cal dairy chemistry.manual

ANALYTICALDAIRY

CHEMISTRY

INDEXNo. 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

INDEXNo. 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 columnand thin layer Chromatography

34 Determination of lysine

35 Determination of phospholipids content by thin layerChromatography

36 Estimation of cholesterol by colorimetric method

EXERCISE 1

DETERMINATION OF VISCOSITY OF MILK

IntroductionViscosity 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. Theviscosity of the fluid is a measure of the resistance to flow. The rate of flow is decreased byinternal frictional forces.

There are some liquids like water, milk etc. which can be poured easily from onecontainer to another. Such fluids are said to have a low viscosity. On the other hand, thereare certain liquids that do not flow easily like condensed milk, glycerol and honey. They aresaid to have a high viscosity.

The resistance to flow of layer of liquid molecules over another depends on variousfactors:

- 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 toViscosity of solutionViscosity 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 p8 µ l

Where l and r stands for the length and radius of the capillary tube in which µ ml ofthe given liquid flows in time t under driving pressure p.Or n = k p t where k = ∏ r4/ 8 µ lThus 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.

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 dgWhere D and d stand for the density of material of the ball and liquid respectively; g is theacceleration due to gravity.F = 4/3 ∏ r3 g (D-d) ˙ . µ = 4/3 ∏ r3 g (D-d) __1___ 6 ∏ n r

= 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 ofproteins, in addition to their concentration, the number of their particles, size and degree ofhydration also affects the viscosity of milk. In the case of fat, the globular size and the extentof clustering determination their influence on the viscosity of milk. The unit of viscosity ispoise while smaller unit is centipoises.

Poise: is defined as the force in dynes/cm2 required to maintain a relative velocity of1 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.

E.g. Hopler viscometer3. 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 ofcapillary tube, the upper part of which is connected to the bulb. Two marks, x and y areplaced on the bulb above the capillary tube. The time taken for a fixed volume ofsolution to know past x and y on the capillary tube is noted. A number of more elaboratedesigns have been used to maintain a constant head of liquid, or by varying the positionof 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 directlyproportional to the time taken for a certain volume of liquids to flow out under a certainpressure gradient, and also is proportional to the density of liquid. A comparison ofviscosity of milk is done to that standard liquid, distilled water, according to the aboveprinciple, 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 theviscometer 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 watersurface.

3. Transfer with the pipette a fixed volume of distilled water (10 to 25 ml) as perrequired 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.

6. Close the rubber tube with fingers and release the pressure slowly to allow thewater to flow down.

7. Start the stop watch when the meniscus of water just leaves the upper mark andmeasure 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 sin 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 thelower reservoir is too high and driving head is reduced by that amount.

2. Kinetic energy corrections primarily expansion losses at the entrance and exit ofthe capillary.

3. Apparent increase in the capillary length owing to the tendency to the fluid as itemerges from the capillary to retain the shape of the capillary for a finite distanceinto the fluid medium.

4. Drainage errors arising from the fact that all liquids do not drain from a surfacewith 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 theviscosity of highly viscous fluids such as condensed milk, glycerol etc. as they areunable 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 severaldegrees from the vertical, so that the ball is positioned closed to one side of the tube duringits fall. The sample tube is encased in a bigger glass cylinder with a device for forming waterbath around it, which can maintained at constant temperature (figure 1.2).

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

Variables in measurement can be eliminated by measuring the relative viscosity ofmilk with respect to distilled water for which the times of fall for a particular sphere aremeasured 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 followingFor temperature from solution

10060 to + 1˚c methanol+ 1 to 98˚c distilled water+ 100˚c distilled water + 20% glycerin100 – 150˚c glycerin (8p. gr. 1.26)

Leave an air space of approximately 10mm. above the liquids in the jacket. Under nocondition 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 orwithout a thermostat. The precision model of the equipment can be used over a temperaturerange from -60˚c to 150˚c.

Cleaning of the tube – the glass tube, balls, plug and washer must be perfectly dryand 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 atthe bottom with plug and washer. Unscrew the cap while applying slight pressure .fill inliquid approximately 20 min. (3/4”) below the upper end of the tube and insert the ball byusing ball pincers. If air bubble adheres to the ball remove it by knocking at it with the glassrod. Selection of balls –A plug guage is supplied for sorting the balls which differ in size ormaterial .as the coefficient of expansion of the standard set of balls is matched to theirdiameter 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 forkeeping in a liquid layer of 1 cm2 area and 1 cm height of layer the upper one is motion

relative to the lower one and parallel there to at a speed of 1 cm /sec. The unit of dynamicviscosity 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. Fixthe instrument in standard position. By means of an accurate stop watch determine thebeginning of the dropping period as soon as the lower periphery of ball appears to the touchthe upper annular marks, which should appear as a straight line. Similarly, dropping period isdetermined by lower mark.

Placing the instrument upside down the ball returns to its initial position a series ofdeterminations of dropping period (3 to 4) are made.

In dark and opaque liquids, the lower periphery of the ball is usually difficult todistinguish. The reading of the passage of the ball through the marks is therefore carried outat 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 theviscosity of the liquid. It employs a cylindrical vessel, 3 cm in diameter ,and acylindrical plunger ,1 cm in diameter and marcler for 1,2,3 or 4 cm immersion .theplunger is suspended by a wire and attached by a wire and attached to the plungerassembly , there is a disc, the circumference of which is graduated into 300 units .thesample cup is filled accurately to control the depth of immersion of the plunger .asthe sample turns ,the resistance between sample and plunger causes a torsion on thewire .readings are made on a scale attached to the plunger shaft. Scale units arearbitrary, “degrees M” each of which is 1/300 of a circle.By standardizing the instrument against solutions of known viscosity, the readingscan be converted to centipoises. A thermostat allows control of sample temperatureupto 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 frictionwhich in turn is related to friction which in turn is related to the viscosity of the testfluid.Brookfield viscometer (figure 1.5) is an instrument that measures the torque on arotating spindle by means of a calibrated spring. Readings are made from 0-100 scaleon the dial. Calculations are made by multiplication by factors that are dependent oninstrument model, size of spindle and speed of rotation. Models of the instrumentare available with upto 7 spindles and 8 speeds, giving 56 ranges encompassing 0 to 8millions centipoises. For exact control of sample temperature for meaningful

viscosity reading, a modified sample holder has been devised that serves to stabilizerreadings 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 slowlythrough the test material, makes possible consistency measurements on products likemashed products.

EXERCISE 2

SPECIFIC GRAVITY BY PYKNOMETER

Apparatus:1. A specific gravity bottle or any other type of pyknometer2. analytical balance and weight box3. Thermometer.

Reagents:1. Experimental liquid-milk2. 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 themass of a certain volume of the substance to the mass of an equal volume of a standardsubstance at a certain temperature and pressure. For solids and liquids the standardsubstance is taken to be water at 4˚ C which has density of unity at that temperature .thedensity of milk is determined by directly weighing a certain volume of milk taken ispyknometer (figure 2.1) and by determining the volume taken. Specific gravity of milk isdetermined by finding out the weights of a certain volume of milk and of the same volumeof 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 wateravoiding 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 thetemperature of the bath.

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

EXERCISE 3

DETERMINATION OF SURFACE TENSION OF MILK

INTRODUCTIONSurface 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 watermixture, then the resultant area of contact is known as interface.There exists a phenomenon of intermolecular attraction in liquid. The molecules inside theliquid experience a force of attraction from all the sides. I.e. they are attracted equallytowards both the sides. However, the surface molecules are attracted only downwards andsidewise. Therefore, due to imbalance of force of attraction at the surface, the liquid behavesas if there is tension and its surface remains stretched or in tension. This phenomenon isknown 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. Themolecules 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 attractionis equalized, but at the surface the balance is broken and a tension results. Surface tensionthus may be defined as the state of stress at the surface of the liquid due to the attraction ofthe molecules for each other. It is expressed in dynes/ an. A dyne is the force that is actingon a mass of one gram giving it an acceleration of one centimeter per second. Imbalance offorces 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 aspherical shape as among the different shapes like square, triangular, rectangular,minimum area is occupied by spherical shape i.e. Sphere will have a minimumvolume 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 tensioni.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 toobtain 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 forceof surface tension.

3. Yet another application of this phenomenon is in the cleaning of utensils, storagetanks, equipments and other greasy surfaces in the butter and ghee sections of a dairyplant. For efficient cleaning, water must come in contact and take away grease whichis 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 tensionbetween contact materials.

4. During transportation of milk, agitation takes place in the tanker and due to thissome of the membrane of ruptured fat globally membrane goes in the serum andlowers the surface tension. Hence the measurement of surface tension can be used asan index for determining the severity of agitation while transportation of milk.

The surface tension value of some liquids is as underWater 72 – 75 dynes/ cmSkim milk 52 – 52.5 dynes/ cmWhole milk 46 – 47.5 dynes/ cmCream (25% fat) 42 – 45 dynes/ cmSweet cream 39 – 40 dynes/ cmButter milk

The surface tension value of cow’s milk is 51.84 – 51.93 dynes/ cm and that ofbuffalo 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 containssurface active components such as proteins, fat, phospholipids and free fatty acids. Variousfactors such as fat content, temperature, liperysis, homogenization, aging affect the surfacetension 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 thesurface is carefully ground flat and polished. Two marks are etched on the tube, onejust above and the other just below the bulb, the volume of the liquid used in theexperiment being thus defined. In the determination of the surface tension, thenumber of drops which fall from the end of the liquid falls from the upper to thelower mark. Since, however, the passage of the meniscus past these marks does notin general coincide with the fall in of a drop; the tube is calibrated for a shortdistance 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 onedrop. 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.

