't be replaced by specialized equipment which can only support it.
The most important rules are listed below, not necessarily in order to importance:
1- Mouth pipetting should be prohibited.
2- Eating, Drinking, Smoking and applying cosmetics should not be permitted
in the laboratory work area.
3- The laboratory should be kept neat, clean and free of materials not pertinent
to the work.
4- Work surface should be decontaminated at least once a day, and after any
spill of potentially dangerous material.
5- Cleaning the glassware after use.

BASIC MEDICAL BIOCHEMISTRY- Practical Notes

HAZZARDOUS PROPERTIES OF LABORATORY CHEMICALS

Incompatible Chemicals:
Many chemicals commonly found in the laboratory undergo dangerous reactions
if allowed to come into contact during storage or else where, some of which are
listed below:
Chemical subs.
Acetic acid

Acetylene
Alkali metal

Ammonium
anhydrous
Ammonium
nitrate
Aniline
Bromine

Carbon acetate
Chlorates
Chromic acid
Chlorine dioxide

Reacts with
With chromic acid, hydroxy-contaning compounds,
ethylene plycol, perchloric acid peroxides and
permanganates.
With copper (tubing). Fluoring, bromine, chloride,
iodine, silver, mercury, carbon
Such as calcium potassium and sodium-with water,
carbon dioxide, carbon tertrachloride and other
chlorinated hydrocarbons.
With mercury, halogens, calcium hypochlorite
hydrogen fluoride.
With acids, metal powders, Flammable liquids,
chlorates nitrates, sulfur and finely divided organics
or combustible.
With nitric acid, hydrogen peroxide.
With ammonia, acetylene, butadiene, butane,
hydrogen, sodium carbide.Turpentine and finely
divided metals.
With calcium hypochlorate with all oxidizing
agents.
With ammonium salts, acids, metal powders. Sulfur
finely divided organics or combustibles carbon.
With acetic acid, naphthalene caniper, alcohol,
glycerol turpentine and other flammable liquids.
With ammonia, methan, phosphate, hydrogen sulfide.

BASIC MEDICAL BIOCHEMISTRY- Practical Notes

Chemical subs.
Chlorine

Copper
Cyanides
Liquids
Hydrogen
peroxide
Hydrogen sulfide
Hydrocarbons
general
Iodine
Mercury
Nitric acid

Oxygen
Perchloric acid
Phosphorus
pentoxide
Potassium
permanganate
Silver
Sodium peroxide

Sodium
Sulfuric acid

Reacts with
With ammonia, acetylene, butadiene, benzene and
other petroleum fractions, hydrogen, sodium carbide,
turpentine and finely divided powdered metals.
With acetylene, hydrogen peroxide.
With acids and alkali.
With ammonium nitrate, chromic acid, hydrogen
peroxide, nitric acid, sodium peroxide and halogens.
With copper, chromium, iron, most metal or their
respective salts, flammable fluids, and other
combustible materials, aniline and nitromethane.
With fuming nitric acid, oxidizing gases.
With fluorine, chlorine, formine, chromic acid and
sodium peroxide.
With acetylene, ammonia.
With acetylene, fulminic acid hydrogen.
With acetic, chromic and hydrocyanic acids, aniline,
carbon, hydrogen sulfide, fluids or gases and
substances that are readily nitrated.
With oils, grease, hydrogen, flammable liquids, solids
and gases.
With acetic anhydride, bismuth and its alloys, alcohol
paper, wood and other organic materials.
With water.
With glycerol, ethylene, glycol, benzaldehyde,
sulfuric acid.
With tartaric acid, ammonium compounds.
With any substance, for instance, methanol, acetic
acid, acetic anhydride, benzaldehyde, carbon
disulfide, glycerol, ethylene, glycol, ethyl acetate,
furfural.
With carbon tetrachloride, carbon dioxide, water.
With chlorates, perchlorates, permanganates and
water.

BASIC MEDICAL BIOCHEMISTRY- Practical Notes

Other chemicals have serious acute and chronic effects, some of which are listed
below:
Chemical

Reported effects
Acute

Chronic

Trichloroethylene
(ethylene trichloride)

Narcotic effects

Liver damage, nonspecific

neurological impairment.

m-Xylene (1,3dimethylbenzene)

Narcotic effects,
Nonspecific neurological
headache, dizziness, impairment.
fatigue, nausea

o-Xylene (1,2dimethylbenzene)

Narcotic effects,
Nonspecific neurological
headache, dizziness, impairment.
fatigue, nausea

p-Xylene (1,4dimethylbenzene)

Narcotic effects,
Nonspecific neurological
headache, dizziness, impairment.
fatigue, nausea

BASIC MEDICAL BIOCHEMISTRY- Practical Notes

CHECKOUT PROCEDURE:
Checking out of the laboratory may count as one experiment. You will be graded
on how thoroughly you complete the following items.
1- clean your portion of the desktop, scrubbing it with soap and rinsing it if
necessary. Clean through your desk and the sink at the end.
2- Clean and dry all apparatus, leave no marks or labels on glassware other
than unavoidable scratches and etches.
3- Oil the screws on the lamp and iron ring.
4- Empty the desk completely and line the drawer with a paper towel.
5- Arrange the apparatus to be checked in on top of your desk in
approximately the order in which they appear on the check-in sheet.
6- Place all chipped or cracked items to one side. Discard (or otherwise
segregate) non-returnable.
7- Make a list of all missing returnable equipment, after checking with
neighbors first to see if they have extras. Then obtain missing item.
8- Return safety glasses, if you were issued them.
9- Return paper items to desk drawer as you check in.
10- Lock desk.
11- Additional cleaner assignment.

