SUNANDAN DIVATIA SCHOOL OF SCIENCE

Bachelors
In
Biomedical Science

Practical Journal
Semester 2
2023-2024

Name of Student: Anushka Gupta

Roll No.: 7538200039

1
 CERTIFICATE
This is to certify that Ms. Anushka Gupta of bachelor’s in biomedical
science course has satisfactorily completed and documented the
practical work in Practical II during the Second Semester of the year
2023-2024 in the Journal.

Signature of Teacher In-charge

Date: College Stamp/Seal

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 Semester I
Practical II

INDEX

Expt. Expt. Title Page Date

No. No.
Section A: Physical Chemistry
1. Determination of the surface tension of a liquid or a dilute 3-7 21/3/24
solution using a stalagmometer.
2. Determination of the relative and absolute viscosity of a liquid 8-13 18/1/24
dilute solution using an Ostwald’s viscometer.
3. Study of the kinetics of the following reaction by integrated 14-19 28/3/24
rate m e t h o d : Acid hydrolysis of methyl acetate
with
hydrochloric acid volumetrically.
Section B: Bioanalytical Chemistry
1. To study protein purification using ion exchange 20-28 24/1/24
chromatography.
2. To study protein purification using gel filtration/gel exclusion 39-42 18/1/24
chromatography.
3. Qualitative analysis of drug by TLC. 29-32 1/2/24
4. Estimation of a drug by HPLC. 33-38 7/3/24
5. To perform Native and SDS Polyacrylamide gel 43-51 8/2/24
electrophoresis (PAGE).

2
 DETERMINATION OF SURFACE TENSION OF A LIQUID

AIM:
To determine surface tension of a liquid using stalagmometer.

INTRODUCTION:
In the drop number method, the number of drops formed by equal volumes of two liquid is
counted. Suppose m1 and m2 are the masses of one drop of each of the liquid having densities
ρ1 and ρ2 respectively. If n1 and n2 is the number of drops formed by volume v of the two
y1 p1 n2
liquids, then their surface tensions are related as = ×
y2 p2 n1
One of the liquids is water. Its surface tension and density are known. Then surface tension of
the given liquid can be calculated.

THEORY:
Surface tension is the force that causes the molecules on the
surface of a liquid to be pushed together and form a layer. It is
the tension of the surface film of a liquid caused by the
attraction of the particles in the surface layer by the bulk of the
liquid, which tends to minimize surface area. The surface
tension arises due to cohesive interactions between the
molecules in the liquid. The stronger the cohesive force, the stronger the surface tension.

Surface tension has the dimension of energy per unit area or a measure of work per unit area
or force per wetted length, surface tension has the unit Nm/m2 or dynes/cm and is designated
by the symbol σ (lower case sigma). If the phase is solid, the equivalent term surface free
energy is normally used. If the adjacent phase is a liquid or a solid, reference is made to
interfacial tension. Interfacial tension is defined as the work which must be expended to
increase the size of the interface between two adjacent phases which do not mix completely
with one another. The term relates to the liquid/liquid and liquid/solid phase boundaries,
while for the liquid/gaseous interface we refer to surface tension and for the solid/gaseous
interface we refer to surface free energy.

3
Surface tension is the tendency of any liquid to shrink into the minimum surface area
possible. Every liquid has surface tension which can be measured. Some examples: Water,
liquid paraffin, oil and other liquids.

Measuring SFT
Various methods are available to measure surface tension of liquids. A few of them are as
follows:
● Drop count method: The drop count method or stalagmometric method is one of the
most common methods for measuring surface tension. It applies the principle of
measuring the weight of drops of the fluid of interest falling from a device known as a
stalagmometer and thereby calculating the surface tension of the fluid. A
stalagmometer is a capillary glass tube whose middle section is widened and the
lower end of the tube is narrowed to force the fluid to fall out as a drop. During the
experiment, the drops of fluid slowly fall from the tube in a vertical direction which is
then counted.
● Bubble pressure method: In this method, the bubble pressure tensiometer is used
which produces gas bubbles at a constant rate and blows them through a capillary
which is submerged in the sample liquid and whose radius is already known. The
pressure inside of the gas bubble continues to increase and the maximum value is
obtained when the bubble has a completely hemispherical shape whose radius
corresponds exactly to the radius of the capillary.
● Spinning drop method: Another method used to measure surface tension.
Measurements are carried out in a rotating horizontal tube which contains a dense
fluid. A drop of a less dense liquid or gas bubble is placed inside the fluid. Since the
rotation of the horizontal tube creates a centrifugal force towards the tube walls, the
liquid drop will start to deform into an elongated shape; this elongation stops when
the interfacial tension and centrifugal forces are balanced. The surface tension
between the two liquids (for bubbles: between the fluid and the gas) can then be
derived from the shape of the drop at this equilibrium point. A device used for such
measurements is called a "spinning drop tensiometer".

4
Relation of Surface Tension and Density
The magnitude of the surface tension of a liquid is a function of the distance between the
molecules and is therefore dependent on the density. Hence, surface tension is directly
proportional to density. Liquid with higher density has higher surface tension while liquid
with lower density has lower surface tension.

Applications of Surface Tension

Surface tension has a huge role in daily life, health and many industrial processes. There are
so many techniques that have been developed to modify surface tension.
1. Small insects such as the water strider can walk on the surface of the water because
their weight is very less so they can’t penetrate the water.
2. Soaps and detergents also work on the basis of surface tension. They lower the
surface tension of the water so that the soaps and detergents easily soak into the pores
and holes.
3. Disinfectants are mainly the solution of low surface tension so that when we use them
in the field they can float on the water and spread out on the cells to destroy them.
4. Surface tension is also important for characterization for food, pharmaceutical and
packaging products.

REQUIREMENTS:
A) Chemicals needed:
1. Distilled water
2. Methanol
B) Apparatus:
1. Stalagmometer
2. 25 ml pipette
3. Two beakers
4. Density bottle
5. Rubber bulb
6. Stop watch

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PROCEDURE:
1. Clean the stalagmometer with distilled water.
2. Attach a piece of clean rubber tubing with a screw clip to the top of the stalagmometer.
3. Immerse the lower end of the stalagmometer in distilled water and suck the water 1–2 cm
above mark A. adjusts the pinch cork so that 10–15 drops fall per minute
4. Clamp the stalagmometer allow the water drops to fall and start counting the number of
drops when the meniscus crosses the upper mark A and stop counting when the meniscus
passes mark B
5. Repeat the exercise to take three to four readings
6. Rinse the stalagmometer with alcohol and dry it
7. Suck the given liquid in the stalagmometer and count the drops as in case of water.

OBSERVATIONS:
Temperature of water bath = room temperature
Density of water = 0.997 g/cm3
Density of Methanol = 0.7913 g/cm3 Surface
tension of water = 72.00 dynes/cm
OBSERVATION TABLE:
Liquid sample Number of drops from fixed volume Mean number of drops, n
Trial 1 Trial 2 Trial 3
1 Water 32 31 NA n1 = 31.5
2 Methanol 88 103 NA n2 = 95.5

CALCULATIONS:
Surface tension of given liquid, γ2
p2×n1 0.7913×31.5
γ =γ ( ) = 72 × = 18.8488 !"#$%/&'
2 1 p1×n2 0.997×95.5

ρ1 and ρ2 are the densities of liquids 1 and 2

n1 and n2 are the number of drops of water and given liquid

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γ1 and γ2 are the surface tension of water and given liquid

RESULTS:
Surface tension of
1. Methanol= 18.8488dynes/cm

CONCLUSION:
SFT is the tension of the surface film of a liquid due to the cohesive attractions of the particles
in the surface layer. To determine the SFT of liquid paraffin sample, the drop-count method
using a stalagmometer was used. It is an easy and simple method, not requiring any expertise.
The SFT of liquid paraffin was found to be 18.8488 dynes/cm which is lower than that of
distilled water (72.00 dynes/cm).

7
 DETERMINATION OF RELATIVE AND ABSOLUTE VISCOSITY

AIM:
To determine relative and absolute viscosity of a liquid solution using Ostwald’s viscometer.