(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, howeversimultaneously the force of gravity acts upon the liquid to push it in downward direction. Ata certain stage the force of surface tension equals the force of gravity, and hence the liquidwill 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 ∏ rWhere r = radius of the capillary.

Force of gravity pulling the liquid downwards= w × g …………… (1)

Where w = weight of liquidAnd 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 hSubstituting the value of µ in (2) we getF (s) = ℓ × ∏ r2 h × gAt balanced position

γ 2 ∏ r = ℓ × ∏ r2h × g

γ = ℓ × ∏ r2h × g 2 ∏ r

γ = ℓ r h g 2

Thus by measuring the height of liquid in capillary tube and the diameter of capillary tube bymoving 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 waterγ w = r h m × ℓm × g 2γm = rhm × ℓm × g × 2γw 2 rhw × ℓw ×g

γm = hm × ℓmγw 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 temperature2. adjust the temperature of distilled water as well as milk sample to 30 ˚ c3. 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 thedifference. This will give the level to which water rises in the capillary.

4. similarly repeat with milk and note the rise in capillary5. 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. stalagmometer2. small bottle3. beaker4. cork5. thermometer6. Rubber tubing with a screw pinch cock.7. hair dryer

Reagents:1. milk sample2. standard liquid3. sodium hydroxide4. chromic acid mixture5. alcohol

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 interfacialtension 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 issurrounded by a flat area (round) in a horizontal position, then a drop will form. If the flatglass area is properly cleaned and polished, the liquid will spread over this area and the pointof support of the drop will be the circumference of this flat area. The drop is held there bysurface tension. As the size of the drop is increased, it will finally attain a mass so that forceof gravity acting on it will just exceed the weight of the drop the surface tension can becalculated from the following formula: surface tension

= wt. Of the dropping × 980.3652∏ r

The size of drop issuing from a capillary orifice is governed by the surface tension of theliquids .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 externalcircumference of the end of the capillary tube, w the weight of the drop, µ its volume and dits density.

In actual use the above method is seldom employed in the manner just explainedbecause flow continues as the successive drops form and the drop that does not representthe weight supported by the surface tension. For this reason, instead of determining theweight of a single drop or a number of falling drops, it is usual to determine the number ofdrops formed when a definite volume of a liquid is allowed to flow slowly out of a capillarytube. With two different liquids the weight of equal volumes are proportional to theirdensities. 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 liquidThus, 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 thoseof the standard liquid can be known from the tables and substituted in the equation tocalculate the surface – tension of milk. Care should be taken that the measurements arecarried out at the same temperature.

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 upperend of the stalagmometer .the rubber tubing with the screw pinch cock on it isused 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 airvent.

4. Clean the stalagmometer thoroughly with NaoH to remove grease if any, thenwith chromic acid mixture and finally with distilled water. Then rinse it withalcohol and dry it by passing a current of air.

5. fill up the stalagmometer by immersing the dropping tip in the distilled water andsucking on the tubing until the water has risen above the mark A. then close thescrew pinch cock and insert the rubber stopper in the small bottle and keep it ina one liter beaker containing water which is maintained at the requiredtemperature (thermostat).

6. The small bottle carrying the stalagmometer must be dipped into water until thewater 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. Themeasurement of this number of drops is repeated thrice. Differentdeterminations 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 alcoholand dry it by passing a current of air. Then attach the rubber tube tostalagmometer and fill it with milk, by sucking on the open end of the rubbertube as before, and replace the stopper into the bottle which is then kept in thebeaker containing water at required temperature .repeat the procedure ofadjusting the pressure and counting the drops as before. Determinations of thedrops are carried out thrice.

13. Determine the specific gravity of the liquid by means of pyknometer or specificgravity 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 slighttraces of grease will markedly alter the size of the drops formed.

2. This apparatus should not be taken while the experiment is being carried out; asthe drops of the liquid may be caused to fall before they have attained theirmaximum size.

3. The surface is affected by the temperature, hence the determinations are to becarried out at a constant temperature in thermostat.

4. The rate of flow of the liquid through the tip should not be rapid but it shouldbe 10 to 15 drops per minute. hence, if the natural rate of dropping is grater, it isretarded by attaching a piece of rubber tubing with a screw pinch cock to theopen 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 theexperiment for the same period.

The actual practical was performed using chromatography column in thefollowing 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 thesurface a film of liquid is pulled up along with it. If the constricted surface of the filmbecomes vertical before it breaks, the downward force (P) due to ST at the constriction istwice the product of the circumference of the ring ® and ST. this force is balanced by theupwards force exerted on the ring, less the weight of the liquid above the vertical portion ofthe 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 isconnected 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, drysuitably.

ii. Take the milk in a clean dry watch glass or Petri dish, and bring it under the ringplacing on the movable platform of the tensiometer.

iii. Bring the beam of the balance to the horizontal position, which must coincidewith the pointer measuring the tension at zero position on the circular scale ofthe disc.

iv. Gently raise the movable platform containing milk till the platform ring justtouches the surface of the milk (the arm side slightly dips downwards at thispoint).

v. Rotate the lever of the balance (placed on the graduation disc) very gently till theplatinum ring snaps off the milk surface; take the reading of one position oflever on the circular scale of the disc.

vi. Repeat the experiment 5-6 times, each time changing the milk surface by addingfresh 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 unduedelay to allow. accumulation of fat globules on the surface

Among all the different methods used for determining ST, this method ismore rapid, accurate and reliable.

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 ofacceptance and can be used as a basis for establishing satisfactory methods for harvesting,processing, packaging, shipping and storing while sensory assessments may provideevaluations closest and expense makes it a difficult way to obtain objective data for universaluse. Objective text methods, once correlated with sensory assessment, provide a method oftexture measurement which can be used constantly with constant reproducibility if thecorrect instrumentation is used.

Texture is defined as the property which represents the way in which the structuralcomponents of a food are arranged in a micro and macro structure and the externalmanifestations of this structure. If is related to the behavior of foods in mechanical testingmachines as well as to their behavior when eaten (sensory aspects).

Machines like Instron universal testing machine can be used for texture testingproviding:I. Constant reproducible deformation rates are maintained at all the test forces,II. Force deformation and time are recorded accurately, andIII. 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 theinstrument.

2. Get the initial requirements like units for use either ENG or MET and the typeof test to be done, either Tension or Compression. (Where cross head goes upand 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 thecalibrating weight on the low capacity load transducers and its weight mustbe balanced out initially).

d) While holding the ZERO/CAL switch in the ZERO position, unlock theCOARSE and FINE balance controls (turn locking ring on each control

about ¼ turn counterclockwise) and then adjust these controls until theLOAD 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 theload transducer is nulled in its coil contained in the cross heed .this readingshould be loss than 5% of full scale of the load RANGE (10/50) for the loadtransducer in use.

When calibrating 1000 lb capacity load transducer, always set RANGEswitch to 10/50 position before pushing the ZERO/CAL switch to CALposition. Otherwise, an erroneers calibration read out will occur due to amplifiersaturation.

If the load reading is greater than 5% of load transducer range, uses thescrewdriver provided and adjust the LVDT core extender shaft in cross-headuntil a null (a minimum reading) occurs. Rotate the adjustment clockwise tolocate 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 onload transducer. Use the number shown for testing mode (tension orcompression) 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 2/10position. Then with the ZERO/CAL. Switch in centre position, unlock balancedcontrols until the LOAD readout shows zero; lock the controls. Hook thecalibration weight (5 kg) provided on to the pin in the grip coupling. Turn theADJ trimmer until the LOAD readout displays the value shown (i.e. 1/10th ofload transducer used) for units in use (English, metric or SI). Remove calibrationweight.

iii. To calibrate 10 lb (5kg/50 N) load transducer, set RANGE switch to 2/10position then with the ZERO/CAL switch in center position, unlock the balancecontrols and adjust these controls until the LOAD readout shown zero, lock thecontrols. Hook the calibration weight (500 g.) provided onto the pin in the gripcoupling. 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 willshow tare weight of grip or anvil. Adjust balance controls to set LOAD readoutto zero.

(ii) When using 100-lb (50 kg/500 N) load transducer with a grip installed, repeatstep f (ii) of calibration procedure except hang the calibration weight directlyfrom grip. Then remove the weight.

(iii) When using 100 lb (50 kg/500 N) load transducer with an anvil installed or the10 lb (5 kg/50 N) load transducer, remove the grip or anvil. Then insert aretaining pin in grip coupling and repeat step f (ii) and f (iii) of calibrationprocedure, depending upon load transducer in use.

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 adiscontinuity in loading applied to specimen.

4) Decide the length of specimen between contact faces of upper and lower gripsat the start of the test (gage length) and set the gage length between two gripsby moving crosshead up and down.

5) To return the grip spacing to gage length at the end of a test, crosshead travellimit stops must be set as follows:

a) Bring lower limit stop up so it contacts the actuator on crosshead at gagelength set. Tighten the stop securely on limit switch red.

b) Reset EXTENSION readout to zero. Press the UP push button and runcrosshead up about 1 inch (25 mm). Then press RETURN push button. Thecross 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 EXTENSIONread out displays same reading as a positive number.

e) Bring the lower limit stop up so it again contacts the actuator and tighten itsecurely. Then raise the crosshead again about 1 inch (25 mm) and press theRETURN push button. The crosshead should stop when EXTENSION readout 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 fortension 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 theanvil firmly into the load transducer by hand.

