ANALYTICAL

DAIRY
CHEMISTRY
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INDEX
No. Experiment Page No.
1 Determination of viscosity of milk 4
2 Determination of specific gravity by pyknometer
3 Determination of surface tension of milk
4 Demonstration of Instron universal testing machine
5 Study of bomb-calorimeter
6 Determination of refractive index of skim milk & whey
7 Determination of electrical conductivity of milk
8 Demonstration of fraction collector
9 Demonstration of autotitrator
10 Demonstration of lyophilizer
11 Demonstration of pH meter
12 Demonstration of colorimeter
13 Demonstration of spectro-photometer
14 Demonstration of high speed & ultra centrifuge
15 Method to measure water activity
16 Demonstration of polarimeter
17 Demonstration of manometer
18 Demonstration of flame photometer
19 Polyacrylamide gel electrophoresis
20 Starch gel electrophoresis
21 Paper strip electrophoresis
22 Chromatography
23 Column chromatography
24 Paper Chromatography
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INDEX
No. Experiment Page No.
25 Thin layer Chromatography
26 Gel filtration
27 Ion exchange Chromatography
28 Affinity Chromatography
29 Gas liquid Chromatography
30 High performance (pressure) liquid Chromatography
31 Determination of free fatty acids in ghee
32 Determination of peroxide value of ghee
33 Determination of aflatoxins from food & feeds by column
and thin layer Chromatography
34 Determination of lysine
35 Determination of phospholipids content by thin layer
Chromatography
36 Estimation of cholesterol by colorimetric method
 4

EXERCISE 1

DETERMINATION OF VISCOSITY OF MILK

Introduction
Viscosity is an important physical property of food system. It is related to flow
properties of fluids which in turn controls manufacturing operation in many cases. The
viscosity of the fluid is a measure of the resistance to flow. The rate of flow is decreased by
internal frictional forces.
There are some liquids like water, milk etc. which can be poured easily from one
container to another. Such fluids are said to have a low viscosity. On the other hand, there
are certain liquids that do not flow easily like condensed milk, glycerol and honey. They are
said to have a high viscosity.
The resistance to flow of layer of liquid molecules over another depends on various
factors:
- Intermolecular attractive forces.
- Molecular weight or mass of the molecule of liquid.
- Structure and shape of the molecules of a liquid.
- Increase in temperature decrease the viscosity of liquids.
- Increase of pressure, viscosity increase due to strengthening of cohesive forces.

Certain terms used in relation to viscosity are as follows:

1 Fluidity: it is the reciprocal of viscosity.
Fluidity = 1/ viscosity

2 relative viscosity: is equal to

Viscosity of solution
Viscosity of solvent

3 Specific viscosity: is equal to Relative viscosity – 1.

Determination of viscosity is based on poiseulle and stoke’s equations.

Poiseulle (1844) gave a fundamental equation:

n = ∏ r4 t p
8µl
Where l and r stands for the length and radius of the capillary tube in which µ ml of
the given liquid flows in time t under driving pressure p.
Or n = k p t where k = ∏ r4/ 8 µ l
Thus coefficient of viscosity (n) under given conditions is, n 2 d and n 2 t.

Strokes law: strokes studied the rate of fall of a heavy ball through various liquids and stated:
The rate of fall of a spherical ball µ through a liquid is directly proportional to the
coefficient of viscosity of the liquid n and radius of the sphere, r.
 5

µ α force
α 1/ n r

µ = ___F__ where 1/6 ∏ is constant

6∏ n r

When the ball is falling under the force of gravity,

F = wt. Of ball- wt of liquid displaced
= 4/3 ∏ r3. Dg – 4/3 ∏ r3 3 dg
Where D and d stand for the density of material of the ball and liquid respectively; g is the
acceleration due to gravity.
F = 4/3 ∏ r3 g (D-d) ˙ . µ = 4/3 ∏ r3 g (D-d) __1___
6∏nr

= 2/9 r3 g (D-d).
µ

n = 2/9 r3 g (D-d)
µ

Viscosity of milk and water:

Viscosity of milk is mainly due to the milk proteins and the fat present as a colloidal
system and to a minor extent due the lactose and salts in solution with water. In the case of
proteins, in addition to their concentration, the number of their particles, size and degree of
hydration also affects the viscosity of milk. In the case of fat, the globular size and the extent
of clustering determination their influence on the viscosity of milk. The unit of viscosity is
poise while smaller unit is centipoises.
Poise: is defined as the force in dynes/cm2 required to maintain a relative velocity of
1 cm per second between two parallel planes 1 cm a part.
Centipoises: is one hundredth of a poise.
1 poise = 100 cp.
At 20˚c the viscosity data of milk and related fluids is as under:
- whole milk 2.127 cp
- skim milk 1.79 cp
- whey 1.2 cp
- 5% lactose solution 1.15 cp, and
- Water 1.005 cp

Methods used for determining viscosity of milk & milk products:

Different methods used for determining the viscosity of milk and milk products are
listed in table 1.1.
These methods can be classified into three main types as under:
1. Capillary tube viscometer method.
E.g. Ostwald viscometer.
2. Falling sphere viscometer.
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E.g. Hopler viscometer

3. co axial cylinder viscometer
E.g. Mc Michael viscometer & Brookfield viscometer.

1. Capillary type viscometer (ostwald viscometer):

The constructions of capillary type viscometer are depicted in figure 1.1. It consists of
capillary tube, the upper part of which is connected to the bulb. Two marks, x and y are
placed on the bulb above the capillary tube. The time taken for a fixed volume of
solution to know past x and y on the capillary tube is noted. A number of more elaborate
designs have been used to maintain a constant head of liquid, or by varying the position
of the capillary to gain access to a range of shear rates.

Principle:
When a liquid flows through a capillary tube the viscosity of the liquid is directly
proportional to the time taken for a certain volume of liquids to flow out under a certain
pressure gradient, and also is proportional to the density of liquid. A comparison of
viscosity of milk is done to that standard liquid, distilled water, according to the above
principle, and absolute viscosity pf milk can be obtained knowing the viscosity of water.

Apparatus:
1. Ostwald viscometer with viscosity range from 1 to 3 centipoises.
2. Stands with clamps to fix the viscometer.
3. Thermometer with gradation of 1.0c.
4. Stopwatch.
5. Water bath maintained at 20˚c + 0.1˚c.
6. Pipette – 10 or 25 ml (according to the bulb of the viscometer is of volume of 15
to 20 ml or 30 to 40 ml).
7. Hair dryer.

Reagents:
1. Milk of cow and buffalo.
2. Distilled water.
3. Sodium hydroxide.
4. Chromic acid.

Procedure:
1. Clean the viscometer successively with the sodium hydroxide solution, tap water,
chromic acid, tap water and finally four to five times with distilled water; dry the
viscometer by drawing a current of cold dust free air.
2. Fix the clean dry viscometer in the constant temperature 20˚c (+ 0.1˚c) or 30˚c
(+ 0.1˚c) water bath, so that the marking above the bulb remains below the water
surface.
3. Transfer with the pipette a fixed volume of distilled water (10 to 25 ml) as per
required to full about two third of the viscometer bulb (the reservoir bulb).
4. Allow the viscometer with water to stand for 15 min. at 20˚c + 0.1˚c.
5. Attach soft rubber tubing to the arm of the viscometer containing the marked
bulb and draw distilled water over the upper mark.
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6. Close the rubber tube with fingers and release the pressure slowly to allow the
water to flow down.
7. Start the stop watch when the meniscus of water just leaves the upper mark and
measure the time for the meniscus to reach the lower marking.
8. Repeat the steps 5 to 7 for at least 5 times and record the times correct upto 0.5 s
in tabular form.
9. Take out the viscometer, drain out the water and dry it by drawing dust free air.
10. Replace the viscometer to the bath and repeat the experiment – fro steps 2 to 8
with milk sample.
11. Record the times for at least 5 observations in the tabular form.
12. Measure the density of the milk sample relative to the distilled water used in the
experiment at 20˚c (or 30˚c) as may be the case.

Precaution:
1. Only those results obtained from the same viscometer are suitable for
comparison.
2. Viscometer should be properly aligned during measurement of viscosity.

Limitations/ Disadvantages/ Errors:

1. Loading errors which arise from the fact that the driving fluid head is dependent
on the amount of liquid in the instrument, if too much liquid is charged into the
lower reservoir is too high and driving head is reduced by that amount.
2. Kinetic energy corrections primarily expansion losses at the entrance and exit of
the capillary.
3. Apparent increase in the capillary length owing to the tendency to the fluid as it
emerges from the capillary to retain the shape of the capillary for a finite distance
into the fluid medium.
4. Drainage errors arising from the fact that all liquids do not drain from a surface
with equal ease.
5. Error resulting from improper alignment of viscometers.
6. Errors resulting from variation in temperature and gravitational constants.
7. The most serious error, however, is caused when the viscometer is applied to
liquids that are not truly viscous in their flow i.e. We cannot determine the
viscosity of highly viscous fluids such as condensed milk, glycerol etc. as they are
unable to flow through the capillary tube.

Hopler viscometer (falling sphere viscometer):

Apparatus:
It consists of a glass or steel ball of suitable diameter which falls in a close fitting
precision bore cylindrical glass tube filled with the experimental liquid and held several
degrees from the vertical, so that the ball is positioned closed to one side of the tube during
its fall. The sample tube is encased in a bigger glass cylinder with a device for forming water
bath around it, which can maintained at constant temperature (figure 1.2).
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Principle:
This method is based on the equation relating the functional force offered by the
liquid to a slow moving sphere. Under ideal condition, when the flow of the liquid around
the falling sphere could be assumed laminar, the following equation holds good:

n = 2/9 gr2 (p – p’) (stock’s law)

V

Where p and p’ densities of the sphere and the medium respectively

V = velocity of the fall of the sphere.
G = acceleration due to gravity.

Variables in measurement can be eliminated by measuring the relative viscosity of

milk with respect to distilled water for which the times of fall for a particular sphere are
measured in milk and in distilled water, under similar condition.

Operating instruction:
Temperature jacket – before filling the caps to the intake and discharge connections,
fill through the threaded aperture for the thermometer, the following
For temperature from solution
100 60 to + 1˚c methanol
+ 1 to 98˚c distilled water
+ 100˚c distilled water + 20% glycerin
100 – 150˚c glycerin (8p. gr. 1.26)
Leave an air space of approximately 10mm. above the liquids in the jacket. Under no
condition use oil.
Temperature adjustment – A pneumatic stirrer serves for agitating the temperature bath.
When heating, loosen the vent screw. Temperature may be adjusted by – thermostat or
without a thermostat. The precision model of the equipment can be used over a temperature
range from -60˚c to 150˚c.
Cleaning of the tube – the glass tube, balls, plug and washer must be perfectly dry
and free from grease or moistened with the liquid to be measured. For aqueous solutions
(e.g. Glues) rinse with water clean with a brush and a mixture of 50% menthylalcohol, 5%
ammonia. Flush and wipe.
Filling the tube –approximately 30 -40 ml liquid is required .close the glass tube at
the bottom with plug and washer. Unscrew the cap while applying slight pressure .fill in
liquid approximately 20 min. (3/4”) below the upper end of the tube and insert the ball by
using ball pincers. If air bubble adheres to the ball remove it by knocking at it with the glass
rod. Selection of balls –A plug guage is supplied for sorting the balls which differ in size or
material .as the coefficient of expansion of the standard set of balls is matched to their
diameter that of the tube, the balls are made partly of apparatus glass, of a Ni alloy or a G –
steel alloy.

Determination of absolute viscosity from the dropping periods:

The viscosity of different liquids is compared by means of the value of the viscosity
factors or coefficient of internal friction, this factor indicates the power in dynes required for
keeping in a liquid layer of 1 cm2 area and 1 cm height of layer the upper one is motion
 9

relative to the lower one and parallel there to at a speed of 1 cm /sec. The unit of dynamic
viscosity is poise.

Determination of dropping period:

Place the instrument against a bright background on a horizontal, strudy vibration
free table. If artificial light is needed, use florescence lighting which is practically cold. Fix
the instrument in standard position. By means of an accurate stop watch determine the
beginning of the dropping period as soon as the lower periphery of ball appears to the touch
the upper annular marks, which should appear as a straight line. Similarly, dropping period is
determined by lower mark.
Placing the instrument upside down the ball returns to its initial position a series of
determinations of dropping period (3 to 4) are made.
In dark and opaque liquids, the lower periphery of the ball is usually difficult to
distinguish. The reading of the passage of the ball through the marks is therefore carried out
at the moment when the ball equator passes the annular mark.

(3) Coaxial cylinder viscometer:

In this method one cylinder is suspended in the liquid with the help of tortion wire
as shown in figure 1.3.
There are two different types of coaxial cylinder viscometers used commonly.

(1) Mc Michael viscosimeter:

In figure 1.4 this type of viscosimeter the liquid under examination is roated.
Tortion/ twisting created due to this movement on the wire are proportional to the
viscosity of the liquid. It employs a cylindrical vessel, 3 cm in diameter ,and a
cylindrical plunger ,1 cm in diameter and marcler for 1,2,3 or 4 cm immersion .the
plunger is suspended by a wire and attached by a wire and attached to the plunger
assembly , there is a disc, the circumference of which is graduated into 300 units .the
sample cup is filled accurately to control the depth of immersion of the plunger .as
the sample turns ,the resistance between sample and plunger causes a torsion on the
wire .readings are made on a scale attached to the plunger shaft. Scale units are
arbitrary, “degrees M” each of which is 1/300 of a circle.
By standardizing the instrument against solutions of known viscosity, the readings
can be converted to centipoises. A thermostat allows control of sample temperature
upto 300˚ F.

(2) Brookfield viscometer:

In this type of viscometer, the test fluid is kept stationary and cylinder is related.
Thus there will be twisting on the wire and the torsion created is related to friction
which in turn is related to friction which in turn is related to the viscosity of the test
fluid.
Brookfield viscometer (figure 1.5) is an instrument that measures the torque on a
rotating spindle by means of a calibrated spring. Readings are made from 0-100 scale
on the dial. Calculations are made by multiplication by factors that are dependent on
instrument model, size of spindle and speed of rotation. Models of the instrument
are available with upto 7 spindles and 8 speeds, giving 56 ranges encompassing 0 to 8
millions centipoises. For exact control of sample temperature for meaningful
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viscosity reading, a modified sample holder has been devised that serves to stabilizer
readings on products that tend to separate .this instrument fitted with a special bar-
type spindle and mounted on a helipath stand that will lower the spindle slowly
through the test material, makes possible consistency measurements on products like
mashed products.
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EXERCISE 2
SPECIFIC GRAVITY BY PYKNOMETER

Apparatus:
1. A specific gravity bottle or any other type of pyknometer
2. analytical balance and weight box
3. Thermometer.

Reagents:
1. Experimental liquid-milk
2. distilled water

Principle:
The density of a substance is its mass per unit volume and it is usually expressed in
grams per ml. The specific gravity or the relative density of a substance is the ratio of the
mass of a certain volume of the substance to the mass of an equal volume of a standard
substance at a certain temperature and pressure. For solids and liquids the standard
substance is taken to be water at 4˚ C which has density of unity at that temperature .the
density of milk is determined by directly weighing a certain volume of milk taken is
pyknometer (figure 2.1) and by determining the volume taken. Specific gravity of milk is
determined by finding out the weights of a certain volume of milk and of the same volume
of distilled water at the same temperature taken in a pyknometer.

Procedure:
1. Clean a specific gravity (S.G) bottle/pyknometer and dry it carefully with a
current of cold dry air.
2. Weigh accurately the S.G. bottle/pyknometer with the stopper.
3. fill the S.G. bottle/pyknometer with milk and place the stopper tight,
overflowing some milk; take care not to leave any air bubble in the milk.
4. Wipe off milk from outside the bottle, with paper strips or absorbent cotton.
5. Weigh accurately the S.G. bottle/pyknometer, with milk.
6. Take out the milk from S.G. bottle/pyknometer, wash thoroughly with detergent
solution, dilute acid and distilled water and finally fill it with distilled water
avoiding air bubbles.
7. Wipe off water from outside the bottle and weigh accurately.
8. Note down the temperature of milk and water in case there is a constant
temperature water bath, keep the milk and water in the bath for about 15 min.
and fill the S.G. bottle/ pyknometer with these liquids and record the
temperature of the bath.

Precautions:
S.G. bottle /pyknometer should not be heated so dryness.
 12

EXERCISE 3

DETERMINATION OF SURFACE TENSION OF MILK

INTRODUCTION
Surface is an area of contact between two phases. Phase may be of three types viz
liquid, solid or gaseous.
If out of the two phases which contact, one is gaseous then the resultant area of
contact is called surface. But if for example, both the phases are liquid like oil and water
mixture, then the resultant area of contact is known as interface.
There exists a phenomenon of intermolecular attraction in liquid. The molecules inside the
liquid experience a force of attraction from all the sides. I.e. they are attracted equally
towards both the sides. However, the surface molecules are attracted only downwards and
sidewise. Therefore, due to imbalance of force of attraction at the surface, the liquid behaves
as if there is tension and its surface remains stretched or in tension. This phenomenon is
known as surface tension. (Fig. 3.1).

Definition:
Surface tension may be defined as force in dynes acting at right angles on the surface
of liquid along 1 cm length of the surface. It is usually represented by the Greek letter γ. The
molecules in a liquid are attracted to each other, and this creates a pull from the surface.
Beneath the surface, the molecules are surrounded by the other molecules and the attraction
is equalized, but at the surface the balance is broken and a tension results. Surface tension
thus may be defined as the state of stress at the surface of the liquid due to the attraction of
the molecules for each other. It is expressed in dynes/ an. A dyne is the force that is acting
on a mass of one gram giving it an acceleration of one centimeter per second. Imbalance of
forces acting at the surface causes the surface to act as though covered with a film or skin.
We experience the phenomenon of surface tension frequently in our day to day life.
The effect of surface can be seen as:
1. Reduced area of the surface to the minimum e.g. Falling drop of liquid acquires a
spherical shape as among the different shapes like square, triangular, rectangular,
minimum area is occupied by spherical shape i.e. Sphere will have a minimum
volume for its given mass.
2. Rise of liquid in a capillary tube.
3. Disc/ ring/ needle floats on the surface of liquid if put without disturbing the
surface.

Applications:
In the dairy field this phenomenon has the following applications:
1. Emulsion of fat in liquid is stabilized by the forces of surface and interfacial tension
i.e. the fat does not separate out from the plasma.
2. During the preparation of ice-cream used for icing of cakes etc. air is incorporated to
obtain over run and fluffiness. Here, too, two phases exists i.e. Liquids – air and solid
– air respectively in both the products. Stability of the two phases is due to the force
of surface tension.
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3. Yet another application of this phenomenon is in the cleaning of utensils, storage

tanks, equipments and other greasy surfaces in the butter and ghee sections of a dairy
plant. For efficient cleaning, water must come in contact and take away grease which
is possible only if the difference in surface tension of the contact liquids is reduced.
Detergents used in the cleaning section reduce the difference in surface tension
between contact materials.
4. During transportation of milk, agitation takes place in the tanker and due to this
some of the membrane of ruptured fat globally membrane goes in the serum and
lowers the surface tension. Hence the measurement of surface tension can be used as
an index for determining the severity of agitation while transportation of milk.

The surface tension value of some liquids is as under

Water 72 – 75 dynes/ cm
Skim milk 52 – 52.5 dynes/ cm
Whole milk 46 – 47.5 dynes/ cm
Cream (25% fat) 42 – 45 dynes/ cm
Sweet cream 39 – 40 dynes/ cm
Butter milk

The surface tension value of cow’s milk is 51.84 – 51.93 dynes/ cm and that of
buffalo milk is 50.40 as respectively reported dynes/ cm by a research work.
Thus, pure water has a higher surface tension than milk, because milk contains
surface active components such as proteins, fat, phospholipids and free fatty acids. Various
factors such as fat content, temperature, liperysis, homogenization, aging affect the surface
tension values.

Measurement of surface tension:

Method used to measure surface tension is based on the following effects of surface
tension.
1. Height of rise of liquid in a capillary tube (fig. 3.2)
2. Weight or volume of drops formed by liquid flowing from a capillary outflow tube,
the end of which is flattened out (in order to give a larger dropping surface) and the
surface is carefully ground flat and polished. Two marks are etched on the tube, one
just above and the other just below the bulb, the volume of the liquid used in the
experiment being thus defined. In the determination of the surface tension, the
number of drops which fall from the end of the liquid falls from the upper to the
lower mark. Since, however, the passage of the meniscus past these marks does not
in general coincide with the fall in of a drop; the tube is calibrated for a short
distance above and below the upper and the lower marks. With the help of this scale,
fraction of a drop, by first determining how many scale divisions correspond to one
drop. The construction of stalagmometer is shown in fig 3.3.
3. Force required to pull a ring or plate out of a surface by du noisy tensiometer.
4. Maximum pressure required to force a bubble of gas through a nozzle immersed in
liquid.
5. Shape of a pendant drop hanging from a capillary.
 14

(1) Measurement of surface tension by capillary tube method:

If a capillary tube is dipped in liquid there will be rise of liquid, in the capillary tube.
Due to surface tension there is a pull of liquid towards upwards direction, however
simultaneously the force of gravity acts upon the liquid to push it in downward direction. At
a certain stage the force of surface tension equals the force of gravity, and hence the liquid
will cease to rise in the capillary tube.
Force of surface tension pulling the liquid upward.
F (st) = γ (surface tension value of liquid)
×
Inside circumference of the capillary

=γ×z∏r
Where r = radius of the capillary.

Force of gravity pulling the liquid downwards

= w × g …………… (1)
Where w = weight of liquid
And g = gravitational constant
w = density (ℓ) × volume (µ)
(d = weight
Volume)
Substituting the value of w in (1) we get
F (g) = ℓ × µ ×… (2)
Again µ =∏ r2 h
Substituting the value of µ in (2) we get
F (s) = ℓ × ∏ r2 h × g
At balanced position
γ 2 ∏ r = ℓ × ∏ r2h × g

γ = ℓ × ∏ r2h × g
2∏r

γ=ℓrhg
2

Thus by measuring the height of liquid in capillary tube and the diameter of capillary tube by
moving telescope, the surface tension value can be calculated.

Alternatively, surface tension of milk, Ym = r h m × ℓ m × g and

2

Surface tension of water

γ w = r h m × ℓm × g
2
γm = rhm × ℓm × g × 2
γw 2 rhw × ℓw ×g
 15

γm = hm × ℓm
γw hw × ℓw

γm = hm × ℓ × γw … (3)
hw × ℓw
Where γw = constant value (at particular temperature)

Apparatus:
1. A stand for fixing capillary.
2. Capillary tube.
3. Glass beaker.
4. Thermometer.
5. Scale.

Procedure:
1. temper the milk sample at 45˚c for 30 seconds and bring it to room temperature
2. adjust the temperature of distilled water as well as milk sample to 30 ˚ c
3. Take distilled water in a beaker, insert the capillary tube of a given diameter and note
the initial level rise through the capillary, mark the level to which it rises and take the
difference. This will give the level to which water rises in the capillary.
4. similarly repeat with milk and note the rise in capillary
5. calculate the density of milk by hydrometer/lactometer (ℓ m =l + C L R)
1000
Or pyknometer.
6. Substitute the values in equation (3) to obtain the S.T. value of milk sample.

(2) Determination of surface tension of milk using stalagmometer:

Apparatus:
1. stalagmometer
2. small bottle
3. beaker
4. cork
5. thermometer
6. Rubber tubing with a screw pinch cock.
7. hair dryer

Reagents:
1. milk sample
2. standard liquid
3. sodium hydroxide
4. chromic acid mixture
5. alcohol
 16

Principle:
Surface tension exists at the interface where two phases come in contact with each
other. The values ordinarily given for the surface tensions of liquids refer to interfacial
tension between liquid and air (the effect of air, however, is negligible).
If a liquid is allowed to flow slowly vertically through a small opening that is
surrounded by a flat area (round) in a horizontal position, then a drop will form. If the flat
glass area is properly cleaned and polished, the liquid will spread over this area and the point
of support of the drop will be the circumference of this flat area. The drop is held there by
surface tension. As the size of the drop is increased, it will finally attain a mass so that force
of gravity acting on it will just exceed the weight of the drop the surface tension can be
calculated from the following formula: surface tension
= wt. Of the dropping × 980.365
2∏ r
The size of drop issuing from a capillary orifice is governed by the surface tension of the
liquids .the moment at which, the drop just breaks away, force drawing it upwards is the (2∏
r γ = w = µ d) weight of the drop acting downwards, where 2∏ r is the external
circumference of the end of the capillary tube, w the weight of the drop, µ its volume and d
its density.
In actual use the above method is seldom employed in the manner just explained
because flow continues as the successive drops form and the drop that does not represent
the weight supported by the surface tension. For this reason, instead of determining the
weight of a single drop or a number of falling drops, it is usual to determine the number of
drops formed when a definite volume of a liquid is allowed to flow slowly out of a capillary
tube. With two different liquids the weight of equal volumes are proportional to their
densities. If a volume µ of one liquid produces n1 drops and another liquid n2 drops then.
2 ∏ r γ1 n1 = µ d1
2 ∏ r γ2 n2 = µ d2

γ1 = n2 d1 therefore Y2 = n1/ n2 × d2/ d1 × γ1

γ2 n1 d2

Where,
γ1 = surface tension of standard liquid
γ2 = surface tension of unknown liquid
n1 = no. of drops of standard liquid
n2 = no. of drops of unknown liquid
d1 = density of standard liquid
d2 = density of unknown liquid
Thus, surface tension of unknown liquid such as milk can be determined by counting
the number of drops and determining the specific gravity of the unknown (milk) and those
of the standard liquid can be known from the tables and substituted in the equation to
calculate the surface – tension of milk. Care should be taken that the measurements are
carried out at the same temperature.
 17

Procedure:
1. Support the stalagmometer by a rubber stopper with two holes, which fits tightly
in a small wide mouthed bottle.
2. Attach a small piece of clean rubber tubing free from dust and grease to upper
end of the stalgamometer. The rubber tubing free from dust and grease to upper
end of the stalagmometer .the rubber tubing with the screw pinch cock on it is
used to control the roose of flow of liquid by limiting the influx of air.
3. Insert a piece of glass tubing in the other hole of the stopper, to save as an air
vent.
4. Clean the stalagmometer thoroughly with NaoH to remove grease if any, then
with chromic acid mixture and finally with distilled water. Then rinse it with
alcohol and dry it by passing a current of air.
5. fill up the stalagmometer by immersing the dropping tip in the distilled water and
sucking on the tubing until the water has risen above the mark A. then close the
screw pinch cock and insert the rubber stopper in the small bottle and keep it in
a one liter beaker containing water which is maintained at the required
temperature (thermostat).
6. The small bottle carrying the stalagmometer must be dipped into water until the
water level in the beaker is above the mark A on the stalagmometer.
7. Allow the stalagmometer to attain the temperature of water.
8. Open the pinch cock and adjust it until the rate of fall of drops is 10 to 15 drops
per min. from the end of the capillary tube.
9. Remove the assembly from the beaker, slip the stopper out of the bottle and
refill the stalagmometer with distilled water as above without altering pressure.
10. Close the open end of the tube with the tip of the finger, replace it in the beaker
and determine the number of scale divisions corresponding to one drop.
11. Refill the stalagmometer. Then start counting the drops, when the meniscus
passes the upper mark A and stop when it just passes the lower mark. The
measurement of this number of drops is repeated thrice. Different
determinations of this number should not vary by more than 0.3 to 0.5 drops.
12. Remove the rubber tube from the stalagmometer, clean it by rising with alcohol
and dry it by passing a current of air. Then attach the rubber tube to
stalagmometer and fill it with milk, by sucking on the open end of the rubber
tube as before, and replace the stopper into the bottle which is then kept in the
beaker containing water at required temperature .repeat the procedure of
adjusting the pressure and counting the drops as before. Determinations of the
drops are carried out thrice.
13. Determine the specific gravity of the liquid by means of pyknometer or specific
gravity bottle.