INTRODUCTION:
The force of friction which one part of the liquid offers to another part of the liquid is called
viscosity. For measuring the viscosity coefficient, Ostwald viscometer method is used which
is based on Poiseuille’s law. According to this law, the rate of flow of liquid through a capillary
π.r4tP
tube having viscosity coefficient, η, can be expressed as: η =
8vl
Where, v = vol. of liquid (in ml)
T = flow time (in sec.) through capillary
r = radius of the capillary (in cm)
l = length of the capillary (in cm)
P = hydrostatic pressure (in dyne/sq.cm)
η = viscosity coefficient (in poise).

Since, the hydrostatic pressure (the driving force) of the liquid is given by P = ρgh (where h
is the height of the column and ρ is the density of the liquid)
η α Pt (or) η α ρght
η1 and η2 are the viscosity coefficients of the liquids under study, ρ1 and ρ2 are densities and
t1 and t2 are times of flow of equal volume of liquids through the same capillary respectively.
η1 α ρ1ght and η2 α ρ2ght
p2×t2
52 = 51
p1×t1

THEORY:

When a layer of liquid is subjected to move upon a surface or another layer of the same
liquid, the fluid particles tend to oppose such movement; this resisting force developed by a
liquid is called viscosity.

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Types of Fluids

A Newtonian fluid's viscosity remains constant, no matter the amount of shear applied for a
constant temperature. These fluids have a linear relationship between viscosity and shear
stress.

Examples:

● Water

● Mineral oil

● Gasoline

● Alcohol

When shear is applied, the viscosity of non-Newtonian fluids decreases or increases,

depending on the fluid.

Examples:

● Quicksand

● Cornflour and water

● Silly putty

Apparent (shear) viscosity: Apparent, or shear, viscosity refers to the relationship between
viscosity and shear rate. In Newtonian fluids, this value doesn’t change, and it can be
calculated by dividing shear stress by shear rate. Viscometers are used to measure apparent
viscosity

Relative viscosity: Relative viscosity is important for non-Newtonian fluids. It refers to the
relationship between molar mass (the mass of a chemical compound divided by total amount)
and viscosity. It’s calculated by dividing the polymer viscosity by the viscosity of the pure
solvent. Rheometers are used to measure relative viscosity

Types of viscosity

• Dynamic viscosity/Absolute viscosity

o Dynamic viscosity is the relationship between velocity gradient and shear

stress. It is more commonly associated with non-Newtonian fluids.

• Kinematic viscosity
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 o Kinematic viscosity refers to the ratio of dynamic viscosity to the density,

o The formula of kinematic viscosity is given as:

o v=η/ρ

• Intrinsic viscosity

o Intrinsic viscosity is a measure of a solute’s contribution to the viscosity of a

solution.

• Inherent viscosity

o Inherent viscosity is the ratio of the natural logarithm of the relative viscosity
to the mass concentration of the polymer.

• Relative viscosity

o Relative viscosity is the ratio of the viscosity of a solution to the viscosity of

the solvent used.

Applications of Viscosity

The viscosity of liquids is a significant property that must be measured accurately in some
industries. Some of its applications are as follows:

● To determine their molecular weights

● To help us choose a suitable lubricant for specific machines. In light machinery thin
oils (for example, lubricant oil used in clocks) with low viscosity is used. In heavy
machinery, highly viscous oils (example: grease) are used

● Drug companies manufacture medicines, such as cough syrup, that have a high
viscosity yet are still drinkable, in order to coat the throat.

● used in brake fluids. Brake fluid transmits force through the braking system, and it
would not operate properly if it had a different viscosity.

Devices used to measure Viscosity:

Viscometer

Other types of viscometers also exist. U-tube viscometers employ a U-shaped tube and
determine the viscosity of a material by measuring the time it takes the material to flow
through the apparatus.

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Bubble viscometers measure the amount of time it takes a bubble of gas to move through the
material whose viscosity is being tested.

A falling sphere viscometer measures viscosity by capturing the time it takes for a sphere of a
known dimension and density to make its way from the top of a material sample to the
bottom.

Rheometer

1) The capillary rheometers

2) Torque rheometers

3) Dynamic Rotational Rheometers

Measuring viscosity

Viscosity of a fluid (Newtonian) is measured using a viscometer. In this experiment, an

Ostwald’s viscometer (aka U-tube) was used which measures the flow rate of the sample.

An Ostwald’s viscometer is not used for highly viscous fluids as

it can be difficult to suck the liquid and it takes a long time for
them to flow freely.

1. Measuring time taken to flow between upper (x) and

lower (y) marks (Ostwald’s viscometer)

2. Calculating ρ2 (Specific gravity flasks)

3. Calculating viscosity η2 in centipoise

Use the formula

!2 × "2
52 = 51 ×
!1 × "1

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REQUIREMENTS:
A) Chemicals needed:
1. Distilled water
2. Methanol
B) Apparatus:
1. Ostwald’s viscometer
2. 25 ml pipette
3. Two beakers
4. Density bottle
5. Rubber bulb
6. Stop watch

PROCEDURE:
1. Clean the viscometer with distilled water.
2. Attach a piece of clean rubber tubing to the left limb and clamp the viscometer exactly in a
vertical place in a water bath. Record the temperature of water bath.
3. By means of a pipette of suitable capacity introduce required volume (as marked on the
viscometer) of liquid into the right limb and allow it to attain the temperature of water bath.
4. With the help of a rubber bulb, suck the liquid in the capillary arm of the viscometer till the
meniscus of the liquid is above the mark A. Allow the liquid to flow back by releasing the
pressure. Start the stop watch when meniscus passes the mark A and stop when it reaches
the mark B. Note down the time of flow of liquid from A to B.
5. Repeat till constant readings are obtained.
6. Empty the viscometer. Rinse it with ethyl alcohol and dry it.
7. Repeat the above procedure by using the required volume of the given liquids.

OBSERVATIONS:
Temperature of water bath = room temperature
Density of water = 0.997 g/cm3
Density of turpentine oil = 0.791 g/cm3
Viscosity of water = 0.8937 cp (centipoise)

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OBSERVATION TABLE:
Liquid sample Flow time in seconds Mean t (sec)
Trial 1 Trial 2 Trial 3
1 Water 100 130 115 t1 = 115
2 Methanol 50 59 68 t2 = 65
CALCULATIONS:
Viscosity of given liquid, η2
5 =5 p2×t2 = 0.8937 × 0.791 ×65 = 0.9240 !"
2 1
p1×t1 0.997×115

ρ1 and ρ2 are the densities of liquids 1 and 2

t1 and t2 are the time of flow of water and given liquid
η1 and η2 are the viscosity of water and given liquid

RESULTS:
Relative viscosity of
1. Methanol = 0.447 cp

CONCLUSION:
Viscosity is the measure of a fluid’s resistance to flow or any objects passing through it. To
determine the relative viscosity of Methanol sample, Ostwald’s viscometer was used; it was
found out to be 0.447 cp at room temperature which is lesser than the relative viscosity of
distilled water (0.8937 cp). Thus, methanol has a lower resistance to flow. Since a
viscometer was used, methanol is a Newtonian fluid (viscosity remains constant).

13
 STUDY OF KINETICS OF ACID CATALYSED HYDROLYSIS OF METHYL
ACETATE WITH HYDROCHLORIC ACID

AIM:
To study the kinetics of the acid-catalysed hydrolysis of methyl acetate with hydrochloric acid
volumetrically by integrated method.

INTRODUCTION:
The rate of hydrolysis of an ester (i.e., methyl acetate) is found to be directly proportional to
the concentration of H+ ions of the acid used as a catalyst. Hydrolysis of methyl acetate takes
place according to the following reaction in the presence of H+ ion.

H+
CH3COOCH3+H2O ––––––→ !+ CH3COOH + CH3OH

Even though the reaction is bimolecular, due to a large excess of water which also acts as a
solvent, the kinetics of the reaction depends only on the concentration of methyl acetate. Hence
the reaction is of the first order. Such a reaction is called, pseudo molecular reaction.
2.303 a
k= log
t (a –x)

where a = initial concentration, x = amount reacted in given time t.