2) When the anvil is installed, the LOAD read out increase. This is tare weight andshould be balanced out. Unlock the COARSE and FINE balance controls and adjustthese 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 crossheadcontrol panel and measure the spacing between anvil and table with a ruler or bringthe anvil and table together carefully at a slow crosshead speed, stop crosshead, thenpress RESET pushbutton to zero the EXTENSION readout. Drive crosshead upuntil required spacing is displayed on EXTENSION read out (inches/mm).

5) To return the anvil and table spacing to gage length at the end of a test, crossheadtravel limit stops must be set. To accurately set the upper limit stop, proceed asfollows:

a) Bring upper limit stop down so it contracts actuator on crosshead at gagelength set in 4. Tighten stop securely on limit switch rod.

b) RESET the EXTENSION readout to zero. Press the DOWN pushbuttonand run the crosshead down about 1 inch (25mm). Press RETURNpushbutton. Crosshead should drive up and limit switch should stop thecrosshead.

c) Note the number on EXTENSION readout. It should be approx. 0.2 inches(5mm).

d) Lower the crosshead, using DOWN pushbutton, press STOP whenEXTENSION readout displays the same reading as a negative number.

e) Bring upper limit stop down so it again contacts the actuator, tighten itsecurely. Then lower the crosshead again about 1 inch (25 mm) and press theRETURN pushbutton. The crosshead should stop when EXTENSIONreadout displays zero. This is an accurate, respectable gage length.

6) Set lower limit stop-just beyond the expected maximum compression. Tighten thestop securely on the limit switch rod.

7) The preload nuts, located beneath moving crosshead, should be hand-tightened forcompression testing. This is necessary to prevent discontinuities in loading applied tothe specimen.

Final pretest setup:1) Set RANGE SWITCH2) Set load readout decimal point3) Set readout mode switch4) Set crosshead speed5) 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

Recording of results:Chart magnification:

When using a chat recorder, the chart speed should be selected to give a convenientrecorded 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 thechart time axis to indicate the displacement of the crosshead in either a reduced ormagnified 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 speedto the crosshead speed, that is:

Magnification ratio (M) = chart speed Crosshead speed

Operation of strip chart recorder:1) Preparation: make the following initial settings:

a) POWER switch: OFFb) Pen lift lever: UPc) CHART DRIVE switch: STOPd) 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, therecorder START button must be pressed but the chart will start and stopin relation to the moving crosshead. Also the chart speed will beproportional to crosshead speed as determined by the setting of the chartmagnification 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 acrosshead 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 enablesamples to be compared or monitored for quality control, storage and processingrequirements.

Previous use of sensory assessment panels has provided the necessary productevaluation but to use the interpretation of stress/strain plots which can be correlated withsuch sensory assessments provides numerous benefits for continuous and long termrequirements.

Texture profile analysis have been performed on a wide variety of foods includingfruits, vegetables, meats, cheese, confectionery, desserts and baked goods. It usually involvesthe compression of standard sized specimens (i.e. cubes, cylinders etc.) depending upon theproduct under test. The specimen is subjected to two successive compressive cycles (referredto as ‘bites’), which are between the same limits of displacement, and a force/displacementcurve for each bite is recorded.

In most cases a crosshead speed is selected in the range 5 to 50 mm/min. but thisdependent upon the material under test.

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 × cohesiveness6) Chewiness (kg. mm) gumminess × elasticity7) 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.

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 sampleburns, 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 heatabsorbed by the calorimeter assembly, knowledge of the water equivalent of the calorimeterassembly, and of the rise in temperature enables one to calculate the heat of combustion ofthe 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. Bomb2. Water Jacket3. Offset stirrer4. Calorimeter vessel5. Bomb firing unit, vibrator, timer and illuminator.6. Pressure gauge on stand.7. Crucible8. 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 bypellet 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.

7) Before detaching the bomb from the oxygen supply, close the O2 supply value usingstopper value.

8) Fill the caloriemeter with sufficient quantity of water so that the cover of the bombcaloriemeter is submerged to a depth of atleast two centimeters vessel to the waterjacket.

9) Load the bomb into caloriemeter vessel and electrical terminal for subsequent firingof the charge.

10) Adjust the stirrer and thermometer at their respective positions. Keep the stirringmechanism in continuous mechanism of operation.

11) Note the rate of change of temperature at equal intervals till it again becomesconstant.

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

TWhere 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

WhereW E = water equivalentT = temperature of firing (˚ c)E1 = caloriefic value of the wireL1 = length of the unfired wireE2 = caloriefic value of the threadL2 = length of the unfired thread, andW = 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 bombwhen it is submerged in water.

d) Stand back from the calorimeter for at least 15 seconds after firing.

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 requiredturbulence during combustion.

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 refractiveindex (n) of the second medium with respect to the first is given by the equation.

n = sin i/ sin rIt is a constant quantity for two given media for the same wave lengths of light at the sametemperature. The refractive index of a solution depends upon the individual moleculespresent 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 inthe older methods of measurements of refractive index.

Refractive index of milk itself is somewhat difficult to determine because ofits opacity, but by using a refractometer, such as the able instrument, which employs athin 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 formilk. The values of infractive index of milk at 40˚c are 1.3460 (1.3450 – 1.3472) for cowmilk and 1.3477 (1.3460 to 1.3490) for buffalo milk. Fat in milk does not contribute tothe 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 tothe refractive index (n) of milk:Casein = 0.0049 - 0.0060Serum protein = 0.0021 – 0.0035Lactose = 0.0063 – 0.0067Miscellaneous constituents = 0.0013 – 0.0022Fat = nothing

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

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 tubes6. Test tubes7. Beakers8. Spatulas9. 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 chloride5. 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 chillin ice contained in a beaker to facilitate the solidification of fat layer at thetop. Remove carefully with the help of a thin spatula the fat plug and takeout the skim milk carefully with the help of pipesse milk from the tube bysuction by the rubber bulb. Adjust the temperature of skim milk to 40˚cbefore 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 CuSO4in water and diluting to 1000 ml. For the coagulation, 20 ml of milk is mixedwith 5 ml of CuSO4 solution in a 6” × 1” test –tubes. The mixture is wellshaken and filtered through filter paper.b) Calcium chloride method:30 ml of milk is solution is thoroughly mixed with 0.25 ml of solution ofCaCl2 (Sp. Gr. 1.1375) in a tube, which is then closed with a cork through

which is passed a short piece of glass tubing to act as a condenser. The tubeis heated in a boiling water bath for 15 min. and placed in cold water. Anywater 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 andwhole diluted to 100ml. 20 ml of milk is mixed with 5 ml of the solution, thewhole well shaken and then filtered through filter paper and the filterate isused 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 onfilteration. The preliminary thickening should be ignored, as it is not usuallypossible 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 ofmost instruments never reach the temperature indicated by the registering thermometer, andat temperatures greatly removed from the standard temperature for the instrument, there is asmall 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 andapply 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 airbubbles. The reading shall be taken after sample has been kept in the prism for 2 to 5minute. And after it has been ensured that it has attained constant temperature by taking twoor more readings. Take care that the sample has reached the temperature of instrumentbefore the reading is taken. Before commencing through prisms a stream of water attemperature 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 afew drops of the sample on prism or if preferred, open prisms slightly by turning screw headand put a few drops of sample into a funnel shaped aperture between prisms. Close prismsfirmly by tightening the screw head. Allow instrument to stand for a few minutes beforereading is taken so that temperature of sample and instrument will be same.

Method of measurement is based upon observation of position of borderline of totalrefraction in relation to the faces of the prism of flint glass figure 6.2. Bring this border lineinto the field of vision of telescope by rotating the double prism by means of alidade in thefollowing manner.Hold sector firmly and move alidade backward or forward until field of vision is divided intolight and dark portion. Line dividing these positions is “borderline” and as a rule will not bea sharp line but a band of color. The colors are eliminated by rotating screw head ofcompensator until sharp, colorless line is obtained. Adjust borderline so that it falls on pointof 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 waterat 20˚c is 1.330. Any correction found necessary should be made on all readings.

EXERCISE 7

DETERMINATION OF ELECTRICAL CONDUCTIVITY OFMILK

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 meansmore resistance and vise-versa. In other words, conductivity = 1/R or R-1.the unit ofconductivity is mho or ohm-1.Specific resistance: (e) it is the resistance offered by conductor of 1cm in length and 1cm incross section. It is the resistance of a substance or conductor which is proportional to itscross sectional area (α) and directly proportional to its length (l). It is expressed as specificresistance = α R/IWhere R is the measured resistance (E/I). The unit of specific resistance is ohm. Cm specificconductance (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 bemeasured with the help of a Wheatstone bridge or conductivity meter.Electrical conductivity of milk: since milk contains various kinds of ions, it can conduct anelectrical current about 60 to 80 % of conductivity is due to Na, K and cl. The most normalmilk samples have conductivity in the range of 0.0040 to 0.0055 ohm at 25˚c mastitis udderhas abnormally high concentration of Na and cl.Relationship between % chloride and conductivity of milk at 18˚c can be used as a simplephysical 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 electricalconductivity varying from 4.2 to 6.9 milli mhos at 25˚c.