Precautions:
1. Ensure that the dropping tip does not come in contact with the hand, desk top
or anything else which might contaminate it with a trace of grease. Even slight
traces of grease will markedly alter the size of the drops formed.
 18

2. This apparatus should not be taken while the experiment is being carried out; as
the drops of the liquid may be caused to fall before they have attained their
maximum size.
3. The surface is affected by the temperature, hence the determinations are to be
carried out at a constant temperature in thermostat.
4. The rate of flow of the liquid through the tip should not be rapid but it should
be 10 to 15 drops per minute. hence, if the natural rate of dropping is grater, it is
retarded by attaching a piece of rubber tubing with a screw pinch cock to the
open end and adjusting the pressure, until the rate is 10 to 15 drops per minute.
Once this adjustment of pressure is made, it should not be altered during the
experiment for the same period.
The actual practical was performed using chromatography column in the
following manner.
1. The flow rate of 10-20 drops per min. was adjusted with the help of distilled
water.
2. The column was filled with distilled water at 30˚c and the number of drops
fallen while collecting 3ml water in calibrated tube was counted.
3. The same procedure was repeated for milk at 30˚c.
4. Five such readings were taken.
5. The γm value was calculated by substituting the obtained readings in the
formula.

Measurement surface tension by ring detachment or tensiometer method:

A convenient method for the measurement of surface tension (ST) is to measure the
force necessary to detach a wire ring from the surface of liquid .as the ring is lifted from the
surface a film of liquid is pulled up along with it. If the constricted surface of the film
becomes vertical before it breaks, the downward force (P) due to ST at the constriction is
twice the product of the circumference of the ring ® and ST. this force is balanced by the
upwards force exerted on the ring, less the weight of the liquid above the vertical portion of
the film. It can be shown that under such conditions of the ST (γ) is: γ = _P__
4∏ R

Apparatus:
For experimental purposes the Du Noüy tensiometer, or one of the modified forms,
is used; it essentially consists of a sturdy platinum ring suspended from an arm which is
connected to a delicate balance. (Fig. 3.4).

Procedure:
i. Degrease the platinum ring by dipping in 10% NaoH solution and washing under
running tap water: clean successively with chromic acid solution and water, dry
suitably.
ii. Take the milk in a clean dry watch glass or Petri dish, and bring it under the ring
placing on the movable platform of the tensiometer.
iii. Bring the beam of the balance to the horizontal position, which must coincide
with the pointer measuring the tension at zero position on the circular scale of
the disc.
 19

iv. Gently raise the movable platform containing milk till the platform ring just
touches the surface of the milk (the arm side slightly dips downwards at this
point).
v. Rotate the lever of the balance (placed on the graduation disc) very gently till the
platinum ring snaps off the milk surface; take the reading of one position of
lever on the circular scale of the disc.
vi. Repeat the experiment 5-6 times, each time changing the milk surface by adding
fresh milk to the container.
vii. Measure the temperature of milk each time by immersing a thermometer.
viii. The average of the multiple readings, expressed in dynes/cm., represent ST of
the milk at the averaged temperature.

Precautions:
i. The platinum ring and also the milk container should be free of fat or similar
matter.
ii. Readings should be taken as quickly as possible to avoid accumulation of fat on
the surface of milk.
iii. The lever of the pointer should be rotated as gently as possible particularly at the
point of break-off of the ring from the liquid surface but without any undue
delay to allow. accumulation of fat globules on the surface
Among all the different methods used for determining ST, this method is
more rapid, accurate and reliable.
 20

EXERCISE 4
INSTRON UNIVERSAL TESTING MACHINE
(DEMONSTRATION)

Introduction:
With texture a primary criterion for consumer acceptance of food products, there is
a need for accurate, reproducible texture measurements which can become standards of
acceptance and can be used as a basis for establishing satisfactory methods for harvesting,
processing, packaging, shipping and storing while sensory assessments may provide
evaluations closest and expense makes it a difficult way to obtain objective data for universal
use. Objective text methods, once correlated with sensory assessment, provide a method of
texture measurement which can be used constantly with constant reproducibility if the
correct instrumentation is used.
Texture is defined as the property which represents the way in which the structural
components of a food are arranged in a micro and macro structure and the external
manifestations of this structure. If is related to the behavior of foods in mechanical testing
machines as well as to their behavior when eaten (sensory aspects).
Machines like Instron universal testing machine can be used for texture testing
providing:
I. Constant reproducible deformation rates are maintained at all the test forces,
II. Force deformation and time are recorded accurately, and
III. The texture text cells can adapt to the machine.

Operation:
The various components of Instron Universal testing Machine and the function of
its controls and indicators have been depicted in fig. 4.1 & 4.2 respectively.
The machine is operated in the following manner:
1. Press the POWER switch on. This switch and the STOP push-button should
light. A warm-up time of 15 min. is recommended before calibrating the
instrument.
2. Get the initial requirements like units for use either ENG or MET and the type
of test to be done, either Tension or Compression. (Where cross head goes up
and down respectively for Tension and Compression).
3. Calibration: the instrument is zeroed, balanced and calibrated as follows:
a) Set read out mode switch to TRACK position.
b) Set RANGE switch to 1/5 position.
c) If using 100 1b or 10 lb capacity lead transducer, insert the retaining pin
providing into the grip coupling. (This pin is required when hanging the
calibrating weight on the low capacity load transducers and its weight must
be balanced out initially).
d) While holding the ZERO/CAL switch in the ZERO position, unlock the
COARSE and FINE balance controls (turn locking ring on each control
 21

about ¼ turn counterclockwise) and then adjust these controls until the
LOAD readout shows zero. Lock the controls.
e) Release the zero/CAL switch then change RANGE switch to 10/50 position.
The LOAD readout is how indicating how well the core of the LVDT in the
load transducer is nulled in its coil contained in the cross heed .this reading
should be loss than 5% of full scale of the load RANGE (10/50) for the load
transducer in use.
When calibrating 1000 lb capacity load transducer, always set RANGE
switch to 10/50 position before pushing the ZERO/CAL switch to CAL
position. Otherwise, an erroneers calibration read out will occur due to amplifier
saturation.
If the load reading is greater than 5% of load transducer range, uses the
screwdriver provided and adjust the LVDT core extender shaft in cross-head
until a null (a minimum reading) occurs. Rotate the adjustment clockwise to
locate the null.
i. To calibrate 1000 lb (500 kg/5KN) load transducer, hold the ZERO/CAL.
Switch in CAL .position. Then turn ADI trimmer using screwdriver provided,
until the LOAD readout matches the proper calibration number stamped on
load transducer. Use the number shown for testing mode (tension or
compression) and the type of units (English, metric or SI) to be used.
ii. To calibrate 100 lb (50 kg/500 N) load transducer, set RANGE switch to 2/10
position. Then with the ZERO/CAL. Switch in centre position, unlock balanced
controls until the LOAD readout shows zero; lock the controls. Hook the
calibration weight (5 kg) provided on to the pin in the grip coupling. Turn the
ADJ trimmer until the LOAD readout displays the value shown (i.e. 1/10th of
load transducer used) for units in use (English, metric or SI). Remove calibration
weight.
iii. To calibrate 10 lb (5kg/50 N) load transducer, set RANGE switch to 2/10
position then with the ZERO/CAL switch in center position, unlock the balance
controls and adjust these controls until the LOAD readout shown zero, lock the
controls. Hook the calibration weight (500 g.) provided onto the pin in the grip
coupling. Turn the ADJ trimmer until the LOAD readout displays the value
(1/10th of load transducer value) for the units in use (English, metric or SI).
Remove the calibration weight.

(9) To recheck calibration at anytime after the upper grip or anvil is installed:
(i) When using 1000 lb (500 kg/5 KN) load transducer, repeat steps a,b,d and f (i).
Of calibration procedure. After checking calibration, the LOAD readout will
show tare weight of grip or anvil. Adjust balance controls to set LOAD readout
to zero.
(ii) When using 100-lb (50 kg/500 N) load transducer with a grip installed, repeat
step f (ii) of calibration procedure except hang the calibration weight directly
from grip. Then remove the weight.
(iii) When using 100 lb (50 kg/500 N) load transducer with an anvil installed or the
10 lb (5 kg/50 N) load transducer, remove the grip or anvil. Then insert a
retaining pin in grip coupling and repeat step f (ii) and f (iii) of calibration
procedure, depending upon load transducer in use.
 22

Preparations for TENSION testing:

1) Decide the type of grips and grip faces to be used. Then upper grip is held in
coupling by a returning pin supplied with coupling.
2) When upper grip is installed, the LOAD readout increases. This is tare weight
and should be balanced out. Unlock the COARSE and FINE balance controls.
Then adjust these controls until LOAD readout shows zero. Lock the controls.
3) The base grip adapter includes a retaining. Pin and a compression (preload)
spring. The spring supports the weight of lower grip and prevents a
discontinuity in loading applied to specimen.
4) Decide the length of specimen between contact faces of upper and lower grips
at the start of the test (gage length) and set the gage length between two grips
by moving crosshead up and down.
5) To return the grip spacing to gage length at the end of a test, crosshead travel
limit stops must be set as follows:
a) Bring lower limit stop up so it contacts the actuator on crosshead at gage
length set. Tighten the stop securely on limit switch red.
b) Reset EXTENSION readout to zero. Press the UP push button and run
crosshead up about 1 inch (25 mm). Then press RETURN push button. The
cross head should drive down.
c) Note the number on the EXTENSION read out. If should be approximately -
0.2 inches (-5mm).
d) Raise crosshead, using UP pushbutton, and press STOP when EXTENSION
read out displays same reading as a positive number.
e) Bring the lower limit stop up so it again contacts the actuator and tighten it
securely. Then raise the crosshead again about 1 inch (25 mm) and press the
RETURN push button. The crosshead should stop when EXTENSION read
out displays zero. This is an accurate, repeatable gage length.
6) The upper limit stop should be set just beyond expected maximum extension.
Tighten the stop securely on limit a switch rod.
7) The preload nuts, located beneath moving crosshead, should be loosened for
tension testing. This prevents unnecessary bearing wear.

Preparations for compression testing:

1) Install compression anvil. For this, remove the grip coupling from the load
transducer by removing the socket head cap screw located in its centre. Tread the
anvil firmly into the load transducer by hand.
2) When the anvil is installed, the LOAD read out increase. This is tare weight and
should be balanced out. Unlock the COARSE and FINE balance controls and adjust
these controls until the LOAD readout shows zero. Lock the controls.
3) Install the compression table.
4) Decide the gage length i.e. height of specimen initially. set the gage length by driving
the moving crosshead up or down, as required, using push button on crosshead
control panel and measure the spacing between anvil and table with a ruler or bring
the anvil and table together carefully at a slow crosshead speed, stop crosshead, then
press RESET pushbutton to zero the EXTENSION readout. Drive crosshead up
until required spacing is displayed on EXTENSION read out (inches/mm).
 23

5) To return the anvil and table spacing to gage length at the end of a test, crosshead
travel limit stops must be set. To accurately set the upper limit stop, proceed as
follows:
a) Bring upper limit stop down so it contracts actuator on crosshead at gage
length set in 4. Tighten stop securely on limit switch rod.
b) RESET the EXTENSION readout to zero. Press the DOWN pushbutton
and run the crosshead down about 1 inch (25mm). Press RETURN
pushbutton. Crosshead should drive up and limit switch should stop the
crosshead.
c) Note the number on EXTENSION readout. It should be approx. 0.2 inches
(5mm).
d) Lower the crosshead, using DOWN pushbutton, press STOP when
EXTENSION readout displays the same reading as a negative number.
e) Bring upper limit stop down so it again contacts the actuator, tighten it
securely. Then lower the crosshead again about 1 inch (25 mm) and press the
RETURN pushbutton. The crosshead should stop when EXTENSION
readout displays zero. This is an accurate, respectable gage length.
6) Set lower limit stop-just beyond the expected maximum compression. Tighten the
stop securely on the limit switch rod.
7) The preload nuts, located beneath moving crosshead, should be hand-tightened for
compression testing. This is necessary to prevent discontinuities in loading applied to
the specimen.

Final pretest setup:

1) Set RANGE SWITCH
2) Set load readout decimal point
3) Set readout mode switch
4) Set crosshead speed
5) Reset EXTENSION readout.
6) Install test specimen – if the specimen does not fit, either trim it or use a different
gage length.
7) Press the DOWN pushbutton for compression test or UP push button for tension
test.

Recording of results:
Chart magnification:
When using a chat recorder, the chart speed should be selected to give a convenient
recorded length of chart for a test. A chart record of 5 to 15 inches (125 to 375mm)
is sufficient but it can be as long as desired. Selecting a chart magnification allows the
chart time axis to indicate the displacement of the crosshead in either a reduced or
magnified manner. The test results appear on the recorder chart as a load –
displacement curve. The chart magnification ratio (M) is the ratio of the chart speed
to the crosshead speed, that is:
Magnification ratio (M) = chart speed
Crosshead speed
 24

Operation of strip chart recorder:

1) Preparation: make the following initial settings:
a) POWER switch: OFF
b) Pen lift lever: UP
c) CHART DRIVE switch: STOP
d) INPUT switch: MEAS
2) Measurement and recording:
i. Press the POWER pushbutton into turn the recorder on.
ii. With input set to zero at the source, adjust the position knob to zero the
pen at a reference point on the chart.
iii. Set the chart speed with the CHART DRIVE pushbuttons, if the TIME
drive is to be used.
iv. When using recorder in time drive mode, the chart must be controlled by
the recorder START/ STOP switch. In the proportional drive mode, the
recorder START button must be pressed but the chart will start and stop
in relation to the moving crosshead. Also the chart speed will be
proportional to crosshead speed as determined by the setting of the chart
magnification selector switch.
For this select TIME drive or PROP drive mode.
v. Position chart to a reference point, if required, using manual advance
wheel.
vi. To start recording proceed as follows:
a) If in the TIME mode, set the pen lift lever down (per cap off),
and press recorder SRART pushbutton.
b) If in the PROP mode, set the pen lift lever down (pen cap off)
and press recorder START pushbutton start the test with a
crosshead UP or DOWN command.

Interpreting results:
Figure 4.3 is an example of the results of a typical first and second bite compression
curves for a food sample.
The texture profile analysis provides objective measurements of texture by a proven
and accepted method. If is vital to be able to obtain repeatable texture assessment to enable
samples to be compared or monitored for quality control, storage and processing
requirements.
Previous use of sensory assessment panels has provided the necessary product
evaluation but to use the interpretation of stress/strain plots which can be correlated with
such sensory assessments provides numerous benefits for continuous and long term
requirements.
Texture profile analysis have been performed on a wide variety of foods including
fruits, vegetables, meats, cheese, confectionery, desserts and baked goods. It usually involves
the compression of standard sized specimens (i.e. cubes, cylinders etc.) depending upon the
product under test. The specimen is subjected to two successive compressive cycles (referred
to as ‘bites’), which are between the same limits of displacement, and a force/displacement
curve for each bite is recorded.
In most cases a crosshead speed is selected in the range 5 to 50 mm/min. but this
dependent upon the material under test.
 25

Determination of textural parameters:

1) Brittleness (fracturability) (kg) = the first peak recorded during first lite.
2) Hardness (firmness) (kg) = second peak recorded during first lite.
3) Cohesiveness = ration of area of forces/ displacement curves. Second lite area: first
lite area.
4) Elasticity (mm) (springiness) = total crosshead movement to the increase in
compressive load at start of second lite.
5) Gumminess (kg) = hardness × cohesiveness
6) Chewiness (kg. mm) gumminess × elasticity
7) Adhesiveness = the area in arbitrary instrumental units, (A3) of the negative peaked
formed when the plunger is pulled from the sample.
Definitions of textural characteristics have been given in table 4.1.
 26

EXERCISE 5

STUDY OF BOMB CALORIMETER

Introduction & Principle:

A bomb calorimeter will measure the amount of heat generated when matter is burnt
in a sealed chamber (Bomb) in an atmosphere of pure oxygen gas.
A known amount of the sample is burnt in a sealed chamber (later on referred to as
‘bomb’). The air is replaced by pure oxygen. The sample is ignited electrically. As the sample
burns, heat is produced. The rise in temperature is determined. Since, barring loss of heat,
the amount of heat produced by burning the sample must be equal to the amount of heat
absorbed by the calorimeter assembly, knowledge of the water equivalent of the calorimeter
assembly, and of the rise in temperature enables one to calculate the heat of combustion of
the sample.

If, W = water equivalent of the calorimeter assembly in calories per degree centigrade.
T = rise in temperature (registered by a sensitive thermometer) in degrees centigrade.
H = heat of combustion of material in calories per gram.
M = mass of the sample burnt in grams.

Then WT = HM
‘H’ is calculated easily since W, T and M are known.

The bomb calorimeter (fig 5.1) consists of the following parts:

1. Bomb
2. Water Jacket
3. Offset stirrer
4. Calorimeter vessel
5. Bomb firing unit, vibrator, timer and illuminator.
6. Pressure gauge on stand.
7. Crucible
8. Ignition wire.

Procedure:
1) Grind a known amount of sample to powder in pestle and motor and pass through I
S sieve 20 (2110 microns). Compress the powder material into a cylindrical pallet by
pellet press. Weigh about 1 gm of pellet in stainless stell crucible.
2) Stretch a piece of 8 cm. long firing wire across the electrodes within the calorimeter.
3) Tighten specific sewing cotton thread of 10 cm. around this wire.
4) Place the crucible in the position so that the loose end of thread remains in contracts
with the pellet.
5) Prepare the bomb, by introducing 2 ml of distilled water into the body of the bomb
and reassemble and tightly screw it.
6) Charge the bomb with O2 from a cylinder at a pressure of 25 atm. Without displacing
its original air content.
 27

7) Before detaching the bomb from the oxygen supply, close the O2 supply value using
stopper value.
8) Fill the caloriemeter with sufficient quantity of water so that the cover of the bomb
caloriemeter is submerged to a depth of atleast two centimeters vessel to the water
jacket.
9) Load the bomb into caloriemeter vessel and electrical terminal for subsequent firing
of the charge.
10) Adjust the stirrer and thermometer at their respective positions. Keep the stirring
mechanism in continuous mechanism of operation.
11) Note the rate of change of temperature at equal intervals till it again becomes
constant.
12) Calculate the rate of change of temperature before and after firing.
The water equivalent is calculated using 0.9 grams of benzoic acid as a
standard substance from the following formula:
W = HM + EL + E2
T
Where
W = energy equivalent of calorimeter in calories per degree centigrade.
H = heat of combustion of standard benzoic acid in calories per gram.
M = mass of standard benzoic acid in calories per gram
T = corrected temperature rise in degrees c.
E1 = correction for heat formation of nitric acid in calories, and
E2 = correction for heat of combustion of firing wire, in calories.

The caloriefic value of sample can be calculated using the following formula:

(W E) × T – [E1 L1 + E2 L2]
W

Where
W E = water equivalent
T = temperature of firing (˚ c)
E1 = caloriefic value of the wire
L1 = length of the unfired wire
E2 = caloriefic value of the thread
L2 = length of the unfired thread, and
W = weight of the sample taken.

Precautions:
a) Do not use two much sample. The weight of combustible material should not exceed
1.10g.
b) Do not charge the bomb with more oxygen than is necessary.
c) Keep all parts of the bomb, especially the insulated electrode assembly in good
repair, at all times. Do not fire the bomb if gas bubbles are leaking from the bomb
when it is submerged in water.
d) Stand back from the calorimeter for at least 15 seconds after firing.
 28

Causes of poor combustion:

An incomplete combustion in the oxygen bomb may be due to one or more of the
following causes:
a) Excessively rapid admission of gas to the bomb during charging causing part of the
sample to be blown out of the cup.
b) Loose or powdery condition of the sample in the cup prior to ignition, causing
ejection due to violence of combustion.
c) The use of sample containing coarse particles which cannot burn readily, with coal,
such particles are usually too large to pass through 60 mesh screen.
d) The use of a sample in the form of a pellet which has been made too hard causing
spilling and the ejection of fragments during heating.
e) Use of an ignition current too low to ignite the charge or too high causing the fuse to
break before combustion is well under way.
f) Insertion of the fuse wire loop below the surface of a loose sample. Best results are
obtained on even hanging the wire slightly above the surface.
g) Use of not enough oxygen to burn the charge completely or conversely the use of a
very high initial gas pressure which may retard development of the required
turbulence during combustion.
 29

EXERCISE 6

DETERMINATION OF REFRACTIVE INDEX OF SKIM MILK

& WHEY
Introduction:
If a ray of light passes from a less dense medium to a more dense medium. As from
air to water, it is reflected towards the normal; so that the angle of incidence; I, the refractive
index (n) of the second medium with respect to the first is given by the equation.
n = sin i/ sin r
It is a constant quantity for two given media for the same wave lengths of light at the same
temperature. The refractive index of a solution depends upon the individual molecules
present and upon their concentration I.e.
1. Refractive index of substances containing dissolved material (milk) is higher than water.
2. Of various solid constituents, proteins contribute the maximum towards the refractive
index of milk serum, while lactose ranks next.
3. since the at and calcium caseinate hinder in the passing of light through milk, they are
removed from the milk by precipitation before refractive index of milk is determined in
the older methods of measurements of refractive index.
Refractive index of milk itself is somewhat difficult to determine because of
its opacity, but by using a refractometer, such as the able instrument, which employs a
thin layer it is possible to make satisfactory measurement of R.I.of milk.
The values of infractive index at 20˚c are 1.33299 for water and 1.4527 to 1.4600 for
milk. The values of infractive index of milk at 40˚c are 1.3460 (1.3450 – 1.3472) for cow
milk and 1.3477 (1.3460 to 1.3490) for buffalo milk. Fat in milk does not contribute to
the R.I. milk because refraction occurs at the interface of air and the continuous phase
(milk serum / plasma). The individual constituents make the following contribution to
the refractive index (n) of milk:
Casein = 0.0049 - 0.0060
Serum protein = 0.0021 – 0.0035
Lactose = 0.0063 – 0.0067
Miscellaneous constituents = 0.0013 – 0.0022
Fat = nothing

Milk heated to 100˚c shows difference in the R.I. o about 2.0 units in the reading on
immersion refractometer scale of copper serum of milk determining before and after
heating. This difference is probably due to the fact that by heating, the whey proteins
have been converted into a form which is precipitated by copper sulphate.
 30

Determination of refractive index of skim milk and whey:

Objective:
1. To get familiar with the use of able refractometer.
2. To defect adulteration especially watering of milk.
3. To know the total solid content of milk.

Apparatus:
1. Refractometer – filled with an accurate thermometer (reading from 40 to 50˚c)
optical system of abbe refractometer is shown in fig. 6.1.
2. Lab. Certificate.
3. Hot water circulating device – to maintain a temperature of prism at 40 ± 1˚c.
4. Sodium lamp or day light can also be used if the refractometer has a chromatic
compensator.
5. Centrifuge tubes
6. Test tubes
7. Beakers
8. Spatulas
9. Pipette with rubber bulb.

Reagents:
1. Standard fluids for checking accuracy of the instrument i.e. Distilled water.
2. Test sample (skim milk/ whey)
3. Copper sulphate.
4. Sodium chloride
5. Phosphatungstic acid.
6. Concentrated hydrochloric acid.