In this reaction amount of acetic acid produced can easily be obtained by titrating the reaction
mixture with NaOH solution.

THEORY:
Hydrolysis is a chemical reaction in which a molecule of water is responsible for the breaking
of one or more chemicals bonds in the reaction.
The general formula of a hydrolysis reaction is:
AB+H2O→AH+BOH
There are 5 different types of hydrolysis reactions. They are:

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 a. Salts: This is the most common type of hydrolysis. During hydrolysis, a salt
breaks down to form ions, completely or partially depending upon the
solubility factor and the water spontaneously ionizes hydroxide anions (OH-)
and hydronium cations (H30+).
b. Acid and Base: Acid–base-catalysed hydrolysis can be found during the
hydrolysis of esters or amides. Here, the process of hydrolysis occurs when
water or hydroxyl ion (OH-) reacts with the carbon of the carbonyl group (-
C=O) of the ester or amide where new compounds are formed. The products
of both hydrolysis are compounds with carboxylic acid groups (-COOH).
c. ATP: Most biochemical reactions that occur in living organisms are in the
form of ATP hydrolysis which takes place with the help of enzymes
acting as catalysts.
d. Metal aqua ions: Metal ions have a positive charge on them which makes them
Lewis acids. These ions, in aqueous solutions, form metal aqua complexes of
the general formula M(H2O)nm+ . These aqua ions undergo hydrolysis to give
ions with the general formula M(H2O)n−1(OH)(m−1)+
e. Polysaccharides: Polysaccharides are Monosaccharides linked together by glycosidic
f. bonds, which can be cleaved by hydrolysis. The hydrolysis of
polysaccharides to soluble sugars is called as saccharification.

In the hydrolysis of methyl acetate, Amberlyst 15 can be used as a catalyst. A common

feature of the hydrolysis of esters and any other organic compounds is that another substance
like any acid or a base, like H+ ions, increases the rate at which the chemical change occurs.
In the contrary, the addition of acids like nitric acid can help in slowing down or supressing
the reaction.

15
The indicators used in titration are:

1. Litmus: Blue litmus paper turns red under acidic conditions while Red litmus paper turns
blue under alkaline conditions.

2. Phenolphthalein: Phenolphthalein turns colourless to pink under Basic conditions while it

remains colourless under Acidic conditions.
3. Methyl orange: In Basic condition Methyl Orange gives yellow colour while under
Acidic conditions it gives red colour.

4. Litmus: Blue litmus paper turns red under acidic conditions while Red litmus paper turns
blue under alkaline conditions.

5. Phenolphthalein: Phenolphthalein turns colourless to pink under Basic conditions while it

remains colourless under Acidic conditions.
6. Methyl orange: In Basic condition Methyl Orange gives yellow colour while under
Acidic conditions it gives red colour.

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The choice of indicator varies from titration to titration:

Strong Acid with Strong Base:

Using Phenolphthalein, Methyl orange, Methyl red or Bromothymol blue can be used to
determine the endpoint.
Strong Acid with Weak Base:
Using Methyl Orange or Methyl red or Bromothymol blue can be used as an indicator.
Weak Acid with Strong Base:
Using Phenolphthalein is a suitable indicator, as it gives accurate colour.
Weak Acid with Weak Base:
No indicator is found to function satisfactorily.

Examples of Hydrolysis:
1) The dissolution of a salt of a weak acid or a base in water is an example of a hydrolysis
reaction. Strong acids can also be hydrolysed. For example, the solution of sulfuric acid in
water produces hydronium and bisulphate.
2) The hydrolysis of a sugar, for example, sugar sucrose may undergo hydrolysis to break
down into its component sugars, glucose and fructose.
3) An example for Acid-Base catalysed hydrolysis is amide hydrolysis.
4) In biological systems. A good example is the hydrolysis of the ATP energy molecule.

While studying chemistry it is important to know hydrolysis as it means setting apart of

chemicals using water. In the metabolism of cell hydrolysis is of prime importance as
Hydrolysis breaks down polymers and is used to form linkages in DNA. Hydrolysis is also
used to produce proteins and synthesis new macromolecules. It is an important part of how
your body breaks down food into its nutritious components. The food you eat is enters the
body as polymers and is broken down to monomers through many hydrolysis reactions. In
biological systems reactions like hydrolysis of the energy molecule adenosine triphosphate,
or ATP is vital for the living.

16
REQUIREMENTS:
1. Glass stoppered bottles
2. Water bath
3. Burette
4. Conical flask
5. Pipette
6. 0.5 N HCl
7. 0.1 N NaOH
8. Methyl acetate
9. Ice
10. Phenolphthalein indicator

PROCEDURE:
1. Pipette out 5 cm3 of methyl acetate in a dry glass stoppered bottle.
2. Take 100 cm3 of 0.5 N HCl using measuring cylinder in another dry glass stoppered
bottle
3. Keep both the bottles in water bath to attain room temperature.
4. Fill the burette with 0.1 N NaOH solution.
5. At a desired time, add the contents of the first bottle i.e., HCl solution to methyl acetate.
Note the time of mixing as t0.
6. Shake the reaction mixture and immediately pipette out 5 cm3 of reaction mixture in a
conical flask containing few ice pieces and 1 or 2 drops of 1% phenolphthalein indicator.
7. Titrate the reaction mixture rapidly with constant shaking against 0.1 N NaOH till the
colour changes from colourless to pink.
8. Record this reading in the observation table against T0.
9. Similarly titrate 5 cm3 of reaction mixture at the time intervals of 10, 20, 30, 40 and 50
minutes against 0.1 N NaOH. These burette readings correspond to Tt.

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OBSERVATION TABLE:
a = (T∞ –To) = 11.8 cm3 T∞ = 11.8 cm3 (given)
2.303 a
Time, t Burette x = Tt – T0 a – x = T∞ – Tt a/a-x log (a/a – x) k= log
t (a –x)
(min) reading, Tt (cm3) (cm3)
(min-1)
(cm3)
00 17 0.0 33 2.9411 0.4685 0.1078

20 19.5 2.5 30.5 2.564 0.4089 0.0470

40 21 4 29 2.3809 0.3767 0.0216

∞ 50 33 0.0 ∞ ∞ ∞

Mean k = 0.1115 min–1

Where, a = initial concentration of ester.
x = amount of ester reacted at time t.
(a – x) = amount of ester unreacted at time t.
T0 = titration reading at zero time i.e., when hydrolysis has not started.
Tt = titration reading at time t.
T∞ = titration reading when hydrolysis is completed.

CALCULATION:
2.303 a
k= log
t (a –x)

2.303 (T∞–To)
mk = log
t (T∞–Tt)

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GRAPH:
Plot a graph of Rate constant against Time t

rate constant
0.12
0.1
Rate Constant

0.08
0.06
0.04
0.02
0
0 5 10 15 20 25 30 35 40 45
Time(min)

RESULTS:
Value of rate constant (k)
1. By calculation = 0.115 min–1
CONCLUSION:
The rate constant of the hydrolysis of methyl acetate was studied by titrating HCl acid (and
ethanoic acid mixture) against 0.1N NaOH.
H+
The hydrolysis reaction CH3COOCH3+H2O ––––––→ !+ CH3COOH + CH3OH uses a HCl

acid catalyst (H+). Other examples of acid catalysts include H2SO4 acid and 1-butylsulfonic-
3-methylimidazolium trifluoromethanesulfonate. Hydrolysis can also occur when methyl
acetate is in alkaline medium. Acid hydrolysis of methyl acetate is a pseudo first order
reaction. This is because even though the rate of reaction theoretically depends on the
concentration of ester and water (bimolecular – second order), [H2O] remains constant
because it is in a large excess, thus the experimental rate of reaction now only depends on
[CH3COOCH3] making it a pseudo first order reaction. Everyone calculated the k values and
a graph was plotted of k against t. The graph shows that k increases from 0 to 0.013 as time
increases from 0 mins to 50 mins (directly proportional). Thus, the hydrolysis of methyl
acetate shows that its rate constant increases with time. It also justifies that the acid catalyst
increases k with time. For the applications, methyl acetate is used to flavour foods due to its
pleasant smell (like whisky). As for hydrolysis, it is an important reaction, for e.g., in
hydrolysing ATP molecules to release energy.