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 diluentsskim milk has higher electrical conductivity than whole milk.

iii. Acidity: electrical conductivity increases with an increase in acidity because thecollerdel minerals are brought into the soluble state.

iv. Dilution: electrical conductivity decrease with concentration but is slightlycompensated by increased dissociation of electrolytes.

v. Concentration of milk: electrical conductivity increases upto 30% concentration ofelectrolytes. Beyond 30% concentration, electrical conductivity decrease due toincrease in viscosity.

Determination of electrical conductivity of milk:Operation:

1) The indicator lamp glows put the ‘CAL/READ’ switch in ‘CAL’ position2) 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 tobe measured.Before dipping the cell in any solution, keep the range switch in extreme clockwisedirection (position) otherwise in the wrong range the digital panel meter will startblinking due to overflow. Reading of 112 or 113 with flickering or ‘1’ (cone) indicatesoverflow.

8) To get the specific conductivity of any solution, dip the cell in the solution (aftercleaning the cell by rising with distilled water) and turn the range switch to get thereading on the digital panel meter.

· For better results with improved accuracy it is advisable to select a range so thatreading is now full scale ie.199.

1. CAL/READ SWITCH2. ‘CAL’ control3. range switch4. 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 prepared2. 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.

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 KC l as well as to milk.

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 doquantitative 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 extentover a period of week or more, and to collect such a small amount with less degree ofattention 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 byapplying a number of vessels consecutively to the column outlet. The rate at which thevessels 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 ofchemically similar materials/substances.

- Saves chemicals, time for analysis etc. as we can select appropriate liquids/solutescontaining tubes.

- No attention of operator required. It can be operated throughout the might withoutany 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).

- In almost all the case, if last tube is over the column effluent is drained out, if we donot attain it (change it with new empty tubes) in time the whole experiment goes invain.

Design:1. A column- column with column holder is supplied. According to the type ofchromatographic 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 areavailable. These are arranged in the circular fashion or in many concentric circles or instraight through conveyor.4. Motor activating mechanism: - it is a mechanism which records the signal given from thesiphon to place the empty test-tube under siphon. It is most important part; its failure cancause mixing of two fractions or losses.5. motor- simple motor following orders from above mechanisms is installed to rotate thetest-tube rack.

The various designs of fraction collector differ mainly in the nature of the signal usedto initiate the measurement of the collection tube. Five groups may be distinguished on thisbasis.1). Time2). Volume3). Weight4). Drop counting5). 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 differentcolumn flow rates.

Usually, the container are arranged in a circle near the circumference of a circulardisc, the disc being turned at intervals by torque from an electric motor, or a clock- workmechanism, or a weight and pulley arrangement. The timing device can be an electric timeswitch 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.

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 thesedesigns 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. Itsuffers from the disadvantage that the collected liquid may remain in contact with mercuryfor 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 centralspindal lie in horizontal planes. The discs are each performed with a ring of holes near theircircumferences so that a glass specimen tube can slide through each pair of holes. This partof the apparatus is carried in a vessel containing water. The specimen tubes are constrainedin the two horizontal dimensions but they can float freely on the water at depths governedby the weight of liquid dropped into the specimen tubes when empty, the tubes ride highand are prevented from moving by the adjustable knife edge (A). Torque is applied to thecentral 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 rimeventually escapes under the knife edge and the next empty specimen tube moves up to theknife 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 apreselected number of drops. (Figure.8.4).ADVANTAGES:

- Can be used to collect very small number/ volume of fractions. G (0.5 ml).

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 forcollection of very small sample .we have installed Frakel fraction collector in thedairy chemistry department of our college. (figure.8.5).

It is a volume based fraction collector which collects predetermined volumesof liquid in a batch of test-tubes. This is achieved either with the help of a siphonbalance or a timer.

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. Thestandard solution is called the titrent, and the process of determining the volume required iscalled titration. During titration, the active component of the titrent reacts with a quantity ofsome specific substance in the solution being titrated, and the reaction in question proceedsto 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 assumedthat this point will be located at, or as close as possible to, the equivalence point of thetitration. The equivalence point can be defined as that point in tration where an amount ofthe active component of the titrent exactly equivalent, stoichiometrically speaking to theamount 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 themethod, and the means of attaining the analytical result from the method.1. Potentiometry2. Coulometry3. Electrolytic methods

a. electrode positionb. internal electrolysisc. electrosolution (electrographic) techniques.

4. Amperometry5. Conductometry6. Impedimentary7. PolarographyPotentiometry and coulometry are commonly used for automatic titrations.

Application of autotitrator 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 andbutter.2. The automated titrators are used for determination of ascorbic acid in milk and iodinevalue 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 usingtitrino 716 or metrohm 686 titroprocessor.

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 themeasurement of a constant current for a known time or of direct measurement of the totalcharge by means of an electromechanical or electrolytic coulometer. Coulometric titrationsare especially adaptable to very small quantities in circumstances where ordinary titrationswould be inappropriate or impossible, particularly where titrant is being consumed as it isgenerated 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 theabove range with a precision of + 0.1 to + 0.2 percent. The other accessories includepotentiometer, indicating electrode, reference electrode, magnetic stirrer, coulometricopposing 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 titrantsolutions. In addition to this, the method permits the use of titration reactions involvingtitrant 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 aconsiderable change in potential at the end point of the titration reaction. Polarizedelectrodes not in equilibrium with electrolyte represent a sufficient potential change at theend point to be satisfactory indicators.

Automatic potentiometric instruments can be divided into three classesi) The titration curve is simply recorded automatically and the end point is assured to be theinflections point of this curve.ii) Some provision is made for stopping the flow of titration when indicator electrodepotential reaches a certain pre-selected value.iii) An electronic circuit that essentially differentiates the titration curve and stops the flow oftitrent 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 titratorse). Malmstadt automatic titrator.

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 theprevious 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 titrationsis set on the precisior-potential dial. This potential “opposes” the potential of the electrodepair immersed in the sample solution. If the two potential are equal, the null meter will be atzero; if they are unequal the meter will deflect to the right or left depending upon whichpotential is larger. The electrode potential can be determined simply by adjusting theopposing potential with the potential dial until the meter is zeroed, and then reading thepotential 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 thispre-set value, causing the meter to deflect. This difference is amplified by integrated circuitamplifier and fed to a control circuit, which activates a solenoid value allowing the titrent todispense into the sample beaker. As titration precedes the differences between the electrodepotential and pre-set potential will narrow until close to the end point, they become equalwith no difference in potential appearing at the controller input, the solenoid getsdeactivated 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 electrodemaintains a fixed potential and all measurements are made with respect to it. The indicatingelectrode potential will change as the titration progresses.

The electrodes most commonly used in potentiometric titrations are classified aseither 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 consideredand 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 UNITi). Power switchii). MV- PH switchiii). Potential dialiv). POL –STD By – USE switch

v). Null adjust controlvi). Null metervii). Auto switchviii). Temperature controlB. STIRRERi). Power switchii). Pilot lampiii). JOGiv). 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 referencescale reading and corresponding voltage range in which potential determination can bemade. Since the titrator may be used for either millivolt or PH measurements, a separatecalibration 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 230v AC outlet. Be sure that the power cable from stirrer is plugged into the receptacle on therear of the control unit.

The end point detection for potentiometric titrations is carried out by any of thefollowing way.A. graphical determinationB. titration to the equivalence point potential.C. pinkhof –treadwell methodD. differential titrationsE. bimetallic electrodeF. 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 titrationa. silver electrode

i. titration of helide mixtureii. Chloride determination in presence of large amount of bromide or iodide.iii. Determination of silver.

b. mercury electrode- Determination of alkaloids

c. iodine electroded. platinum electrodee. Third class electrodef. membrane electrode

3). Complexometric titrationa. silver electrodeb. platinum electrodec. mercury electrode

- Titration of metal ion with E.D.T.A.

4). Oxidation –reduction titration reactionsa. potassium permagnate

- Titration of manganeseb. potassium dichromate

- Determination of carbohydrates and other organic substances.c. ceric cells

- Determination of vanadium in steelsd. potassium iodated and bromination of organic compounde. other oxidants.f. teravalent arsenic

- Determination of chromium and vanadium in steelg. bivalent iron

- Determination of manganese, cerium and chromiumh. titanous ion

- Preparation of titanous chloride from titanium hydridei. chromous ion

- Preparation of standard chromous solution- Determination of nitrate ion

j. other reductants.

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 likevaccines, blood plasma, sera, living bacterial and virus suspensions, cultures, therapentic andprophylactic preparations, antibiotic, fruitlets and food stuff etc. can be dried to lowmoisture contents; thus increasing their shelf life, and preserved.

Lyophilization or freeze drying is the total process of freezing and sublimation of thewater from the frozen preparation. The product is frozen and then exposed to anatmosphere to an of low relative humidity when the contained ice sublimes that is, it changesdirectly from the solid to the vapour state without melting, thereby avoiding the chemicaland enzymatic changes usually associated with other forms of drying. The term lyophilizer isapplied 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 plugfreezing, shell freezing, evaporative freezing and pelletizing.the different types oflyophilizes in common use are of the glass, metal, do – it- yourself and commerciallyavailable types. The lyophilization process for preservation of microorganisms isdepicted 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 fordrying polysaccharides to obtain a surface active form.