Procedure:
(A) preparation of sample:
1. Preparation of skim milk:
Take a representative sample of raw, whole milk and centrifuge in lab.
Centrifuge at 2000 rpm for 5 to 10 min. remove the centrifuge tubes and chill
in ice contained in a beaker to facilitate the solidification of fat layer at the
top. Remove carefully with the help of a thin spatula the fat plug and take
out the skim milk carefully with the help of pipesse milk from the tube by
suction by the rubber bulb. Adjust the temperature of skim milk to 40˚c
before determining the refractive index.
2. Preparation of whey:
This may be prepared by any one of the following four methods.
a) Copper sulphate method:
The copper sulphate solution is prepared by dissolving 71.5g of AG CuSO4
in water and diluting to 1000 ml. For the coagulation, 20 ml of milk is mixed
with 5 ml of CuSO4 solution in a 6” × 1” test –tubes. The mixture is well
shaken and filtered through filter paper.
b) Calcium chloride method:
30 ml of milk is solution is thoroughly mixed with 0.25 ml of solution of
CaCl2 (Sp. Gr. 1.1375) in a tube, which is then closed with a cork through
 31

which is passed a short piece of glass tubing to act as a condenser. The tube
is heated in a boiling water bath for 15 min. and placed in cold water. Any
water condensed in the tube is added and serum decanted filtered.
c) Phosphotungstic acid method:
7 g of Phosphotungstic acid is dissolved in water, 2.5 ml of conc. added and
whole diluted to 100ml. 20 ml of milk is mixed with 5 ml of the solution, the
whole well shaken and then filtered through filter paper and the filterate is
used for determination of R.I.
d) Souring method:
The milk is allowed to sour probably at 21˚c (the temp. of cool incubator),
until the milk has coagulated sufficiently for a clear serum to be obtained on
filteration. The preliminary thickening should be ignored, as it is not usually
possible to obtain a clear liquid until coagulation is fairly complete.
(B) Checking the instrument:
The correctness of the instrument shall be tested before taking reading by carrying
out tests with fluid of known refractive index. At temperature of 40˚c or more, the prisms of
most instruments never reach the temperature indicated by the registering thermometer, and
at temperatures greatly removed from the standard temperature for the instrument, there is a
small error due to the change of refractive index of the glass. At these high temperatures,
check the instrument experiment. Ally with a liquid of known temperature coefficient and
apply the correction thus found to instrument readings given by the sample.
(C) Measurement of R.I. of skim milk/whey:
Sample shall completely fill the space between the two prisms, and shall show no air
bubbles. The reading shall be taken after sample has been kept in the prism for 2 to 5
minute. And after it has been ensured that it has attained constant temperature by taking two
or more readings. Take care that the sample has reached the temperature of instrument
before the reading is taken. Before commencing through prisms a stream of water at
temperature at which the readings are taken.
(D) Use of abbe refractometer:
To charge the instrument open double prism by means of screw head and place a
few drops of the sample on prism or if preferred, open prisms slightly by turning screw head
and put a few drops of sample into a funnel shaped aperture between prisms. Close prisms
firmly by tightening the screw head. Allow instrument to stand for a few minutes before
reading is taken so that temperature of sample and instrument will be same.
Method of measurement is based upon observation of position of borderline of total
refraction in relation to the faces of the prism of flint glass figure 6.2. Bring this border line
into the field of vision of telescope by rotating the double prism by means of alidade in the
following manner.
Hold sector firmly and move alidade backward or forward until field of vision is divided into
light and dark portion. Line dividing these positions is “borderline” and as a rule will not be
a sharp line but a band of color. The colors are eliminated by rotating screw head of
compensator until sharp, colorless line is obtained. Adjust borderline so that it falls on point
of intersection of cross hairs. Read refractive index of substance directly on scale of sector.
Check correctness of instrument with water at 20˚c, the theoretical refractive index of water
at 20˚c is 1.330. Any correction found necessary should be made on all readings.
 32

EXERCISE 7
DETERMINATION OF ELECTRICAL CONDUCTIVITY OF
MILK
Introduction:
Resistance (R): it is the ratio of potential difference (E) to the rate of electric current
(I).
Therefore, R = E/I. Resistance is measured in terms of ohms.
Conductivity: conductivity is the inverse of resistance. A low conductivity means
more resistance and vise-versa. In other words, conductivity = 1/R or R-1.the unit of
conductivity is mho or ohm-1.
Specific resistance: (e) it is the resistance offered by conductor of 1cm in length and 1cm in
cross section. It is the resistance of a substance or conductor which is proportional to its
cross sectional area (α) and directly proportional to its length (l). It is expressed as specific
resistance = α R/I
Where R is the measured resistance (E/I). The unit of specific resistance is ohm. Cm specific
conductance (K): it is the reciprocal of specific resistance K= L/e = 1/ α R/1 = 1/ α R.
Unit of specific conductance is ohm-1 cm-1 or mho.cm. Electrical conductivity can be
measured with the help of a Wheatstone bridge or conductivity meter.
Electrical conductivity of milk: since milk contains various kinds of ions, it can conduct an
electrical current about 60 to 80 % of conductivity is due to Na, K and cl. The most normal
milk samples have conductivity in the range of 0.0040 to 0.0055 ohm at 25˚c mastitis udder
has abnormally high concentration of Na and cl.
Relationship between % chloride and conductivity of milk at 18˚c can be used as a simple
physical method of measuring the chloride content.

Conductivity 40 50 60 70
× 10 mho
% chloride 0.075 0.125 0.175 0.225

Conductivity 80 90 100
× 10 mho
% chloride 0.275 0.325 0.375

In another report, whole milk of buffalo and cow is reported to give an electrical
conductivity varying from 4.2 to 6.9 milli mhos at 25˚c.
 33

Factors affecting the conductivity:

i. Temperature: the electrical conductivity increase due to increase in desecration of
electrolyses and decrease in viscosity with the increase in temperature.
ii. Fat: electrical conductivity decrease with the increase in fat in milk because the fat
globules impede the mobility of ions and the fat phase constitutes an insert diluents
skim milk has higher electrical conductivity than whole milk.
iii. Acidity: electrical conductivity increases with an increase in acidity because the
collerdel minerals are brought into the soluble state.
iv. Dilution: electrical conductivity decrease with concentration but is slightly
compensated by increased dissociation of electrolytes.
v. Concentration of milk: electrical conductivity increases upto 30% concentration of
electrolytes. Beyond 30% concentration, electrical conductivity decrease due to
increase in viscosity.

Determination of electrical conductivity of milk:

Operation:
1) The indicator lamp glows put the ‘CAL/READ’ switch in ‘CAL’ position
2) Adjust the ‘CAL’ control to full scale reading (199) on the digital panel meter (DPM)
3) Turn the range switch to extreme clockwise position.
4) Immerse the cell in the standard solution, the specific conductivity of which is
accurately known at the temperature of solution.
5) Put the ‘CAL/READ’ switch at ‘READ’ position and turn the range switch to get
the reading in the corresponding range.
6) Now adjust the ‘CEL CONSTANT’
7) Control so that the digital panel shows the right value of the specific conductivity of
the solution. Now the instrument is ready for measuring conductivity of solution to
be measured.
Before dipping the cell in any solution, keep the range switch in extreme clockwise
direction (position) otherwise in the wrong range the digital panel meter will start
blinking due to overflow. Reading of 112 or 113 with flickering or ‘1’ (cone) indicates
overflow.
8) To get the specific conductivity of any solution, dip the cell in the solution (after
cleaning the cell by rising with distilled water) and turn the range switch to get the
reading on the digital panel meter.
· For better results with improved accuracy it is advisable to select a range so that
reading is now full scale ie.199.
1. CAL/READ SWITCH
2. ‘CAL’ control
3. range switch
4. cell constant control

Procedure:
That was followed for measuring the electrical conductivity milk is as follows.
1. standard 0.1N K C l solution was prepared
2. The milk was heated to 45˚c for 30 seconds and then it was cooled to 25˚c.
3. The temperature of milk sample was adjusted to 25˚c.
4. Electrical conductivity meter was standardized with 0.1 N K C l solutions.
 34

5. The reading of the given milk sample was taken thereafter.

NOTE: - the electrode (disc) is to be washed with distilled water before changing to 0.1 N K
C l as well as to milk.
 35

EXERCISE 8
DEMONSTRATION OF FRACTION COLLECTOR
Introduction:
There are various chromatographic methods like column chromatography, gel
filtration, ion exchange or fractional distillation where there is elution of effluent and to do
quantitative or qualitative analysis we have to carry out various tests.
These are of two types.
(i) On line testing
- Refractometric method
- Ultraviolet spectrometric method.
(ii) Fraction collection
- manual
- fraction collector
There are various chromatographic techniques where separations carried out may extent
over a period of week or more, and to collect such a small amount with less degree of
attention is highly impossible. Thus fraction collector comes into the picture.

Fraction collector:
An automatic fraction collector is a device to divide the effluent into small fractions by
applying a number of vessels consecutively to the column outlet. The rate at which the
vessels are changed can be varied to give.
(a) Fractions of equal volume or
(b) Fractions corresponding to particular sample peaks in the effluent.

Advantages:
- lower flow rate of elution can be kept (as it is advantageous)
- accurate measurement of samples is possible (from 0.5 ml to 500 ml)
- Large number of small fractions can be collected and analyzed for profiles of zonal
of frontal boundaries which is necessary in the case of incomplete resolution of
chemically similar materials/substances.
- Saves chemicals, time for analysis etc. as we can select appropriate liquids/solutes
containing tubes.
- No attention of operator required. It can be operated throughout the might without
any problems.

Disadvantages:
- If a motor is slow/slippage of belts or gears or failure of automatic action of rotation
of rock there are chances of spillage of effluent.
- Electricity failure may cause losses in electrically operated test tube racks.
- For samples, it is difficult to have error free operations (generally below 5 ml of
fractions).
 36

- In almost all the case, if last tube is over the column effluent is drained out, if we do
not attain it (change it with new empty tubes) in time the whole experiment goes in
vain.

Design:
1. A column- column with column holder is supplied. According to the type of
chromatographic technique install the column.
2. A siphon- siphon is must for transfer of effluent to the test-tube. It is of various kinds.
3. Test tube rack holder-test tube rack holder upto the capacity of 250 test tubes per rack are
available. These are arranged in the circular fashion or in many concentric circles or in
straight through conveyor.
4. Motor activating mechanism: - it is a mechanism which records the signal given from the
siphon to place the empty test-tube under siphon. It is most important part; its failure can
cause mixing of two fractions or losses.
5. motor- simple motor following orders from above mechanisms is installed to rotate the
test-tube rack.
The various designs of fraction collector differ mainly in the nature of the signal used
to initiate the measurement of the collection tube. Five groups may be distinguished on this
basis.
1). Time
2). Volume
3). Weight
4). Drop counting
5). Miscellaneous.

1). Time based:

It includes those forms in which the effluent container is changed at fixed time
intervals. The time interval between transfers can usually be varied for use with different
column flow rates.
Usually, the container are arranged in a circle near the circumference of a circular
disc, the disc being turned at intervals by torque from an electric motor, or a clock- work
mechanism, or a weight and pulley arrangement. The timing device can be an electric time
switch or a clock wise mechanism.

ADVANTAGES:
- simple
- reliable
- Use where other mechanism cannot be used or is impossible to use.
- In expensive
- Greater flexibility

DISADVANTAGES:
- Column condition should remain constant
- Less effluent occurs
- Unequal proportions of effluent following is suggested
- Use flexible tips of delivery
- Use test tubes with inserting the piece as shown in figure 8.1.
 37

2).volume based:
These are most satisfactory type of fraction collectors, particularly for analytical
purposes. The apparatus collects equal volume of volumes of column effluent. Most of these
designs depend on the use of some form of siphon for the measurement of liquid volume.
Many of the siphons described are suitable only for fractions of 5 ml or greater. Figure 8.2.
Describes a siphon in which the weight of the liquid collected operates a mercury switch. It
suffers from the disadvantage that the collected liquid may remain in contact with mercury
for sometime.

ADVANTAGES:
- most satisfactory type
- Accuracy high for smaller fractions.

DISADVANTAGES:
- Less than 0.5ml fraction collection difficult.
- Siphoning difficult for smaller fraction.
- Liquid may remain in contact of mercury for sometime.

3). Weight based:

This principle was very much in use earlier but now only few are available. First
principle as shown in figure 8.3 was given by Mr. Philip. Here two discs fixed to a central
spindal lie in horizontal planes. The discs are each performed with a ring of holes near their
circumferences so that a glass specimen tube can slide through each pair of holes. This part
of the apparatus is carried in a vessel containing water. The specimen tubes are constrained
in the two horizontal dimensions but they can float freely on the water at depths governed
by the weight of liquid dropped into the specimen tubes when empty, the tubes ride high
and are prevented from moving by the adjustable knife edge (A). Torque is applied to the
central spindal by a small weight, cord and pulley as shown. Effluent from the column (B)
dips into the specimen tube(C) which gradually sinks lower in the water until its rim
eventually escapes under the knife edge and the next empty specimen tube moves up to the
knife edge. (D) Is an overflow tube.
ADVANTAGES:
- Some of the fraction collectors operate without electric current.
DISADVANTAGES:
- The tubes of approximate equal weight must be used for balancing.

4). Drop counting method:

It is a fraction collector which changes the collecting tubes after a predetermined
number of drops of effluent have fallen from the column.
The falling drop interrupts a light beam focused on a photoelectrical cell. The
resulting variations in output voltage operate a stepping relay-type counter- mechanism,
which can be set to given as actuating signal to the turntable transport mechanism after a
preselected number of drops. (Figure.8.4).
ADVANTAGES:
- Can be used to collect very small number/ volume of fractions. G (0.5 ml).
 38

DISADVANTAGES:
- Evaporation of volatile substances can occur with deposition of solutes on the
column outlet.
- The adjustment of the mechanism is also more critical than in other types
- The electronic and mechanical design is necessarily more complex.
- The drop size is also critically dependent on the density and surface tension of the
column effluent, which can lead to serious error in certain cases.
Thus many disadvantages of this type appear to outweigh the advantages except for
collection of very small sample .we have installed Frakel fraction collector in the
dairy chemistry department of our college. (figure.8.5).
It is a volume based fraction collector which collects predetermined volumes
of liquid in a batch of test-tubes. This is achieved either with the help of a siphon
balance or a timer.
 39

EXERCISE 9
DEMONSTRACTION OF AUTOTITRATOR
Introduction:
Volumetric techniques of analysis are in general those involve as the basic step the
measurement of the volume of some standard solution of known concentration. The
standard solution is called the titrent, and the process of determining the volume required is
called titration. During titration, the active component of the titrent reacts with a quantity of
some specific substance in the solution being titrated, and the reaction in question proceeds
to some point of completion which has significance in the quantitative analytical sense.
The point of completion is called end point of the titration and it is usually assumed
that this point will be located at, or as close as possible to, the equivalence point of the
titration. The equivalence point can be defined as that point in tration where an amount of
the active component of the titrent exactly equivalent, stoichiometrically speaking to the
amount of the reacting substance in the titrated solution has been added.
Volumetric methods can be classified as acid-base or neutralization, precipitation,
complexation, and oxidation- reduction methods.
Electrochemical techniques are classified according to the processes employed in the
method, and the means of attaining the analytical result from the method.
1. Potentiometry
2. Coulometry
3. Electrolytic methods
a. electrode position
b. internal electrolysis
c. electrosolution (electrographic) techniques.
4. Amperometry
5. Conductometry
6. Impedimentary
7. Polarography
Potentiometry and coulometry are commonly used for automatic titrations.

Application of auto- titrator in dairy industry:

Several research workers have reported the following applications of auto titrator in
dairy industry:
1. Automated titration system can be used for the determination of Na C l in cheese and
butter.
2. The automated titrators are used for determination of ascorbic acid in milk and iodine
value of butter fat.
3. The Karl-Fischer automatic titrators are used for high moisture food.
4. The voltametric automatic titrators are used for acidity determination of cream and milk.
5. Lipase activity can be determined by use of metrohm colorimetric titration assembly.
6. The free fatty acids in non-aqueous medium of the milk or cream can be measured using
titrino 716 or metrohm 686 titroprocessor.
 40

Automatic coulmetric titrations:

Principle: conlometry has as its basis faraday’s laws of electrolysis, so that the total
charge transfer gives a measure of the amount of material transferred. It involves the
measurement of a constant current for a known time or of direct measurement of the total
charge by means of an electromechanical or electrolytic coulometer. Coulometric titrations
are especially adaptable to very small quantities in circumstances where ordinary titrations
would be inappropriate or impossible, particularly where titrant is being consumed as it is
generated so that no losses occur.

Apparatus:
The constant- current generators used in coulometric titrimetry must be capable of
providing precise and accurate constant currents ranging in steps from about 1 to 20 m A.
electronic instruments available commercially are able to provide constant currents in the
above range with a precision of + 0.1 to + 0.2 percent. The other accessories include
potentiometer, indicating electrode, reference electrode, magnetic stirrer, coulometric
opposing electrode etc. (figure 9.1).

Advantages:
The technique is capable of determining small quantities of a reactant species
precisely and all other factors being equal, accurately.
The techniques of coulometric titrimetry eliminate the need for the preparation and
standardization, and subsequent periodic restandardization, of a large variety of titrant
solutions. In addition to this, the method permits the use of titration reactions involving
titrant substance normally unstable enough to render impossible or at least most difficult,
their preparation, storage and use in volumetric titrimetry.

Potentiometry:
Potentiometry can be used in end-point determinations for titrations. Here, the
indicator electrode, usually in reversible equilibrium with one of the constituent undergoes a
considerable change in potential at the end point of the titration reaction. Polarized
electrodes not in equilibrium with electrolyte represent a sufficient potential change at the
end point to be satisfactory indicators.
Automatic potentiometric instruments can be divided into three classes
i) The titration curve is simply recorded automatically and the end point is assured to be the
inflections point of this curve.
ii) Some provision is made for stopping the flow of titration when indicator electrode
potential reaches a certain pre-selected value.
iii) An electronic circuit that essentially differentiates the titration curve and stops the flow of
titrent when a large change in d E/ dµ or d2E/ d2µ occurs.
There are many potentiometric automatic titration devices.
a). beckmen auto-titrator model K.
b). Radiometer (figure 9.2)
c). The pge automatic titrator (figure9.3).
d).Diffentrial titrators
e). Malmstadt automatic titrator.
 41

Automatic Karl Fisher Titrator:

In our college we have automatic Karl Fisher titrator. (figure 9.4) it can be perform
all types of potentiometric titrations- acid –base, oxidation reduction, precipitation titrations,
complex formation and Karl Fisher moisture, addition of titrant and stop the process at the
previous titrations more rapidly and more accurately than the ordinary visual methods.

Principle of operation:
The electronic system of the titrator uses a null balance type of circuitry to make
potential measurements. The exact potential equivalent to the end point of a given titrations
is set on the precisior-potential dial. This potential “opposes” the potential of the electrode
pair immersed in the sample solution. If the two potential are equal, the null meter will be at
zero; if they are unequal the meter will deflect to the right or left depending upon which
potential is larger. The electrode potential can be determined simply by adjusting the
opposing potential with the potential dial until the meter is zeroed, and then reading the
potential value from the dial.
In performing the titration with the automatic titrator, the end point potential is pre-
set on the dial. The potential of the electrodes immersed in the sample will differ from this
pre-set value, causing the meter to deflect. This difference is amplified by integrated circuit
amplifier and fed to a control circuit, which activates a solenoid value allowing the titrent to
dispense into the sample beaker. As titration precedes the differences between the electrode
potential and pre-set potential will narrow until close to the end point, they become equal
with no difference in potential appearing at the controller input, the solenoid gets
deactivated and the value closes. The meter reads near zero signifying the end point.

Electrodes:
A pair of electrodes is always required in any potentiometric titration-one is termed
an indicating electrode and the other a reference electrode. The other a reference electrode
maintains a fixed potential and all measurements are made with respect to it. The indicating
electrode potential will change as the titration progresses.
The electrodes most commonly used in potentiometric titrations are classified as
either indicating or reference electrodes.

Anticipation:
To achieve results with the titrator it is necessary to obtain proper anticipation so as
not to oversheet the end point.
To achieve proper chemical anticipation with the titrator, the factors such as delivery
tip, stirring rate, titration curve and the rate of titrent addition should be carefully considered
and adjusted to meet the requirement of particular titrating situation.

Operating controls:
The operating controls for the AUTO TITRATOR are located on the control unit
and stirrer. They are listed below (figure 9.5 & 9.6).
A. CONTROL UNIT
i). Power switch
ii). MV- PH switch
iii). Potential dial
iv). POL –STD By – USE switch
 42

v). Null adjust control

vi). Null meter
vii). Auto switch
viii). Temperature control
B. STIRRER
i). Power switch
ii). Pilot lamp
iii). JOG
iv). Pilot light (valve)
v). speed control

Operating procedure:
Calibration: - the first step in the operation of the titration is the calibration of the
potential dial. The term “CALIBRATION” as used here refers to the choice of a reference
scale reading and corresponding voltage range in which potential determination can be
made. Since the titrator may be used for either millivolt or PH measurements, a separate
calibration must be performed for the type of titration desired.

Calibration for millivolt range:

To calibrate the potential dial for milivolt measurements, follow the steps given
below:
1). Make certain the grounded three prong plug on the titrator line cord is plugged into a 230
v AC outlet. Be sure that the power cable from stirrer is plugged into the receptacle on the
rear of the control unit.
The end point detection for potentiometric titrations is carried out by any of the
following way.
A. graphical determination
B. titration to the equivalence point potential.
C. pinkhof –treadwell method
D. differential titrations
E. bimetallic electrode
F. potentiometry with polarized indicator electrodes.

Applications:
1). Acid- base titration
- Hydrogen electrode
- Oxygen electrode
- Metal electrode
- Ruinhydrone electrode
- Glass electrode
- Non –aqueous titration.

2). Precipitation titration

a. silver electrode
i. titration of helide mixture
ii. Chloride determination in presence of large amount of bromide or iodide.
iii. Determination of silver.
 43

b. mercury electrode
- Determination of alkaloids
c. iodine electrode
d. platinum electrode
e. Third class electrode
f. membrane electrode

3). Complexometric titration

a. silver electrode
b. platinum electrode
c. mercury electrode
- Titration of metal ion with E.D.T.A.

4). Oxidation –reduction titration reactions

a. potassium permagnate
- Titration of manganese
b. potassium dichromate
- Determination of carbohydrates and other organic substances.
c. ceric cells
- Determination of vanadium in steels
d. potassium iodated and bromination of organic compound
e. other oxidants.
f. teravalent arsenic
- Determination of chromium and vanadium in steel
g. bivalent iron
- Determination of manganese, cerium and chromium
h. titanous ion
- Preparation of titanous chloride from titanium hydride
i. chromous ion
- Preparation of standard chromous solution
- Determination of nitrate ion
j. other reductants.
 44

EXERCISE 10
DEMONSTRATION OF LYOPHILIZER
Introduction:
The technique of freeze drying is very widely used for preserving and increasing the
self life of biological materials. By using this process sensitive thermolabile materials like
vaccines, blood plasma, sera, living bacterial and virus suspensions, cultures, therapentic and
prophylactic preparations, antibiotic, fruitlets and food stuff etc. can be dried to low
moisture contents; thus increasing their shelf life, and preserved.
Lyophilization or freeze drying is the total process of freezing and sublimation of the
water from the frozen preparation. The product is frozen and then exposed to an
atmosphere to an of low relative humidity when the contained ice sublimes that is, it changes
directly from the solid to the vapour state without melting, thereby avoiding the chemical
and enzymatic changes usually associated with other forms of drying. The term lyophilizer is
applied to the equipment which freeze- dries the substance or material. (Figure 10.3).

General procedure:
The basic steps in this process involve
- Preparation of a dense suspension of cells in a selected fluid.
- Freezing the suspension of cells in an ampules in a dry ice-bath.
- Evacuating the vials while frozen until the contents are completely desicated,
- And then hermetically sealing the evacuated containers.
The brief steps of lyophilization are shown in figure 10.1.
The different methods employed for freezing the substance or material include plug
freezing, shell freezing, evaporative freezing and pelletizing.the different types of
lyophilizes in common use are of the glass, metal, do – it- yourself and commercially
available types. The lyophilization process for preservation of microorganisms is
depicted in figure 10.2.

Uses:
1. Lyophilization has been extensively used for the study of biological macromolecules
in solution.
2. Several species of microorganisms are preserved for longer period of time with the
help of lyophilization.
3. Freeze drying is useful for the concentration of solution of biological substance and
tissue specimens which are highly sensitive to water even at low temperatures and for
drying polysaccharides to obtain a surface active form.
4. For preparing biological objects for election microscopy. Freezing makes it possible
to fix the preparation instantly without any change of the initial conditions.
5. Freeze drying, freeze fracturing and etching and low temperature electron
microscopy of frozen hydrated specimens are methods for studying cytofixed
material. In case of screen lipoproteins, human chylomicrons, VLDL, LDL, apo
LDL, HDL and normolipidemic and hyperlipidemic rhesus men key LDL have been
the subject of freeze – fracture and freeze – etching electron microscopic studies.
 45

6. Freeze – drying is used commonly for preservation of cheese, yoghurt and kefir,
hence improving their keeping quality.
7. Applications of freeze – drying in chemical analysis include defection and estimation
of added rennet whey solids to dried whole milk using microscopic method and
determination of lactose in milk.

We have installed Toshniwal lyophilizer as well as Herysan shelf freeze dryer (fig.
10.3) at the dairy Microbiology department of the college.
 46

EXERCISE 11

DEMONSTRATION OF PH METER
Introduction:
Definitions
1. Acid and base:
The modern concept of acids and bases developed by Bronsted, Lowry and others
defines acids as proton donors and bases as proton accepters. Each acid therefore has a
conjugate base.
Acid Base + H+

2. Alkali:
The term alkali is reversed for those compounds that yield hydroxyl ion on
dissociation
-
KOH K+ + OH

3. Ampholytes:
Some ionic species can act as both acids and bases and these are known as
ampholytes or are said to be amphoteric.

4. Strong acids or bases:

These compounds are completely ionized in solution, so that the concentration of
+ -
the free H or OH is same as the concentration of the acids or bases.

5. Week acids or bases:

These compounds dissociate only to a limited extent and the concentration of
+ -
free H and OH depends on the value of their dissociation constants.

Hydrogen ion concentration and PH:

The hydrogen ion concentration of most solution is extremely low and, in 1909,
Sorenson introduced the term PH as a convenient way of expressing hydrogen ion
concentration instead of the use of cumbersome numbers. PH is strictly defined as the
negative logarithm of the hydrogen ion activity, but in practice the hydrogen ion
concentration is usually the same as the activity except in strongly acid solutions.
From conducting measurements, water has been shown to be very weekly ionized
and at 25˚c the concentration of hydrogen ion is only 10-7 moles/ litre.
-
H2O - - - H+ + OH
The equilibrium constant for the dissociation of water is given by
-
K = [H+] [OH ]
H2O
Now the concentration of water to all intents is constant so we can write:
-
KW = [H+] [OH ]
 47

Table iii: the ionic product of water and PH of neutrality at various temp.