BASIC MEDICAL BIOCHEMISTRY- Practical Notes

How to protect yourself in the laboratory

SAFETY RULES:
Every experiment is designed to minimize hazards, but the following rules are a
necessary adjust to that design.
1- Wear safety glasses all times when you are in the laboratory. Those who
wear prescription glasses have considerable protection already. At the very
least, others should wear inexpensive plastic nonprescription glasses. By
"safety glasses" we mean industrial quality eye protective devices meeting
the standards of the American standard safety code for head, eye, and
respiratory protection. Those who were contact. Lenses are warned of a
special problem. if a chemical splashes onto the eyes, it may seep under the
edge of the contact lenses. If you wash the eye with the lens in place you
will not the certain of flushing away all of the material, The lens must be
removed as soon as practicable, so that both lens and eye can be thoroughly
washed.
2- Learn the exact locations of eyewash fountains, fire extinguishers, fire
alarms, fire blankets, and other safety features in your laboratory, as will as
how to use these devices. sketch the laboratory and indicate their location.
3- Work only during the scheduled laboratory periods and perform only
authorized experiments. Your instructor will
advise you about local
regulations. An important safety rule, however is never work alone in
laboratory. If an accident occurs, the other person may able to aid you.
4- If you feel faint, sit down right away.
5- If you burned and require the attention of a doctor, has someone accompany
you to the doctor's office. Do not apply salves or ointments on the burned
areas, let the doctor decide the treatment. Prompt cooling of a burned area
with cold water markedly reduces subsequent pain and facilitates heating of
the area.
6- Some accidents happen when lapels are not read carefully. Get in the habit
of reading out loud (but softly) the label of bottle you intend to use.
7- To avoid contamination;
(a)Discard unused chemicals : do not return them to reagent bottles ;claen
up anything you spilled..
(b)Never put a medicine cropper or a pipette from your desk into a regent
bottle but, instead pour a very small amount of the regent quantities.
(c)Try to keep inner walls of bottle stoppers or corks from touching tops of
desks or shelves where they might pick up dust or other chemicals.

BASIC MEDICAL BIOCHEMISTRY- Practical Notes

8-Discard all waste solids-water-insoluble chemicals, litmus paper, used matches,

broken glass, paper towels-into crocks at the end of your laboratory bench.
When sinks are used as wastebaskets they may over flow.
9-Your shoes should cover your feet to protect them from spilled chemicals or
dropped objects.
10-If your hair is long, fluffed with chemicals, it is quite flammable. At least pin
or tie it back in some way while you work around benzene burner flames.
11-Neither food not beverage is allowed in the laboratory.
12-Every time you select a flask, cylinder, or test tube for some experiment,
examine it for cracks and broken edges. Some times a broken edge can
be tolerated, but under no circumstances use a cracked container.
13-Never taste chemical. Check odors only very cautiously.
14-Always pour concentrated acid into water (never water into acid).

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BASIC MEDICAL BIOCHEMISTRY- Practical Notes

Laboratory Safety Signs

Sign

Meaning
Toxic:
Materials causing immediate and serious
toxic effects.
Flammable:
Flammable and combustible materials.

Explosive:
Explosive materials.

Corrosive:
Corrosive materials.

Radiation:
Danger radiation.

Biohazard:
Biohazard materials.

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BASIC MEDICAL BIOCHEMISTRY- Practical Notes

II- Introduction to the commonly used instruments in the

laboratory
Objective:
For student to be familiar with the use of the following:
a) Pipettes and pipetting techniques. b) pH meter.
c) Spectrophotometer.
1- Pipettes
Types of pipettes:
In general, two main types of pipettes are used in the biochemistry
laboratory.
1- Volumetric (Bulb) pipettes:
These are designed to deliver the exact
amount of solution. Has only one point
of graduation, i.e. it is used to deliver only
one volume such as 0.5 ml, 1.0 ml,
2.0 ml, etc. Volumetric pipettes contain a
bulb in the middle of the stem. They are
considered very accurate types of pipettes and
are usually used in preparing standard
solutions or in measuring blood or serum.
When delivering the solution of the
volumetric pipette, the remaining drop at the
tip should not be blown out.
2- Graduated pipettes:
Which are graduated along
the stem, but not at the tip.
They are less accurate
than the volumetric pipettes,
but can deliver small
fractions of the total capacity
of the pipette.

12

BASIC MEDICAL BIOCHEMISTRY- Practical Notes

3- Other types of pipettes:

These include the micropipette, which are used to measure small
quantities
that the regular pipettes cannot measure, i.e. micro litter amounts
(ul.). these types of pipettes are usually used in techniques that
require high accuracy such as in working with enzymes and
hormonal assays.
Automatic pipettes are two types:
1. Fixed volume pipette:
This delivers only the exact volume which is
written on the top of the pipette (e.g. 20 ul,
100 ul, 1000 ul, etc.).
2. Adjustable or variable volume pipette:
This can be calibrated to deliver any amount
of solution which is written on the side of the pipette, (e.g. 20 ul.
Pipette can be used to deliver volumes from 1 to 20 ul, and 100 ul.
Pipette can be used to deliver volumes from
10 to 100 ul.).
Use of pipettes:
1. Never use mouth, use pipette bulbs or pipette filters in
aspirating solutions.
2. Pipettes must be clean from any dirt or grease that might be in or
outside the pipettes.
3. After aspirating the required volume, wipe off the surface of the
pipette with tissue paper.
4. Always read the bottom of the meniscus for clear solutions, and
top for
colored or viscous ones.
5. The graduation point and your eyes should be in a horizontal
position.
6. When delivering solutions, let the tip of the pipette touch the
inner surface of the container, and let the solution flow by capillary
action.
7. Always choose the proper type of pipette in measuring the
required volume, i.e. use smaller pipette for small volumes and vice
verse.

13

BASIC MEDICAL BIOCHEMISTRY- Practical Notes

Other types of glassware in the laboratory include, but not limited to:
Beakers:
Available
in
different volumes.

Conical flasks:
Used in titration
procedures
and
other different uses,
also available in
different volumes.
Graduated
cylinders:
For measuring the
volume of solutions
and
dilution
procedures.

Burette:
For addition
volumes
titration
procedures.

of
in

14

BASIC MEDICAL BIOCHEMISTRY- Practical Notes

2- pH meter
The pH meter is an instrument that is used to measure the pH
(Acidity or alkalinity) of any solution. It is composed of the
following:
1- Glass-bulb electrode.
2- Reference electrode.
3- Sensitive meter or measuring device.

pH measurement procedure:
1. Leave the pH meter in STD BY mode to eliminate warm up and
increase component life. Allow one half hour warm up if meter has
been disconnected.
2. Securely connect pH and reference electrodes, or combination pH
electrode into INPUT (pH) on the rear panel.
3. Verify that reference filling solution level in the electrode is
adequate. The level of filling solution should be higher than the
sample level.
4. Stir buffer and sample during analysis with a magnetic stirrer, if
possible.
5. Rinse electrodes with distilled water between measurements.