4. For preparing biological objects for election microscopy. Freezing makes it possibleto fix the preparation instantly without any change of the initial conditions.

5. Freeze drying, freeze fracturing and etching and low temperature electronmicroscopy of frozen hydrated specimens are methods for studying cytofixedmaterial. In case of screen lipoproteins, human chylomicrons, VLDL, LDL, apoLDL, HDL and normolipidemic and hyperlipidemic rhesus men key LDL have beenthe subject of freeze – fracture and freeze – etching electron microscopic studies.

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 estimationof added rennet whey solids to dried whole milk using microscopic method anddetermination 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.

EXERCISE 11

DEMONSTRATION OF PH METER

Introduction:Definitions1. Acid and base:

The modern concept of acids and bases developed by Bronsted, Lowry and othersdefines acids as proton donors and bases as proton accepters. Each acid therefore has aconjugate base.Acid Base + H+

2. Alkali:The term alkali is reversed for those compounds that yield hydroxyl ion on

dissociationKOH 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 ionconcentration instead of the use of cumbersome numbers. PH is strictly defined as thenegative logarithm of the hydrogen ion activity, but in practice the hydrogen ionconcentration is usually the same as the activity except in strongly acid solutions.

From conducting measurements, water has been shown to be very weekly ionizedand 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 byK = [H+] [OH-]

H2ONow the concentration of water to all intents is constant so we can write:

KW = [H+] [OH-]

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 7.9725 1.03 × 10-14 = 10-14.00 7.0037 2.51 × 10-14 = 10-13.60 6.8040 2.95 × 10-14 = 10-13.53 6.7175 16.90 × 10-14 = 10-12.77 6.39100 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 theconjugate 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) = 10.010 mol/ lit NaOH, [H+] = KW

= 10-14 = 10-12

Therefore PH = - log10 (10-12) = 12

Weak acids:The Henderson – Hasselbath equation:

Weak acids are only slightly ionized in solution and a true equilibrium is establishedbetween the acid and the conjugate base. It HA presents a weak acid, thenHA H+ + A-

According to the laws of mass action Ka, the acid dissociation constant is definedas:Ka = [H+] [A-]

[HA]And [H+] = Ka [HA]

[A]Taking negative logarithm- log10 [H+] = - log10 Ka + - log10 [HA]

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

[Acid]

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 FHAWhere 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 termof apparent dissociation constant pka’PH = pka’ + log CA

CHAThis 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 ionicconcentration.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. Theseare 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 equationPH = 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 baseequilibrium 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 neededinclude:

1. Reference electrode2. Indicator electrode (PH sensitive).3. Voltmeter or amplifier4. The sample being analyzed (fig. 11.1).

Hydrogen ion concentration is determined by the voltage that develops between the twoelectrodes, the nearest equation relating electrode response to activity.

The saturated calomel electrode is usually used as the reference electrode, while theglass electrode is used as an indicator electrode for PH measurements. Combinationelectrodes (reference + indicator) are also available, being of value when the volume of

sample is limited. Department of dairy chemistry of our college has the following instrumentfor PH measurements.

1. Systronic PH meter type 3312. Systronic PH meter type 3223. ELICOS digital PH meter, model Li – 1204. 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 repeatability7. 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 withthat of PH needle.

4. Rinse the electrodes with distilled water, immerse in the test solution, set properrange (0-8 or 6 to 14), note the PH, put the switch on ‘nent’ and remove theelectrodes.

EXERCISE 12

DEMONSTRATION OF COLORIMETER

Introduction: Many biological experiments involve the measurement of a compound or group ofcompounds present in a complex mixture colorimetry is the most widely used methodfor determining the concentration of biological compound and makes the use of theproperty that when light passes through a colored solution, some wavelengths areabsorbed more than others. May compounds are not colored but are made to absorblight in the visible region by reaction with suitable reagents. The big advantage is thatcomplete isolation of the compound is not necessary and the constituents of complexmixtures such as blood can be determined after little treatment. Colorimetric techniques may be used for either qualitative or quantitativemeasurements. Qualitative measurements are based on the premise that each analyte hasa 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 emissionspectrum of the analyte. In contrast, quantitative assays are based on measuring theabsorbance and/or florescence of the analyte at one wavelength. Quantitative absorptionassays are based on the premise that the absorbance of the test solution will be afunction of the solution analyte concentration. Under optimum conditions, there is a direct linear relationship between a solution’sabsorbance and its analyte concentration. The equation describing this linear relationshipis 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 orenergized by a constant voltage transformer forms the light source. This light passesthrough a selected filter and a centrally opening shatter, controlled by a wheel, the dm ofwhich protrudes out. This light passes through the test-tube containing the solution andfalls on a sensitive photoelectric cell. The current generated by the photo electric cell isamplified by a transistor amplifier which drives 50 micro ammeter calibrated in terms ofpercent transmission and optical density. The transistor amplifier is in corporate mainlyto 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 isillustrated in figure 12.3.

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

Colors of solution colors of filter

Orange blue greenYellow bluePurple greenRed blueViolet yellow-greenGreen purpleBlue-green red-orangeBlue 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 filter4. Insert a test-tube containing ‘blank’ solution in the sample socket5. 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.

EXERCISE 13

DEMONSTRATION OF SPECTROPHOTOMETER

Spectrophotometer consists of a light source, a monochromator (device to produce amonochromatic beam of light from any point on the spectrum), and a vessel containing thesolution and positioning it in the light beam, and a detector with its amplifier. The lightsource used for the visible spectrum is the tungsten lamp, usually a single filament carheadlamp 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 isprovided by an accumulator or car battery. A constant voltage transformer or stabilizedpower pack is used in most instruments. The source of u.v.light is the hydrogen ordeuterium discharge tube which supplies light of 180-350 nm. A stabilized power supply isessential for these lamps. The tube has a quartz window and when adjustments are made thisshould not be touched. If a more intense source is required the mercury arc or xenon lamp isused.

Infra-rod radiation is supplied by a Nemst filament, rod of oxides of zirconium andthorium, which requires a heater winding for starting, the operating temperature being about1350˚c .a stabilized voltage is required.

The visible or u.v.type employs either a glass or quartz prism or a diffraction gratingin the monochromator. Some instruments are both prism and grating to improve resolvingpower. When working with the u.v.region, silica cells must be used as glass is not transparentto 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’ controlswhich allow the balancing of the electronics with no light entering the photo cell. This isdone after a wavelength has been selected. With a reagent blank in the light path thegalvanometer or potentiometer is set at zero. The blank is then replaced by the test solutionand the absorbance or transmission is noted. The instrument should be zeroed with theblank in position with each successive wavelength.

In the dairy chemistry department of our college, following spectrophotometers havebeen installed.1. Gystronics photoelectric colorimeter type 101, and2. Spectronic -20- colorimeter- spectrophotometer, (figure 13.3).

Types of spectroscopy:1. visible spectrophotometry2. ultraviolet spectrophotometry3. infra –red spectrophotometry4. circular dichroism spectrophotometry5. spectrofluorimetry6. luminometry7. atomic/flame spectrophotometry8. electron spin resonance spectrophotometry

9. Nuclear magnetic resonance spectrophotometry10. Mass spectrometry etc.

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 theseinstruments are available ranging in capacity from those handling 0.2 ml and less to thoseaccommodating thousands of liters with relative case. Some are crudely controlled withregard to speed and temperature whereas in other these parameters are regulated withinlimits of less than 5%. In its simplest form a centrifuge is composed of a metal rotor with holes in it toaccommodate a vessel of liquid and a motor or other means of spinning the rotor at aselected speed. All the other parts found in today’s modern centrifuge are merelyaccessories used to perform various useful tasks and maintain the environment withinwhich the rotor operates.

Principle: Centrifugation separation techniques are based upon the behavior of particles in anapplied centrifugal field. Particles which differ in density, size or shape sediment atdifferent rates in a centrifugal field. The rate of sedimentation of a spherical particle isdependent upon the applied centrifugal field, density, radius of the particle and theviscosity of the suspending medium. These relationships are expressed by stoke’s law. U= 2/9 r2p (ℓp - ℓm) × g

nWhereu = sedimentation rate or velocity of sphereℓp = density of the particleℓm = density of the suspending mediumr p = radius of the particlen = viscosity of the suspending mediumg = gravitational field2/9 = shape factor constant for a sphere.

If the density of medium and density of particle are equal, then the sedimentation rate will bezero. But this sedimentation rate will also depend upon its sedimentation rate. Hence, even ifthe particles have similar density, slight differences in their size can make large differences intheir sedimentation velocity.

Classification:Centrifugation techniques can be mainly classified into two types namely

a. preparative centrifugation andb. analytical centrifugation

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 fromtissue culture and plasma from blood. Relatively large amounts of cellular particles may alsobe isolated in order to study their morphology, composition and biological activity. It is alsopossible to isolate such biological macromolecules as DNA and proteins, from preparationswhich 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 furtherclassified into four groups.

i. differential centrifugationii. Rate zonal centrifugationiii. Equal density or isopycniciv. 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 fractionsby the step wise increase of applied centrifugal field. The centrifugal field at each stage ischosen so that a particular type of material sediments, during the pre-determined time ofcentrifugation to give a pellet. At the end of each stage the pellet is washed to obtain a purefraction. The sedimentation of particles during centrifugation varies depending upon theirsize and shape.