˚c KW PH
0 0.12 × 10-14 = 10-14.94 7.97
25 1.03 × 10-14 = 10-14.00 7.00
37 2.51 × 10-14 = 10-13.60 6.80
40 2.95 × 10-14 = 10-13.53 6.71
75 16.90 × 10-14 = 10-12.77 6.39
100 48.00 × 10-14 = 10-12.32 6.16

-
At 25˚c, [H+] = [OH ] = 10-7
Therefore PH = - log [10-7] = 7

Dissociation of acids and bases:

Strong acids:
These are compound in which complete dissociation to hydrogen ions and the
conjugate base occurs, so that the hydrogen ion concentration is the same as that of the acid.
The PH of such solution can therefore be very easily calculated:
E.g. 0.01 mol/ lit HCL, PH = - log 10(10-2) = 2
0.1 mol/ lit HCL, PH = - log 10(10-1) = 1
0.010 mol/ lit NaOH, [H+] = KW
-
[OH ]

= 10-14 = 10-12
10-2
H -12
Therefore P = - log10 (10 ) = 12

Weak acids:
The Henderson – Hasselbath equation:
Weak acids are only slightly ionized in solution and a true equilibrium is established
between the acid and the conjugate base. It HA presents a weak acid, then
-
HA H+ + A
According to the laws of mass action Ka, the acid dissociation constant is defined
as:
-
Ka = [H+] [A ]
[HA]
And [H+] = Ka [HA]
[A]
Taking negative logarithm
- log10 [H+] = - log10 Ka + - log10 [HA]
-
[A ]

In general terms:
PH = pka + log10 [conjugate base]
[Acid]
 48

- -
The activities of A and HA are not always known, so it is convenient to express A
and HA concentration in terms thus:
- -
PH = pka + log C A + log F A
CHA FHA
- -
Where FA and FHA are activity coefficient of A and HA respectively. Since log (FA/FHA)
is constant for a given acid, these activity coefficients can be incorporated in the pka in term
of apparent dissociation constant pka’
PH = pka’ + log CA
CHA
This relationship is know as the Handerson Hasselbath equation and is valid over PH
range 4.10 where the hydroxyl ions do not contribute significantly to the total ionic
concentration.
PH = pka + log 101
Therefore PH = pka

Measurement of PH:
1. PH indicators:
An approximate idea of the PH of a solution can be obtained using indicators. These
are organic compounds of natural or synthetic origin whose colour is PH dependent.
Indicators are usually weak acids which dissociate in solution.
Indicators = Indicator - + H+
Applying the Handerson – Hasselbath equation
PH = pkIn + log10 [Indicator -]
[Indicator]
The greatest colour change occurs around the pkIn and this is where the indicator is
most useful, however limitations are:
1. Colour change occurs over a wide PH range.
2. Indicators are affected by oxidizing agents, reducing agents, salt concentration,
protein etc.
A small quantity of indicator to the solution should be used, otherwise the acid base
equilibrium of the test solution may be displaced and the PH changed.

PH Meters:
The most convenient and reliable method of measuring PH is by the use of PH meter
which measures the emp. The four major parts of the PH system that are always needed
include:
1. Reference electrode
2. Indicator electrode (PH sensitive).
3. Voltmeter or amplifier
4. The sample being analyzed (fig. 11.1).
Hydrogen ion concentration is determined by the voltage that develops between the two
electrodes, the nearest equation relating electrode response to activity.
The saturated calomel electrode is usually used as the reference electrode, while the
glass electrode is used as an indicator electrode for PH measurements. Combination
electrodes (reference + indicator) are also available, being of value when the volume of
 49

sample is limited. Department of dairy chemistry of our college has the following instrument
for PH measurements.
1. Systronic PH meter type 331
2. Systronic PH meter type 322
3. ELICOS digital PH meter, model Li – 120
4. Bechman PH meter, model H2
The operation of ELICO digital PH meter and Bechman PH meter model H2 is narrated.
ELICO digital PH meter (fig. 11.2)
This is a PH meter in which the glass and reference electrodes are combined and PH
value is displayed in digits.
1. Connects power line to mains.
2. Wash the electrode with distilled water and wipe with tissue paper.
3. Dip the electrode in standard buffer and set temp. Knob to the temp. of buffer.
4. Set PH knob to PH and adjust the PH to the above PH value of buffer.
5. Set the knob to ‘std – by’ and remove the electrode.
6. Check repeatability
7. Dip the electrodes in test solutions.
8. Repeat steps 9 & 5.
9. Wash the electrode with d/w.

Beachmen PH meter model H2:

1. Set range switch on start, connect to mains and allow to warm up for 10 mins.
2. Dip the electrodes in buffer, set temp., set proper range and adjust PH.
3. A reference point is established to correct the instrument without restandardizing.
With range switch at ‘nent’ position set the check pointer so that it coincides with
that of PH needle.
4. Rinse the electrodes with distilled water, immerse in the test solution, set proper
range (0-8 or 6 to 14), note the PH, put the switch on ‘nent’ and remove the
electrodes.
 50

EXERCISE 12

DEMONSTRATION OF COLORIMETER

Introduction:
Many biological experiments involve the measurement of a compound or group of
compounds present in a complex mixture colorimetry is the most widely used method
for determining the concentration of biological compound and makes the use of the
property that when light passes through a colored solution, some wavelengths are
absorbed more than others. May compounds are not colored but are made to absorb
light in the visible region by reaction with suitable reagents. The big advantage is that
complete isolation of the compound is not necessary and the constituents of complex
mixtures such as blood can be determined after little treatment.
Colorimetric techniques may be used for either qualitative or quantitative
measurements. Qualitative measurements are based on the premise that each analyte has
a unique set of energy spacing that will dictate its absorption/emission spectrum. Hence,
qualitative assays are generally based on the analysis of the absorption and/or emission
spectrum of the analyte. In contrast, quantitative assays are based on measuring the
absorbance and/or florescence of the analyte at one wavelength. Quantitative absorption
assays are based on the premise that the absorbance of the test solution will be a
function of the solution analyte concentration.
Under optimum conditions, there is a direct linear relationship between a solution’s
absorbance and its analyte concentration. The equation describing this linear relationship
is known as beer’s law.

Principle of operation:
Principle operation of photoelectric colorimeter type 101.
The different parts of a colorimeter are shown in figure12.1. A low voltage lamp or
energized by a constant voltage transformer forms the light source. This light passes
through a selected filter and a centrally opening shatter, controlled by a wheel, the dm of
which protrudes out. This light passes through the test-tube containing the solution and
falls on a sensitive photoelectric cell. The current generated by the photo electric cell is
amplified by a transistor amplifier which drives 50 micro ammeter calibrated in terms of
percent transmission and optical density. The transistor amplifier is in corporate mainly
to replace the delicate meter with a relatively robust 50 micro ammeter power supply.
The model of photo-electric colorimeter is shown in figure 12.1. Whereas its principle is
illustrated in figure 12.3.

Choice of filters:
Selecting the approximate filter and obtaining reproducible photometric readings are the
principle considerations in the measurement process. The color of the filter should be
complementary so that of the solution to be measured.
 51

Colors of solution colors of filter

Orange blue green

Yellow blue
Purple green
Red blue
Violet yellow-green
Green purple
Blue-green red-orange
Blue yellow

Operation of instrument:
1. Set the ‘increase light’ control wheel to maximum i.e. rotate it to left extreme.
Keep the lid closed.
2. Switch ON; bring the meter to oc optical density (zero on percent transmission
scale).
3. Select and insert filter
4. Insert a test-tube containing ‘blank’ solution in the sample socket
5. Close the lid, adjust the ‘increase light’ control until the meter reads zero on the
O.D. scale.
6. Remove the ‘blank’ solution, insert the test sample, close the lid and note the
meter reading.
 52

EXERCISE 13
DEMONSTRATION OF SPECTROPHOTOMETER
Spectrophotometer consists of a light source, a monochromator (device to produce a
monochromatic beam of light from any point on the spectrum), and a vessel containing the
solution and positioning it in the light beam, and a detector with its amplifier. The light
source used for the visible spectrum is the tungsten lamp, usually a single filament car
headlamp bulb. This gives a peak energy output at about 950nm and coves the range 320-
3000 nm. The electric supply must be constant as the radiant energy must not vary. This is
provided by an accumulator or car battery. A constant voltage transformer or stabilized
power pack is used in most instruments. The source of u.v.light is the hydrogen or
deuterium discharge tube which supplies light of 180-350 nm. A stabilized power supply is
essential for these lamps. The tube has a quartz window and when adjustments are made this
should not be touched. If a more intense source is required the mercury arc or xenon lamp is
used.
Infra-rod radiation is supplied by a Nemst filament, rod of oxides of zirconium and
thorium, which requires a heater winding for starting, the operating temperature being about
1350˚c .a stabilized voltage is required.
The visible or u.v.type employs either a glass or quartz prism or a diffraction grating
in the monochromator. Some instruments are both prism and grating to improve resolving
power. When working with the u.v.region, silica cells must be used as glass is not transparent
to u.v.light.

Operation:
The instrument is turned on and allowed to warm up for a few minutes. The
illumination must be stable before starting. Most instruments have ‘dark current’ controls
which allow the balancing of the electronics with no light entering the photo cell. This is
done after a wavelength has been selected. With a reagent blank in the light path the
galvanometer or potentiometer is set at zero. The blank is then replaced by the test solution
and the absorbance or transmission is noted. The instrument should be zeroed with the
blank in position with each successive wavelength.
In the dairy chemistry department of our college, following spectrophotometers have
been installed.
1. Gystronics photoelectric colorimeter type 101, and
2. Spectronic -20- colorimeter- spectrophotometer, (figure 13.3).

Types of spectroscopy:
1. visible spectrophotometry
2. ultraviolet spectrophotometry
3. infra –red spectrophotometry
4. circular dichroism spectrophotometry
5. spectrofluorimetry
6. luminometry
7. atomic/flame spectrophotometry
8. electron spin resonance spectrophotometry
 53

9. Nuclear magnetic resonance spectrophotometry

10. Mass spectrometry etc.
 54

EXERCISE 14
HIGH SPEED CENTRIFUGE & ULTRACENTRIFUGE
(DEMONSTRATION)
Introduction:
The physical techniques most responsible for current understanding of cellular
makeup and operation are those involving the centrifuge. Wide varieties of these
instruments are available ranging in capacity from those handling 0.2 ml and less to those
accommodating thousands of liters with relative case. Some are crudely controlled with
regard to speed and temperature whereas in other these parameters are regulated within
limits of less than 5%.
In its simplest form a centrifuge is composed of a metal rotor with holes in it to
accommodate a vessel of liquid and a motor or other means of spinning the rotor at a
selected speed. All the other parts found in today’s modern centrifuge are merely
accessories used to perform various useful tasks and maintain the environment within
which the rotor operates.

Principle:
Centrifugation separation techniques are based upon the behavior of particles in an
applied centrifugal field. Particles which differ in density, size or shape sediment at
different rates in a centrifugal field. The rate of sedimentation of a spherical particle is
dependent upon the applied centrifugal field, density, radius of the particle and the
viscosity of the suspending medium. These relationships are expressed by stoke’s law.
U= 2/9 r2p (ℓp - ℓm) × g
n
Where
u = sedimentation rate or velocity of sphere
ℓp = density of the particle
ℓm = density of the suspending medium
r p = radius of the particle
n = viscosity of the suspending medium
g = gravitational field
2/9 = shape factor constant for a sphere.
If the density of medium and density of particle are equal, then the sedimentation rate will be
zero. But this sedimentation rate will also depend upon its sedimentation rate. Hence, even if
the particles have similar density, slight differences in their size can make large differences in
their sedimentation velocity.

Classification:
Centrifugation techniques can be mainly classified into two types namely
a. preparative centrifugation and
b. analytical centrifugation
 55

a. preparative centrifugation:-
This is concerned with the actual isolation of biological material for subsequent
biochemical investigations. Very large amounts of material may be involved when harvesting,
for example, microlical cells from batch or continuous culture, plant and animal cells from
tissue culture and plasma from blood. Relatively large amounts of cellular particles may also
be isolated in order to study their morphology, composition and biological activity. It is also
possible to isolate such biological macromolecules as DNA and proteins, from preparations
which have received some preliminary purification, for example fractional precipitation.
b. analytical centrifugation:-
It is used for the study of pure or virtually pure macromolecules requires for e.g.:
ribosome. It requires very small amount of material.
Preparative centrifugations are the more commonly used ones. This can be further
classified into four groups.
i. differential centrifugation
ii. Rate zonal centrifugation
iii. Equal density or isopycnic
iv. Equilibrium isodensity centrifugation.
i. differential centrifugation:
This is based on the difference on sedimentation rate of particles of different size
and density. The material to be separated is centrifugally divided into a number of fractions
by the step wise increase of applied centrifugal field. The centrifugal field at each stage is
chosen so that a particular type of material sediments, during the pre-determined time of
centrifugation to give a pellet. At the end of each stage the pellet is washed to obtain a pure
fraction. The sedimentation of particles during centrifugation varies depending upon their
size and shape.
Differential sedimentation of a particular suspension in a centrifugal field is shown in
figure 14.1.
It is the most commonly used method for the isolation of cell organells from
homogenized tissue. The main demerit is that the liquid density gradient has to be
performed when it is required and this may be a time consuming process.
ii. Rate –zonal centrifugation:-
This is also called as s- zonal centrifugation. This involves carefully layering a sample
solution containing particles to be separated, on top of a performed liquid density gradient
whose density continuously increases towards the bottom of the centrifuge tube. The sample
is prevented from pre-mature sedimentation by the steep positive density gradient beneath it.
The sample is then centrifuged until the desired degree of separation is affected i.e.
Complete sedimentation should not be there, but the particles should form discrete zones or
bands consisting of particles characterized by their sedimentation rate (figure 14.2). Here, to
achieve separation density of particles must be higher than the density gradient.
This method is used for the separation of RNA-DNA hybrids, ribosomal subunits
and other cell components.
iii. Equal density (isopycnic) centrifugation:-
Here density gradient may or may not be used. In the absence of a continuous
density gradient, the sample is initially centrifuged to sediment particles heavier than the
ones required. After separation and rejection of heavier particles the sample is suspended in
a medium having a density equal to the fraction to be isolated. After further centrifugation
the lighter and heavier particles than the required ones will be at the meniscus and base of
the tube respectively while the required one will be in an intermediate position (figure 14.3).
 56

In the presence of density gradient it will be same as that of rate zonal method. But the main
difference between the two methods is that for equal density centrifugation, the
centrifugation is continued until the desired particles have reached their equal density or
isopyenic position in the gradient. This is not required in rate separation.
Equal density or isopycnic position: - means where the buoyant density of the particle
and the density of the gradient are equal. At this point no further sedimentation occurs.
iv. Equilibrium isodensity centrifugation: -
In this method, for the preparation of gradient, salt of heavy metals like Co or
rubidium or sucrose is used. The sample is mixed homogeneously. The sample is mixed with
a concentrated solution of cell and centrifuged. This gives a concentration gradient of Cs c l
and hence a density gradient due to large mass of Cs ion. Redistribution of particles then
takes place in this gradient.
This is most commonly used in analytical centrifugation. Also used to separate and
analyze human plasma lipoproteins.

Types of preparative centrifuge:

Preparative centrifuge can be classified into three major groups:
1. General purpose centrifuge
2. High speed centrifuge, and
3. Ultra centrifuge

1. General purpose centrifuge

General purpose centrifuge has a maximum speed of 6000 rpm. Used for collecting
substances that sediment rapidly, like yeast cells, RBC’s etc…..
2. High speed centrifuge
These can operate up to speeds of 20,000 to 25,000 rpm. These are generally
equipped with refrigeration equipment to cool the rotor chamber. Two types are there. The
first is relatively simple, high capacity, continuous flow centrifuge. If consists of a very fast
electric motor, which is connected to a long tubular rotor. This is used for the harvesting of
yeast or bacteria from large cultures.
The culture is siphoned or pumped into the bottom of the spinning rotor. As the
cultures move up towards the top of the rotor, microorganisms are sedimented against the
rotor walls and the clarified culture medium exits through an effluent part. After all the
medium has been passed through the centrifuge the rotor is disassembled and the compared
cells are removed with long spactula.
The second type consists of lower capacity refrigerated equipment. A broking device
is also used here to decrease the time required for rotor declaration at the end of
centrifugation, it is used to collect microorganisms, cellular debris, cells, large cell organelles,
immunoprecipitates, etc…
The main demerit is that it cannot sediment viruses, small organells like ribosome’s.
3. Ultra centrifuge
It can operate at a speed of 75,000 rpm. It has four major parts.
a. drive and speed control
b. temperature control
c. vacuum system, and
d. rotors.
USES
 57

1. It permits fractionation of sub cellular organells which can be seen only with the help of
electron micrographs.
2. It can be used for the isolation of viruses.
3. DNA, RNA and protein can be carefully analyzed.
 58

EXERCISE 15
METHODS TO MEASURE WATER ACITIVITY
Introduction:
Moisture content is very often the only parameter used to define moisture condition
in hygroscopic products. It influences many of the physical and mechanical properties of
material and even their selling price.
Water activity (aw) or equilibrium relative humidity (% ERH) expresses the active
part of moisture content or “free water “as opposed to the total moisture content which also
includes “bound water”.
Water activity (aw or % ERH) not only determines the stability of moisture content
(and of the parameters it influences) but is also a decisive factor in various problems where
moisture content is of less interest.
There are various techniques to measure aw. a commonly used approach relies on measuring
the amount of moisture in the equilibrated headspace above a sample of the food product,
which correlates directly with sample aw. A sample for such analysis is placed in a small
closed chamber at constant temperature, and a relative humidity sensor is used to measure
the ERH of the sample atmosphere after equilibrium. A simple and accurate variation of this
approach is the chilled mirror technique, in which the water vapour in the head space
condenses on the surface of a mirror that is cooled in a controlled manner. The dew drop is
determined by the temperature at which condensation takes place, and this determines the
relative humidity in the headspace. Two other general approaches to measuring aw are (i)
using sample freezing point depression and moisture content to calculate aw and (2)
equilibrating a sample in a chamber held at a constant relative humidity and then using the
water content of the sample to calculate aw.

Moisture content and water activity:

The moisture content of a product sample is usually defined as the percentage weight
of water content in relation to the dry weight of sample.
The water activity (aw) can be defined in any number of ways. It can be related to the
ratio of the vapour pressure of a solution (p) to that of pure water (po) at specified
temperature.
aw = p/po
Water activity can also be defined in terms of solute concentration through its relation to
Raoult’s law
aw = p/po = n2/n1 + n2
In which n1 & n2 are number of moles of solute and solvent respectively. Water activity also
is related to the osmotic pressure (o.p).
o.p = -RT log e aw/v
Where R is the gas constant, T is the absolute temperature and v is the partial molar volume
of water.
aw is the relationship between this parameter and equilibrium relative humidity or ERH.
ERH (%) = Aw × 100
Water activity (or % ERH) indicates the degree of freedom of water absorbed in a material
and shows better than moisture content does, the effect of this water on physical properties,
 59

such as dimensions, structure, cohesion, agglomeration properties, electrical and chemical

properties.

aw in the food industry:

water activity exerts a decisive influence on such phenomena as change in color, taste
and aroma, food poisoning and spoilage, loss of vitamins etc. (total moisture content has
very little to do with this).
Water activity in foods can be controlled by using various additives (e.g. Salts, sugars
etc.), by using satisfactory packing materials, by maintaining favorable maturation and
storage conditions. Water activities measurements are increasing in food research and
development as well as in ‘production quantity control’ on line measurements are possible to
a certain extent.

The importance of aw in foods is an follows:

1. It affects the growth of micro-organisms. Therefore, by measuring the aw value of food
stuff, it is possible to determine which micro-organism will not be able to develop on it.
(Figure 15.1).
2. The control of water activity is important for chemical stability of food.
3. Enzymatic stability is also dictated by aw values. Most enzymatic reactions are slowed
down at aw values below 0.8.
4. Calculating and controlling drying processes also requires the knowledge of the
relationship between aw and % water (sorption – isotherm). The moisture content and aw of
some food stuffs is depicted in table 15.1.
The desired characteristics of aw measurement techniques include accuracy,
reproducibility, speed, low cost, portability, case of use and durability.

Methods of measuring water activity:

1. graphic interpolation
The aw peramoter is the equilibrium relative humidity or aw at which a substance neither
gains nor looses moisture at a specific temperature. This concept may be used to
estimate, with a reasonable precision, the water activity of an unknown material.
The weight of sample water loss or gain in different RH chamber is measured for a
given period of time, usually 1 or 2hrs. If the amounts of water gained or lost by the
several aliquots of the sample maintained in different RH are then plotted against aw, this
plot will interest with the line representing zero moisture change. It is this point of
interpolation that represents aw of sample.
2. Bithermal equilibration
I this technique, the vapour pressure of a solution am measured by a method that
depends on the vapour phase equilibrium of the solution at 25˚c with pure water at
lower temperature. This device is shown in figure. It consists of a thin walled,
moderately flexible tube, which joins two “bells” each submerged in its own bath. The
immersed in the 25˚c water bath contains the sample and the other bell at a lower
temperature, surrounds a dish of pure water. The temperature of each water bath is
measured with great accuracy by means of a copper constant thermocouple. The
difference in temperature of two baths is calculated following equilibrium and this is
taken as the temperature of sample and pure water. The concentration of water in the
solution to be determined and vapours pressure of both solution are read from
 60

established tables.aw is then calculated from the ratio of two vapours pressure as noted
above.
The method is cumbersome and lengthy. It was originally used to determine the aw
of various saturated solutions. Presently, this method is seldomly used.
3. Vapour pressure manometer (manometry)
The moisture content of food related directly to its vapour pressure at constant
temperature. This pressure may be measured accurately by manometric procedures.
Relatively high precision (±0.002 aw) has been claimed for this technique.
The sample to be measured is ground and introduced into flask, which is attached
through a trap to a simple manometer. The manometer assembly (figure 15.2) is then
evacuated and during this time the sample chamber is maintained out approximately -
80˚c. Following evacuation, the sample is warmed to room temperature, while one side
of manometer is maintained at essentially zero pressure. The fluid level in the sample
arm of the manometer is then deflected by increase in pressure caused by vapour
pressure of the sample itself. Air entrapped in the sample also contributes to deflection
of the manometer, but this can be compensated for relatively easily. Humidity
determination can be carried out on the same sample at various moisture levels by
allowing the sample moisture to distill into freeze trap by reweighing the sample.
4. Hair hygrometer
The principle underlying this instrument is that the keratinaceous protein of hair
absorbs moisture from the atmosphere with commensurate stretching. It the hair strands
(usually 3 or more strands are breided) are fixed at one end, attached to an indicating
lever arm on the other end and allowed to come into equilibrium with the substance to
be measured, ERH can be read directly.
5. Isopiestic hygrometry
(Isopiestic equilibration) like hair hygrometry, this technique requires only very
inexpensive equipment. With this procedure, the weight gain of some absorptive material
such as microcrystalline cellulose or a protein is measured at various established aw levels.
The equilibration usually is carried out in a series of desiccators, each containing a
saturated solution and each at different aw levels. From this the standard sorption
isotherm is calculated. The unknown to be measured then is placed in desiccators with
one of the absorptive material for a given time, usually 24 hr and the weight gain at the
end of this period is compared to the standard isotherm.
Comparisons between aw levels of various food determined by this technique and by
electric hydrometric procedure has given good correlation at aw levels > 0.09 and
superior precision has been claimed at aw levels < 0.90.
6. Electric hygrometry
Ratronic electric hygrometer (in our college) has operation similar to above (figure15.3)
one. In this, A.C. current can be passed through as saturated solution of LiCl2 suspended
into an insert carrier such as glass wool. A current potential difference of 25v, which
heats the cell, is provided across the solution. The water vapour pressure (WVP) of
solution rises and upon reaching the WVP of environment, water evaporation occurs.
The dried lithium chloride residues remaining after evaporation no longer conducts
current and heating ceases. As the residue cools, water is once again taken up from the
environment and the cycle is repeated at reduced amplitude. Eventually, a temperature is
reached at which the WVP of environment. This temperature is then measured and
related to the WVP of saturned lithium chloride and hence the environment from which
the ERH can be calculated.
 61

The other method used for measuring water activity includes:

7. Freezing point depression
8. Dew point methods, and
9. Chemical methods
 62

EXERCISE 16
DEMONSTRATION OF POLARIMETER
Principle:
Certain organic substance possesses the property of rotating the plane of polarized
light. Such substances are said to be optically active. In ordinary light, the vibrations are
confined to a single direction or plane. Polarized light may be obtained by passing light
through a nicol prism. The instrument by which optical activity of a liquid is determined by
inserting nicol prism (figure 16.1) in the path of a ray of light before and after passing
through a liquid is called polarimeter. This instrument is chiefly used to determine the optical
rotation of a solution. A beam of monochromatic light is passed through nicol prism to
produce plane polarized light, and through a tube containing the optically active material
(e.g. Sugar solution) and finally through a second nicol prism (figure16.2). When these
prisms are placed at right angles with no optically active material intervening, the only light
passed by first prism is stopped by the second prism and the rear field appears dark. When
optically active material is introduced between the two prisms, light appears and the field
becomes bright. The second prism must be rotated in order to prevent the passage of light.
The number of degrees through which the 2nd prism must be rotated to the rear prism must
be rotated to the right. For leavo rotatory substances it must be turned to the left. The extent
of optical rotation depends upon several factors including the nature of the substance, its
concentration, the length of column through which light passé, wave length of light, and
temperature of liquid. The measurement of rotation can be employed as a method for
analyzing the sugar the optical rotation of a known concentration of a particular sugar is
determined and is expressed as a physical constant in terms of specific rotation. Specific
rotation is defined as rotation in angular degrees of the plane of polarized monochromatic
light produced by a solution of optically active substance having a concentration of 1gm/ml
solution 1 decimeter length. Specific rotation may be expressed as
[α] = 100 × 9
1.0
[α ] 20. = specific rotation at 20˚c referred to the D line of spectrum.
a = observed angular rotation in degrees
l = length of column of solution in decimeter
c = concentration in gms per 100 ml

Instrument: (figure 16.3)

Polarimeter model A is installed in the department of dairy chemistry of our college
which is operated as follows:
1. Set the polarimeter on a triople stand or on a table, app 4” away from the edge with eye
piece facing the operator.
2. Open the hinged trough, remove the drum illuminator packed there in, and fit to the
socket provided adjacent to the drum.
3. Also remove any other packing and adjusting key from the trough.
4. Focus the top eye piece to show the scale and index line.
5. Remove the transit spring on the loss of the control to allow it to engage with the scale
within the head.
 63

6. Looking through the telescope turn the scale control wheel till it reads zero.
7. Without disturbing, focus the field telescope to obtain a disc of light. Turn the control
wheel to back and forth and obscure the darkening halves. The balanced position between
these two halves is used in all measurements. At this position the scale should read zero and
micrometer should be set at zero. If not the procedure given in “adjusting zero” should be
followed.
8. Fill the sample into the tube, place in the through, close the cover and observe in the field
telescope. The field will be disturbed. Now set it to balance position by the control wheel.
9. Obscure through scale telescope and note the reading.