15

BASIC MEDICAL BIOCHEMISTRY- Practical Notes

3-Spectrophotometer
Spectrophotometer is one of the most commonly used instruments in
the biochemistry laboratory. Its main function is to measure the
absorbance or concentration of any substance (Carbohydrates,
proteins, etc.) in a solution.
Beer-Lambert Law:
Consider a ray of light of initial intensity (I0) passing through a
solution in a transparent vessel. Some of the light is absorbed, so that
the intensity of the transmitted light (I) is less than (I0). The ratio of
(I) to (I0) is known as the transmittance (T) and depends upon the
path length of the light through solution.

So, transmittance is a measure of the amount of light that is allowed

to pass through a solution. The Lamberts law states that:
When a ray of monochromatic light (single wavelength) passes
through an absorbing medium, its intensity decreases as the
length of the medium
increases.
Similarly, Beers Law states that:
When a ray of monochromatic light passes through an absorbing
medium, the intensity decreases as the concentration of the absorbing
medium increases.
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BASIC MEDICAL BIOCHEMISTRY- Practical Notes

These two laws are combined in the form of Beer-Lambert

Law and expressed as: A = abc. Where:
A = Absorbance.
a = Extension coefficient (constant).
b = Length of light path. = 1cm.
c = Concentration of the substance in the solution.
It is clear from this law, that the absorbance of any colored solution
is directly proportional to the concentration of the substance
producing the color.
3- components of the spectrophotometer:
Spectrophotometer is generally composed of the following major
parts:
i) Light source:
Emits visible or UV light depending on the source itself.
ii) Monochromator:
Filter which functions in isolating only one single light. i.e. one
wavelength.
iii) Slit:
This part functions in passing a very fine beam of isolated
wavelength.
iv) Cuvette holder:
This is where the sample of the colored solution is inserted. When
measuring absorbance in the visible region the cuvette is usually
made of glass, while in measuring absorbance at the low UV region
cuvettes are made of quartz.
v) Photo cell:
This part converts light energy to electric energy so it can be
measured by the measured by the measuring device.
vi ) Galvanometer:
This is where the electric pulses are received and converted on a
scale either to absorbance or to transmittance units.

17

BASIC MEDICAL BIOCHEMISTRY- Practical Notes

18

BASIC MEDICAL BIOCHEMISTRY- Practical Notes

Methods Expressing Concentration

Objectives:
By the end of this topic the students should be able to:
Identify the molarity, normality and molality.
Ideal preparation of different types of solutions (Standard,
Saturated, Unsaturated and Percent solutions).
Definitions:
Normality (g eq/L): The concentration of a solution in equivalents per
liter.
Molarity (W/V): The concentration of a solution in units of moles of
solute per liter of solution.
Molality (W/W): Mole (mol.): A mass of a pure substance equal to its
formula weight in grams (g).
In case of solids:
1 molar solution = M.W in gms. x 100/ purity% g/l.
Example :
1 molar solution of NaOH = 40 x 100/96.0 g/l.
In case of liquids:
1 molar solution = M.W x 100/purity x Sp.Gr. g/l.
Example:
1 molar solution of acetic acid = 60.05 x 100/99.5 x 1.049 g/l.
Experiment 1: Assessment of Normality and Molarity
Read the given details of the reagent from its bottle:
M.W.
Sp. Gr. / or wt/v at 20 c.
Purity %
Prepare the given table:
Table: Estimates of Normality and Molarity of analytical reagents:
Compound M.W
Sp. Gr.
Purity % Amount /L
1N
1M
CH3COOH
HCl
H2SO4
H3PO4
NH3
NaOH
KOH
NaCl
KH2PO4
K3HPO4
Liquids in ml. and solids in grams.
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BASIC MEDICAL BIOCHEMISTRY- Practical Notes

Questions:
1. Calculate the formula weight of each of the following:
(NH4)2SO4 , K2HPO4 , NaCl.
2. How many grams in 2.5 mol of each of the following:
NaOH , Na2CO3 , H2SO4.
3. How many moles in 75 g of each of the following:
NaOH , Na2CO3 , H2SO4.
4. To prepare each of the following solutions would require how many
grams of the solute in each case:
a) 100 ml. of 0.1N HBr.
b) 250 ml. of 0.1N H2SO4.
c) 500 ml. of 1N Na2CO3.
5. How many milliliters of 2.5M solution of NaOH are required to
make 100 ml. of 0.25M solution?

20

BASIC MEDICAL BIOCHEMISTRY- Practical Notes

III-

CARBOHYDRATES

Introduction:
Sugars can be defined as polyhydroxy aldehydes or ketones. Hence the
simplest sugars contain at least three carbons. The most common are the aldoand keto-trioses, tetroses, pentoses, and hexoses. The simplest 3C sugars are
glyceraldehye and dihydroxyacetone
All carbohydrates can be classified as either monosaccharides,
oligosaccharides or polysaccharides. Anywhere from two to ten
monosaccharide units, linked by glycosidic bonds, make up an oligosaccharide.
Polysaccharides are much larger, containing hundreds of monosaccharide units.
The presence of the hydroxyl groups allows carbohydrates to interact with the
aqueous environment and to participate in hydrogen bonding, both within and
between chains. Derivatives of the carbohydrates may contain nitrogens,
phosphates and sulfur compounds. Carbohydrates also can combine with lipid
to form glycolipids or with protein to form glycoproteins.
Monosaccharides:
The monosaccharides commonly found in humans are classified according to
the number of carbons they contain in their backbone structures. The major
monosaccharides contain four to six carbon atoms.

Cyclic Fischer Projection of

Haworth Projection of

-D-Glucose

-D-Glucose

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BASIC MEDICAL BIOCHEMISTRY- Practical Notes

Disaccharides:
Covalent bonds between the anomeric hydroxyl of a cyclic sugar and the
hydroxyl of a second sugar are termed glycosidic bonds, and the resultant
molecules are glycosides. The linkage of two monosaccharides to form
disaccharides involves a glycosidic bond. Physiogically important
disaccharides are sucrose, lactose and maltose.