Differential sedimentation of a particular suspension in a centrifugal field is shown infigure 14.1.

It is the most commonly used method for the isolation of cell organells fromhomogenized tissue. The main demerit is that the liquid density gradient has to beperformed 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 samplesolution containing particles to be separated, on top of a performed liquid density gradientwhose density continuously increases towards the bottom of the centrifuge tube. The sampleis 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 orbands consisting of particles characterized by their sedimentation rate (figure 14.2). Here, toachieve separation density of particles must be higher than the density gradient.

This method is used for the separation of RNA-DNA hybrids, ribosomal subunitsand other cell components.iii. Equal density (isopycnic) centrifugation:-

Here density gradient may or may not be used. In the absence of a continuousdensity gradient, the sample is initially centrifuged to sediment particles heavier than theones required. After separation and rejection of heavier particles the sample is suspended ina medium having a density equal to the fraction to be isolated. After further centrifugationthe lighter and heavier particles than the required ones will be at the meniscus and base ofthe tube respectively while the required one will be in an intermediate position (figure 14.3).

In the presence of density gradient it will be same as that of rate zonal method. But the maindifference between the two methods is that for equal density centrifugation, thecentrifugation is continued until the desired particles have reached their equal density orisopyenic position in the gradient. This is not required in rate separation. Equal density or isopycnic position: - means where the buoyant density of the particleand 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 orrubidium or sucrose is used. The sample is mixed homogeneously. The sample is mixed witha concentrated solution of cell and centrifuged. This gives a concentration gradient of Cs c land hence a density gradient due to large mass of Cs ion. Redistribution of particles thentakes place in this gradient.

This is most commonly used in analytical centrifugation. Also used to separate andanalyze human plasma lipoproteins.

Types of preparative centrifuge:Preparative centrifuge can be classified into three major groups:

1. General purpose centrifuge2. High speed centrifuge, and3. Ultra centrifuge

1. General purpose centrifugeGeneral 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 generallyequipped with refrigeration equipment to cool the rotor chamber. Two types are there. Thefirst is relatively simple, high capacity, continuous flow centrifuge. If consists of a very fastelectric motor, which is connected to a long tubular rotor. This is used for the harvesting ofyeast or bacteria from large cultures.

The culture is siphoned or pumped into the bottom of the spinning rotor. As thecultures move up towards the top of the rotor, microorganisms are sedimented against therotor walls and the clarified culture medium exits through an effluent part. After all themedium has been passed through the centrifuge the rotor is disassembled and the comparedcells are removed with long spactula.

The second type consists of lower capacity refrigerated equipment. A broking deviceis also used here to decrease the time required for rotor declaration at the end ofcentrifugation, 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 controlb. temperature controlc. vacuum system, andd. rotors.USES

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

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 ofmaterial and even their selling price.

Water activity (aw) or equilibrium relative humidity (% ERH) expresses the activepart of moisture content or “free water “as opposed to the total moisture content which alsoincludes “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 wheremoisture content is of less interest.There are various techniques to measure aw. a commonly used approach relies on measuringthe 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 smallclosed chamber at constant temperature, and a relative humidity sensor is used to measurethe ERH of the sample atmosphere after equilibrium. A simple and accurate variation of thisapproach is the chilled mirror technique, in which the water vapour in the head spacecondenses on the surface of a mirror that is cooled in a controlled manner. The dew drop isdetermined by the temperature at which condensation takes place, and this determines therelative 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 thewater 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 specifiedtemperature.

aw = p/po

Water activity can also be defined in terms of solute concentration through its relation toRaoult’s law

aw = p/po = n2/n1 + n2In which n1 & n2 are number of moles of solute and solvent respectively. Water activity alsois related to the osmotic pressure (o.p).o.p = -RT log e aw/vWhere R is the gas constant, T is the absolute temperature and v is the partial molar volumeof water.aw is the relationship between this parameter and equilibrium relative humidity or ERH.ERH (%) = Aw × 100Water activity (or % ERH) indicates the degree of freedom of water absorbed in a materialand shows better than moisture content does, the effect of this water on physical properties,

such as dimensions, structure, cohesion, agglomeration properties, electrical and chemicalproperties.

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 hasvery little to do with this).

Water activity in foods can be controlled by using various additives (e.g. Salts, sugarsetc.), by using satisfactory packing materials, by maintaining favorable maturation andstorage conditions. Water activities measurements are increasing in food research anddevelopment as well as in ‘production quantity control’ on line measurements are possible toa 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 foodstuff, 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 sloweddown at aw values below 0.8.4. Calculating and controlling drying processes also requires the knowledge of therelationship between aw and % water (sorption – isotherm). The moisture content and aw ofsome 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 interpolationThe aw peramoter is the equilibrium relative humidity or aw at which a substance neithergains nor looses moisture at a specific temperature. This concept may be used toestimate, 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 agiven period of time, usually 1 or 2hrs. If the amounts of water gained or lost by theseveral aliquots of the sample maintained in different RH are then plotted against aw, thisplot will interest with the line representing zero moisture change. It is this point ofinterpolation that represents aw of sample.2. Bithermal equilibrationI this technique, the vapour pressure of a solution am measured by a method thatdepends on the vapour phase equilibrium of the solution at 25˚c with pure water atlower 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. Theimmersed in the 25˚c water bath contains the sample and the other bell at a lowertemperature, surrounds a dish of pure water. The temperature of each water bath ismeasured with great accuracy by means of a copper constant thermocouple. Thedifference in temperature of two baths is calculated following equilibrium and this istaken as the temperature of sample and pure water. The concentration of water in thesolution to be determined and vapours pressure of both solution are read from

established tables.aw is then calculated from the ratio of two vapours pressure as notedabove. The method is cumbersome and lengthy. It was originally used to determine the awof 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 constanttemperature. 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 attachedthrough a trap to a simple manometer. The manometer assembly (figure 15.2) is thenevacuated and during this time the sample chamber is maintained out approximately -80˚c. Following evacuation, the sample is warmed to room temperature, while one sideof manometer is maintained at essentially zero pressure. The fluid level in the samplearm of the manometer is then deflected by increase in pressure caused by vapourpressure of the sample itself. Air entrapped in the sample also contributes to deflectionof the manometer, but this can be compensated for relatively easily. Humiditydetermination can be carried out on the same sample at various moisture levels byallowing 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 hairabsorbs 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 indicatinglever arm on the other end and allowed to come into equilibrium with the substance tobe measured, ERH can be read directly.5. Isopiestic hygrometry(Isopiestic equilibration) like hair hygrometry, this technique requires only veryinexpensive equipment. With this procedure, the weight gain of some absorptive materialsuch 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 asaturated solution and each at different aw levels. From this the standard sorptionisotherm is calculated. The unknown to be measured then is placed in desiccators withone of the absorptive material for a given time, usually 24 hr and the weight gain at theend of this period is compared to the standard isotherm. Comparisons between aw levels of various food determined by this technique and byelectric hydrometric procedure has given good correlation at aw levels > 0.09 andsuperior precision has been claimed at aw levels < 0.90.6. Electric hygrometryRatronic 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 suspendedinto an insert carrier such as glass wool. A current potential difference of 25v, whichheats the cell, is provided across the solution. The water vapour pressure (WVP) ofsolution rises and upon reaching the WVP of environment, water evaporation occurs.The dried lithium chloride residues remaining after evaporation no longer conductscurrent and heating ceases. As the residue cools, water is once again taken up from theenvironment and the cycle is repeated at reduced amplitude. Eventually, a temperature isreached at which the WVP of environment. This temperature is then measured andrelated to the WVP of saturned lithium chloride and hence the environment from whichthe ERH can be calculated.

The other method used for measuring water activity includes:7. Freezing point depression8. Dew point methods, and9. Chemical methods

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 areconfined to a single direction or plane. Polarized light may be obtained by passing lightthrough a nicol prism. The instrument by which optical activity of a liquid is determined byinserting nicol prism (figure 16.1) in the path of a ray of light before and after passingthrough a liquid is called polarimeter. This instrument is chiefly used to determine the opticalrotation of a solution. A beam of monochromatic light is passed through nicol prism toproduce 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 theseprisms are placed at right angles with no optically active material intervening, the only lightpassed by first prism is stopped by the second prism and the rear field appears dark. Whenoptically active material is introduced between the two prisms, light appears and the fieldbecomes 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 mustbe rotated to the right. For leavo rotatory substances it must be turned to the left. The extentof optical rotation depends upon several factors including the nature of the substance, itsconcentration, the length of column through which light passé, wave length of light, andtemperature of liquid. The measurement of rotation can be employed as a method foranalyzing the sugar the optical rotation of a known concentration of a particular sugar isdetermined and is expressed as a physical constant in terms of specific rotation. Specificrotation is defined as rotation in angular degrees of the plane of polarized monochromaticlight produced by a solution of optically active substance having a concentration of 1gm/mlsolution 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 degreesl = length of column of solution in decimeterc = 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 eyepiece facing the operator.2. Open the hinged trough, remove the drum illuminator packed there in, and fit to thesocket 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 scalewithin the head.

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 controlwheel to back and forth and obscure the darkening halves. The balanced position betweenthese two halves is used in all measurements. At this position the scale should read zero andmicrometer should be set at zero. If not the procedure given in “adjusting zero” should befollowed.8. Fill the sample into the tube, place in the through, close the cover and observe in the fieldtelescope. 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 bubblesenter. 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 nowreleased from the screw. Turn the screw by means of knurled wash until the index linecoincides 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 errorwithin the scope of micrometer drum.