Reading the scale:

The scale at top is divided at 1˚ interval ranging from 1˚ to 360˚. Down scale is
international sugar scale (ISS) may be used directly in the evaluation of sugar solution.
Micrometer drum is used to subdivide the angular and ISS scales. Angular on the left
subdivided at 0.05˚ and ISS on right subdivided at 0.1.

Sample tube filling:

Remove caps from both the ends and carefully clean the glass windows. Screw one
end cap, invert the tube and fill the tube. Screw the other cap taking care that no air bubbles
enter. Tubes with air bubbles trap are also available.

Adjusting zero:
An error upto 1/2˚ can be controlled by micrometer drum as follows:
1. With no sample in trough, set the field to balanced position.
2. Set micrometer scale to zero and observe scale in telescope.
3. Grasp the knurled washer and with other hand stacken the knurled nut. The drum is now
released from the screw. Turn the screw by means of knurled wash until the index line
coincides both zero on the scale.
4. Retighten it ensuring that scale still reads zero at balanced position of field.
For error more than ½˚, the optical aliment must be readjusted to bring the error
within the scope of micrometer drum.
 64

EXERCISE 17
MANOMETER (demonstration)
Manometry can be used to measure either uptake or evolution of both co2 and o2
whereas o2 and co2 electrodes simply monitor changes in o2 and co2 levels, respectively, albeit
with greater sensitivity. Manometry also has the distinctive feature of allowing the
simultaneous determination of o2 and co2 exchange and has the advantage that the
magnitude of the exchange is independent of the partial pressure of the gas at the beginning
of the experiment manometric studies are carried out in a small flask attached to some of
manometer that measures changes in the flask. In all types of manometer, the flasks,
immersed in a water bath with a temperature control of + 0.5 deg c, are shaken 100 to 120
oscillations min-1 to ensure that respiratory gas exchange is not limited by diffusion of gas
into the liquid phase. The total volume of liquid in the flask should not generally exceed 4
cm3 because of gas diffusion limitations. Two principle types of manometer are the Warburg
constant volume manometer and Gilson constant pressure manometer, which are illustrated
in figure 17.1.

Applications:
The biological applications of manometery are extensive. Respiratory quotients (RQ),
defined as the relationship between the volume of co2 produced and the volume of o2
consumed during respiration i.e.
R Q = co2 evolved
o2 absorbed

May give an indication of the nature of the endogenous substrate being metabolized.
Manometric techniques have been applied in studies on tissue slices and homogenates, with
attendant problems of homogeneity of gas supply and artifacts, in studies of respiratory
control, and the effect of inhibitors on mitochondrial respiration. When used for
photosynthetic studies a control determination in darkness should be performed and the
partial pressure of the gas kept constant during the experiment.
 65

EXERCISE 18
FLAME PHOTOMETRY (demonstration)
Flame photometry is a technique by which concentration of ions (Na+, K+) of some
elements in the given solution is determined by the measurement of light emission on
introducing. The ionic material into a non luminous flame. Emission of such characteristic
radiation by each element forms the basis of flame photometry. The test solutions
containing these elements are introduced as fine sprays into the flame. A flame photometer
essentially consists of a burner, an atomizer which disperses the solution as a fine spray into
the flame, a means o isolating from the spectrum (specific filter system or diffraction grating)
that portion of the emitted light which is specific characteristic of the substance under
examination, a photocell converting the light energy into electric current, an amplifier to
amplify the feeble current and a galvanometer to measure it.
 66

ELECTROPHORESIS
Many important biological molecules such as amino acids, peptides, protein,
nucleotides and nucleic acids possess ionizable groups and can therefore be made to exist in
solution as electrically charged species, either as cations (+) or anions (-). Even typically non-
polar substances such as carbohydrates can be given weak charges by derivatization, for
example as borates or phosphates. Moreover, molecules which have a similar charge will
have different charge/mass ratios when they have inherent differences in molecular weight.
In combination these differences form a sufficient basis for a differential migration when the
ions in solution are subjected to an electric field. This is the principle of electrophoresis.
 67

EXERCISE 19
POLY ACRYLAMIDE GEL ELECTROPHORESIS
Introduction:
Electrophoresis is an electrochemical process in which substances with a net electric
charge migrate under the influence of an electric current. This migration occurs in an agar
gel or in a liquid-filled buffered matrix such as cellulose acetate. Positively charged
substances travel toward the cathode (negative electrode); while negatively charged
substances go toward the anode (positive electrode). Different substances move at different
rates depending on their charge. This movement is called electrophoretic mobility.
Proteins are charged molecules. They have a positive as well as negative charge.
Among proteins, particularly caseins have different content in them. The charge density also
varies depending on the molecular weight. Proteins have a net negative charge at a pH above
4.6 and hence they move towards the anode (positive electrode) when subjected under an
electric field under alkaline conditions. Due to differences in the charge of individual protein
fractions, their migration rates also differ and hence separate out as distinct bands. These
bands are stained with dye amido-black, destained with 7% acetic acid and observed for the
electrophoretic pattern.

Principle:
The gel is prepared by polymerizing acryl amide and a small quantity of cross-linking agents
(CH2 = CH C O N H2)2 CH2 methylene bis acryl amide in the presence of a catalyses,
ammonium persulphate (N H4)2 S2 08. Tetramethylethylenediamide (TEMED) is also present
to initiate and control the polymerization. The gel mixture is allowed to polymerize in small
tube (or Perspex) bottles with rubber cap. Layer of water is placed on the top of the gel to
ensure flat surface and also to exclude O2 which inhibits the polymerization.
Davis and Ornstein (1959) and Raymond and Weintranb (1959) were the first to use
PAGE instead of starch gel electrophoresis. The material used had a trade name of cyan
gum 41. it consisted of a mixture of acryl amide in proportions such that a gel could be
formed from 3-10 percent of n-N1 methylene bis acryl amide most appropriate for gel
formation was composed of 60 parts of acryl amide to 1.0- 1.013 parts of N –N1 methylene
bisacrylamide. This composition has been given by cyanogum - 41.
Polyacrylamide gel electrophoresis (PAGE) is superior to other electrophoretic
processes such as paper and starch gel electrophoresis due to the following reasons:
- Transparency of gel permits direct measurement of pattern by Tranquitting light
photometry through gels.
- Gels can be dried to thin flexible films. Rehydration to original volume is possible by
immersion in water.
- Gels are chemically inert which facilities differential scanning.
- Gels are non-ionic
- Pore sizes of the gels can be controlled by the choosing various concentrations of
acryl amide and methylenebisacrylamide at the time of polymerization (figure 19.2).
 68

- By incorporating a detergent SDS (sodium dodecyl sulphate) proteins can be

separated and their molecular weights can also be calculated. Hence heterogeneity of
proteins can be studied using SDS – PAGE.
PAGE is most commonly used to separate out various fractions of milk proteins like α
lactallumin, whey proteins and caseins (α, β,K & Y) under alkaline conditions (pH 8.0.
8.4 or 8.8). However, some of the components separated are not homogenous. They are
better separated under acidic conditions, particularly the β – casein variants A1, A2, A3
etc.

Protocol for performing PAGE:

1. Preparation of acryl amide stock solution
Acrylamide 5.55 gm
Bisacrylamide 0.15 gm
Dissolve in 25 ml distilled water.
Acryl amide stock solution is also available as cyanogum 41. The ratio of acrylamide
to bisacrylamide is 60: 1.013 which is the most preferred one by several workers.
1. Acrylamide 22.20 gm
2. Bisacrylamide 0.6 gm
Dissolve in 100 ml distilled water. Hence a ratio of 60: 1.013 is obtained.
2. tris- glycine buffer (pH 8.3)
Buffer solution
1. 0.025 M tris 6.0 gm
2. 0.192 M glycine 28.8 gm
Make the volume upto 2 litres. Glass distilled water is preferred for adding to make up
the volume to a litres.
3. Ammonium persulphate (1 % solution)
100 mg ammonium persulphate/ 10ml for vertical gel electrophoresis 30-50 mg
ammonium persulphate solution for cross electrophoresis/ 10 ml prepare the solution
freshly.
4. Preparation of 8 % acrylamide
The following reagents are taken in prescribed amounts in a glass beaker:
1. 10.5 ml of acrylamide stock solution.
2. 18 ml of tris – glycine buffer (gel buffer)
Gel buffer contains the following ingredients
Glycine 1.44 g
NH2 Tris 0.3 g
C=0 urea 42 g

Make the volume to 100 ml with water.

Add 0.3 ml mercaptoethanol CH2 – CH2 – oH.
3. 0.1 ml of TEMED
4. 1.5ml of ammonium persulphate solution
5. Preparation of protein samples
1.25 mg casein
+
2. 1 ml solubilizing buffer of the composition:
Glycine1.44 gm
 69

Tris 0.3 gm
Urea 42 gm
Dilute to 100 ml with water
+
3. 0.5 ml glycerol. Glycerol is added to make the protein solution heavy so that it does
not float
+
4. 0.05 ml mercaptoethanol
+
5. A drop of 0.7 % (wt. / vol.) amido black in water bromophenol blue can also be used
instead of amido-black as a tracking dye.
6. Electrophoresis
It is done by applying 0.02 to 0.04 ml of protein solution and passing a current of 1
milli ampere per sample.
7. Staining of gel
Is done in 1 % amido black prepared in 7 % acetic acid.
1 % amido black 100 ml
7 % acetic acid 100 ml
Both are mixed together
8. Destaining in 7 % acetic acid
Glacial acetic acid 35 ml
Distilled water 5oo ml
The gel portion containing protein fraction will retain the dye, whereas the other
portion of the gel will get decolorized by treatment with 7 % acetic acid. For speeding up the
decolorization rate, methanol acetic acid can be used instead of 7 % acetic acid as a
decolorizer.

Procedure:
1. Prepare all the reagents as per the procedure given in protocol.
2. Fill the small glass capillary tubes with 8 % acryl amide gel rubber stoppers are inserted at
the end. Fill the remaining space left at the top with water.
3. Allow the gel to polymerize. It takes approximately 30 -45 minutes for the gel to form.
4. Assemble the electrophoretic apparatus. Apply the protein samples in adjusted
concentration at the top of the tubes with the help of a pipette after filling electrode buffer
on both the trough containing the +ve and –ve electrodes.
5. pass current of 1 miliampere/ sample till the tracking dye comes out from the capillaries
into the buffer from below. It took approximately 60-70 minutes for the cheese, K-casein
and whey protein samples to migrate from the –ve to the +ve pole as indicated by the
movement of the tracking dye, amido black.
6. Dissemble the capillary tubes after stopping the current. Take out the gel column from the
glass tubes with the help of syringe and needle assembly in a trey filled with water. Transfer
the gels into labeled test-tubes and fill the individual test-tubes containing the gel column
with staining solution i.e. 1 % amido black in 7 % acetic acid. Ideally, the staining is done for
1 hour, however in our case staining was done overnight.
7. Remove the staining solution after the prescribed period and decolorize with 7 % acetic
acid in the same test-tubes. Change the decolorizer at regular intervals as indicated by the
 70

removal of color from gels. Overnight decolorization is sufficient, but in the present exercise
decolorzation took about 72 hours as the staining period had been increased.
8. Examine the pattern of the obtained bands.
 71

EXERCISE 20
STARCH GEL ELECTROPHORESIS

Electrophoresis is the study of the movement of charged molecules in an electric

field. Electrophoretic techniques can be classified as either moving boundary electrophoresis
or zone electrophoresis.
Starch gel electrophoresis is a zonal eletrophoretic technique wherein a buffer-
saturated solid matrix is employed as the support medium. Starch gel as a supporting
medium was the first gel medium to receive attention. In these techniques, a slab gel is
prepared from potato starch paste or hydrolyzed starch and is layered on a horizontal glass
plate or Petri plate. The sample is placed in a well cut in the slab, and the plate is subjected
to a voltage. After electrophoresis, the starch slab is stained for visualization of the sample
components. The enchanted resolution achieved by starch gels is probably due to molecular
sieving effect and to reduced diffusion because of a more rigid network. Starch gels are still
used for some protein and isozyme analysis.

Reagents:
1. tris- citrate buffer, pH 8.6.
This buffer is prepared by using tris. Tris means three groups of hydroxyl methyl
amine. The buffer has the following composition:
1. Tris hydroxyl methyl amine
H2N (CH3 OH)3 = 6.897 g
2. Citric acid = 0.7881g
3. Distilled water = 750 ml
For preparing 50 ml buffer, the quantity of chemicals mentioned above required is:
1. Tris (hydroxyl methyl) methylamine
= 6.897 g for 750 ml
= 0.4798 g for 50 ml
2. Citric acid = 0.05254 g
3. Distilled water = 50 ml.
2. Electrode buffer /NaOH boric acid buffer, pH 8.0.
This buffer is used for separation of caseins. Are negatively charged under
alkaline conditions and they separate out on the basis of difference in charge and a better
resolution is obtained.
However, some proteins like immunoglobulin, whey proteins etc. are denatured at
the alkaline pH employed. Hence, their mobility is reduced due to denaturation and
aggregation and thus NaOH boric acid buffer, pH 8.0 is not used for their separation.
Caseins, being naturally denatured proteins, do not show such an effect and thus the buffer
is used for their separation.
The composition of the buffer is as follows:
1. Boric acid = 18.552 g
2. NaOH = 2.0 g
3. β- mercaptoethanol = 2 to 4 drops per 100 ml
4. Water to make the total volume to 1000 ml
The pH of the buffer is checked after preparation by pH sensitive electrode or pH strip.
 72

The function of β- mercaptoethanol added in the buffer is to keep K-CN and β-

lactoglobulin in a reduced condition so that they do not form disulphide linkages.
β Mercaptoethanol has the following chemical structure
β α
H S – CH2 – CH2 OH
3. Amido –black dye solution:
It is prepared as 0.1 % concentrated solution in washing solution.
4. Washing solution:
Washing solution is a mixture of methanol, acetic and distilled water in 5: 1: 5 ratio
volume /volume respectively.

Preparation of starch gel:

Potato starch was used earlier for demonstration purpose. However, recently,
hydrolyzed starch is used for controlled gelling purpose. It is prepared by lysing starch by
amylases to a certain required size. Better reproducibility and accuracy are obtained and
hence hydrolyzed starch is preferred to ordinary starch for research work. Starch is taken in
the vacuum/suction flask.

Preparation of starch gel plates:

The following ingredients are used in the specified amount for preparing two starch
gel plates of small (8.6 cm diameter) size.
1. Tris citrate buffer (pH 8.6) = 4.5 ml
2. Water = 17 ml
3. Urea = 9.7 g
4. 2 – mercaptoethanol = 0.12 ml
Firstly, water and tris – cirate buffer are mixed in a small beaker. Half of this mixture
is added to the suction. Flask and mixed thoroughly and quickly while heating over a flame.
The other half of the mixture is added to the suction flask when the contents just start
boiling. The flask is further heated fill the solution becomes clear, transparent and viscous.
Then, urea is added to the flask and mixing is done. Urea prevents aggregation of caseins
while they are passing through the gel. Further, air bubbles are removed by suction pump.
Finally mercaptoethanol (0.12 ml) is added to the suction flask and through mixing is carried
out. Then the prepared gel is added uniformly to Petri dishes and is allowed to solidify for
overnight period of time.

Preparation of casein for starch gel electrophoresis:

1. Take 10 ml milk sample in a centrifuge tube.
2. Centrifuge for 10 minutes. The fat plug formed at the top is removed with a
spactula after keeping the tube in a cold water bath for a few minutes. Alternatively, to free
fat from milk, the fat plug is pierced with the help of a pointed and of glass rod and milk is
taken out from side.
3. to the skim milk obtained this, 1 ml of 10 % acetic acid and 1 ml of 1N sodium
acetate is added to bring the pH to 4.5. On shaking the contents, precipitation of casein
occurs. Keep the tube as such for 10 minutes and then centrifuge the contents for 5 minutes.
4. Wash the precipitated casein 2-3 times with cold distilled water to remove excess
acid. Then wash casein 2 times with 5 ml acetone. Acetone in 5 ml quantity is added to the
 73

casein, the curd formed is broken, contents are centrifuged and acetone is decanted. This
process is repeated. Dry the obtained casein powder on a filter paper. The casein obtained
thus can be used immediately.
5. For prolonged preservation, casein is washed with acetone as well as either two
times each, and then it is dissolved in veronal buffer which contains urea.
Composition of veronal buffer:
1. Sodium barlitone = 20.618 g
2. Barlitone = 3.684 g
3. Distilled water = 2 litres
Veronal is a trade name for barlitol.
(C2H5)2 CONH CONH CO Barlitone (mel. Wt 184.20)

(C2H5)2 CONH CONH CO sodium barlitone (mel. Wt 206.18)

Composition of urea- veronal buffer

2 gm urea is dissolved in 5 ml veronal buffer, pH 8.6.
Seventy milligram of casein in 1 ml of urea veronal buffer is dispersed/dissolved and to this,
2-3 drops of β – mercaptoethanol are added. Small filter paper strips are taken and they are
hanged on thread with help of U pins. Casein is applied on to these filter paper strips and is
dried with a hair- dries. Casein layer is applied thus for three times. And subsequently the
strips are stored.

Electrophoresis:
Small bits of strips are put vertically at one end of the Petri dish. Then
electrophoresis is carried out using sodium boric acid buffer, pH 8.0, at 300 volts and 3 milli
amperes current. From the power supply given.
After 1 ½ hours, (as detected by the movement of tracking dye put on to the filter
paper initially) the plates are removed and amide black staining solution is added to the
plates and kept for 10 minutes. Then the dye is replaced with the washing solution
containing methanol, acetic acid and water in the ratio of 5:1: 5 until the background is clear.
Subsequently, the components separated are identified.
 74

EXERCISE 21
PAPER STRIP ELECTROPHORESIS
Introduction:
Recent advances in analytical techniques have effectively been used in obtaining and
identifying the substances in the high state of purity. Physical methods like fractional
precipitation, distillation, and crystallization have been used in the separation and
purification of chemical compounds. These methods worked quite successfully in many
cases but some difficulties arose where the individual components of the compounds had
similar physical and chemical properties. For example, fractional distillation method could be
safely used in case of mixture of liquids which have a good range of boiling point
differences. However, this method could not be used in the case of liquids as well as rare
gases where the boiling points of individual components are very close to each other.
Likewise, after treating the biological material by the usual classical methods, one is usually
left with a number of compounds such as a mixture of amine acids which are similar in
properties to each other. In such cases, the extreme of temperature, pH, organic solvents
and the use of oxidizing and reducing agents are avoided as these may irreversibly change the
structure of the molecules and destroy their biological activity.
Such complicated separations were techniques of chromatography and paper
electrophoresis. These methods resolve the individual components under relatively mild
conditions and utilize differences in the basis physical properties of the individual molecules.
Such as their mass, size, shape, charge and absorption properties. Paper electrophoresis
method has an edge over paper chromatographic methods, as in the latter, substances with
low distribution coefficients are not properly separated. Such difficulties are not faced with
the techniques of paper electrophoresis which involves migration of charged substances
under the influences of electric current. Paper electrophoresis is an incomplete form of
electrolysis which is widely used in the separation of biological material and many other rare
and costly substances in the compounds or a mixture of compounds where some ionize and
others do not, the degree of separation can be effectively made by the use of paper
electrophoresis.
This method owes its development to the painstaking research of Arne Wilhelm
Tiselius of Sweden who was awarded the Nobel Prize in 1948.

Techniques:
The paper to be used for electrophoresis is first wetted with an electrolytic solution
which is normally a buffer solution. Barlitone buffer at pH 8.6 is used. The wet paper is
placed in a vessel made up of anodic and catholic compartments in such a manner that the
two ends dip inside the solution. Test solutions are put on the middle portion of the paper,
which is dried by pressing in between filter papers and the test solutions are put at the
middle portion of the order of 2-10 V/cm. Is then passed through the solution. Buffer
solutions serve the purpose of conductors and the wet paper as a connecting bridge between
the two compartments. The substances move either to the cathode or anode, depending on
the nature of the charge which they possess as shown in figure.
The movement of the substances is expressed by eletrophoretic mobility (µ) which is
defined as.
 75

µ = distance moved/ unit time

Electrical field strength

Whose unit is = cm. /sec

Volt/ cm.

= cm.2 volt sec-1

The movement of the substance depends on many factors which are listed below.
1. Size and nature of charge.
2. Environment factors like the concentration of electrolyte, ionic strength, dielectric
properties, chemical properties, temperature, viscosity etc.
3. pH effect
4. Diffusion, and
5. electro- osmosis
Various procedures commonly used for performing paper electrophoresis include
1. Sandwich
2. Ridge pole
3. Solvent immersion, and
4. Horizontal strip techniques.
Paper electrophoresis has been widely used in the field of large molecules such as proteins,
enzymes and nucleic acids etc. now it is also used in the separation of peptides, nucleotides,
amino acids, and abnormal hemoglobin.

Reagents:
Composition
1. Veronal buffer, pH 8.6 containing 10 % urea
Sodium barlitone 20.618 gm
(C2 H5)2 CONH CONa CO

Barlitone
(C2 H5)2 CONH CONa CO 3. 684 gm

Distilled water 2.0 litres

Add 10 mg urea/ 100 ml veronal buffer.
2. Bromophenol blue solution, 0.1 % preparation.
100 mg bromophenol blue,
pH 2.4 – 4.6
+
100 ml ethanol
+
Saturated mercurous chloride
(Hg2Cl2)
3. Two percentage casein
Add 5 ml of veronal buffer with 10 % urea to 100 mg of dry (pH 8.6) casein.
4. Acetic acid, 0.5 % solution
 76

Apparatus and arrangement for paper electrophoresis is illustrated in figure 21.1.

Procedure:
1. Cut strips of 37 × 4 cm size from 3mm sheets of what men filter paper no. 31.
Soak these strips in veronal buffer pH 8.6 containing 10 % urea blot the excess buffer by
blotting paper and place the strips on eletrophoretic tank horizontally. Apply samples at a
distance of 8 cm from the cathode end.
Two percentage casein in 0.1 ml quantity is spread in the central portion of the
starting line of paper strips.
2. Run these samples for 6 hours at room temperature, keeping the voltage constant
at 220 in veronal buffer, pH 8.6. Six strips can be run at a time. Keep the current flow
between 15-17 milli amperes.
3. After completion of the run, dry the strips at 37˚c in an oven.
4. Stain the strips with 0.1 % solution of bromophenol by soaking them in it for 30
minutes.
5. Wash the excess dye three times with 0.5 % acetic acid subsequent to staining.
6. Dry the stained stripes in an oven.
7. Identify the separated components in the test sample by comparison with the
pattern obtained for pure fractions like αs and K- casein prepared from whole milk.
Paper strip electrophoresis has been used for the resolution of both acid and micellar
casein into α and β components by the procedure of aschaffenberg.
 77

EXERCISE 22
CHROMATOGRAPHY
Introduction:
Chromatography is a relatively new techniques which was first invented by
Michal Tswett, a Russian botanist in 1906 in Warsaw, for the separation of colored
substances into individual components. Since then, the techniques has undergone
tremendous modifications so that now a days various types of chromatography are in use to
separate almost any given mixture, whether colored or colorless, into its constituents and to
test the purity of these constituents.
The name chromatography (Greek chrome, color and graphy, writing) means color
writing.
Chromatography may be defined as a technique is the separation of the components
of mixtures by a continuous distribution of the components between two phases, one of
which is moving part the other (figure 22.1). The systems associated with this definition are
as follows:
i. a solid stationary phase with a liquid or gaseous mobile phase and
ii. A liquid stationary phase with a liquid or gaseous mobile phase.
The system (i) gives rise to adsorption chromatography whiles the system (ii) to
partition chromatography.
Chromatography differs from other methods of separation in that a wide variety of
materials, equipments, and techniques can be used. It is basically dependent on two
principles viz. adsorption and partition.

Classification:
The moving phase may be a liquid or a gas. Based on the nature of the fixed and
moving phase, different types of chromatography are as follows:
a. Adsorption chromatography:
It is refers to the use of a stationary phase or support, such as an ion exchange resin,
that has a finite number of relatively specific binding sites for solute molecules. It is
based on the differences in the adsorption coefficients. In this the fixed phase is a
solid, e.g. alumina, magnesium oxides, silica gel etc. the solutes are adsorbed in
different parts of the adsorbent column. The adsorbed components are then eluted
by passing suitable solvents through the column. Adsorption techniques, represented
by ion – exchange chromatography, are most effective when applied to the
separation of macromolecules including proteins and nucleic acids.
b. partition chromatography:
It is the distribution of a solute between two liquid phases, one fixed and the other
mobile. It operates by mechanism analogous to counter – current distribution. Partition
chromatography may involve direct extraction using two liquids, or it may use a liquid
immobilized on a solid support as in the case of paper, thin- layer, and gas –liquid
chromatography. For partition chromatography, the stationary phase in figure 22.2. Consists
of inert solid particles coated with liquid absorbent. The distribution of solutes between the
two phases is based primarily on solubility differences. The distribution may be quantified by
using the partition coefficient, KD.
 78

KD = concentration of solute mobile phase

Concentration of solute in stationary phase
Also, the similarity or identity of unknown compound as compared to standard can be
established by color development or retardation value (RF value).