Sucrose: prevalent in sugar cane and sugar beets, is composed of glucose

-glycosidic bond.

Sucrose

Lactose:
galactose

is found exclusively in the milk of mammals and consists of

-(1,4) glycosidic bond.

Lactose

Maltose: the major degradation product of starch, is composed of 2 glucose

-(1,4) glycosidic bond.

Maltose
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BASIC MEDICAL BIOCHEMISTRY- Practical Notes

Polysaccharides:
Most of the carbohydrates found in nature occur in the form of high molecular
weight polymers called polysaccharides. The monomeric building blocks used
to generate polysaccharides can be varied; in all cases, however, the
predominant monosaccharide found in polysaccharides is D-glucose. When
polysaccharides are composed of a single monosaccharide building block, they
are termed homopolysaccharides. While the Polysaccharides composed of
more than one type of monosaccharide are termed heteropolysaccharides.
Preparation of reagents
1. Molischs reagent
5% naphthal in alcohol, i.e., 5g of naphthal dissolved in 100ml of
ethanol.
2. Iodine solution
0.005% in 3% KI, i.e., 3g of KI dissolved in 100ml water and then 5mg of
iodine is dissolved.
3. Benedicts solution
17.3g of sodium citrate and 10g of sodium carbonate are dissolved in 75ml
of water. 1.73g of CuSO4.5H2O is dissolved in 20ml of water. Mix the
CuSO4 solution with alkaline citrate with constant stirring, finally the whole
volume is made up to 100ml with water.
4. Barfoeds reagent
13.3g of copper acetate in 200ml of water and add 2ml of glacial acetic acid.
5. Bials reagent
Dissolve 300mg of orcinol in 100ml of concentrated HCl.
6. Seliwanoffs reagent
Dissolve 50g of resorcinol in 100ml of con.HCl in the ratio of 1:2.
7. Concentrated HCl
8. Concentrated H2SO4
9. Osazone Reagent
Phenyl hydrazine hydrochloride
Sodium acetate
Acetic acid

23

BASIC MEDICAL BIOCHEMISTRY- Practical Notes

Mechanistic principles of qualitative identification of Carbohydrates

PRINCIPLE OF REACTIONS:
1. Molischs test:
Con. H2SO4 dehydrates carbohydrates to form furfural and its derivatives.
This product combines with sulphonated naphthal to give purple colour.
2. Anthrone test:
The same principle outlined above, except that the furfural reacts with
anthrone (10-keto-9,10-dihydroanthracene) to give a blue-green complex.
3. Iodine test:
Iodine forms a coloured absorption complex with polysaccharides due to
the formation of micellae aggregate. Iodine will form a polysaccharide
inclusion complex.
4. Benedicts test:
Carbohydrates with a potential aldehyde or ketone group have reducing
property when placed in an alkaline solution. Cupric ions present in the
solution will be reduced to cuprous ion. This will give a red coloured
precipitate. Moreover, this test is more specific for reducing sugars.
5. Barfoed test:
Barfoeds reagent is weakly acidic and it is only reduced by
monosaccharides. Prolonged boiling may hydrolyze the disaccharide to
give false positive test.
6. Bials test:
When pentose is heated with con.HCl, furfural, which condenses with
orcinol in the prescence of ferric ion to give a blue green colour.
7. Seliwanoffs test:
Ketoses are dehydrated more rapidly than aldose to give a furfural
derivatives, which then condenses with resorcinol to form a red colour
complex.

24

BASIC MEDICAL BIOCHEMISTRY- Practical Notes

GENERAL PROCEDURE FOR QUALITATIVE ANALYSIS OF CARBOHYDRATES

No.
1.

2.

3.

EXPERIMENT

OBSERVATION

Molischs test
Violet coloured ring is
To 1ml of test solution, add 2
formed at the junction of
drops of Molischs reagent.
the 2 layers.
Then add con. H2SO4 carefully
along the sides of the test tube.
Blue green color complex
Anthrone test
is formed
To 5 drops of sugar solution
add 2 ml. Anthrone reagent.
Iodine test
To 1ml of the test solution, 2
drops of iodine is added and
observe the colour change.

(i) Deep blue colour is

obtained.
(ii)Dark brown colour is
obtained.
iii)No colour change.

4.

Benedicts test
2ml of Benedicts reagent is
mixed with 0.5ml of test
solution and the contents are
boiled for a few minutes.

(i)Orange red precipitate

is obtained.
(ii)No characteristic
colour change.

25

INFERENCE
Presence of
carbohydrate.

Presence of
carbohydrate.

Presence of
polysaccharide.
Presence of
polysaccharide.
(Glycogen)
Absence of
polysaccharide.
Presence of
reducing sugar.
Absence of
reducing sugar.

BASIC MEDICAL BIOCHEMISTRY- Practical Notes

5.

6.

7.

8.

(i)Brick red precipitate is

Barfoeds test
obtained at the bottom of
To 2ml of test solution, 2ml of
test tube.
Barfoeds reagent is added and
(ii)No colour change.
boiled for 3 minutes and the
colour change is noted.
Bials test
(i)Blue green colour
To 1ml of the test solution,
5ml of Bials reagent is added.
is obtained.
The contents are boiled and
cooled.
(ii)No colour change.

Seliwanoffs test
To 1ml of the test solution, (i)Cherry red colour
3ml of Seliwanoffs reagent is
is obtained.
added and the contents are
boiled
(ii)No colour change.

Presence of
reducing
monosaccharide
Absence of
reducing
monosaccharide.

Presence of
pentose sugar.
Absence of
pentose sugar.

Presence of
fructose.
Absence of
fructose.

FEARON'S TEST
To 1ml of the test solution,
2ml of Fearons reagent is
added and the content is
heated. Then NaOH was
added to the cold mixture.

i)Red coloration appear.

ii)no color change.

26

Presence of
reducing
disaccharide.
Absence of
reducing
disaccharide.