EXERCISE 17

MANOMETER (demonstration)

Manometry can be used to measure either uptake or evolution of both co2 and o2whereas o2 and co2 electrodes simply monitor changes in o2 and co2 levels, respectively, albeitwith greater sensitivity. Manometry also has the distinctive feature of allowing thesimultaneous determination of o2 and co2 exchange and has the advantage that themagnitude of the exchange is independent of the partial pressure of the gas at the beginningof the experiment manometric studies are carried out in a small flask attached to some ofmanometer 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 120oscillations min-1 to ensure that respiratory gas exchange is not limited by diffusion of gasinto the liquid phase. The total volume of liquid in the flask should not generally exceed 4cm3 because of gas diffusion limitations. Two principle types of manometer are the Warburgconstant volume manometer and Gilson constant pressure manometer, which are illustratedin 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, withattendant problems of homogeneity of gas supply and artifacts, in studies of respiratorycontrol, and the effect of inhibitors on mitochondrial respiration. When used forphotosynthetic studies a control determination in darkness should be performed and thepartial pressure of the gas kept constant during the experiment.

EXERCISE 18

FLAME PHOTOMETRY (demonstration)

Flame photometry is a technique by which concentration of ions (Na+, K+) of someelements in the given solution is determined by the measurement of light emission onintroducing. The ionic material into a non luminous flame. Emission of such characteristicradiation by each element forms the basis of flame photometry. The test solutionscontaining these elements are introduced as fine sprays into the flame. A flame photometeressentially consists of a burner, an atomizer which disperses the solution as a fine spray intothe 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 underexamination, a photocell converting the light energy into electric current, an amplifier toamplify the feeble current and a galvanometer to measure it.

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 insolution as electrically charged species, either as cations (+) or anions (-). Even typically non-polar substances such as carbohydrates can be given weak charges by derivatization, forexample as borates or phosphates. Moreover, molecules which have a similar charge willhave 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 theions in solution are subjected to an electric field. This is the principle of electrophoresis.

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 agargel or in a liquid-filled buffered matrix such as cellulose acetate. Positively chargedsubstances travel toward the cathode (negative electrode); while negatively chargedsubstances go toward the anode (positive electrode). Different substances move at differentrates 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 alsovaries depending on the molecular weight. Proteins have a net negative charge at a pH above4.6 and hence they move towards the anode (positive electrode) when subjected under anelectric field under alkaline conditions. Due to differences in the charge of individual proteinfractions, their migration rates also differ and hence separate out as distinct bands. Thesebands are stained with dye amido-black, destained with 7% acetic acid and observed for theelectrophoretic 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 presentto initiate and control the polymerization. The gel mixture is allowed to polymerize in smalltube (or Perspex) bottles with rubber cap. Layer of water is placed on the top of the gel toensure flat surface and also to exclude O2 which inhibits the polymerization.

Davis and Ornstein (1959) and Raymond and Weintranb (1959) were the first to usePAGE instead of starch gel electrophoresis. The material used had a trade name of cyangum 41. it consisted of a mixture of acryl amide in proportions such that a gel could beformed from 3-10 percent of n-N1 methylene bis acryl amide most appropriate for gelformation was composed of 60 parts of acryl amide to 1.0- 1.013 parts of N –N1 methylenebisacrylamide. This composition has been given by cyanogum - 41.

Polyacrylamide gel electrophoresis (PAGE) is superior to other electrophoreticprocesses such as paper and starch gel electrophoresis due to the following reasons:

- Transparency of gel permits direct measurement of pattern by Tranquitting lightphotometry through gels.

- Gels can be dried to thin flexible films. Rehydration to original volume is possible byimmersion 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).

- By incorporating a detergent SDS (sodium dodecyl sulphate) proteins can beseparated and their molecular weights can also be calculated. Hence heterogeneity ofproteins can be studied using SDS – PAGE.

PAGE is most commonly used to separate out various fractions of milk proteins like αlactallumin, 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 arebetter separated under acidic conditions, particularly the β – casein variants A1, A2, A3etc.

Protocol for performing PAGE:1. Preparation of acryl amide stock solution Acrylamide 5.55 gm Bisacrylamide 0.15 gmDissolve in 25 ml distilled water. Acryl amide stock solution is also available as cyanogum 41. The ratio of acrylamideto bisacrylamide is 60: 1.013 which is the most preferred one by several workers. 1. Acrylamide 22.20 gm 2. Bisacrylamide 0.6 gmDissolve 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 gmMake the volume upto 2 litres. Glass distilled water is preferred for adding to make upthe volume to a litres.3. Ammonium persulphate (1 % solution) 100 mg ammonium persulphate/ 10ml for vertical gel electrophoresis 30-50 mgammonium persulphate solution for cross electrophoresis/ 10 ml prepare the solutionfreshly.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 gNH2 Tris 0.3 gC = 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 TEMED4. 1.5ml of ammonium persulphate solution5. Preparation of protein samples1.25 mg casein +2. 1 ml solubilizing buffer of the composition: Glycine 1.44 gm

Tris 0.3 gm Urea 42 gmDilute 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 usedinstead 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 together8. Destaining in 7 % acetic acidGlacial acetic acid 35 mlDistilled 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 thedecolorization rate, methanol acetic acid can be used instead of 7 % acetic acid as adecolorizer.

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 atthe 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 adjustedconcentration at the top of the tubes with the help of a pipette after filling electrode bufferon both the trough containing the +ve and –ve electrodes.5. pass current of 1 miliampere/ sample till the tracking dye comes out from the capillariesinto the buffer from below. It took approximately 60-70 minutes for the cheese, K-caseinand whey protein samples to migrate from the –ve to the +ve pole as indicated by themovement of the tracking dye, amido black.6. Dissemble the capillary tubes after stopping the current. Take out the gel column from theglass tubes with the help of syringe and needle assembly in a trey filled with water. Transferthe gels into labeled test-tubes and fill the individual test-tubes containing the gel columnwith staining solution i.e. 1 % amido black in 7 % acetic acid. Ideally, the staining is done for1 hour, however in our case staining was done overnight.7. Remove the staining solution after the prescribed period and decolorize with 7 % aceticacid in the same test-tubes. Change the decolorizer at regular intervals as indicated by the

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

EXERCISE 20

STARCH GEL ELECTROPHORESIS

Electrophoresis is the study of the movement of charged molecules in an electricfield. Electrophoretic techniques can be classified as either moving boundary electrophoresisor 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 supportingmedium was the first gel medium to receive attention. In these techniques, a slab gel isprepared from potato starch paste or hydrolyzed starch and is layered on a horizontal glassplate or Petri plate. The sample is placed in a well cut in the slab, and the plate is subjectedto a voltage. After electrophoresis, the starch slab is stained for visualization of the samplecomponents. The enchanted resolution achieved by starch gels is probably due to molecularsieving effect and to reduced diffusion because of a more rigid network. Starch gels are stillused 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 amineH2N (CH3 OH)3 = 6.897 g2. Citric acid = 0.7881g3. Distilled water = 750 mlFor 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 g3. 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 underalkaline conditions and they separate out on the basis of difference in charge and a betterresolution is obtained.

However, some proteins like immunoglobulin, whey proteins etc. are denatured atthe alkaline pH employed. Hence, their mobility is reduced due to denaturation andaggregation 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 bufferis used for their separation.

The composition of the buffer is as follows:1. Boric acid = 18.552 g2. NaOH = 2.0 g3. β- mercaptoethanol = 2 to 4 drops per 100 ml4. 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.

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 structureβ α

H S – CH2 – CH2 OH3. 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 ratiovolume /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 byamylases to a certain required size. Better reproducibility and accuracy are obtained andhence hydrolyzed starch is preferred to ordinary starch for research work. Starch is taken inthe 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 ml2. Water = 17 ml3. Urea = 9.7 g4. 2 – mercaptoethanol = 0.12 mlFirstly, 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 startboiling. 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 caseinswhile 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 carriedout. Then the prepared gel is added uniformly to Petri dishes and is allowed to solidify forovernight 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 freefat from milk, the fat plug is pierced with the help of a pointed and of glass rod and milk istaken out from side.

3. to the skim milk obtained this, 1 ml of 10 % acetic acid and 1 ml of 1N sodiumacetate is added to bring the pH to 4.5. On shaking the contents, precipitation of caseinoccurs. 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 excessacid. Then wash casein 2 times with 5 ml acetone. Acetone in 5 ml quantity is added to the

casein, the curd formed is broken, contents are centrifuged and acetone is decanted. Thisprocess is repeated. Dry the obtained casein powder on a filter paper. The casein obtainedthus can be used immediately.

5. For prolonged preservation, casein is washed with acetone as well as either twotimes each, and then it is dissolved in veronal buffer which contains urea.Composition of veronal buffer:1. Sodium barlitone = 20.618 g2. Barlitone = 3.684 g3. Distilled water = 2 litresVeronal 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 buffer2 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 arehanged on thread with help of U pins. Casein is applied on to these filter paper strips and isdried with a hair- dries. Casein layer is applied thus for three times. And subsequently thestrips 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 milliamperes current. From the power supply given.