RF value = distance moved by the solute

Distance moved by the solvents

The RF value would be constant for a similar compound of similar structure, and hence the
identity of test sample can be established by finding out its RF value.
A partition system is manipulated by changing the nature of the two liquid phases,
usually by combination of solvents or pH adjustment of buffers. Usually, the more polar of
the two liquids is held stationary on the inert solvent is used to elute the sample components
(normal- phase chromatography). Reversal of this arrangement, using a non-polar stationary
phase and a polar mobile phase, is known as reversed – phase chromatography. Polar
hydrophilic substances, such as amino- acids, carbohydrates, and water – soluble plant
pigments, are separable by normal –phase partition chromatography. Lipophilic compounds,
much as lipids and fat – soluble pigments, may be resolved with reversed- phase systems.
Partition chromatography has been widely used for the separation and identification
of amino acids, carbohydrates, and fatty acids.
c. gas chromatography:
When the moving phase is a mixture of gases, it is called gas chromatography.
Depending on whether the compound of interest has to be separated or identified,
chromatographic techniques are classified as preparative or analytical.
A preparative procedure is one that can be applied to the purification of a relatively
large amount of a biological material. The purpose of such an experiment would be to obtain
purified material for further characterization and study, in this type of technique the layer of
gel or paper is kept thick e.g. what Mann paper no. 3 is used.
Analytical procedures are used most often for the determination of purity of a
biological sample; however, they may be used to evaluate any physical, chemical, or
biological characteristic of a biomolecule or biological system. Here, the layer of gel or paper
used is kept thin e.g. what men filter paper no. 1 is used.
On the basis of solute material used, chromatography has been classified as
1. Liquid – liquid chromatography
2. Gas – liquid chromatography
3. Solid – liquid chromatography and so on.
Depending on the support material used chromatography has been classified as paper, this
layer, column chromatography or gel permeation chromatography etc. the column used in
column chromatography may be calcium carbonate, sephadex, resins, DEAE cellulose,
hydroxyl methyl cellulose alumina etc.
Depending on the method of development used, chromatography can be classified
as ascending or descending chromatography.
If chromatography is run in one direction then it is called unidirectional
chromatography, and if run in two dimensional chromatography.
Depending upon the type of spot material, chromatography can be classified as
circular, paper strip and so on.
Thus, there are numerous chromatographic techniques based on the principle of
either adsorption or elution.
 79

Different methods for separation as well as type’s chromatographic techniques are

listed in table 22.3.
Chromatographic separations in practice may take one of three forms: column
chromatography in which the stationary phase is packed into glass or metal columns; thin
layer chromatography in which the stationary phase is thinly coated onto glass, plastic or foil
plates; and paper chromatography in which the stationary phase is supported by the cellulose
fibers of a paper sheet. Each of these three forms of chromatography has their specific
advantages, applications and mode of operation.
Column chromatography is the method used for the isolation and preparation of
compounds, whereas paper or thin layer chromatographic techniques are used for analytical
work.
 80

EXERCISE 23
COLUMN CHROMATOGRAPHY
Separation of compounds by column chromatography must be one of the most
widely used techniques in biochemical work.

Columns:
Chromatography columns are usually glass and, generally, long columns give good
resolution of components but wide columns are better for dealing with large quantities of
material. The essential features of a chromatography column are shown in figure 23.1.

Preparation of the material:

A wide range of materials are used in chromatographic separations and all need to be
equilibrated with the solvent before preparing the column. In addition, some form of
pretreatment is often required; for example, some gel filtration materials need to be swollen,
adsorbents need to be ‘activated’ by heating or acid treatment, and ion exchange resins have
to be obtained in the required ionized form by washing.
During the equilibrium with solvent the material is allowed to settle and the fine
particles remaining in suspension are removed by decantation (figure 23.2). it this is not
carried out, the flow rate of solvent down the column will be considerably reduced due to
clogging by these fine particles.

The poring of the column:

The chromatography column is packed with material by filling it about one- third full
with solvent and slowly adding slurry of the material in the solvents, this is carefully poured
down a glass red, as shown, to stop air bubbles being trapped in the column. (Figure 23.2)
the suspension is allowed to settle and excess solvent run off. This process is repeated until
the column is the required height. The column is then washed thoroughly with solvent and
the level of the liquid kept just above the surface of the material.

Application of the sample:

The sample is first dissolved in the solvent or dialyzed against the eluting buffer
before loading it on to the column. In most class experiments the concentrated sample is
carefully pipetted onto the surface and the tap opened until the top of the column is just
below the level of the meniscus. The solvent reservoir is connected, and a constant head of
liquid maintained at the top of the column from a pressure reservoir.

Elution:
The next stage is to remove the materials from the column in order by eluting with
an appropriate solvent.
 81

The collection and analysis of fractions:

The effluent from the column is collected into a series of test tubes, either manually
or with a fraction collector. Each fraction is then analyzed for the presence of the
compounds being examined and an elution profile prepared of the amount eluted against the
effluent volume.
 82

EXERCISE 24
PAPER CHROMATOGRAPHY
Principle:
Cellulose in the form of paper sheets makes an ideal support medium where water is
adsorbed between the cellulose fibers and forms a stationary hydrophilic phase.
The mixture is spotted on to the paper, dried and the chromatogram developed by
allowing the solvent to flow along the sheet. The solvent front is marked and, after drying
the paper, the portions of the compounds present in the mixture are visualized by a suitable
staining reaction. The ratio of the distance moved by a compound to that moved by a
compound to that moved by the solvent is known as the RF value and is more or less
constant for a particular compound, solvent system and paper under carefully controlled
conditions of solute concentration, temperature and pH. (Figure 24.1).

Paper:
What men no.1 is the paper most frequently used for analytical purpose what men
no.3 is a thick paper and is best employed for separating large quantities of material; the
resolution is, however, inferior to what man no.1. For a rapid separation, what man nos 4
and 5 are convenient, although the spots are less well defined. The paper may be
impregnated with a buffer solution before use or chemically modified by acetylation. Ion
exchange papers are also available commercially for separation of lipids and similar
hydrophilic molecules, silica –impregnated papers are available commercially.

Solvents:
This choice, like that of the paper, is largely empirial and will depend on the mixture
investigated. The pH may also be important in a particular separation, and many solvents
contain acetic acid or ammonia to create a strongly acidic or basic environment.

Ascending chromatography:
The sheet of paper is supported on a frame with the bottom edge in contact with a
trough filled with a solvent. Alternatively, the paper can be rolled into a cylinder, fastened
with a paper clip and stood in the solvent. The arrangement is contained in an air tight tank
lined with paper saturated with the solvent to provide a constant atmosphere and separations
are carried out I a constant temperature room. (Figure 24.2).

Descending chromatography:
The method is convenient for compounds which have similar Rf values since the
solvents drips off the bottom of the paper, thus giving a wider separation. In this case, the
Rf values cannot be measured with a standard reference compound such as glucose, for
example in the case of sugars.

Rg = distance moved by compounds ×

Distance moved by glucose
 83

Two- dimensional chromatography:

The mixture is separated in the first –solvent, which should be volatile: then after
drying, the paper is turned through 90˚ and separation is carried out in the second solvent.
After location, a map is obtained and compounds developed under the same conditions
(figure.24.3).

Detection of spots:
Most compounds are colorless and are visualized by specific reagents. The location
reagent is applied by spraying the paper or rapidly dipping it in a solution of the reagent in a
volatile solvent. Viewing under ultraviolet light is also useful since some compounds which
absorb strongly show up as dark spots against the fluorescent background of the paper.
Other compounds show a characteristic fluorescence under ultraviolet light.
 84

EXERCISE 25
THIN LAYER CHROMATOGRAPHY
Introduction:
Thin layer chromatography is adsorption chromatography performed on open layers
of adsorbent materials supported on glass plates. It is a separation method in which uniform
thin layers of sorbent or selected media are used as a carrier medium. The sorbent is applied
to a backing as a coating to obtain a stable layer of suitable size. The most common supports
such as plastic sheets and aluminum foil are also used. The sorbents most commonly used
are: silice gel, alumina, kieselglhr (diatomaceous earth), and cellulose.
The term sorbent is used in a general sense to include layer materials that may be
used for either adsorption or partition chromatography. The standard size for TLC plates is
20 × 20 cm. For most separations, the mobile phase is allowed to travel on the layer for a
distance of 15 cm. Other plate sizes used are 5 × 20 cm 10 × 20 cm, and 20 × 40 cm.
“Micro” plates have been made from microscope slides.
In practice, a sample to be separated is applied on the layer 1-2 cm from one end of
the plate. The furthermost edge of the application is called the starting point or origin.
Separation is achieved by passing a solvent, the mobile phase, through the layer. The layer,
with the sample zones at the bottom, is placed on a slight angle from the vertical into a
closed tank containing a small amount of the mobile phase.
The nature and chemical composition of the mobile phase is determined by the type
of substance to be separated and the type of sorbent to be used for the separation. The
composition of a mobile phase can be as simple a single, pure solvent (such as benzene used
to separate dyes on alumina) or as complex as a three – or four –component mixture
containing definite proportions of chemically different substances, such as a 1: 1: 1: 1
solution of n –butanolethyl acetate –acetic acid –water used to separate amino acids on silica
gel.
Capillary action causes the mobile phase to travel through the medium in a process
called development. Ascending development is the most common, but horizontal,
descending, and centrifugal methods have also been reported. After the plate is dried, the
separated spots can be visualized in a number of ways such as viewing under an ultraviolet
light or spraying with one of a wide variety of reagents. The apparatus for performing TLC is
depicted in figure.25.1. The entire TLC process is summarized in figure. 25.2.
The Rf value is a convenient way to express the position of a substance on a
developed chromatogram. It is calculated as the ratio:
Rf = distance of compound from origin
Distance of solvent from origin
Rf values are between 0 and .999, and without units. Distance is measured to the centre of
the sample zone or spot.
According to the types of compounds being investigated, the separations on the
plate may then be evaluated by a number of instrumental techniques. If the substances are
radioactive or suspected to be so, the plate can be examined by a radioisotope scanner to
locate these substances. Under proper conditions, it is also possible to measure both the area
of a spot and its density, using a photo densitometer. These characteristic are related to the
quantity of a given substances in the spot and can thereby be used to assess the amount of
 85

the substance in the sample. A quantitative calibration or reference curve should be

established beforehand using known amounts of the substance to be quantitated.
Thin layer chromatography (TLC) has found wide applicability in separation and
identification of both organic and inorganic substances.
The main advantage of TLC lies in the fact that the technique can carry out
separation and identification of unknown substances in very small amounts and in a very
short time. None of the other methods can match it in this regard. Even paper
chromatography which has earned the reputation of carrying out separation in very small
amounts has been exceeded by this method. For example, TLC can carry out separation or
identification in still smaller amounts and in much less time than is taken by paper
chromatography.
The credit of discovering these techniques goes to two Russian workers,
N.A.Izmailor and M.S.Schraiber, who in the year 1938 attempted the separation of plant
extracts on alumina-coated glass plates and got a satisfactory separation. This discovery did
not attract the attention of other workers till 1949. In that year, J.E.Meinhard and N.F.Hall
showed some satisfactory separation with the new method. They were also the first to
describe an apparatus for coating glass plates with adsorbents. But the technique could not
gain popularity because of the difficulties involved in the preparation of suitable adsorbents
and applying them in uniform layers on glass plates.
The method which was practically given up was reinvented by the untiring efforts of
Egan Stahl, a German worker, who not only found practical equipment for coating glass
plates with adsorbents, but also demonstrated the usefulness of this technique on a wide
range of materials. Since 1958, the use of TLC has been so widespread that it has entered
almost every phase of analysis.
TLC has been used in separation and identification of a large variety of organic
compounds: alkaloids, terpenes, vitamins, hormones, dyes, drugs, sugars, fat, pigments,
alcohols, aldehydes, acids, and many others. TLC has also found useful application in the
analysis and identification of inorganic compounds.

Techniques and materials:

Absorbents and applicators are the two essential items of the TLC techniques,
although other accessories like glass plates, aligning tray, activating oven, developing
chamber, etc. play their individual roles. The efficiency of the former two plays a very
important part in the success of the technique.

Applicator:
There are various types of applicators for spreading the adsorbent in the form of
slurry over the glass plates which are used in chromatography. Adjustable applicators are
available for spreading layers of various degrees of thickness ranging from 250µ to 2 mm.
Over glass plates.

Adsorbents:
The character of the adsorbent is of the greatest importance in this technique. Silica
gel and alumina are mostly used and have proved to be quite suitable. They are used along
with some binding materials like plaster of Paris to fix the thin and porous layer on the glass
plates. In addition to this kiesel gel has also been used for making thin layers. Cellulose
 86

powders with and without binding materials is also in use. Various types of cellulose powder,
including ion- exchange cellulose powder, are much in use.

Preparation of thin layers:

The adsorbents are mixed thoroughly with requisite quantities of water or mixtures
of water and alcohol with the help of water mechanical stirrer for about two minutes to form
homogenous slurry. Then, immediately it is spread over well cleaned glass plates of standard
size (20 × 20 cm. Or 20 × 5 cm.) Arranged in a row on an aligning tray with the help of the
applicator. The applicator is first adjusted according to the thickness of the layer, after which
the slurry is put inside the applicator and the latter is drawn by handover the glass plates
arranged on the aligned tray. The coated glass plates are then allowed to dry at room
temperature for 10 minutes to 2 hours during which the Linder present in the adsorbent sets
the adsorbent firmly over the glass plates.
After air drying, the coated glass plates are activated by drying in an oven at a
suitable temperature depending on the adsorbent. The heating period similarly varies. After
activation the plates are stored in vacuum desiccators until they are used. If the plates are not
stored in desiccators and exposed to air or kept for a long time without being used, the
efficiency of the plates decreases.
The samples to be analyzed are applied in the same way as in paper chromatography.
Usually, a small amount, e.g., 4 microlitres of the solution of the substance or a mixture in a
micro- pipette is applied to the starting point near one of the edges (narrow edge) of the
chromatoplates in such a manner that the drop becomes as well as particable, and diameter
of the spot, the better is the resolution, 5-10 micrograms are sufficient for one operation.
Substances are usually applied about 2 cm. From one side of the plate.
The plates are then put in the chromatography chamber, usually much smaller than
the paper chromatography chamber. The chamber should be saturated with the solvent
vapour sufficiently ahead of the actual operation. Its walls should be lined up with filter
paper soaked with the solvent to maintain steady saturation inside the chamber. The solvent
is put inside the chamber to a thickness of 1 cm. The spotted chromatoplates are developed
by the ascending techniques. The plates are held vertically inside chamber. They can also be
developed by the descending technique with certain modifications of the chamber. The
development takes much less time than in the case of paper chromatography. When the
development is complete, the plates are taken out and allowed to dry in the air inside a clean,
dust- free chamber at room temperature. Then developed plates are activated by keeping
them in an oven adjusted at specified temperature (100˚ - 250˚c).
Detection of the movement of spots and determination of Rf values are similar to
those in paper chromatography. Detection of spots can be made by spraying the
chromatogram with visualizing reagents (e.g. ninhydrin) or in some by seeing the
chromatogram in U.V.light.
Partition and ion exchange TLC can be performed in the same way. Two-
dimensional thin- layer chromatography has also been achieved with success by developing
the single plate in two directions at right angles to each other utilizing two different solvent
systems. Thin –layer electrophoresis has shown good success in separation of mixtures of
various types of compounds.
 87

Separation of milk fat components by TLC:

PROCEDURE
PREPARATION OF TLC PLATES:
Silica gel G taken in quantity of 4 Gms, 11 ml of distilled water added and slurry was
made with pastel and grinder. Further, the slurry was poured over glass plates of 10 × 20 cm
size uniformly and spread evenly with the help of a glass rod or special apparatus available
for the purpose. The plates were then air dried and water evaporated. The plates, after
completion of drying, were activated at 110˚c for 20 minutes. During activation much of the
moisture gels evaporated and silica gel gets properly charged. These plates, after activation
were spotted with the ghee sample.

Preparation of sample:
11 ml of fat sample was solubilized in 1 ml of hexane and spotted on TLC plates
with the help of glass capillary. Spotting is done in a manner that the gel is not pulled out.
The amount of the sample to be analyzed should also be standardized. E.g. if the area of
spotting is kept too large, overlapping of components occur and a poor resolution between
molecules which retards their migration.

Development:
After spotting the standard as well as unknown, plates were put in chromatographic chamber
containing the solvent system. To aid saturation of the developing chamber atmosphere, the
inner walls are normally lined with blotting paper or a thick chromatography paper such as
what men 3 MM. This is conveniently done by taking a single large sheet of paper and
placing it into the dry tank so that the back and ends are lined. The front is left open so the
plate may be observed. The paper should come to within 1 cm of the top of the tank. The
chromatography was allowed to proceed for a given period of time and then the plates were
removed from other end, air dried and taken for color development of plates can be done
using any one of the two solvent systems A & B.
Composition of solvent A:
Hexane, diethyl ether and glacial acetic acid in the ratio of 70:30:1.
Composition of solvent B:
Petroleum ether (boiling point 250˚c), diethyl ether and glacial acetic acid in the ratio
of 80:20:1.

Detection of the separated pattern:

The plates were air dried and sprayed with 50 % H2SO4 and heated at 110˚c for 30
minutes. The components were tentatively identified according to their mobility of various
lipid components as long chain triglycerides, > short chain triglycerides, > free fatty acids, >
diglycerides, > cholesteror > 1.2 diglycerides > 2 monoglycerides > phospholipids. (Figure
25.3).

Separation of pigments:
PROCEDURE
In a mortar place 5 ml of petroleum ether + ether and a few green leaves, crush the
leaves with pastel and transfer the extract to a separating funnel by means of a pipette. Swirl
the extract with equal volume of water. Discard the lower aqueous layer. Repeat washing two
times with water. Discard the lower aqueous layer. Take petroleum ether layer to a small
 88

aluminums flask, dry over 2 gm of and sodium sulphate or if concentrated gel is used, by a
stream of dry nitrogen. Place a small spot with the help of a capillary tube on a 10 cm TLC
plate. Allow the spot to dry. Use acetone as developing solvent. Develop the chromatogram
using a wide mouth bottle or a conical flask. A folded piece of filter paper is placed into
bottle to moisten/ saturate the atmosphere with water. Dry the plates again ad it will be
possible to observe as many as eight colored spots. In order of decreasing Rf value, the
following spots will be developed of different separated pigment compounds.

Caroteins two orange colored spots

Chlorophyll A one blue- green colored spot

Chlorophyll B one green spot

Xanthophills four yellow colored spots

Pates used for performing chromatography are either silica gel or alumina of 0.25 mm
thickness.
 89

EXERCISE 26
GEL FILTRATION
The technique of separating molecules of different size by passage through a gel
column is called gel filtration. Small molecules can enter the gel but larger molecules are
excluded from the cross- linked network (figure 26.1). This means that the accessible volume
of solvent is very much less for molecules totally excluded from the gel than for small
molecules which are free to penetrate. The separation of molecules by gel filtration is
illustrated in the diagram. First the mixture of large and small molecules is placed on top of
the column. (figure 26.2(a)). As they pass down the column, the small molecules diffuse into
the gel ad follow a longer path than the large molecules, which are completely excluded from
the gel particles (figure 26.2 (b)) eventually, complete separation occurs, with the large
molecules leaving the column first and the smaller ones last (figure 26.2 (c)).
A gel filtration separation can be performed in the presence of essential ions or
cofactors, detergents, urea, at high or low ionic strength, at 37˚ c or in the cold room
according to the requirements of the experiment.
Pharmacia manufacture cross- linked dextrans (sephadex) and agarose (sepharose),
and cross –linked agarose (sepharosecl) and sephacryl, while bio-rad laboratories make
polyacrylamide (biogel p), agarose (biogel A), porous glass (bio-glas), and polystyrene (bio
leads). The degree of cross- linking etc. is carefully controlled to give a range of products
able to fractionate molecules over a limited size range.

Principle:
The technique of separating molecules of different size by passage through a gel
column is called gel filtration. Sephadex is the most popular of the gel materials. A
polysaccharide, dextran, is carefully cross- linked to give small beads of a hydrophilic,
insoluble nature which when placed in water swells considerably to form an insoluble gel.
Sephadex is the commercial name for the gel prepared thus.
Sephadex is prepared from polysaccharide dextran which has been synthesized by
the action of a bacterium on sucrose. Each glucose residue contains 3 hydroxyl groups
giving the dextran a polar character. The cross linking reaction (figure 26.3). Is accompanied
with epichlorohydrine.

CH2 CH CH2Cl
O
Epoxy group

By adjusting the conditions, the amount of cross- linking, and thus the size of pores, can be
carefully controlled. Sephadex gels are insoluble in all solvents and are stable in bases, weak
acids, water and mild reducing and oxidizing agents. Long exposure to 0.1N HCl or strong
oxidizing agents will cause breakdown of the gel granules. Temperature of about 120˚c
should be avoided, too. In addition to water in which they are commonly soluble, sephadex
gels swell in glycel, dimethyl sulphoxide and form amide but not in glacial acetic acids,
methanol or ethanol.
 90

These gels are classified by the amount of “water regained” which is a function of
the looseness of the structure or the extent of cross linking. The classification of sephadex
gels is given in table 26.1. By sephedex gel, the useful fractionation range of molecules is
only approximate since separation depends upon the shape, charge to a minor extent and
size of the molecules. Sephadex G- 25 and G-50 are made in several particle sizes and we
find coarse, medium and fine grades. Fine grades are suitable for most laboratory work
requiring high resolution, while the coarse material is convenient for preparative work with
large columns since this gives a higher flow rate.
Various other materials which give improved resolution have been developed e.g.
sepharose. Sepharose is stable from pH 4.0 to 10.0 and can be used over the temperature
range 0-30˚c. Three different types of sepharose are available which have different
concentrations of agarose. (Table 26.2) sepharose CL is stable over pH 3.0 to 14.0 and can
be used upto temperatures of upto 70˚c. These are some porens gels and are used to
fractionate very large molecules such as nucleic acids and viruses. After use, the columns can
be washed in water and stored in the cold room for quite a time provided traces of
preservative such as phenol or chloroform is added to prevent bacterial growth. Another
material used for column chromatography is ‘sephacryl’. It is a polymer of allyldextran
covalently crossed lambda linked with N – N’ methylene –bisacrylamide (figure 26.4) and
can be obtained in a preswollen form. The material gives much faster flow rates than
sephadex or biogel and 5200 can be used to separate proteins with a molecular weight
between 5000 and 2, 50,000. The molecular weight of proteins can be determined from
elution volume after calibration of column with known molecular weight markers.
Another gel chromatography material that is used for molecular extrusion
chromatography is Biogel-P of different types (table 26.3). They are made from
polyacryamide.

Principle of separation:
Theory of gel filtration:
The total volume of the column (Vt) is the sum of volume of the gel matrix (Vg), the
volume of water inside the gel particle (Vi) and the volume of the water outside the gel
grains (Vo), as illustrated in figure 26.5.
I.e. VT = Vo + Vg + Vi
Vo i.e. void volume is the volume of the liquid required to elute compounds that are
completely excluded from the gel grains, and is known. Vi can be calculated from the
knowledge of dry weight of the gel (a) and the water regained (Wr) e.g. Vi = a × Wr. The
elution volume Ve of a compound is the volume required to elute that compound from a
column i.e. Ve = Vi + Kd Vi where Kd indicates the fraction of the inner volume accessible
to a particular compound and is independent of the geometry of the column. I.e. Kd = Ve -
Vo
Vi
Substituting the value of Vi in the above equation
Kd = Ve- Vo
aWr
If Kd = 0, then Ve = Vo i.e. the elution volume would be the void volume.
If Kd = 1
Ve –Vo = Vi
Or Ve = Vi + Vo
 91

If a molecule is completely eluded from the gel, then Kd = 0 and Ve= Vo, while if a
molecule has complete accessibility to the gel, then Kd = 1.
Molecules therefore have a Kd value between 0 and 1. If Kd >1, then adsorption of
the compound on the gel occurs.
By plotting a graph of elution volume against molecular weight taking different
markers and comparing the elution volume of the test compound with the standard graph,
its corresponding molecular weight can be known. (Figure 26.7).

Applications:
Gel chromatography has been widely used by biochemists to separate proteins,
peptides, nucleic acids, polysaccharides, enzymes, and hormones alike and by polymer
chemists to characterize molecular weight distribution in polymeric mixtures. The other
applications of gel filtration chromatography are as follows:
1. Desalting:
One of the common separations of salts and small molecules from macromolecules.
The large difference in distance coefficient for this separation makes it possible to use simple
columns with high flow rates.e.g. With sephadex G-25, solute molecules having molecular
weights over 5000 are eluted in a volume Vo, smaller molecules with molecular weights
below 1000 will be eluted following the macromolecules inversely in order of their size. One
of the advantages of this method of desalting is that the macromolecules are eluted with
essentially no dilution.
2. Concentrating:
The diluted solution of macromolecules with molecular weights higher than the
exclusion limit may be readily concentrated by utilizing the hygroscopic nature of dry gels.
Sephadex G 200 absorbs 20 times its weight of water, although G-25 is preferred
because of its more rapid action. Since salt molecules are imbibed to the same extent as
water, their concentration in supernatant solvent remains essentially unchanged and ionic
strength and pH of macromolecular solution remains essentially unaltered. This is an
important advantage in separation of proteins which are easily denatured by change in
temperature.
3. Fractionation:
Components separation of mixture of closely related materials having small
difference in K- values will require long columns, slow flow rates and long times. The
difference of molecular weights in a complex mixture is often sufficient information to
check the solution or to give a rough idea of its composition. It has been observed and can
be predicated that elution volume is related to the molecular weight in a simple fashion. Ve
= (A – B) log molecular weight. Where A and B are constants whose values are determined
from a plot of V versus log molecular weight for several known compounds. The equation is
valid over a considerable range of molecular weights, provided all compounds are closely
related. Values for A and B must be redetermined for each column in each set of specific
conditions and for different classes of compounds. Table 26.4 lists the applications of gel
filtration.
 92

The separation of proteins by gel filtration:

S -200 can be used to separate proteins with a molecular weight between 5000 and 2,
50,000. The molecular weight of a protein can be determined from the elution volume after
calibrating the column with known molecular weight markers.
Materials:
1. Sephacryl S-200 columns (20 cm × 2 cm) equilibrated with the elution buffer-5.
2. Elution buffer (0.5 mol/litre NaCl in 0.1 mol/litre tris-HCl buffer,-3 litre pH 8.0).
3. Buffer reservoirs 5
4. Fraction collectors 5
5. Molecular weight standards (5 mg of each protein per ml of elution buffer)

Standard Mol.wt. Log mol.wt.