BASIC MEDICAL BIOCHEMISTRY- Practical Notes

Quantitative analysis of Carbohydrates

Objectives:
- To know the difference between Qualitative and Quantitative analysis.
- To know the different methods for estimation of blood glucose.
Qualitative analysis:
determination of the presence or absence of a substrate in the given sample.
Quantitative analysis:
determination of the concentration of a substrate in the given sample.
Introduction:
The importance of testing the blood glucose level comes from the fact that the
brain cells depend on the extracellular glucose concentration for their energy
supply; hypoglycemia is likely to impair cerebral functions as well as
dysfunction by affecting extracellular osmolarity.
Methods:
Many methods were developed to estimate the glucose level in body fluids
among which the commonly used nowadays, the enzymatic methods. These
methods can be summarized and categorized into
A) Reduction methods:
These methods depend on the reductive property of glucose (aldose)
1-Ferriccyanide( Hoffmans) method: using ferricyanide which is
reduced by the glucose .
Fe+++
Fe++
(color change from yellow to colorless solution , measured photometerically )
2-Copper sulfate methods:
Benedict: The reagent contains Na-citrate &Na carbonate with
CuSO4 . It gives color according To conc. of glucose (green-----yellow----brown-----red).
Fehling : using KOH &Na/K tartrate with CuSO4
3-Smogi-Nelson method: using Arsenomolybdate
N.B. The reduction methods need alkaline medium &heat
These methods are qualitative & semi-quantitative.

27

BASIC MEDICAL BIOCHEMISTRY- Practical Notes

B) Enzymatic methods:
1-Hexokinase method (The reference method).
Glucose +ATP +HKADP+G6P
G6P +NAD +G6PD 6 P-gluconolactone +NADH+H (measured at 340)
2- Glucose Dehydrogenase Method:
Glucose +NAD

GDH

Gluconolactone +NADH+H (measured at 340)

3- Glucose oxidase method:

Glucose Oxidase for blood glucose estimation (Experiment #1)
PRINCIPLE OF THE METHOD
Glucose oxidase (GOD) catalyses the oxidation of glucose to gluconic acid.
The formed hydrogen peroxide (H2O2), is detected by a chromogenic
oxygen acceptor, phenol-aminophenazone in the presence of peroxidase
(POD):
Principle: (Trinders method )
-D-glucose

Mutarotase

-D-glucose +H2O+O2

-D-glucose
Glucose oxidase

H2O2+ 4-aminophenazone + phenol

D-gluconic acid+H2O2
Peroxidase

Quinonemine +4 H2O

The intensity of the color formed is proportional to the glucose concentration

in the sample.
CLINICAL SIGNIFICANCE
Glucose is a major source of energy for most cells of the body; insulin
facilitates glucose entry into the cells. Diabetes is a disease manifested by
hyperglycemia; patients with diabetes demonstrate an inability to produce
insulin. Clinical diagnosis should not be made on a single test result; it should
integrate clinical and other laboratory data.

28

BASIC MEDICAL BIOCHEMISTRY- Practical Notes

PREPARATION
Working reagent (WR):
Dissolve the contents of one vial R 2 Enzymes in one bottle of R 1 Buffer.
Cap and mix gently to dissolve contents.
Instrumentation:
-Photometer adjusted on wavelength 540 nm
-Cuvette (light path) 1 cm
-Water bath at 37 C
-Automatic pipettes, disposable test tubes , racks and disposable tips.
PROCEDURE
1. Assay conditions:
Wavelength: . . . . . . . . . . . . . .. . 505 nm (490-550)
Cuvette: . . . . . . . . . . . . . . . . . . . . .. 1 cm light path
Temperature. . . . . . . . . . . . . . . . . . . 37C / 15-25C
2. Adjust the instrument to zero with distilled water.
3. Pipette into a cuvette:
Sample
1.0
-10

Standard
1.0
10
--

Blank
1.0
WR (mL)
-Standard (L)
-Sample (L)

4. Mix and incubate for 10 min at 37C or 15-20 min at room temperature (1525C).
5. Read the absorbance (A) of the samples and standard, against the Blank.
The colour is stable for at least 30 minutes.
CALCULATIONS
(A) Sample
x 100 (Standard conc.) = mg/dL glucose in the sample
(A) Standard

29

BASIC MEDICAL BIOCHEMISTRY- Practical Notes

Discussion:
*Physiological & Biochemical Background:
Glucose metabolism, Insulin action and other hormonal effects on glucose in the
human body.
*Pathological & Disease Correlation:
Diabetes Mellitus, Cushing syndrome ,Hyperthyroidism ..etc
Questions:
1- What are the basis of reduction methods for glucose
estimation. ?
2- Give short notes on Trinders method for glucose estimion.

30

BASIC MEDICAL BIOCHEMISTRY- Practical Notes

QUALITATIVE ANALYSIS OF LIPIDS

Introduction :
Lipids are naturally occurring compounds that are esters of long chain fatty acids.
They are insoluble in water but soluble in fat solvents such as acetone, alcohol,
chloroform or benzene.
Alkaline hydrolysis (known as saponification) gives rise to the alcohol and the Na
or K salts of the constituent fatty acids.
Chemically, lipids can be divided into two main groups:
1- Simple lipids.
2- Compound lipids.
Steroids and the fat soluble vitamins are also considered as lipids because of their
similar solubility characteristics and are known as derived lipids.
Major Roles of Biological Lipids:
Lipids of physiological importance for humans have four major functions:
1. They serve as structural components of biological membranes.
2. They provide energy reserves, predominantly in the form of
triacylglycerols.
3. Both lipids and lipid derivatives serve as vitamins and hormones.
4. Lipophilic bile acids aid in lipid solubilization.
1- Simple lipids:
Esters of glycerol and fatty acids are known as acylglycerols or glycerides.
The acylglycerols are known as
neutral lipids, they are called fats or
oils, depending on whether they are
solid or liquid at room temperature.
Shown to the right is the structure of
Triglycerides.