19
 ION EXCHANGE CHROMATOGRAPHY

AIM: To study protein purification using ion exchange chromatography.

INTRODUCTION:
Ion-exchange chromatography (IEC) is a process that allows the separation of ions and polar
molecules based on the charge of the molecules. IEC is a form of adsorption chromatography
in which ionic solutes display reversible electrostatic interactions with a charged stationary
phase. Stationary phase is a synthetic resin tagged with positively or negatively charged
functional groups. Resin binds oppositely charged ions. Elution of bound ions requires
similarly-charged competing ions:
Resin and charge of matrix bound ion eluting ion
anion-exchange: (+) anion anion
cation-exchange: (-) cation cation

IEC is further subdivided into Cation Exchange Chromatography and Anion Exchange
Chromatography. Cation Exchange Chromatography (CEC) retains positively charged
cations, as the stationary phase displays a negatively charged functional group such as a
phosphoric acid. Commonly used cation exchange resins are S-resin, sulfate derivatives; and
CM resins, carboxylate derived ions. Anion exchange chromatography (AEC) retains
negatively charged anions, using positively charged functional group such as a quaternary
ammonium cation. Commonly used anion exchange resins are Q-resin, a Quaternary amine;
and DEAE resin, Di Ethyl Amino Ethane. AEC is used as a primary chromatography. Uses of
AEC include initial cleanup of crude slurry, separation of proteins from each other,
concentrating a protein, and the removal of negatively charged endotoxin from protein
preparations. Uses of CEC include initial cleanup of crude slurry, separation of proteins from
each other, concentrating a protein. Proteins carry both positive and negative charges and the
overall net charge are dependent on the pH. Below its pI (iso-electric point), a protein has more
positively charged amino acids and therefore an overall positive charge and bind to cation
exchangers. Conversely, above its pI, a protein has more negatively charged amino acids and
an overall negative charge; the protein will bind to anion exchangers. At its pI, a protein will
not bind to either a cationic or an anionic exchanger.

20
 Anion exchanger

Cation exchanger

THEORY:
Ion exchange chromatography is commonly used to separate charged biological molecules
such as proteins, peptides, nucleotides, or amino acids. The two types of ion exchange
chromatography are -
1. Anion exchange chromatography is used to separate molecules based on their net
surface charge. It uses a positively charged ion exchange resin with an affinity for
molecules having net negative surface charges. It is used both for preparative and
analytical purposes and can separate a large range of molecules, from amino acids and
nucleotides to large proteins. A protein’s net surface charge changes with pH in a
manner that is dictated by a protein’s isoelectric point, or pI. At a pH equal to a
protein’s pI, the protein carries no net charge. At a pH below the pI, the protein
carries a net positive charge. If the buffer pH is raised above a protein’s pI, it carries a
net negative charge.
2. Cation exchange chromatography is the separation of proteins on the basis of their
negative charge for cation exchange resins are negatively charged, which reflects the
number and nature of charged amino acid residues on the protein as well as the pH of
the buffer.
In this type of chromatography, the mobile phase is negatively charged, possessing an
affinity for molecules of net positive charge that are packed to be attracted to it.

21
 There are two resins depending on their strength:
1. CM (Carboxymethyl group, weak cation exchange)
2. SP (sulfopropyl group, strong cation exchange)

Examples Of Buffers used in EIC:

Cationic Buffers (Positively charge) are used for anion exchange chromatography. For
example: Alkyl amines, Tris, Amino ethyl alcohol
Anionic Buffers (Negatively charge) are used for cation exchange chromatography. For
example: Phosphate, acetate, Citrate

Ion exchange chromatography resins are composed of positively or negatively charged

functional groups that are covalently bound to a solid matrix. Common matrices are cellulose,
agarose, polymethacrylate, polystyrene, and polyacrylamide. The latter three matrices allow
higher flow rates.
Several factors inform resin choice:
• Anion exchanger or cation exchanger
• Weak vs. strong ion exchanger
• Ionic form of the resin
• Resin particle size
• Permissible flow rate
• Dynamic binding capacity
Anion exchange chromatography, more specifically, uses a positively charged ion exchange
resin with an affinity for molecules having net negative surface charges. Cation exchange
chromatography, more specifically, uses a negatively charged ion exchange resin with an
affinity for molecules having net positive surface charges.

Applications of Ion exchange chromatography:

1. Ion-exchange chromatography is a chromatographic tool for chemical analysis and ion
separation that is commonly used. It is commonly used in biochemistry to isolate
charged molecules such as proteins, for example. Extraction and purification of
biologically derived substances such as proteins (amino acids) and DNA/RNA are
essential aspects of the application.

22
 2. Water treatment is a major industrial application for ion exchange. Hard water is
softened by exchanging calcium and magnesium ions with sodium ions. Hard water is
caused by the presence of calcium and magnesium ions, which form insoluble
precipitates with soaps. To do so, the hard water is passed through a sodium-ion-
containing cation exchanger column. Calcium and magnesium begin to appear in the
water leaving the column after it has been in use for a while. The column must then be
regenerated by slowly passing a concentrated solution of common salt through it; the
excess sodium ions displace the ions that cause stiffness, and the bed of the exchanger
is ready to use again after flushing with water. Natural aluminosilicates were initially
used as heat exchangers, but synthetic resins were eventually substituted.

Cation exchange separates rare earth elements on an industrial scale using a displacement
technique in which each element displaces elements bound less strongly as it progresses
down the column. The elements appear in high purity one after the other (the one with the
weakest bond first).

REQUIREMENTS:
A) Instruments
1. SDS PAGE apparatus
2. Power pack
3. Hot plate to heat the samples
4. Micro-centrifuge
B) Glassware/Plasticware
1. 1.5ml eppendorf vials
2. Columns
3. 4 ml Bottles
4. Micropipette- 1ml and 200µl
5. Staining trays
C) Reagents/culture/media
Chromous Biotech Ion exchange teaching kit that contains the following:
1. Protein Mix
2. Q-sepharose- Anion Exchange Chromatography resin
3. CM- sepharose- Cation Exchange Chromatography resin

23
 4. Equilibration buffer Q
5. Equilibration buffer CM
6. Elution buffer Q
7. Elution buffer CM
8. PBS
SDS-PAGE components that includes the following:
9. Separating Gel Mix (12 %)
10. Stacking Gel Mix (5%)
11. 30% Acrylamide
12. APS Powder
13. TEMED
14. 10XGel Running Buffer
15. Sample Loading Buffer
16. Staining Solution
17. Ready-to-load protein marker
18. Distilled water
D) Miscellaneous
1. Test tube rack
2. Burette stand

PROCEDURE:
Anion Exchange Chromatography:
1. Take a fresh column from the kit provided. Fix the column vertically to a stand. Add 1ml
of distilled autoclaved water to make the column and base moist.
2. Use P-200 Pipette for packing the column material, without any air bubbles trapped in the
Column. Cut the end of the pipette tip to pack the column.
3. Remove the lower cap of the column and pack 0.5 ml of the Q-sepharose column material
provided. Allow the liquid in the column material to flow down.
4. Do not let the column dry. Allow the liquid in the column material to settle down; pack
slowly, keep the buffer volume to ~ 0.3 ml. (to ensure the column does not dry).
5. If Slurry becomes too dense, add respective Equilibration buffer to the Column material
bottle mix and pack the column.