Cytochrome C 12400 4.09
Ovalbumin bovine serum 45000 4.65
Y- Globulin 2, 10,000 5.32
Blue dextran 2,000,000 -
6. Lactate dehydrogenises 1 ml
7. Spectrophotometer 5
8. Reagents for the assay of glucose –
9. Reagents for the assay of lactate dehydrogenises –

Method:
Running the column:
Open the tap on the column and allow the level of the elution buffer to fall until it is just
above the top of the gel. Mix 0.1 ml of lactate dehydrogenises solution and 0.4 ml of the
markers and place this on top of the column. Allow the mixture to enter the gel, add a small
volume of the elution buffer until the markers are clear of the top then connect the buffer
reservoir. Adjust the height of the reservoir to give a flow of about 50 ml/h and collect 3 ml
fractions using the fraction collector. Locate the position of the blue dextran and the glucose
and examine the fractions between these compounds for the presence of the proteins.

Detection of compounds:
Blue dextran: extinction at 625 nm Y-globulin and ovalbumin: extinction at 280nm
cytochrome c: extinction at 412 nm glucose & lactate dehydrogenase: as per the standard
method prescribed.

Molecular weight determination:

Calibrate the column by determining the volume at which the blue dextran is eluted
(Vo) and the volume when the glucose appears (Vo +Vi). Determine the elution volume
(Ve) of the three marker proteins and the LDH and calculate the Kd values. Plot a graph of
Kd against log10 mol.wt. For each marker and calculate the apparent molecular weight of
the LDH.
 93

EXERCISE 27
ION EXCHANGE CHROMATOGRAPHY
Introduction:
Electrostatic attraction of oppositely charged ion on a polyelectrolyte surface forms
the basis of ion exchange chromatography. Typical systems include the synthetic resin
polymers, such as the strongly acidic cation exchanger Dowex -50, a polystyrene sulfonic
acid, and a strongly basic anion exchanger Dowex -1, a polystyrene quaternary ammonium
salt. Cellulose derivates such as carboxymethyl cellulose (CMC) and diethyl amino ethyl
cellulose (DEAE) exchangers have been very successfully used in protein purification.
The basic principle involves an electrostatic interaction with the exchanging ion and
the normal charge on the surface of the resin. These reactions are considered to be
equilibrium processed and involve diffusion of a given ion to the resin surface and then to
the exchange site, the actual exchange, and finally diffusion away from the resin. The rate of
movement of a given ionizable compound down the column is a function of its degree of
ionization, the concentration of other ions, and the relative affinities of the various ions
present in the solution for charged sites on the resin. By adjusting the pH of the eluting
solvent and the ionic strength, the electro statically held ions are eluted differently to yield
the desired separation.
The separation in ion exchange chromatography is obtained by reversible adsorption.
Most ion exchange experiments are performed in two main stages. The first stage is sample
application and adsorption. Unbound substances can be washed out from the exchanger bed
using a column volume of starting buffer. In the second stage, substances are eluted from
the column, separated from each other. The separation is obtained since different substances
have different affinities for the ion-exchanger the to differences in their charge. These
affinities can be controlled by varying. Conditions such as ionic strength and pH. (Table
27.1). Ion- exchange chromatography is capable of separating species with very minor
differences in properties, such as two proteins differing by only one amino acid- it is most
widely used technique for separating, identifying, and quantitating the amounts of each
amino acid in a mixture. The entire procedure has been automated, so that the elution,
collection of fractions, analysis of each fraction, and recording of data are performed
automatically in an amino acid analyzer.
In ion exchange chromatography one can choose whether to, bind the substances of
interest, or to adsorb out contaminants and allow the substance of interest, to pass through
the column. Generally it is more useful to adsorb the substance of interest, since this allows
a greater degree of fractionation.
Ion exchange may be carried out in a column or by a batch procedure. Both methods
are performed in definite stages. These stages are: equilibration of the ion exchanger,
addition and binding of sample substances, change of conditions to produce selective
desorption, and regeneration of the ion exchanger. The step- by- step procedure for ion
exchange chromatography is given in figure 27.1.
 94

Theory:
The matrix many commercial ion- exchangers which have been successfully
employed for separation of biological materials are made by c0-polymerizing styrene with
divinglbenzone, however, cross- linking of molecules occurs and this produces an insoluble
resin. Various degrees of cross linkage may be obtained by co-polymerizing varying
proportions of divinglbenzene and styrene. The higher the amount of divinglbenzene,
employed with respect to the styrene, the greater the degree of cross- linkage obtained.
Resins with a low degree of cross-linking are more permeable to high molecular weight
compounds than are highly cross-linked ones, but they are also less rigid and swell more
when paced in a buffer. These swelling characteristics must be taken into account when a
column is prepared. Sulphonation of cross- linked polystyrene resin such as dowex 50 which
is strong acidic exchangers. The SO3H group is ionized at all except very low pH values. An
analogous basic exchanger may be prepared by reacting cross-linked polystyrene with
chloromethyl ether, and then reacting the chloro groups with tertiary amines. These –
CH2N+ (CH3)3Cl- groups are ionized at all but very alkaline pH values.
Chemically modified celluloses have proved to be a particularly useful alternative to
the polystyrene- based exchangers. Cellulose is a high molecular weight compound which
can be obtained in a highly pure state (arboxymethyl cellulose (CM- CH2 OCH2 COOH
cellulose), and DEAE- cellulose (-CH2OCH2CH2N(CH2CH3)2) are examples of the main
derivates of practical value.

Ionizable groups:
Ionizable groups charged groups are attached to the matrix and the type of group
defines the nature and strength of the ion exchanger. These groups may be either anionic or
cationic, according to the nature of their affinity for either negative or positive ions. For
example, the cation exchanger materials exchange positive ions, so it is the charge carried by
the exchangeable ion which decides whether a material is anionic or cationic and not the
charge carried on the matrix. (figure 27.3) these two types can be further divided into
materials that contain strongly ionized groups, such as –SO3H and –NR3, and the weakly
ionized groups, such as –COOH, -OH and –NH2. The strong ion exchange resins are
completely ionized and exist in the charged form except at extreme pH values:
- SO3H SO-3 + H+
+
-NR3OH NR3 + OH-

The weak ion exchange materials, on the other hand, contain groups whose ionization is
dependent on the pH, and they can only be used at maximum capacity over a narrow pH
range.
-COOH -COO- + H+
-NH3+ -NH2 + H+
As a rough guide, resins containing carboxyl groups have a maximum capacity above pH 6,
while those with amino groups are effective below pH6.
The number of ionizable group determines the capacity of the ion
exchanger. The total capacity is the number of ionizable groups per gram of material,
whereas the available capacity is the amount of a given molecule that can bind under defined
experimental conditions. In the case of some materials, large molecules may be unable to
 95

penetrate the matrix and can only react with charged groups on the surface. In this case, the
available capacity will be considerably less than the total capacity.
The ionizable groups commonly met are given in table 27.3 together with their
abbreviations.
Ion exchange equilibria. The typical way that an ion exchange material function is illustrated
in the following sample, where an anion exchange resin containing amino groups is used to
separate two negatively charged ions X- and Y- (figure 27.4).
Although ion exchange materials are claimed to be monofunctional, in that only the
ion exchange process is used in separation, in practice some molecular sieving and
adsorption can occur. The adsorption is small, but it can sometimes be used to separate
closely related compounds.
Elution of bound ions. The bound ions can be removed by changing the pH of the buffer.
For example, as the pH of a protein moves towards its isoeletric point, the net charge
decreases and the macro-molecule is no longer bound. Separation is achieved as other
charged proteins remain on the column alternatively, ions can be removed by increasing the
ionic strength, when high concentrations of ions in the solvent displace the bound ions by
increasing the competition for the charged groups of the ion exchange material. The pH or
ionic strength can be altered sharply, by changing the eluting buffer, or, gradually, by means
of a gradient.
Preparation of material. Ion exchange materials are first allowed to swell in the buffer and
the fines removed.
The ion exchange material is then obtained in the required ionic form by washing with the
appropriate solution. For example, the H+ form of a cation exchange resin is obtained by
washing the material with hydrochloric acid then water until the washing are neutral.
Similarly the Na+ form is prepared by washing the resin with sodium chloride or sodium
hydroxide then water as above.
The final stage before preparing the column is to equilibrate the material by stirring
with the eluting buffer.

The separation of amino acids by ion exchange chromatography:

MATERIALS
1. Chromatography column (20 cm × 1.5 cm) 5
2. Strongly acidic resin 30 g.
3. Hydrochloric acid (4 ml/ litre) 1 litre.
4. Hydrochloric acid (0.1 ml/ litre) 4 litres
5. Glass wool –
6. Amino acid mixture 10 mg
(Dissolve aspartic acid, histidine of each and lysine in 0.1 mol/ litre HCl to a final
concentration of 2mg/ ml)
7. tris-HCl buffer (0.2 mol/ litre, pH 8.5) 3 litres
8. Sodium hydroxide (0.1 mol/litre) 2 litres
9. Separating funnels (500 ml) 10
10. Acetate buffer (4 mol/ litre, pH 5.5) 250 ml
11. Ninhydrin reagent. (Dissolve 20 g of ninhydrin and 3 g of 1 litre hydrindation in 750 ml
of methyl cello solve and add 250 ml of acetate buffer). Prepare fresh and store in a brown
bottle.
12. Methyl cello solve (ethylene glycol monomethyl ether) 1 litre.
 96

13. Ethanol (50 percent v/v) 1 litre.

14. Ninhyrin (2 g/litre in acetone). (Care: carcinogenic!) 100 ml
15. Oven at 105˚c 1.

Method:
PREPARATION OF THE COLUMN- gently stirs the resin with 4 mol/ litre HCl until
fully swollen (15- 30 ml/ g dry resin). Allow the resin to settle, and then decant the acid.
Repeat the washing with 0.1 mol/ litre HCl, resuspend in this solution, and prepare the
column as previously described.
Elutions of amino acids carefully apply 0.2 ml of the amino acid mixture to the top of the
column, open the tap and allow the sample to flow into the resin. Add 0.2 ml of 0.1 mol/
litre HCl, allow to flow into the column as before, and repeat the process twice. Finally,
apply 2 ml of 0.1 mol/ litre HCl to the top of the resin and connect the column to a
reservoir containing 500 ml of 0.1 mol/ litre HCl. Adjust the height of the reservoir to give a
flow rate of about 1 ml/ min and collect a total of forty 2 ml fractions. Test five of the tubes
at a time for the presence of amino acids by spotting a sample from each tube on to a filter
paper: dip this in the acetone solution of ninhydrin and heat in an oven at 105˚c. If amino
acids are present, they will show up as blue spots on the filter paper. When the first amino
acid has been eluted, remove the reservoir of 0.1 mol/ litre HCl and allow the level of acid
to fall to just above the resin. Run 2 ml of 0.2 mol/ litre tris- HCl buffer (pH 8.5) on to the
column, then connect to a reservoir of this buffer and continue with the elution until the
third amino acid is removed from the column.

Detection of amino acids: Adjust the pH of each tube to 5 by the addition of a few drops of
acid or alkali. Add 2ml of the buffered ninhydrin reagent and heat in a boiling water bath for
15 min. cool the tubes to room temperature, add 3 ml of 50 percent v/v ethanol and read
the extinction at 5570 nm after allowing the tubes to stand for 10 min. set up the appropriate
blanks and standards, and plot the amount of amino acid in each fraction against the volume
eluted.

Applications:
Ion exchange has proved to be one the major methods of fractionation of labile
biological substances. It is used most widely for protein and peptide separations, enzyme
purification, hormone preparation, studies, carbohydrate and lipids separations and vitamin
and co – enzyme work etc. the special characteristic of ion exchange is that the separation is
based on charge, so that when it is combined with techniques which use other criteria for
separation, such as gel filtration (size) or affinity chromatography (bio selectivity), it is a very
powerful analytical and preparative tool.
 97

EXERCISE 28

AFFINITY CHROMATOGRAPHY

Introduction:
Affinity chromatography is a unique separation technique that does not rely on
differences in the physical properties of the molecules to be separated. Instead, it exploits
the unique property of extremely specific biological interactions to achieve separation and
purification. As a consequence, affinity chromatography is theoretically capable of giving
absolute purification, even from complex mixtures, in a single process. The technique was
originally developed for the purification of enzyme, but it has been extended to nucleotides,
nucleic acids, immunoglobulin, membrane receptors and even whole cells and cell fragments.
The technique requires that the material to be isolated is capable of reversible
binding to a specific ligand which is attached to an insoluble matrix.

M + L k + 1 ML
Macro – ligand complex
Molecule (attached to k-1
Matrix)

Under the correct experimental conditions when a complex mixture containing the specific
compound to be purified is added to the insolubilized ligand, generally contained in a
conventional chromatography column, only that compound will bind to the ligand. All other
compounds can therefore be washed away and the compound subsequently recovered by
displacement from the ligand (fig 28.1).

Practical procedure:
The procedure for affinity chromatography is similar to that used in other forms of
liquid chromatography. The ligand – treated matrix is placed into column in the normal way
for the particular type of support. A buffer is used which will encourage the binding
macromolecule to be strongly bound to the ligand. The buffer generally has a high ionic
strength to minimize non – specific adsorption of polyelectrolyte ligand. The buffer must
also contain any co – factors such as metal ions necessary for ligand – macromolecule
interaction. Once the sample has been applied and the macromolecule bound, the column is
eluted with more buffers to remove non – specifically bound contaminants. The purified
compound is finally recovered by either specific or non – specific elution. Non – specific
elution may be achieved by a change in either pH or ionic strength. pH shift elution using
dilute acetic acid or ammonium hydroxide results from a change in the DNA by a similar
procedure. The method has also been used to isolate whole genes by hybridizing practically
single stranded DNA with the complementary mercurated mRNA which is then reacted
with a thioleted matrix.
 98

EXERCISE 29

GAS LIQUID CHROMATOGRAPHY

Introduction:
Gas liquid chromatography has also been to as vapour phase chromatography, gas –
liquid partition chromatography, gas – partition chromatography, gas – chromatography,
vapour fractometry and by several similar designations.
This technique, which is based upon the distribution of compounds between a liquid
and a gas phase, is a widely used method for the qualitative and quantitave analysis of a large
number of compounds size it has high sensitivity, reproducibility and speed of resolution. It
has proved to be most valuable for the separation of compounds of relatively low polarity. A
stationary phase of ‘liquid’ material such as silicone grease is supported on an inert granular
solid. This material is packed into a narrow coiled glass or steel column 1 – 3m long and 2 –
4mm internal diameter through which is passed an inert gas (the mobile phase) such as
nitrogen or argon. The column is maintained in an oven at an elevated temperature which
volatilizes the compounds to be analyzed. The basis for the separation of compound being
analyzed is the difference in the partition coefficient of the volatilized compounds as they are
carried through the column by the gas. As the compounds leave the column they pass
through a detector. This is linked via an amplifier to a chart recorder, which records a peak
as a compound passes through the detector.

Apparatus and working:

Figure 29.1 shows the essential parts of the apparatus. Helium gas is introduced at a
constant rate of flow through value V, passing through detector D then through the column
SP (stationary phase) filled with silicon or other non-volatile liquid, then through lower part
of detector (d), than through gas flow meter M where exact rate of flow of gas is measured.
The sample to be analyzed is dissolved in carrier solvent and injected at the top of the
column with STD. The detector (d) is a thermal conductivity cell called a double
kapharometer. The principle of this detector is Wheatstone bridge. Heat is condensed away
from a hot body by passing a gas over hot body. The rate of heat removal will depend on the
nature of the gas, other factors being constant. Thus 1/ 5th the heat conductivity of air
whereas H2 gas is seven times more effective than air.
In figure 29.2 the identical gas chromato C1 and C2 contains identical pieces of
platinum wire. They are heated by identical quantity of electric current. The resistance of two
wires will be identical if the same gas passes through C1 and C2 at a constant rate, however if
inert gas such as helium passes over C1 and methyl ester of fatty acids is mixed with SP, and
this mixture passes over wire C2 , C2 will be surrounded by a gas with different thermal
conductivity (probably a low conductivity in this case). The C2 wire will not lose as much
heat as the C1 wire, and so will have higher resistance. If the resistance of C1 and C2 are first
just balanced with R3 and R4 resistances of a Wheatstone bridge with only helium flowing,
then a potential would be developed between A and B when the methyl ester was
introduced. This can be measured and taped with an electrical tracing paper recorder.
An example of such a tracing is shown below
 99

Twenty ml of mixture of methyl esters of C10- C20 fatty acids dissolved in methyl
corporate were injected into a column operating at 208˚c and a flow rate of 7500 million/
minute. The separation of esters were recorded with a 10 mV full scale sensitive recorder, a
filament current of 260 mA and charge speed of 15 inches/ hr. this apparatus used was an
aerograph A-100-c.

Solid support:
Since this is used to provide a supporting surface on which is coated the film of
stationary phase, it is important that the support should be inert to the sample. The most
commonly used support is celite (diatomaceous silica) which because of the problem of
support/ sample interaction, is often treated so that hydroxyl groups which occur in celite
are modified. The support particles have an even size which, for the majority of practical
applications, is 60 to 80, 80 to 100 or100 to 120 meshes,

Stationary phase:
The requirements for any stationary phase are that it must be involatile and thermally
stable at the temperature used for analysis. Commonly used stationary phases include the
polyethylene glycols, methyl phenyl- and methylvinylsilicone gums (so called OV phases),
Apiezen L, esters of adipic, succinic and phthalic acids, and squalene.

Preparation and application of sample:

The majority of non-and low polar compounds are directly amenable to GLC, but
other compounds possessing such polar groups as –OH, -NH2, -COOH are generally
retained on the column for excessive periods of time if they are applied directly. This
excessive is inevitably accompanied by poor resolution and peak tailing. This problem can be
overcome by derivatisation of these polar groups. Methylation, silanisation and
trifluoromethyl- silanisation are common derivatisation methods for fatty acids,
carbohydrates and amino acids.
The sample for chromatography is dissolved in a suitable solvent such as ether,
heptanes or methanol. The sample is injected onto the column using a micro-syringe
through a septum in the injection port which is attached to the top of the column. Normally
between 0.1 and 10 mm3 of solution is injected. It is a common practice to maintain the
injection region of the column at a slightly higher temperature than the column itself. This
helps to ensure rapid and complete volatilization of the sample. Sample injection is
automated in many commercial instruments.

Separation conditions:
Nitrogen, helium and oxygen are the three most commonly used carrier gases. They
are passed through the column at a flow rate of 40 to 80 cm3 min-1; the column temperature
must be within the working range of the particular stationary phase and is chosen to give a
balance between peak retention time and resolution. In isothermal analysis a constant
temperature is employed. In the separation of compounds of weight it may be advantageous
to gradually increase the temperature. This is referred to as temperature programming.

Detection systems:
By far the most widely used detector is the flame ionizing detector (FID). It
responds to almost all organic compounds, can detect as low as 1 ng and has a wide linear
 100

response range (106). A mixture of hydrogen and air is introduced into the detector to give a
flame, the jet of which forms one electrode, whilst the other electrode is brass or platinum
wire mounted near the tip of the flame. When the sample components emerge from the
column they are ionized in the flame, resulting in an increased signal being passed to the
recorder. The carrier gas passing through the column and the detector gives a small
background signal, which can be offset electronically to give a baseline.
The nitrogen- phosphorous detector (NPD), which is also called a thermionic
detector, is similar in design to a FID, but has a sodium salt fused onto the electrode system
or a burner tip embedded in a ceramic tube containing a sodium salt or a rubidium chloride
tip
The electron capture detector (CED) responds only to substances which capture
electrons, particularly halogen- containing compounds. This detector is, therefore,
particularly used in the analysis of polychlorinated compounds.
The volatile solvent used to introduce the test sample gives rise to a solvent peak at
the beginning of the chromatogram.
The three main forms of detector respond to this solvent with varying sensitivity
thereby affecting the detection and resolution of rapidly eluting solutes. In cases where
authentic samples of the test compounds are not available for calibration purposes or in
cases where the identity of the compounds is not known, the detector may be replaced by a
mass spectrometer. More recently, GLC has been linked to other types of detector including
an infer-red spectrophometer and to a nuclear magnetic resonance Spectrometer, the
resulting spectra aiding in the identification of unknown compounds.

Applications:
Until the recent developments in HPLC, GLC was probably the most commonly
used form of chromatography. Its use nowadays is confined to volatile, non-polar
compounds which do not need derivatisation; compounds are characterized by their
retention time or preferably by their relative retention time to a standard reference
compound. In the analysis of compounds which form a homologous series, for example the
methyl esters of the saturated fatty acids, there is a linear relationship between the logarithm
of the retention time and the number of carbon atoms. This can be exploited, for example,
to identify an unknown fatty acid ester in a fat hydrolysate. A widely used system for
quantitative analysis is the Retention Index (RI) which is based on the retention of a
compound relative to n- alkanes. The compound is chromatographed with a number of n-
alkanes and a semi- logarithmic plot constructed of retention time against carbon number.
Each n- alkane is assigned an RI of 100 times the number of carbon atoms it contains
(pentane therefore has an RI of 500) allowing the RI for the compound to be calculated.
Many commercially available GLC systems with data processing facilities have the capacity
to calculate RI values automatically.
 101

EXERCISE 30
HIGH PERFORMANCE (PRESSURE) LIQUID
CHROMATIGRAPHY (HPLC)
Principle:
Originally, HPLC was referred to as high pressure liquid chromatography but
nowadays the term high performance liquid chromatography is preferred since it better
describes the characteristics of the chromatography and avoids creating the impression that
high pressure are an inevitable pre- requisite for high performance. This is new known not
to be the case and the term medium pressure liquid chromatography (MPLC) has been
coined for some separations. The components of a HPLC system are shown in figure 30.1.
The resolving power of a chromatographic column increases with column length and
the number of theoretical plates per unit length, although there are limits to the length of a
column due to the problem of peak broadening. As the number of theoretical plates is
related to the surface area of the stationary phase it follows that the smaller the particle size
of the stationary phase, the better the resolution. Unfortunately, the smaller the particle size,
the greater the resistance to eluant flow. All of the forms of column chromatography so far
discussed rely on gravity or low pressure pumping systems for the supply of eluant to the
column. The consequences of this is that the flow rates achieved are relatively low and this
gives greater time for band broadening by simple diffusion phenomena. The use of faster
flow rates is not possible because it creates a back- pressure which is sufficient to damage
the matrix structure of the stationary phase, thereby actually reducing eluant flow and
impairing resolution. In the past decade there has been a dramatic development in column
chromatography technology which has resulted in the availability of new smaller particles
size stationary phases which can withstand these pressures and of pumping systems which
can give reliable flow rates. These developments, which have occurred in adsorption,
partition, ion- exchange, exclusion and affinity chromatography, have resulted in faster and
better resolution and explain why HPLC has emerged as the most popular, powerful and
versatile form of chromatography.

Column packing:
Three forms of column packing material are available based on a rigid solid (as opposed to
gel) structure. These are:
i. Microporous supports where micropores ramify through the particles which are
generally 5 to 10 µm in diameter;
ii. Pellicular (superficially porous) supports where porous particles are coated onto an
inert solid core such as a glass bead of about 40 µm in diameter:
iii. Bonded phases where the stationary phase is chemically bonded onto an inert
support.
For adsorption chromatography, adsorbents such as silica or alumina are available as
microporous or pellicular forms with a range of particle size. Pellicular systems generally
have a high efficiency but low sample capacity, and therefore microporous supports are
preferred where applicable. All forms of HPLC column packing are characterized by their
 102

regular spherical shape which distinguishes them from conventional materials. These small
spheres pack most efficiently and give good flow properties.
In liquid – liquid partition systems, the stationary phase may be coated onto the inert
support. Both microporous and pellicular supports are used for supporting the liquid phase.
One disadvantage of supports coated with liquid phase is that the developing solvent may
gradually wash off the liquid phase with repeated use. To overcome this problem bonded
phases have been developed where the liquid phase has been covalently bonded to the
supporting material which may be silica or a silicone polymer. The silicone polymer bonded
phases have the particular advantage that as well as not being eluted by the developing
solvent, they are chemically, hydrolytically and thermally stable. In normal phase liquid –
liquid chromatography, the stationary phase is a polar compound such as alloys nitride or
alloylamine derivatives and the mobile phase a non – polar solvent such as hexane. For
reverse – phase chromatography, the stationary phase is non – polar compound such as a C8
or C18 hydrocarbon and the mobile phase a polar solvent such as water / acetonitrile or
water / methanol mixtures.
Many different types of ion exchangers are available of which the cross – linked
microporous polystyrene resins are widely used. Pellicular resin forms are also available, as
are bonded phase exchangers covalently bonded to a cross – linked silicone network. These
resins are classed as hard and readily withstand the pressures required during analysis.
The stationary phases for exclusion separations are generally porous silica, beads,
polystyrene or polyvinyl acetate the eluting solvent is an organic system, and the beads are
available in a range of pere sizes. Semi rigid gels such as sephedex or bio – gel P and non –
rigid gels such as sepharese and bio – gel A are only of limited use in HPLC since they can
withstand only low pressure. The supports for affinity separations are similar to those for
exclusion separations. The spacer arm and ligand are attached to these supports by similar
chemical means to those used in conventional low pressure affinity chromatography. Table
30.1 lists some examples of commonly used stationary phases and their applications.

The column:
The columns used for HPLC are generally made of stainless steel and are
manufactured so that they can withstand pressure of up to 5.5 × 107 Pa (8000psi). Straight
column of 20 to 50 cm in length and 1 to 4 mm in diameter are generally used though
smaller capillary columns are available. The best columns are precision bored with an
internal mirror finish which allows efficient packing of the column. Porous plugs of stainless
steel or Teflon are used in the ends of the columns to retain the packing material. The plugs
must be homogeneous to ensure the uniform flow of solvent through the column. It is
important in some separations involving liquid partition and ion – exchange that the column
temperature us thermostatically controlled during the analysis.

Column packing procedure:

Columns may be purchased already packed from commercial companies with
specified packing material structure and dimensions. Many workers, however, prefer to pack
their own columns since this is cheaper than purchasing pre – packed columns. Several
methods are available for packing columns and the method used will depend on the nature
of the packing material and the dimensions of the particles. The major priority in the packing
of a column is to obtain a uniform bed of material with no cracks or channels. Rigid solids
 103

and hard gels should be packed as densely as possible, but without fracturing the particles
the packing process. The most widely used technique for column packing is the high
pressure slurring technique. A suspension of the packing is made in a solvent of equal
density to the packing material. The slurry is then rapidly pumped at high pressure into a
column with a porous plug at its outlet. The resulting bed of packed material within the
column can then be prepared for use by running the developing solvent through the column,
hence equilibrating the packing with the developing solvent. When hard gels are packed, it is
necessary for them to be allowed to swell first in the solvent to be used in the
chromatographic process before packing under pressure. Soft gels cannot be packed under
pressure and have to be allowed to pack from slurry in the column under gravitational
sedimentation only, in a similar way to the packing of columns for conventional column
chromatography.