31

BASIC MEDICAL BIOCHEMISTRY- Practical Notes

Fatty acids:
Fatty acids are long-chain hydrocarbon molecules containing a carboxylic acid
moiety at one end.
Fatty acids that contain no carbon-carbon double bonds are termed saturated fatty
acids; those that contain double bonds are unsaturated fatty acids.
Saturated fatty acids of less than eight carbon atoms are liquid at physiological
temperature, whereas those containing more than ten are solid. The presence of
double bonds in fatty acids significantly lowers the melting point relative to a
saturated fatty acid.
2- Compound lipids:
Complete hydrolysis of a compound lipid yields at least one other component as
well as the usual alcohol and fatty acids. These compounds are essential structural
components of cell membranes.
3- Derived lipids:
Include Steroids and fat soluble vitamins.
Cholesterol:
Cholesterol is an extremely important biological molecule that has roles in
membrane structure as well as being a precursor for the synthesis of the steroid
hormones and bile acids.
The synthesis and utilization of cholesterol must be tightly regulated in order to
prevent over-accumulation and abnormal deposition within the body. Of particular
importance clinically is the abnormal deposition of cholesterol and cholesterolrich lipoproteins in the coronary arteries. Such deposition, eventually leading to
atherosclerosis, is the leading contributory factor in diseases of the coronary
arteries.

cholesterol
32

BASIC MEDICAL BIOCHEMISTRY- Practical Notes

PRINCIPLES:
The main groups of lipids have different solubility characteristics. When fats and
oils are heated with alkali, free fatty acids and glycerol are liberated and this
process is known as saponification. The excess alkali present reacts with the
liberated fatty acids to form the Na or K salts which give the solution a
characteristic soap appearance.
The fatty acids in animal fats are usually fully saturated whereas those found in
vegetable oils contain one or more double bonds. Hydrogenation of the double
bonds converts the oils into solid fats and this is carried out commercially for the
production of margarine.
Halogens also readily add across the double bonds and the decolorize
tion of a solution of bromine or iodine by a lipid indicates the presence of double
bonds.
MATERIALS:
Reagents

Standard liquids

Alco. KOH (100 g/l in ethanol).

HCl. Conc.
NaOH (1N).
NaCl (solid).
KI (10% w/v).

Phospholipids: Lecithin.
Sterols: cholesterol (solid and
0.5% in ethanol).

Ethanol (absolute).
Diethyl ether.
Petroleum ether.
Chloroform.
Benzene.
Copper acetate (1% w/v).

Fatty acids: palmitic acid,

Stearic acid.
Fats: Butter.
Oils: Olive oil.

1SOLUBILITY:
Note the physical state of:
f) Water.
g) Ethanol.
h) Diethyl ether.
i) Petroleum ether.
j) Chloroform.
Carefully observe the differences between the above
remarks.
33

a) Palmetic acid.
b) Stearic acid.
c) Oleic acid.
d) Olive oil.
e) Butter.
groups of lipids, write your

BASIC MEDICAL BIOCHEMISTRY- Practical Notes

2GREASE STAIN TEST:

Place one drop of the above lipids on a filter and leave to dry, observe the
formation of a clear grease spot and give your remarks.
3COPPER ACETATE TEST:
Dissolve a few drops of the oil in 3 ml. of petroleum ether and add equal volumes
of copper acetate (1%), mix once by inversion (Dont shake). Leave the tube until
the emulsion will separate into two layers.
A. Saturated fatty acid: upper layer is clear and precipitate in the lower layer.
B. Unsaturated FA: upper layer is greenish-Blue color and lower layer is
colorless.
C. If the two layers are clear then the test.
4TEST FOR UNSATURATION:
Add one drop of Olive oil, one spatula-point of Butter and one spatula-point of
Lecithin to separate dry test tubes, and dissolve the lipids in about 1 ml. of
chloroform.
Add 1 ml. of chloroform to other test tube to act as Blank.
By means of Pasteur pipette add drop wise a solution of bromine in c hloroform
until define yellow color is produced, Note the number of drops in each case, and
comment on the results.
CH3 (CH2)7 CH=CH (CH2)7 COOH
Oleic acid

Br2
Bromine

H
CH3 (CH2)7 C

H
C (CH2)7 COOH

Br Br
Dibromo-Stearic acid

34

BASIC MEDICAL BIOCHEMISTRY- Practical Notes

5TEST FOR CHOLESTEROL (Liebermans test):

Add 10 drops of acetic anhydride and 2 drops of conc. H2SO4 to 2 ml of each of the
following:
a) Cholesterol solution in chloroform (0.5 %).
b) Egg yolk solution in chloroform (0.5%).
c) Butter solution in chloroform (0.5%).
d) Chloroform.
Give possible interpretation of the reaction in each case.

35

BASIC MEDICAL BIOCHEMISTRY- Practical Notes

IONIC PROPERTIES OF AMINO ACIDS

OBJECTIVES:
1- To investigate the ionization of compounds with two or more dissociable
groups.
2- To demonstrate the particular importance of histidine as a potential buffer at
physiologic pH.
Introduction:
Amino acids serve as basic building blocks for proteins. There are a large number of
chemically possible amino acids, but only a few of these occur naturally.
In case of the 22 or so amino acids found in proteins, nearly all of them are -amino
acids, where the amino group is present on the -carbon atom.
The strong positive charge on the -NH3+ group induces a tendency for the COOH
group to lose a proton, so that amino acids are strong acids. The pKa for (the COOH
of) Glycine, for example (2.3), is much lower than acetic acid (4.75), and other
amino acids containing an aliphatic side chain have similar pK values to Glycine.
Ionic properties of amino acids:
Amino acids are polyprotic, i.e. they all contain at least two dissociable
protons, when dissolved in water, The proton from the -COOH group transfers to
the -NH2 end of the molecule, because the NH2 group is a stronger base than -COOforming the so called (zwitter ion) which contain both positive and negative charges
,while the net charge on the ion will be zero.
If the zwitter ion is treated with acid, H+ will be added to the COO- to form
COOH, the resulting shape is the cation bearing a net positive charge. Similarly,
treating the zwitter ion with base will result in the loss of the removable proton
attached to the NH3+ group to form NH2, and the result anion bears a net negative
charge. The following pH dependent equilibrium can be drawn.
K1
H3N+-CHR-COOH

K2
H3N+-CHR-COO-

(K1 , K2: are the ionization constants of the COOH and NH3 respectively.)