24
6. Equilibrate the column with 2.5 ml of Equilibration Buffer- Q provided with the kit. Allow
the buffer to flow through the column. Use P-200 Pipette for loading buffer to avoid air
bubble formation.
7. Prepare Load for the column by adding 0.9 ml of Equilibration Buffer-Q to 100◻l of
protein mix. Mix it properly before loading on to column.
8. When almost all the buffer is drained out, load the protein sample on to the column.
9. Load 200 µl of the above 1 ml diluted Protein sample (Load) prepared onto the column.
10. All the fractions are collected in 3 ml or 5 ml test tubes, which are not provided in the kit.
11. Allow the sample to flow through the column completely. Collect the flow through coming
out after adding the protein sample. Label it as Flow through-Q.
12. Add 4 ml of Equilibration buffer-Q (for washing the unbound protein) provided onto the
column.
13. Allow the buffer to flow through the column. Collect 4 ml of buffer flowing out in bottle
and label this tube as Wash.
14. Allow the Equilibration buffer-Q to flow out completely, leaving the column almost dry.
15. Add 1ml of Elution buffer-Q. Allow the buffer to pass through the column bed.
16. Collect 1ml of the elution buffer-Q coming out of the column in a tube. Collect as a single
fraction. Label the tube as Elution Q-sepharose.

Cation Exchange Chromatography:

1. Similarly, when performing the experiment with CM-Sepharose, pack column with 0.5 ml
of CM-Sepharose.
2. Slurry becomes too dense add respective Equilibration buffer to the Column material bottle
mix and pack the column.
3. Equilibrate the column with 2.5 ml of Equilibration buffer-CM
4. Load 200µl of protein sample (100 µl of protein mix diluted with 0.9 ml Equilibration
buffer-CM). Collect the flow through in a test tube.
5. Wash the column with 4 ml of Equilibration buffer-CM (for washing the unbound protein).
Collect the wash buffer in a bottle labeled as Wash CM
6. Elute the protein by adding 1ml of Elution buffer-CM. Label it as Elution CM.

25
Sample preparation
1. The samples are prepared in 1.5 ml vials provided in the kit.
2. Add 5µl of Sample Loading Buffer (SLB) with 20 µl of each fraction; boil for 2 minutes
and the samples are ready to load.
3. Load 20µl of Ready-to-load Protein marker, no boiling is required.
4. Load all the samples collected on to the 12 % SDS-PAGE prepared previously.
5. Load the samples in following order:

Amount of sample SLB to be added to

Sample
the sample (in µl)
LOAD 5µl 10µl
Flow through-Q 20µl 5µl
Wash –Q 20µl 5µl
Elution-Q 20µl 5µl
Flow through - CM 20µl 5µl
Wash-CM 20µl 5µl
Elution – CM 20µl 5µl
Ready-to-load Protein marker 20µl -

6. For casting the gel follow the steps given below:

Preparation of SDS-PAGE:
1. Prepare a casting assembly as per instrument instruction manual. Ensure the assembly is
leak proof by filling water between the plates. Measure the gel volume (by pouring and
measuring water). Ensure 6ml of separating gel and 4ml of stacking for 10ml gel volume.
2. Add 1ml of water to 100 mg of APS (provided in tube)
3. Add 60 µl of ammonium persulphate and 2.4 µl TEMED and 2.4ml of 30% Acrylamide to
3.6 ml of separating gel mix and mix thoroughly.
4. Pour the gel solution between the plates 60% of gel volume.
5. Add 200 to 250 µl of water very gently on the top to ensure smooth interface.
6. After the gel set (approximately 30 minutes), wash the top of the separating gel with
distilled water and drain off the water completely.
26
7. Add 40 µl of APS solution and 4 µl TEMED and 0.67ml of 30% Acrylamide to 3.4ml of
stacking gel mix; mixed thoroughly and poured on top of the polymerized separating gel.
8. Insert the comb immediately into the gel solution carefully without trapping air bubbles.
The stacking gel will polymerise in about 30 minutes.
9. After the stacking gel has set, carefully remove the comb and the bottom spacer. Wash the
wells immediately with distilled water to remove non-polymerized acrylamide.
10. Fill the gel running apparatus with 1X Gel Running Buffer.
11. Carefully fix the plate to the PAGE apparatus with the notched plate facing the top reservoir
without trapping air bubbles between the buffer and the bottom of the gel.
12. Load samples into the wells using fresh tips. Note down the order in which the samples
have been loaded.
13. Connect the cords to the power supply, according to the convention, red for anode and
black for cathode.
14. Set current at 20mA and switch on the power supply.
15. When the dye front reaches the separating gel, the current can be increased to 40mA.
Continue to run till the dye-head reaches 1 cm above the bottom of the gel, turn off the
power.
16. Remove the gel plates.
17. Transfer the gel to a tray containing water to wash the gel. Discard the water. Add 25 ml
of staining solution to gel container. Stain it for more than 45 min on a rocker until bands
visible. If the bands appear faint, stain for overnight.
18. De-stain the gel with water.

27
FLOW-CHART:
Pack the column
▼
Equilibrate the column
▼
Load protein mix
▼
Flow through
▼
Wash
▼
Elution
▼

Cast SDS PAGE Load on SDS-PAGE

▼
Stain, de-stain and Observe

CONCLUSION:
A demo practical of IEC of both types : anionic and cationic was observed. The cationic IEC
used a citrate solution as buffer and a mixture of amino acids was the analyte solution.

28
 QUALITATIVE ANALYSIS OF DRUG BY THIN LAYER CHROMATOGRAPHY

AIM:
To perform qualitative analysis of drug by thin layer chromatography

INTRODUCTION:
Chromatography is the collective term for a set of laboratory techniques for the separation of
mixtures. It was first employed in Russia by the Italian-born scientist Mikhail Tsvet in 1900.
Planar chromatography is a separation technique in which the stationary phase is present as or
on a plane. Thin layer chromatography is an example of such type of chromatography.
Thin Layer Chromatography (TLC) is a commonly used analytical technique that allows for
rapid and inexpensive analysis of various mixtures. It is usually used for separation of non-
volatile substances.
The stationary phase of TLC is a glass, plastic or an aluminium sheet, which is coated with
thin layer of adsorbent like silica gel, aluminium oxide or cellulose.
The mobile phase of the system is a solvent or a mixture of solvents which is drawn by the
plate via capillary action. Different analytes ascend the TLC plate at different rates which may
be because of molecular weight, size, porosity of the adsorbent, etc, (Vogel et al, 2002).
Thin-layer chromatography can be used to monitor the progress of a reaction, identify
compounds present in a given mixture, and determine the purity of a substance. Specific
examples of these applications include: analyzing ceramides and fatty acids, detection of
pesticides or insecticides in food and water, analyzing the dye composition of fibers in
forensics, assaying the radiochemical purity of radiopharmaceuticals, or identification of
medicinal plants and their constituents.

REQUIREMENTS:
1. TLC plate
2. Mobile phase: Toluene, Acetone, Methanol, Formic Acid (6.9:2:2:0.15)
3. 10% developing reagent – Methanolic sulphuric acid (90:10)
4. Sample – plant extract (1:10 diluted)
5. capillaries
6. UV chamber
29
PROCEDURE:
1. Cut a strip of 10 cm × 4 cm silica gel TLC plates from the 20 cm × 20 cm sheet.
2. Mark the starting position 1 cm from the bottom of the plate using a pencil. Remember,
your spots must lie above the level of the mobile phase when the plate is placed into the
chamber.
3. Apply the sample as thin bands of approximately 6–7 mm width using capillaries. Be
careful that the spots do not spread and they remain as concentrated as possible.
4. Once all the spots are dried, place the sheets (origin side down) in a TLC chamber with ~
1 cm of mobile phase at the bottom.
5. Allow the mobile phase to travel up to 8 cm.
6. Remove the plate and allow to dry thoroughly.
7. Further, observe the plate under the UV at 254 nm and 366 nm. Click images for
documentation.
8. Dip the plates rapidly in the developing reagent for 1–2 sec and remove the excess reagent
by draining on a tissue paper.
9. Develop the plates by heating at 105°C for 5–10 min.
10. Observe the plates under UV at 254 nm and 366 nm again. Click images for documentation.
11. Measure the distance travelled by each band and the solvent front from the origin and
calculate the Rf values for each band.