Chromatographic solvent (mobile phase):

The choice of mobile phase to be used in any separation will depend on the type
separation to be achieved. Isocratic separations may be more made with a single solvent, or
two or more solvents mixed in fixed proportions. Alternatively a gradient elution system may
be used where the composition of the developing solvent is continuously changed by use of
suitable gradient elution system may be used where the composition of the developing
solvent is continuously changed by use of a suitable gradient programmer. In the majority of
cases this involves the use of two pumps. All solvents for use in HPLC systems must be
specially purified since traces of impurities can affect the column and interfere with the
detection system. This is particularly the case if the detection system is measuring
absorbance at below 200 nm. Purified solvents for use in HPLC systems are available
commercially, but even with these solvents a 1 to 5 µm micro filter is generally introduced
into the system prior to the pump. It is also essential that all solvents are degassed before use
otherwise gassing tends to occur in most pumps. It tends to be particularly bad for aqueous
methanol and ethanol solvents, gassing (the presence of air bubbles in the solvent) can alter
column resolution and interface with the continuous monitoring of the column effluent.
Degassing may be carried out in several ways; by warming the solvent, by stirring it
vigorously with a magnetic stirrer, subjecting it to a vacuum, ultrasonic vibration, or by
bubbling helium gas through the solvent reservoir.

Pumping systems:
The pumping system is one of the most important features of an HPLC system.
There is a high resistance to solvent flow due to the narrow columns packed with small
particles, and high pressures are therefore required to achieve satisfactory flow rates. The
main feature of a good pumping system is that it is capable of outputs of at least 3.4 * 1.07
Pa (5000 p.s.i) and ideally there must be no pulses of flow through the system. There must
be a flow delivery of at least 10 cm3 min-1 for normal analysis, and upto 30 cm3 min-1 for
preparative analysis. All materials in the pump should be chemically resistant to all solvents.
Various pumping systems are available which operate on the principle of constant pressure
or constant displacement.
Constant pressure pumps produce a pulse less flow through the column, but any
decrease in the permeability of the column will result in lower flow rates for which the pump
will not compensate. These pumps will not compensate. These pumps operate by the
introduction of high pressure gas into the pump, and the gas into the pump, and the gas in
 104

turn forces the solvent from the pump chamber into the column. The use of an intermediate
solvent between the gas and the eluting solvent reduces the chances of dissolved gas directly
entering the eluting solvent and causing problems during the analysis.
Constant displacement pumps maintain a constant flow rate through the column
irrespective of changing conditions within the column. One form of constant displacement
pump is a motor-driven syringe type pump where a fixed volume of solvent is forced from
the pump to the column by a piston driven by a motor. Such pumps, as well as providing
uniform solvent flow rates, also yield a pulse less solvent flow which is important as certain
detectors are sensitive to changes in solvent flow of constant displacement pump. The
piston is moved by a motorized creak and entry of solvent from the reservoir to the pump
chamber and exit of solvent to the narrow band onto the column. There are two methods
which are generally used. The first method makes use of a micro syringe designed to
withstand high pressures. The sample is injected through a septum in an injection port,
either directly onto the column packing or onto a small plug of inert material immediately
above the column packing. This can be done the pump may be turned of before injection,
and when the pressure has dropped to near atmospheric, the injection is made and the pump
switched on again. This is termed a stop flow injection. The second method of sample
introduction is by use of a loop injector. This consists of a metal loop of small volume which
can be filled with the sample. By means of an appropriate value, the eluant from the pump is
channeled through the loop, the outlet of which leads directly onto the column. The sample
is column is regulated by check values. On the compression stoke solvent forced from the
pump chamber into the column. During the return stroke the exit check value closes and
solvent is drawn in via. The entry value to the pump chamber, ready to be pumped onto the
column on the next compression stoke. Such pumps produce pulses of flow and pulse
dampness are usually incorporated into the system to minimize this pulsing affect. All
constant displacement pumps have in-built safety cut-out mechanisms so that if the pressure
within the chromatographic systems changes from pre-set limits the pump is inactivated.

Practical procedure:
The correct application sample onto a HPLC column is another particularly
important factor in achieving successful separations. Ideally the sample ought to be
introduced as an infinitely thus flushed onto the column by the eluant, without interruption
solvent flow to the column. Automatic versions of loop injectors are commercially available.
Repeated application of highly impure samples such as sera, urine, plasma or whole
blood, which have preferably been deproteinated, may eventually cause the column to lose
its resolving power. To prevent this occurrence a guard column is installed between the
injector and the analytical column. This guard column is a short (2 to 10 cm) column. Of the
same internal diameter, and packed with similar material to that present in the analytical
column. The packing of the guard column can be replaced at regular intervals.

Applications:
The wide applicability, speed and sensitivity of HPLC have resulted in it becoming
the most popular form of chromatography and virtually all types of biological molecules
have been purified using the approach. Reverse phase partition HPLC is particularly useful
for the separation of polar compounds such as drugs and their metabolites, peptides,
vitamins, polyphenols and steroids. Prior to the advent of this form of chromatography, the
 105

separation of such polar compounds was not easily accomplished and often required pre-
derivatization to less polar compounds.
HPLC has probably had the biggest impact on the separation of oligopeptides and
proteins. Instruments dedicated to the separation of proteins have given rise to the
technique of fast protein liquid chromatography (FPLC). There are no unique principles
associated with FPLC; it is simply based on reverse phase and ion-exchange chromatography
and on chromato – focusing. Microlore glass- lined stainless steel columns 1 mm diameter
and 2.5 cm long have recently been developed which enable very small amounts of sample to
be used with separation taking as little as 10 minutes.
 106

EXERCISE 31

DETERMINATION OF FREE FATTY ACIDS IN GHEE

Milk fats contain a little concentration of free fatty acids in their composition. Upon
passage of time due to splitting of glycerides by the action of lipase the concentrations of
free fatty acids increase resulting into hydrolytic rancidity.
CH2OOC – R1 CH2OH
1 lipase 1
CHOOC – R2 CHOH + R1COOH + R2COOH
1 1
CH2OOC – R3 CH2OH + R3COOH
Triglyceride Glyderide free fatty acids

In milk, and cream, lactic acid is important in contributing major part of acidity,
whereas, in butter and ghee a whole range of fatty acids occur which vary widely in
molecular weight, it is therefore preferable to express the free fatty acids in terms of acids
value. The different terms used to express acidity in ghee are:
1. Acid value: it is the number of mg of KOH required to neutralize of ghee
sample.
2. Free fatty acids (FFA): as oleic acid is major among the other free fatty acids,
the acidity of ghee is frequently expressed as the percentage of the fatty acids
in the sample calculated as oleic acid.
3. Degree of acidity: it is the total titratable acidity present in the sample
expressed as percentage.

Principle:
The free fatty acids presents in the ghee are estimated by titration with alkali using
phenolphthalein as an indicator
o o

R – C – OH + NaOH R – C – O – Na + H2O

Reagents and apparatus:

1. Ethyl alcohol or rectified spirit 95% (V/V); sp. Gr. 0.8160, neutral to
phenolphthalein.
2. Sodium hydroxide or potassium hydroxide 0.1 N aqueous solution.
3. Phenolphthalein indicator.
4. Conical flask, 250 ml capacity.
5. Burette, with soda lime guard tube.

Procedure:
1. Weigh accurately 10g of sample in a conical flask.
2. IN a second flask bring 50 ml of alcohol to boiling point and while still above 70˚c,
neutralize it to phenolphthalein with 0.1 N sodium hydroxide.
3. Pour the neutralized alcohol on ghee in the flask and mix the contents of the flask.
 107

4. Bring them to boil and while it is still hot, titrate with 0.1N sodium hydroxide,
shaking vigorously during.
5. The end point of titration is reached when the addition of a single drop produces a
sight but definite colour change persisting at least 15 sec.

Calculations:
1. Acid degree value
= 5.61 × T/W when 0.1N KOH is used OR
4.0 × T if 0.1NaOH is used
W
2. Free fatty acids
= 2.82 × T/W
3. Degree of acidity
= 100 N/W; where

T = volume of 0.1N alkali required for titration (ml)

N = the quantity of alkali used, expressed as ml of1N solution.
W = weight of sample (gm)
 108

EXERCISE 32

DETERMINATION OF PEROXIDE VALUE OF GHEE

Introduction:
The physico – chemical reactions that occur on processing and storage of fat rich
dairy products bring some undesirable changes in texture, flavour, stability, acceptability and
nutritional characteristics and are of permanent importance from economic, commercial and
functional view points. This deterioration in quality of ghee generally arises from two major
pathways viz hydrolytic rancidity and oxidative rancidity. The other process is ketonic
rancidity.
The oxidative rancidity affects an isolated fat is caused by the action of oxygen. Air
in contact with the milk lipid at the surface will provide oxygen. The term “auto oxidation”
is applied to the phenomenon of oxidative deterioration which is a neutral complex chemical
reaction occurring between atmosphere oxygen and unsaturated lipid components mediated
by a variety catalysts resulting in destruction at valuable nutrients, undesirable flavours and
odors affecting the palatability of foods and even generation of toxic compounds. It exhibits
auto catalytic property, once initiated, cannot be stopped. Oxidation of unsaturated lipids
follows two different oxygen addition mechanisms.
i. Addition of molecular oxygen on methylene group adjacent to double bound i.e.
Auto catalytically radial chain reaction mechanism.
ii. Concentrated addition ‘ene’ reaction: addition of singlet oxygen directly at double
bond in unsaturated lipids.

The free radical auto oxidation is described in terms of initiation propagation

termination process. Auto oxidation may be considered as a two step process:
a. Primary auto oxidation reaction which may lead to the formation of hydro
peroxide.
b. Further reaction of decomposition of hydro peroxides by cleavage of C-C
bonds to form volatile compounds.

Hydrolytic rancidity is caused by hydrolysis of triglycerides in presence of moisture,

with the liberation of free fatty acids ( capric, lauric, myristic)
Ketonic rancidity occurs when fungal attack on food in presence of limited amount
of oxygen and water (short chain fatty acids)
Peroxide value is the measure of oxidative rancidity (peroxide value) of ghee. It is
expressed as ml of 0.002 N sodium thiosulphate solutions per gm of sample, or since 2
electrons are provided by the iodide to reduce one molecule of peroxide; it is expressed as
m. eq. per kg fat or mill mole per kg fat. The peroxide value of normal ghee range from 1.5
to 50.
The other chemical methods to measure oxidative deterioration in lipids and lipid
containing foods products include.
1. Determination of peroxide by modified stamm method.
2. Using loftus – hill method
3. thio- barbituric acid (TBA) value
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4. Determination of carbonyl content

5. Oxygen adsorption method.

Apparatus:
1. Test tube: 50 ml thoroughly rinsed and dried.
2. Rubber bung: to fit in the tube, with a hole in the centre through which is
inserted a small glass rod.
3. Boiling water bath.
4. Conical flask: 250 ml capacity.

Principle:
During the process of auto oxidation molecular oxygen combines with unsaturated
fatty acids and as a result, several peroxide structure compounds are formed. These are then
determined iodometrically, by liberation of iodine from an acidic and sodium shiosulphate.

Reactions:
-CH2 –CH = CH-CH2 (original radical)
-H

-CH0 – CH = CH –CH (free radical)

O2

-CH2 (OO)-CH = CH-CH2 (peroxide radicle)

CH2CH = CH-CH2 (original radical)

-CH COOH-CH = CH-CH2 + (hydroperoxide radical + free radical)

-CH-CH= CH-CH

3I˚ + ROOH RO: +: OH-

+ 2H+
ROH + H2) + I3-

2Na2S2O3 + I2 + starch
Blue
2NaI + starch + Na2S2O6
Colourless

Procedure:
1. Take 1 ml melted ghee sample in a peroxide value tube.
2. To this add 20 ml of solvent mixture consisting of 2:1 parts of glacial acetic acid:
chloroform and 1 gm of potassium iodide.
3. Heat the contents of the tube to boiling within 30 seconds and boil vigorously.
4. Then stopper the tube and cool it immediately under tap water.
 110

5. Transfer the contents to a 250 ml conical flask. Rinse the tube with 20 ml of 5%
aqueous solution of KI and twice with 25 – 30 ml of distilled water.
6. Titrate with 0.002N sodium thiosulphate using 2 ml of 1% starch as an indicator. Do
not add starch until the end point is almost reached.
7. Note the ml of sodium thiosulphate required to change the colour from violet to
colourless and express the result in terms of peroxide value.
 111

EXERCISE 33

DETERMINATION OF AFLATOXINS FROM FOOD AND

FEEDS BY COLUMN AND THIN LAYER
CHROMATOGRAPHY

Importance:
Carcinogenic nature and involvement of aflatoxins in the disease of animals and
humans have collectively urged major public health organizations to stipulate maximum
levels of aflatoxins that could be permitted in food and feeds. Many organizations in our
nation and others, including other import- export agencies, have fixed maximum permissible
limit of aflatoxins in products, whatever the limit may be, it is emphasized that the method,
by which the level of aflatoxins is estimated, plays a very important role, either from the
point of view of the effect on the health of humans and livestock or to make the product
acceptable in the international market.

Principle:
Methods of estimation of aflatoxins are mainly based on the properties of aflatoxins.
The solubility of aflatoxins in organic solvents like chloroform, methanol, ethanol, acetone,
acetonitrite, benzene etc., helps in their quantitative extraction from the commodities. Their
insoluble character in diethyl ether, petroleum ether and hexane affords a method to separate
them from certain interfering pigments and fats. Characteristic fluorescence and adsorption
under long wave ultraviolet light aid detection and estimation.
The steps involved in methods of detection and estimation of aflatoxin are:
1. Sampling and sample preparation.
2. Extraction
3. Purification, and
4. Detection or estimation.

Materials:
1. glass- waves (pipettes, conical flasks, glass stoppered flask, measuring cylinder
etc.).
2. Petroleum ether
3. Chloroform
4. Mechanical shaker
5. Glass distilled water
6. Rough and whatman filter paper
7. Anlnydrous sodium sulphate
8. Glass column, 2.5 cm internal diameter
9. Silica gel G and silica gel for chromatography
10. Hot air oven
11. Cleaning solvent, diethyl ether: hexane (3:1 v/v)
12. Eluting solvent; chloroform: ethanol (97:3 v/v)
13. Flash evaporator
 112

14. Boiling waterbath

15. Benzene: acetonitrite (98:2 v/v)
16. Mobile phase: chloroform: acetone (90:10 v/v)

Procedure:
(A). sample preparation:
Draw 2 kg sample (feed)

Weigh 250 gm sample

Cut in pieces

Grind the sample

(B). extraction
Weigh 50 gm sample in a glass stoppered 500 ml flask

Add 150 ml petroleum ether (for fatty feeds)

Apply stopper and shake

Held it over night.

Shake and filter through rough filter paper and collect retentate and return some to
flask along with filter paper

Add 25 ml glass distilled water

Add 250 ml chloroform (exlar; qualigens)

Shake for 30 min. on mechanical shaker

Filter through rough filter paper and collect filtrate in flask

Add 10 -15 gm of anhydrous sodium sulphate

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Refilter through rough filter paper preparing Na- sulphate (10- 15 gm) bed and collect
filtrate in flask.

(C). Purification
Take 50 ml of above filtrate in a round bottom flask and condense it to 5-7 ml

Prepare column by fixing glass wool at bottom, followed by 5gm Na- sulphate.
Tapper and elute with chloroform. Add 10 gm activated silica gel in form of slurry made
with chloroform of slurry made with chloroform; followed by 7.5 gm sodium sulphate.

Pour condensed extract to column load cleaning solvent: 150 ml (diethyl ether: hexane 3:1)
throw the eluent

At the end add/ pour dissolving solvent (chloroform: methanol: 97:3) 150 ml

Collect the extractant in round bottom flask

Condense it to 5-7 ml and transfer it to small vial and dry under flow of N2.

(D). estimation
Make dilutions like 0.2, 0.4, 0.8, 1.0 ml with benzene: acctonitrite (98:2)

Spot 5µl on prepared thin silica plates along with standards of aflatoxin having
concentration of 2.5, 5.0, 7.5 and 10.0 ng per 5µl spot.

Develop the plate in chromatographic solvent using chloroform: acetone (90:10)

developing agent.

Dry with fan and see fluorescence under long wave uv lamp and compare with
standards.
 114

EXERCISE 34

DETERMINATION OF LYSINE

Overview:
The free amino group on the side chain of lysine can react chemically with many
constituents during food processing and storage to give biologically unavailable lysine
complexes. These lysine complexes can result from reactions with reducing sugars
(producing maillard products), oxidized polyphenols, and oxidized lipids, glutaminyl and
asparaginyl residues, and alkaline solutions. Utilizable lysine is not equal to lysine content.
Essentially, the only source of utilizable lysine in a food is the lysine residue with its є- amino
group free (referred to as reactive lysine)
Reactive lysine can be measured directly by using reagents such as 1-fluoro-2, 4-
dinitrobenzene (DNFB), trinitrobenzene sulfonic acid (TNBS), w-methylisourea, or o-
phthalaldehyde it can be measured indirectly by the DNFB difference method, dye binding
procedure, furesine method, or reduction by NaBH4. Several microbiological assays have
been developed for available lysine, as have in vitro enzymatic hydrolysis methods. In vitro
diestability assays assume that protein digestability of each amino acid, including reactive
lysine. Enzymatic hydrolysis with pepsin plus pancreetin or with pronase makes use of the
fact that these proteolytic enzymes release only reactive lysine.
A water- soluble reagent, trinitrobenzene sulphonic acid (TNBS) can be used for free
lysine measurement. TNBS reacts with lysine derivatives formed early in maillars browning
which may or may not be bioavailable.

Principle:
The terminal (epsilon amino group present in the amino-acids lysine and hydro lysine
is not involved in the peptide linkage of a protein but may sometimes be combined with
some other compound. (E.g. An aldehyde such as an aldose sugar2 in such a way that part of
lysine becomes nutritionally unavailable. If the combination of the є amino group with
another compound such as an aldose sugar also prevents a chemical reaction between the
amino group and an amino coupling reagent, then the extent of combination of the lysine
amino group could be measured, thus giving an indication of the amount of lysine of
biological value (figure 34.1).

Reagents:
1. 0.1 % agar solution
2. 1 M sodium bicarbonate
3. 1 % 2, 4, 6 – trinitrobenzene sulfonic acid (freshly prepared)
4. 11 M HCl
5. diethyl ether

Procedure:
The procedure for the determination of lysine in biscuits given by Hall etal (1973)
and modified by Hall and Henderson (1979) is as follows:
 115

1. for the estimation of 2,4,6- trinitrobenzene sulphonic acid (TNBS) reactive lysine, suspend
250 mg biscuit powder (defatted) in 0.1 % weight/volume agar solution.
2. Homogenize the suspension in a potter- elvehjem all glass homogenizer and make the
volume to 25 ml.
3. Take 0.5 ml of the above solution in a 10 ml culture tube. Add 0.5 ml of 1 M sodium
bicarbonate and 1.0 ml of 1.0 percent solution of 2,4,6-TNBS reagent to the tube.
4. Close the tubes with screw caps and incubate them at 40˚c for 75 minutes.
5. Unscrew the tubes and add 3 ml of approximately 11 M hydrochloric acid.
6. Close the tubes again tightly with the screw caps and keep them in a boiling water bath for
2 hours.
7. After hydrolysis, close the tubes and make the volume to 10 ml with distilled water.
8. Dilute four millilites of the above solution to 8 ml with distilled water, remove the excess
of 2, 4, 6-TNBS and other interfering substances by shaking thrice with 5 ml of diethyl ether
each time.
9. Evaporate the residual ether by placing the tubes in a boiling water bath. Make the volume
of the aqueous phase in the tube to 10 ml with distilled water.
10. Measure the absorbance of the solution at 415 nm in spectronic – 20 against the
respective blank.
11. For blank, cease the reaction by adding first 3 ml of 11 M hydrochloric acid followed by
1 ml of 1 percent (w/v) solution of TNBS reagents; follow all other steps in the same
manner as in the case of samples.
12. Plot standard curve taking lysine (BDH) ranging in concentrations from 50 to 250
mg/ml. All other steps are the same as to be followed in case of samples.
13. Calculate the lysine content on the basis of whole biscuit and express as g/100g protein
in the biscuits.

REFERENCE:
Patel, S.M. (1986). Physico chemical and storage characteristics of biscuits developed
from the blends of casein- whey protein co- prepitates and partially deoiled groundnut meal.
LHD thesis submitted to G.A.U, 112-113.
 116

EXECRISE 35
ESTIMATION OF PHOSPHOLIPIDS BY THIN LAYER
CHROMATOGRAPHY

PROCEDURE:
Phospholipids from sorghum powder were extracted by the method of Folch et al.
(1957).
Ten grams of sorghum powder was shaken with 100 ml of chloroform: methanol
(2:1, v/µ) for 1h and stored overnight. Next day it was filtered through whatman filter paper
No. 41 and the extract so obtained was evaporated to its one third volumes under vacuum.
This extract was used further for resolution of phospholipids by thin layer chromatography.
Silica gel –G (BDH) plates were prepared by the method of Lees and De Muria
(1962).
The glass plates (21.5 × 21.5 cm) were thoroughly cleaned with chromic acid and
dried. Slurry was prepared by mixing 8 g of silica gel –G with 22 ml of water and shaking for
about 2 min. and used promptly. A plate was placed under the tough of an applicator with
leading edge protruding about one inch beyond the gate. The slurry was poured into the
tough to prepare single dimensional plate. Another glass plate was immediately pushed under
gate of tough in a continuous movement. The coated plates were immediately placed on a
uniform surface for superficial drying. After about 30 min. the plates were transferred to an
oven maintained at 50˚c, where the plates were dried overnight.
Before use, the plates were activated at 110˚c for 1h. The sample was applied with a
capillary tube at a line about 2 cm above the end of the plate. After evaporation of the
solvent, the plate was insert in a saturated chromatographic chamber containing a solvent
system consisting of chloroform: methanol: ammonia: water (65:35:5:2.5) as suggested by
Morrison v/v et al. (1980). Total time taken for separation was about 2 h. the
chromatograms, after drying were sprayed with Hanes reagent (prepared by mixing 5 ml of
60 percent w/w perchloric acid, 10 ml of 1N hydrochloric acid and 25 ml of 4 percent
ammonium molyldate in 100 ml acetone) as suggested by stahl (1969),
The chromatograms were incubated at 110˚c until the developments of blue
coloured spots were noticed.

Reference:
Parmar, S.S (1984). Effect of mango seed Kernels on oxidative stability of ghee. Msc.
Thesis submitted to G.A.U.: 63, 64.
 117

EXERCISE 36

ESTIMATION OF CHOLESTEROL BY COLORIMETRIC

METHOD (from cream, butter and ghee by the method of schoenheimer, R and
Sperry, W.M (1934) with slight modifications.)

Procedure:
1. Add 10 ml of 30 % ethanolic KOH solution to about 2 g fat, accurately weighed in a 50
ml test- tube. Shake the mixture until it becomes free of dispensed fat particles.
2. Saponify the mixture at 25-30˚c for 20 -22h with occasional shaking. Extract the
unsaponifiable matter with three successive 50 ml portions of diethyl ether.
3. Then wash the combined extract thrice alternatively with distilled water and 0.1 %
aqueous potassium hydroxide solution and finally with distilled water to make it alkali free.
4. Filter the alkali free extract through whatman No.1 filter paper over anhydrous sodium
sulphate using a little excess of diethyl ether to wash the separating funnel and the filter
paper.
5. Evaporate the ether extract under reduced pressure.
6. Dissolve the obtained residue in glacial acetic acid and make the volume to 10 ml.
7. Take 1 ml of this solution in a clean and dry glass stoppered test-tube. Add 2 ml of
glacial acetic acid and 4 ml of Licker Mann Burchard reagent (freshly prepared by mixing
20 ml acetic anhydride and 1 ml concentrated H2SO4 and then incubating in ice-bath for
27 minutes). Keep the mixture at 25˚c for 35 minutes.
8. Record the absorbance at 650 nm on spectronic 21 using a blank.
9. Prepare standard curve with crystalline cholesterol and calculate concentration of total
cholesterol in fat.
The cholesterol content of milk and milk products is depicted in table 36.1.

Reference:
Schoenheimer, R and Sperry, W.M. (1934). A micromethod for the determination of
free and combined cholesterol. J.Biol. Chem. 106:745.

Estimation of total cholesterol in ghee prepared from milk of cow and buffaloes by
the method of Bindal and Jain (1973).

Materials:
1. milk/butter/ghee
2. Chloroform AR
3. Acetic analydride-H2SO4 AR mixture (30:1) fresh
4. Stock cholesterol solution 2mg/ml in CHCl3
5. working standard cholesterol dilute the above solution to 5ml with CHCl3 (114) to give
0.4mg/ml
6. Alcohol: acetone (1:1)
 118

Method:
1. Place 10ml alcohol – acetone in a centrifuge tube and add 0.5g milk/0.2g butter
2. Immerse the tube in boiling water- bath with shaking until the solvent begins to boil.
3. Remove the tube and continue shaking the mixture for further 5 minutes.
4. Cool to room temperature and centrifuge.
5. Decant the supernatant fluid into a test-tube and evaporate to dryness in boiling
waterbath.
6. Cool and dissolve the residue in 5 ml CHCl3
7. Set at the same time series of standard tubes containing cholesterol and blank with (3) S
chloroform.
8. Add 4 ml of acetic anhydride: H2SO4 mixture to all the tubes and mix thoroughly.
9. Leave the tube in a dark at room temperature for about 12 minutes and measure the O.D.
at extinction at 650 nm.

Reference:
Bindal, M.P. and Jain, M.K. (1973). Estimation of total cholesterol in ghee prepared
from milk of cow and buffaloes. J.Indian Chem. Soc. 50 (1):63-65.