36

H2N-CHR-COO-

BASIC MEDICAL BIOCHEMISTRY- Practical Notes

e.g. : Glycine :
pKa=

2.34

H3N+---CH2---COOH
Gly+

9.60
H++H3N+---CH2---COO-

H++H2N---CH2---COO-

Glyo

Gly-

Buffering:
According to Henderson-Hasselbalch equation:
pH = pKa + log[A-]/[HA]
At the point of the dissociation where the concentration of the conjugate base [A -] is
equal to that of the acid [HA]: pH = pKa + log[1]
The log of 1 = 0. Thus, at the mid-point of titration of a weak acid:
pH = pKa
At this point, when the pH = pKa, the slope of the curve (i.e. the change in
pH with addition of base or acid) is at a minimum, so the buffer solution best resists
addition of either acid or base, and hence has its greatest buffering ability. As a
general rule, buffer solution can be made for a weak acid/base in the range of 1 pH
unit from the pKa of the weak acids.

addition of strong base produces weak conjugate base:

CH3CO2H + OH- ------------> CH3CO2- + H2O
addition of strong acid produces weak acid:
H3O+ + CH3CO2------------> CH3CO2H + H2O

Blood Buffering:
(to understand the role of imidazole ring of Histidine in buffering capacity of
haemoglobin)
The pH of blood is maintained in a narrow range around 7.4. Even
relatively small changes in this value of blood pH can lead to severe metabolic
consequences. Therefore, blood buffering is extremely important in order to
maintain homeostasis.The primary buffers in blood are hemoglobin in erythrocytes
and bicarbonate ion (HCO3-) in the plasma. Buffering by hemoglobin is
accomplished by ionization of the imidazole ring of histidines in the protein.
37

BASIC MEDICAL BIOCHEMISTRY- Practical Notes

EXPERIMENT-1
Preparation of Normal Titration Curve for Glutamic Acid
The dissociation of glutamic acid can be represented as:

We are going to use the pH meter to explore the acid-base behavior of Glutamic
acid .
Materials:
- Burette
- 0.05M HCl.
- Beaker.
- 0.05M Glu.
- pH standards.
- 0.05M Hi.
- 0.05M NaOH.
- Ph meter.
Procedure:
1- Titrate 10 ml. of 0.05M Glutamic acid against 0.05M NaOH. Repeat the
titration with 0.05M HCl.
2- Record your data in the given table.
3- Sketch a curve from your data on a graph paper.
(Plot pH (Yaxis) versus volume of NaOH expended (Xaxis).
Volume (ml.)
NaOH 0.05M
0.0
0.5
1.0
1.5
2.0
2.5
3.0
3.5
4.0
4.5
5.0

pH

Volume (ml.)
HCl. 0.05M
0.0
0.5
1.0
1.5
2.0
2.5
3.0
3.5
4.0
4.5
5.0

38

pH

BASIC MEDICAL BIOCHEMISTRY- Practical Notes

EXPERIMENT-2
Preparation of Normal Titration Curve for Histidine
Repeat the above procedure using 0.05M Histidine.
The dissociation of Histidine can be represented as:

Here , is the ionizable groups of Histidine:

39

BASIC MEDICAL BIOCHEMISTRY- Practical Notes

Protein solubility and separation

PROTEINS:
Proteins are polymers of the bifunctional monomers, amino acids. The twenty
common naturally-occurring amino acids each contain an -carbon, an -amino
group, an -carboxylic acid group, and an -side chain or side group. These side
chains (or R groups) may be either nonpolar, polar and uncharged, or charged,
depending on the pH and pKa of the ionizable group.
Amino acids form polymers, the amino group from one amino acid attached to
the carboxylic group of the adjacent amino acid, the resulting link between them
is an amide link which biochemists call a peptide bond. In this reaction, water is
released. In a reverse reaction, the peptide bond can be cleaved by water
(hydrolysis).
O

C ------ HN

H2 N

R1

R2

OH

Peptide bond
When two amino acids link together to form an amide link, the resulting structure
is called a dipeptide. Likewise, we can have tripeptides, tetrapeptides, and other
polypeptides. At some point, when the structure is long enough, it is called a
protein. There are many different ways to represent the structure of a polypeptide
or
protein.

40

BASIC MEDICAL BIOCHEMISTRY- Practical Notes

Solubility of proteins:
Introduction:
The solubility of protein depends on, among other things, the salt concentration in
the solution.
At low concentrations, the presence of salt stabilizes the various charged groups
on a protein molecule, thus attracting protein into the solution and enhancing the
solubility of protein. This is commonly known as salting-in. However, as the salt
concentration is increased, a point of maximum protein solubility is usually
reached. Further increase in the salt concentration implies that there is less and
less water available to solubilize protein. Finally, protein starts to precipitate when
there are not sufficient water molecules to interact with protein molecules. This
phenomenon of protein precipitation in the presence of excess salt is known as
salting-out.
Precipitation of Proteins at isoelectric Point:
Protein solubility:
The solubility of proteins in aqueous buffers depends on the distribution of
hydrophilic and hydrophobic amino acid residues on the proteins surface.
Proteins that have high hydrophobic amino acid content on the surface have
low solubility in an aqueous solvent.
Charged and polar surface residues interact with ionic groups in the solvent
and increase solubility.
Isoelectric point (pI):
Is the pH-value of a solution at which the total net charge of a protein equals
zero.
At a solution pH that is above the pI the surface of the protein is predominantly
negatively charged and therefore like-charged molecules will exhibit repulsive
forces.
Likewise the surface of the protein is predominantly positively charged at a
solution pH that is below the pI, and repulsion between proteins occurs.
However, at the pI the negative and positive charges are eliminated, repulsive
electrostatic forces are reduced and the dispersive forces predominate.
The dispersive forces will cause aggregation and precipitation.
The pI of most proteins ranges between the pH 4 to 6.
When microorganisms grow in milk, they often produce acids and lower the
pH of the milk.
The phenomenon of precipitation or coagulation of milk protein (casein) at low
pH as milk becomes spoiled is one of the common examples of protein
isolation due to changes in the pH.
41