30
OBSERVATION:

a b c a b c
1 2
1. TLC plate under normal conditions
2. TLC plate under 254nm in UV chamber
3. TLC plate under 366nm in UV chamber
a. Aspirin
b. Ibuprofen
c. Caffeine

RESULT:
Distance of the band from the origin
Rf value =
solvent front

R fvalue (Aspirin) = 0.9091cm = 5/5.5

Rf value (Ibuprofen) = 0.7273cm = 4/5.5

R fvalue (Caffeine) = 0.0909 cm = 0.5/5.5

31
CONCLUSION:
Thin Layer Chromatography was conducted to detect the presence of standard Aspirin ,
Ibuprofen and caffeine.

32
 ESTIMATION OF A DRUG BY HIGH PERFORMANCE LIQUID
CHROMATOGRAPHY (HPLC)

AIM: Estimation of a drug by high performance liquid chromatography (HPLC).

INTRODUCTION:

High-performance liquid chromatography (or high-pressure liquid chromatography, HPLC)

is a specific form of column chromatography generally used to separate, identify, and
quantify the active compounds. HPLC mainly utilizes a column that holds packing material
(stationary phase), a pump that moves the mobile phase through the column, and a detector
that shows the retention times of the molecules. Retention time varies depending on the
interactions between the stationary phase, the molecules being analyzed, and the solvents
used. The sample to be analyzed is introduced in small volume to the stream of mobile phase
and is retarded by specific chemical or physical interactions with the stationary phase. The
amount of retardation depends on the nature of the analyte and composition of both stationary and
mobile phase. The time at which a specific analyte elutes (comes out of the end of the
column) is called the retention time. Common solvents used include any miscible
combinations of water or organic liquids (the most common are methanol and acetonitrile).
Separation has been done to vary the mobile phase composition during the analysis; this is
known as gradient elution. The gradient separates the analyte mixtures as a function of the
affinity of the analyte for the current mobile phase. The choice of solvents, additives and
gradient depend on the nature of the stationary phase and the analyte.

Figure 1: Components of HPLC

33
 Types of HPLC:

Types of HPLC generally depend on phase system used in the process. Following types of
HPLC are generally used in analysis:
Normal phase chromatography: Also known Normal phase HPLC (NP-HPLC), this
method separates analytes based on polarity. NP-HPLC uses a polar stationary phase and
a non-polar mobile phase. The polar analyte interacted with and is retained by the polar
stationary phase. Adsorption strengths increase with increased analyte polarity, and the
interaction between the polar analyte and the polar stationary phase increases the elution
time.
Reversed phase chromatography: Reversed phase HPLC (RP-HPLC or RPC) has a non-
polar stationary phase and an aqueous, moderately polar mobile phase. RPC operates on
the principle of hydrophobic interactions, which result from repulsive forces between a
polar eluent, the relatively non-polar analyte, and the non-polar stationary phase. The
binding of the analyte to the stationary phase is proportional to the contact surface area
around the non-polar segment of the analyte molecule upon association with the ligand in
the aqueous eluent.

REQUIREMENTS:
1. For solvent system (HPLC grade): 0.1% Formic acid and Acetonitrile (30:70)
2. Distilled water
3. Drug sample: Standard Quercetin
4. Column: Kromasil C18 reverse-phase column 4.6×250 mm (5 µm particle size)
5. HPLC system equipped with a UV detector

PROCEDURE:
1. Mobile Phase preparation:
● Mobile phase: 0.1% Formic acid: Acetonitrile (30:70)
● To 300 ml of Distilled water, 300 µl of Formic acid was added, the pH was
adjusted to 2 (using orthophosphoric acid) and was degassed using the bath
sonicator
● Blank/ Mobile Phase was prepared using ACN and 0.1% buffer in the ratio 70:30

34
 2. Standard preparation:
● Stock solution of 1000 ppm Quercetin: 1mg of Quercetin was dissolved in 1 ml of
ethanol to make a standard stock solution of 1000 ppm
● The stock solution was diluted with ethanol to prepare a working stock solution of
100 ppm.
● The working stock was diluted with mobile phase to prepare 1, 5, 10, 15, 20, 25
ppm for testing Linearity parameters. The dilutions were made from the stock
solution as follows:
Conc. Volume of Mobile Total
(ppm) stock (µl) Phase (µl) Volume (ml)
1 1 999 1
5 5 995 1
10 10 990 1
15 15 985 1
20 20 980 1
25 25 975 1

3) Sample preparation:

● 10 ml of 1000 ppm plant extract was prepared by dissolving 10 mg of plant extract in

10 ml of ethanol. It was further diluted to 100 ppm with mobile phase and its
concentration was determined

CHROMATOGRAPHIC CONDITIONS:
• Sample name: Standard drug: Quercetin; Test samples: Plant extract 1, Plant extract 2
• Mobile Phase: 0.1% Formic acid and Acetonitrile (30:70)
• Column: Kromasil C18 reverse-phase column 4.6×250mm (5 µm particle size)
• λabs : 268 nm
• Flow rate: 0.5 ml/min
• Run Time: 30 mins

35
 OBSERVATION:

(a) HPLC chromatogram of quercetin standard at 365 nm; 20 µL of the quercetin standard
(Sigma Aldrich, USA) in the range of 10–1000 µg/mL was injected into a C18, using H 3 PO 4
10 mM in water (solvent A) and acetonitrile (solvent B) with gradient elution at a flow rate of
0.8 mL/min. Quercetin peak appeared at a retention time of 13.64 min. (b) HPLC
chromatogram of plant extract at 365 nm. After acid hydrolysis of 100 mg.

RESULTS:
We performed a Demo Experiment and observed an exemplary sample already running in the HPLC.

CONCLUSION:

We successfully observed the presence of pure compound and sample with mixture of two compound
by comparing the peaks. And where the graph represented concentration by area under the graph
and retention time of the sample.

36
 SIZE EXCLUSION/GEL FILTRATION CHROMATOGRAPHY

AIM: To study protein purification using gel filtration/gel exclusion chromatography

INTRODUCTION:
Size Exclusion Chromatography (SEC) is a chromatographic method in which particles are
separated based on their size, or in more technical terms, their hydrodynamic volume.
Separation is usually achieved with an apparatus called a column, which consists of a hollow
tube tightly packed with extremely small porous polymer beads designed to have pores of
different sizes. A solution of biomolecules (mobile phase) is applied to a column filled with
porous polymer beads or gel (stationary phase). Molecules larger than the largest pores in the
gel are excluded from the gel and come out first. Intermediate-size molecules penetrate the gel
to varying extents depending on their size; this retards their progress through the column.
Larger molecules come out first while small molecules elute last. SEC separates proteins,
carbohydrates and nucleic acids on the basis of their molecular mass (Dalton)/Stokes radius.
Stokes radius: effective radius of a molecule as it tumbles freely in solution; Dalton = unit of
mass equal to 1/12th mass of 12C (atomic mass unit). The main application of gel filtration
chromatography is the fractionation of proteins and other water-soluble polymers, while gel
permeation chromatography is used to analyze the molecular weight distribution of organic-
soluble polymers. Also, it’s used for desalting proteins, e.g., Proteins precipitated by
ammonium sulfate. Important properties of gel filtration: fractionation range (e.g. Sephadex
G100, 4 to 100 kDa), Exclusion limit (G-100 has an upper limit of 100 kDa), Sample loading
volume (for separation 1- 5% of bed volume and for desalting 10-20% of bed volume).

37
REQUIREMENTS:
A) Glassware/Plasticware
1. Columns
2. 1.5ml vials
3. Micropipettes- 1ml and 200µl
B) Reagents/culture/media
1. Protein Mix
2. Column
3. Regeneration buffer
C) Miscellaneous
1. Test tube rack
2. Burette stand

PROCEDURE:
1. Gel filtration column available in the GeNeiTM kit was fixed vertically to the column stand.
2. Top cap of the column was removed and 4 mL of gel filtration buffer was added for
equilibrating the column. Excess of the buffer was drained out completely.
3. In the meantime, sample was prepared as follows: In 1.5ml tube containing the crude
sample, 0.2 ml of gel filtration buffer was added and mixed until the sample was completely
dissolved
4. 0.2 ml of the sample was loaded onto the column, along the sides of the column.
38
5. The sample was allowed to sink completely and then 0.2 ml of buffer was added.
6. The buffer was allowed to flow out completely.
7. The column was filled with buffer, till all the colored biomolecules have eluted out.
8. Three colored fractions were eluted and collected from the column in three separate
centrifuge tubes.
9. The column was washed with the buffer and was sealed by placing the bottom cap first
followed by top cap then and it was stored at 40C for future use.

FLOW-CHART: Pack column with agarose beads

▼
Equilibrate the column
▼
Load protein mix
▼
Collect the fractions
▼
Regenerate the column
▼
OBSERVATION:

Fig. Picture of the gel filtration chromatography column showing dextran, haemoglobin
and vitamin B12 fractions
39
RESULT AND CONCLUSION:
The blue-colored component is dextran having a molecular weight of 20,00,000 Da. These
are large molecules that exit fast from the column and are collected as the first fraction. The
brownish-red colored component collected is hemoglobin having a molecular weight of
64500 Da; they have intermediate molecular weight, so they enter the pores, but their retention
time is low and hence they take less time to exit the column. The pink-colored component is
vitamin B12 having molecular weight of 376 Da. These are very small molecules that enter or
permeate through the pores of the beads and are retained in the beads for a longer time. They
take a long time to exit the column and are collected as the third fraction.

40
 NATIVE AND SDS PAGE

AIM: To perform Native and SDS Polyacrylamide gel electrophoresis (PAGE).

INTRODUCTION:
Electrophoresis can be one dimensional (i.e., one plane of separation) or two dimensional. One
dimensional electrophoresis is used for most routine protein and nucleic acid separations. Two
dimensional separation of proteins is used for finger-printing, and when properly constructed
can be extremely accurate in resolving all of the proteins present within a cell.

Polyacrylamide gels
When separated on a polyacrylamide gel, the procedure is abbreviated as SDS-PAGE (for
Sodium Dodecyl Sulfate Poly Acrylamide Gel Electrophoresis). The technique is a standard
means for separating proteins according to their molecular weight. Acrylamide is the material
of choice for preparing electrophoretic gels to separate proteins by size. Acrylamide mixed
with bisacrylamide forms a crosslinked polymer network when the polymerizing agent,
ammonium persulfate (APS), is added. TEMED (N,N,N,N'-tetramethylenediamine) catalyzes
the polymerization reaction by promoting the production of free radicals by APS.

Fig. – Polyacrylamide gel polymerization

The size of the pores created in the gel is inversely related to the amount of acrylamide used.
A 7% polyacrylamide gel has larger pores than a 12% polyacrylamide gel. Gels with a low
percentage of acrylamide are typically used to resolve large proteins, and high percentage gels

41
are used to resolve small proteins. "Gradient gels" are specially prepared to have low percent
acrylamide at the top (beginning of sample path) and high percent-acrylamide at the bottom
(end), enabling a broader range of protein sizes to be separated. Electrophoresis gels are
formulated in buffers that provide for conduction of an electrical current through the matrix.
The solution is poured into the thin space between two glass or plastic plates of an assembly
called a "cassette". Once the gel polymerizes, the cassette is mounted (usually vertically) into
an apparatus so that opposite edges (top and bottom) are placed in contact with buffer chambers
containing cathode and anode electrodes, respectively. When proteins are added in wells at the
top edge and current is applied, the proteins are drawn by the current through the matrix slab
and separated by the its sieving properties. To obtain optimal resolution of proteins, a stacking
gel is cast over the top of the resolving‖ gel. The stacking gel has a lower concentration of
acrylamide (e.g., 7% for larger pore size), lower pH (e.g., 6.8) and a different ionic content.
This allows the proteins in a loaded sample to be concentrated into a tight band during the first
few minutes of electrophoresis before entering the resolving portion of a gel. A stacking gel is
not necessary when using a gradient gel, as the gradient itself performs this function.

Table 1 – Polyacrylamide gel percentages on basis of protein size

Native PAGE:
In native PAGE, proteins are separated according to the net charge, size and shape of their
native structure. Electrophoretic migration occurs because most proteins carry a net negative
charge in alkaline running buffers. The higher the negative charge density (more charges per
molecule mass), the faster a protein will migrate. At the same time, the frictional force of the
gel matrix creates a sieving effect, retarding the movement of proteins according to their size
42
and three-dimensional shape. Small proteins face only a small frictional force while large
proteins face a larger frictional force. Thus, native PAGE separates proteins based upon both
their charge and mass. Because no denaturants are used in native PAGE, subunit interactions
within a multimeric protein are generally retained and information can be gained about the
quaternary structure. In addition, some proteins retain their enzymatic activity (function)
following separation by native PAGE. Thus, it may be used for preparation of purified, active
proteins. Following electrophoresis, proteins can be recovered from a native gel by passive
diffusion or electroelution. In order to maintain the integrity of proteins during electrophoresis,
it is important to keep the apparatus cool and minimize the effects of denaturation and
proteolysis. Extremes of pH should generally be avoided in native PAGE as they may lead to
irreversible damage to protein of interest, such as denaturation or aggregation.

Polyacrylamide Gel Electrophoresis (SDS-PAGE):

SDS PAGE is an analytical method used to separate components of a protein mixture based
on their size. The biological samples are treated with Sodium dodecyl sulfate (SDS) so that
they acquire uniform charge, then the electrophoretic mobility depends primarily on size.
SDS is a detergent that disrupts the tertiary structure of proteins and coats them with a uniform
negative charge. This changes the folded proteins to linear molecules. The proteins being
covered by SDS are negatively charged and when loaded onto a gel and placed in an electric
field, migrate towards the anode and get separated by a molecular sieving effect based on size.
The gel used is divided into an upper stacking gel of low percentage (with large pore size) and
low pH (6.8), where the protein bands get stacked and a resolving gel (pH 8.8) with smaller
pores where the proteins gets separated on the basis of its molecular size by the sieving effect
of pores in gel.

Principle of SDS-PAGE:
SDS-PAGE separates proteins according to their molecular weight, based on their differential
rates of migration through a sieving matrix (a gel) under the influence of an applied electrical
field.
Difference between a stacking gel and Running gel and its pH:
The system is set up with a stacking gel at pH 6.8, buffered by Tris-HCl, a running gel buffered
to pH 8.8 by Tris-HCl and an electrode buffer at pH 8.3. The stacking gel has a low
43
concentration of acrylamide and the running gel a higher concentration capable of retarding the
movement of the proteins.

Figure – SDS-PAGE stacking and resolving gel

Glycine can exist in three different charge states, positive, neutral or negative depending on the
pH. This is shown in the diagram above. Control of the charge state of the glycine by the
different buffers is the key to the whole stacking gel.
When the power is turned on, the negatively-charged glycine ions in the pH 8.3 electrode
buffer are forced to enter the stacking gel, where the pH is 6.8. In this environment glycine
switches predominantly to the zwitterionic (neutrally charged) state. This loss of charge causes
them to move very slowly in the electric field. The Cl- ions (from Tris-HCl) on the other hand,
move much more quickly in the electric field and they form an ion front that migrates ahead of
the glycine. The separation of Cl- ions from the Tris counter-ion (which is now moving towards
the cathode) creates a narrow zone with a steep voltage gradient that pulls the glycine along
behind it, resulting in two narrowly separated fronts of migrating ions; the highly mobile Cl-
ions front, followed by the slower, mostly neutral glycine front. All of the proteins in the gel
sample have an electrophoretic mobility that is intermediate between the extreme of the
mobility of the glycine and Cl- ions so when the two fronts sweep through the sample well the
proteins are concentrated into the narrow zone between the Cl- ions and glycine fronts. This
procession carries on until it hits the running gel, where the pH switches to 8.8. At this pH the
glycine molecules are mostly negatively charged and can migrate much faster than the proteins.
So the glycine front accelerates past the proteins, leaving them in the dust. The result is that
the proteins are dumped in a very narrow band at the interface of the stacking and running gels

44
and since the running gel has an increased acrylamide concentration, which slows the
movement of the proteins according to their size, the separation begins. Chloride ion (Cl-) is
the only mobile anion present in both gels. Stacking occurs by the differential migration of
ionic species, which carry the electric current through the gel. When an electrical current is
applied to the gel, the Cl- ions with highest charge/mass ratio move fast. SDS coated proteins
has a higher charge/mass ratio than glycine so it moves fast, but slower than Cl- ions. When
protein encounters resolving gel it slows the migration because of increased frictional
resistance, allowing the protein to stack in the gel. When glycine reaches resolving gel it
becomes negatively charged and migrates much faster than protein due to higher charge/mass
ratio. Now proteins act as the main carrier of current and separate according to their molecular
mass by the sieving effect of pores in gel.