BASIC MEDICAL BIOCHEMISTRY- Practical Notes

Procedure:
1. Into a 50 ml volumetric flask add 20 ml of water.
2. Add 0.25 g of pure casein, followed by the addition of 5 ml of 1 N NaOH
solution.
3. Once casein is dissolved, add 5 ml of 1 N acetic acid solution, then dilute with
H2O to 50 ml and mix well. The resulted solution is a 0.1 N casein acetate
sodium.
4. Setup a series of 9 test tubes.
5. In the first test tube put 3.2 ml 1 N CH3COOH, and 6.8 ml H2O and mix
thoroughly.
6. In each of the other test tubes (2-9) put 5 ml H2Od.
7. From the test tube 1 transfer 5 ml to the test tube 2, and mix thoroughly.
8. Repeat step 7 for the rest of test tubes (3 - 9).
9. Now to each test tube (1 -9) add 1 ml of the casein acetate sodium solution, and
shake the test tubes immediately.
10. Let the samples stand for 30 min, and note the turbidity in the 9 test tubes.
11. Use )+( and ) (signs to describe the turbidity in the different test tubes.
12. You should observe the most precipitation in the test tube which has the pH
around 4.7 (close to the isoelectric point of casein).
Results:
TUBE
1
2
3
4
5
6
7
8
9
1N CH3COOH

1.6

0.8

0.4

0.2

0.1

0.05

0.025

0.012

0.006

PH

3.5

3.8

4.1

4.4

4.7

5.0

5.3

5.6

5.9

TURBI-DITY

42

BASIC MEDICAL BIOCHEMISTRY- Practical Notes

PROTEIN SIZE AND SEPARATION

Separation of proteins
The molecular weights of proteins are very high. Due to their wide variety of
amino acid configurations, proteins behave very differently. These differences
constitute the bases of the biochemical function of proteins. And these basic
differences are also the parameters which are used to separate proteins:
1- Physical Size:
Which reflects molecular weight of the protein, Gel filtration uses this
parameter, also referred to as Exclusion chromatography.
2- Electric Charge:
Some amino acid residues are positively charged while others are negatively
charged. Variations in the pH of an amino acid system cause variations in the
charge of amino acid residues. Consequently, the net surface charge of a
protein (comprised of amino acid residues) also varies with its environment.
It is these variations in the net charge of proteins which allow them to be
separated by such techniques as ion exchange chromatography or by
electrophoretic techniques.
3- Hydrophobic character:
Hydrophobic regions available to interact with a hydrophobic stationary
phase provide the important characteristic of proteins which is used in
adsorption chromatography.
4- 3-dimentional substructure:
Provide the basis for very specific separation methods. Bio-specific affinity
chromatography is used to separate proteins according to their biological
functions. Proteins, such as enzymes, antibodies and glycoproteins are
particularly appropriate for separation by affinity chromatography.

43

BASIC MEDICAL BIOCHEMISTRY- Practical Notes

Exclusion Chromatography
Experiment:
Objectives:
a- To demonstrate the principle of molecular exclusion (gel permeation)
chromatography using a bead of G-100 Sephadex gel to separate a mixture
of:
Fluorescin
: Yellow M.wt. = 332 (size of a dipeptide).
Hemoglobin

: Red

M.wt. = 68,000 (protein).

Blue Dextran

: Blue

M.wt. = 200,000 (large protein).

b- To show that the technique can be used to determine the Molecular Weight
of a newly discovered protein.
Principle:
In gel filtration the gel acts as a molecular sieve separating molecules with
differences in molecular size and weight. The gel matrix contains numerous
porous beads (stationary phase) with (mobile phase) in between. If the ample of
mixture is applied at the top of the column, the large molecules in the sample will
not be able to enter the pores in the bead but will pass between them and so be
eluted first, smaller molecules that have access to the pores are retarded in the gel
to a certain extent and will therefore be eluted after the large molecules in order of
decreasing M.wt. and size.
Materials:
Sephadex G-100: 0.5 g of (S.G-100) /100 ml. of 0.3% (w/v) NaCl; leave to
swell for 1 hr. prior to experiment.
Eluant: 0.3% (w/v) NaCl.
Mixture of : Flurescin, hemoglobin and blue dextran (each 0.1 g/10 ml.
H2O). To prepare Hemoglobin dilute 1 ml. of blood to 10 ml. with H2O.
Chromatography column (0.1 to 1.5 cm. diameters).
Microperpox peristaltic pump: Flow rate = 0.5 ml/min.
Disposable syringes (5ml, needle size 20 G).
Method:
1. Set up the column replacing the waist flask by a fraction collector.
2. Pour slurry of Sephadex G-100, into the column and allow the gel beads to
settle. Once about 10 cm. have settled, allow the solvent to run out of the
button of the column. Do not allow the liquid level to fall below the top of
the Sephadex. Continue to add Sephadex until you have a column whose
settled height is at least 15 cm.
44

BASIC MEDICAL BIOCHEMISTRY- Practical Notes

3. Carefully place a small filter paper disc on top of the gel bed. This will
prevent disturbance of the surface when sample or solvent are added.
4. Let the liquid level fall to the filter paper disc. Carefully add 0.2 ml. of the
colored mixture with a pipette to the top of the column and let this run into
the gel. Then add 0.5 ml. of the eluant and run this into the gel.
5. Fill the column with the eluant by connecting it to the reservoir and elute
the sample, collecting fractions of ml. in numbered test tubes (Flow rate =
0.5 ml/min.)
6. Stop collecting fractions when the last visible band has been eluted and
record the appearance of your fractions.
7. Repeat the procedure after adding 1% of protein to the mixture.
Fig.: Exclusion Chromatography.

Separation of the sample

Theory of separation
45

BASIC MEDICAL BIOCHEMISTRY- Practical Notes

References:
1. Skoog, D. A.; Principles of Instrumental Analysis, 6th ed.;
Thompson
Brooks/Cole: Belmont, CA, 2006, Chapter 28.
2. Smith AL (Ed) et al. (1997). Oxford dictionary of
biochemistry and molecular biology.: Oxford University
Press. ISBN 0-19-854768-4.
3. Grisham, Charles M.; Reginald H. Garrett (1999). Biochemistry.
Philadelphia: Saunders College Pub. pp. 4267. ISBN 0-03022318-0.
4. Plummer D. T.: An Introduction to Principle Biochemistry, 2 nd.
ed. 1978.

46