After the visualization by Coomassie Brilliant Blue R-250 staining technique, the size of a
protein can be calculated by comparing its migration distance with that of a known molecular
weight ladder.

Visualization of proteins in gels:

Once protein bands have been separated by polyacrylamide gel electrophoresis, they can be
visualized directly in the gel using various staining or detection methods. Coomassie dye is the
most popular reagent for staining protein bands in electrophoretic gels. In acidic buffer
45
conditions, Coomassie dye binds to basic and hydrophobic residues of proteins, changing from
dull reddish brown to intense blue. As with all staining methods, Coomassie dye reagents detect
some proteins better than others based on their chemistry of action and differences in protein
composition. For most proteins, however, Coomassie dye reagents detect as few as 10
nanograms per band in a mini-gel. Most staining methods involve some version of the same
incubation steps: A water-wash to remove electrophoresis buffers from the gel matrix. An acid-
or alcohol-wash to condition or fix the gel to limit diffusion of protein bands from the matrix.
Treatment with the stain reagent to allow the dye or chemical to diffuse into the gel and bind
(or react with) the proteins. De-staining to remove excess dye from the background gel matrix
As soon as the power is turned off the separated protein bands will begin to diffuse (they are
freely soluble in aqueous solution). To prevent diffusion of proteins the gel is treated with a
40% distilled water, 10% acetic acid, and 50% methanol solution which causes almost all
proteins to precipitate (become insoluble). To visualize the fixed proteins, the gel is placed in
the same mixture of water/acetic acid/methanol but with the addition of 0.25% by weight
Coomassie Brilliant Blue R-250 and incubated either for 4 hours to overnight at room
temperature on a shaker. Later, it is de-stained with the same solution and the Coomassie dye.
The stain will not bind to the acrylamide, and will wash out (leaving a clear gel). However, it
remains strongly bound to the proteins in the gel, and these take on a deep blue colour. Another
popular method for detecting protein bands within a gel is silver staining, which deposits
metallic silver onto the surface of a gel at the location of protein bands. Commercial silver stain
kits are exceptionally robust and easy to use, detecting less than 0.5 nanograms of protein in
typical gels and use glutaraldehyde or formaldehyde as enhancers, and typical formulations
chemically crosslink proteins in the gel matrix.

REQUIREMENTS:
A) Instruments
1. PAGE apparatus – Biorad
2. Power pack
3. Heating plate to heat the samples
4. Microcentrifuge
B) Glassware/Plasticware
1. 1.5 ml eppendorf tubes
2. Staining trays
46
 3. Glass Beakers: 100, 500 and 1000 ml
4. Glass test tubes
5. Glass storage bottles- 1000 ml
6. Measuring cylinder
7. Pipette tips
C) Reagents/culture/media
1. 30% acrylamide mix (29.2gm acrylamide, 0.8 gm bis-acrylamide in 100ml D/w)
2. 1.5M Tris-Cl pH-8.8 (18.15gm Tris base in 100ml D/w, pH-8.8)
3. 1.0M Tris-Cl pH-6.8 (12.1gm Tris base in 100ml D/w, pH-6.8)
4. 10% SDS- (10gm SDS in 100ml D/w)
5. 10% Ammonium persulphate (APS)- (0.05gm APS in 500ul D/w)
6. TEMED
7. Distilled water
8. SDS-PAGE Running buffer (1X) [Tris Base 3.027gm, Glycine 14.4gm, 10% SDS-10
ml. The volume was made up to 1 litre.]
9. Native PAGE Running buffer (1X) [Tris Base 3.027 gm, Glycine 14.4 gm. The
volume was made up to 1 litre]
10. Protein sample (Fractions of Ion exchange chromatography)
11. Pre-stained protein ladder/marker (Puregene)
12. SDS-PAGE gel loading dye (4X) [2.0 ml 1M Tris-HCl pH 6.8, 0.8 g SDS, 4.0 ml
100% glycerol, 0.4 ml 14.7 M β-mercaptoethanol, 8 mg bromophenol Blue] - 10ml
13. Native PAGE gel loading dye (4X) [2.0 ml 1M Tris-HCl pH 6.8, 4.0 ml 100%
glycerol, 8 mg bromophenol Blue]- 10ml
14. Coomassie blue stain (0.25% Coomassie blue, 45% Methanol, 10% glacial acetic
acid)
15. De-staining solution (45% Methanol, 10% glacial acetic acid)
D) Miscellaneous
1. Micropipettes- 20µl, 200 µl and 1000 µl)
2. Eppendorf rack

47
PROCEDURE:
1. Assembled the glass plates in the PAGE apparatus and ensure there is no leakage.
2. Pour the respective resolving gel solution for SDS and native PAGE as per the recipe given
below between the plates till the level is about 2cm below the top edge of notched plate.
10% Resolving Gel
Components of 10% resolving gel (20 ml) Volume (ml)
Distilled water 3.2
30% Acrylamide mix 2.6
1.5M Tris-Cl pH-8.8 2.0
10% SDS (to be omitted for Native, instead replace with D/W) 0.08
10% Ammonium persulphate (APS) 0.08
TEMED 0.008

3. Add ~ 0.2 to 0.3 ml of butanol mixed with water, to prevent oxidation of gel.
4. After the gel is polymerized, drained off the butanol-water layer.
5. Stacking gel is prepared as per the recipe given below and poured directly onto the
polymerized resolving gel.
5% Stacking Gel
Components of 5% stacking gel (5ml gel) Volume (ml)
Distilled water 3.0
30% Acrylamide mix 0.67
1.0M Tris-Cl pH-6.8 1.25
10% SDS (to be omitted for Native, instead replace with D/W) 0.05
10% Ammonium persulphate (APS) 0.05
TEMED 0.005

6. Inserted comb into the gel without air bubbles.

7. After stacking gel is set, carefully removed the comb. Wash the wells with distilled water.
Fixe the plate to PAGE apparatus.
8. Fill the tank with appropriate 1X running buffer, both between the plates and at the bottom
of the gel.
9. Prepare the protein samples by mixing it with appropriate amount of gel loading dye.

48
10. The ones for SDS-PAGE are heated in boiling water bath for 5-10 mins and given a quick
spin to collect the protein mixture that has condensed onto the lid. This is then loaded into
the respective well.
11. For Native PAGE, the samples are mixed with the loading dye and loaded directly on to
the gel.
12. Connect the power supply and set the voltage to 100V.
13. When the dye reaches 0.5cm above the bottom of the gel, switch off power supply.
14. Disassemble the apparatus. Remove gel plates using a spatula gently.
15. Stain the gel for 1 hour or overnight using Coomassie brilliant blue stain.
16. De-stain the gel for 2-3 hours till background is clear.

RESULTS

INTERPRETATION
The simulation was run with a 10% acrylamide gel. Lane 1 consists of 2 standards:
ovalbumin and ß-galactosidase, while Lane 2 consists of Unknown sample #1. An automated
graph of log(Mr) v/s Relative migration was used to find the Mr of the unknown sample. The
relative migration rate of the unknown sample #1 was found out to be 0.08. Thus the log(Mr)
was 4.92 which meant that the experimental Mr was 83259 (which is correct).

49