After 1 ½ hours, (as detected by the movement of tracking dye put on to the filterpaper initially) the plates are removed and amide black staining solution is added to theplates and kept for 10 minutes. Then the dye is replaced with the washing solutioncontaining methanol, acetic acid and water in the ratio of 5:1: 5 until the background is clear.Subsequently, the components separated are identified.

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 fractionalprecipitation, distillation, and crystallization have been used in the separation andpurification of chemical compounds. These methods worked quite successfully in manycases but some difficulties arose where the individual components of the compounds hadsimilar physical and chemical properties. For example, fractional distillation method could besafely used in case of mixture of liquids which have a good range of boiling pointdifferences. However, this method could not be used in the case of liquids as well as raregases 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 usuallyleft with a number of compounds such as a mixture of amine acids which are similar inproperties to each other. In such cases, the extreme of temperature, pH, organic solventsand the use of oxidizing and reducing agents are avoided as these may irreversibly change thestructure of the molecules and destroy their biological activity.

Such complicated separations were techniques of chromatography and paperelectrophoresis. These methods resolve the individual components under relatively mildconditions and utilize differences in the basis physical properties of the individual molecules.Such as their mass, size, shape, charge and absorption properties. Paper electrophoresismethod has an edge over paper chromatographic methods, as in the latter, substances withlow distribution coefficients are not properly separated. Such difficulties are not faced withthe techniques of paper electrophoresis which involves migration of charged substancesunder the influences of electric current. Paper electrophoresis is an incomplete form ofelectrolysis which is widely used in the separation of biological material and many other rareand costly substances in the compounds or a mixture of compounds where some ionize andothers do not, the degree of separation can be effectively made by the use of paperelectrophoresis.

This method owes its development to the painstaking research of Arne WilhelmTiselius 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 isplaced in a vessel made up of anodic and catholic compartments in such a manner that thetwo 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 themiddle portion of the order of 2-10 V/cm. Is then passed through the solution. Buffersolutions serve the purpose of conductors and the wet paper as a connecting bridge betweenthe two compartments. The substances move either to the cathode or anode, depending onthe nature of the charge which they possess as shown in figure.

The movement of the substances is expressed by eletrophoretic mobility (µ) which isdefined as.

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 effect4. Diffusion, and5. electro- osmosis

Various procedures commonly used for performing paper electrophoresis include1. Sandwich2. Ridge pole3. Solvent immersion, and4. 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 % ureaSodium barlitone 20.618 gm(C2 H5)2 CONH CONa CO

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

Distilled water 2.0 litresAdd 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 caseinAdd 5 ml of veronal buffer with 10 % urea to 100 mg of dry (pH 8.6) casein.

4. Acetic acid, 0.5 % solution

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 byblotting paper and place the strips on eletrophoretic tank horizontally. Apply samples at adistance of 8 cm from the cathode end.

Two percentage casein in 0.1 ml quantity is spread in the central portion of thestarting line of paper strips.

2. Run these samples for 6 hours at room temperature, keeping the voltage constantat 220 in veronal buffer, pH 8.6. Six strips can be run at a time. Keep the current flowbetween 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.

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 coloredsubstances into individual components. Since then, the techniques has undergonetremendous modifications so that now a days various types of chromatography are in use toseparate almost any given mixture, whether colored or colorless, into its constituents and totest the purity of these constituents.

The name chromatography (Greek chrome, color and graphy, writing) means colorwriting.

Chromatography may be defined as a technique is the separation of the componentsof mixtures by a continuous distribution of the components between two phases, one ofwhich is moving part the other (figure 22.1). The systems associated with this definition areas follows:

i. a solid stationary phase with a liquid or gaseous mobile phase andii. 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 twoprinciples 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 isbased on the differences in the adsorption coefficients. In this the fixed phase is asolid, e.g. alumina, magnesium oxides, silica gel etc. the solutes are adsorbed indifferent parts of the adsorbent column. The adsorbed components are then elutedby passing suitable solvents through the column. Adsorption techniques, representedby ion – exchange chromatography, are most effective when applied to theseparation 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. Partitionchromatography may involve direct extraction using two liquids, or it may use a liquidimmobilized on a solid support as in the case of paper, thinlayer, and gas –liquidchromatography. For partition chromatography, the stationary phase in figure 22.2. Consistsof inert solid particles coated with liquid absorbent. The distribution of solutes between thetwo phases is based primarily on solubility differences. The distribution may be quantified byusing the partition coefficient, KD.

KD = concentration of solute mobile phase Concentration of solute in stationary phaseAlso, the similarity or identity of unknown compound as compared to standard can beestablished 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 theidentity 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 ofthe 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 stationaryphase and a polar mobile phase, is known as reversed – phase chromatography. Polarhydrophilic substances, such as aminoacids, carbohydrates, and water – soluble plantpigments, 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 identificationof 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 obtainpurified material for further characterization and study, in this type of technique the layer ofgel 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 abiological sample; however, they may be used to evaluate any physical, chemical, orbiological characteristic of a biomolecule or biological system. Here, the layer of gel or paperused is kept thin e.g. what men filter paper no. 1 is used.

On the basis of solute material used, chromatography has been classified as1. Liquid – liquid chromatography2. Gas – liquid chromatography3. Solid – liquid chromatography and so on.

Depending on the support material used chromatography has been classified as paper, thislayer, column chromatography or gel permeation chromatography etc. the column used incolumn chromatography may be calcium carbonate, sephadex, resins, DEAE cellulose,hydroxyl methyl cellulose alumina etc.

Depending on the method of development used, chromatography can be classifiedas ascending or descending chromatography.

If chromatography is run in one direction then it is called unidirectionalchromatography, and if run in two dimensional chromatography.

Depending upon the type of spot material, chromatography can be classified ascircular, paper strip and so on.

Thus, there are numerous chromatographic techniques based on the principle ofeither adsorption or elution.

Different methods for separation as well as type’s chromatographic techniques arelisted in table 22.3.

Chromatographic separations in practice may take one of three forms: columnchromatography in which the stationary phase is packed into glass or metal columns; thinlayer chromatography in which the stationary phase is thinly coated onto glass, plastic or foilplates; and paper chromatography in which the stationary phase is supported by the cellulosefibers of a paper sheet. Each of these three forms of chromatography has their specificadvantages, applications and mode of operation.

Column chromatography is the method used for the isolation and preparation ofcompounds, whereas paper or thin layer chromatographic techniques are used for analyticalwork.

EXERCISE 23

COLUMN CHROMATOGRAPHY

Separation of compounds by column chromatography must be one of the mostwidely 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 ofmaterial. 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 ofpretreatment 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 haveto be obtained in the required ionized form by washing.

During the equilibrium with solvent the material is allowed to settle and the fineparticles remaining in suspension are removed by decantation (figure 23.2). it this is notcarried out, the flow rate of solvent down the column will be considerably reduced due toclogging 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 poureddown 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 untilthe column is the required height. The column is then washed thoroughly with solvent andthe 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 iscarefully pipetted onto the surface and the tap opened until the top of the column is justbelow the level of the meniscus. The solvent reservoir is connected, and a constant head ofliquid 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.

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 thecompounds being examined and an elution profile prepared of the amount eluted against theeffluent volume.

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 dryingthe paper, the portions of the compounds present in the mixture are visualized by a suitablestaining reaction. The ratio of the distance moved by a compound to that moved by acompound to that moved by the solvent is known as the RF value and is more or lessconstant for a particular compound, solvent system and paper under carefully controlledconditions 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; theresolution is, however, inferior to what man no.1. For a rapid separation, what man nos 4and 5 are convenient, although the spots are less well defined. The paper may beimpregnated with a buffer solution before use or chemically modified by acetylation. Ionexchange papers are also available commercially for separation of lipids and similarhydrophilic 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 solventscontain 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, fastenedwith a paper clip and stood in the solvent. The arrangement is contained in an air tight tanklined with paper saturated with the solvent to provide a constant atmosphere and separationsare 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, theRf values cannot be measured with a standard reference compound such as glucose, forexample in the case of sugars.

Rg = distance moved by compounds × Distance moved by glucose

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 avolatile solvent. Viewing under ultraviolet light is also useful since some compounds whichabsorb strongly show up as dark spots against the fluorescent background of the paper.Other compounds show a characteristic fluorescence under ultraviolet light.

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 uniformthin layers of sorbent or selected media are used as a carrier medium. The sorbent is appliedto a backing as a coating to obtain a stable layer of suitable size. The most common supportssuch as plastic sheets and aluminum foil are also used. The sorbents most commonly usedare: silice gel, alumina, kieselglhr (diatomaceous earth), and cellulose.

The term sorbent is used in a general sense to include layer materials that may beused for either adsorption or partition chromatography. The standard size for TLC plates is20 × 20 cm. For most separations, the mobile phase is allowed to travel on the layer for adistance 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 ofthe 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 aclosed tank containing a small amount of the mobile phase.

The nature and chemical composition of the mobile phase is determined by the typeof substance to be separated and the type of sorbent to be used for the separation. Thecomposition of a mobile phase can be as simple a single, pure solvent (such as benzene usedto separate dyes on alumina) or as complex as a three – or four –component mixturecontaining definite proportions of chemically different substances, such as a 1: 1: 1: 1solution of n –butanolethyl acetate –acetic acid –water used to separate amino acids on silicagel.

Capillary action causes the mobile phase to travel through the medium in a processcalled development. Ascending development is the most common, but horizontal,descending, and centrifugal methods have also been reported. After the plate is dried, theseparated spots can be visualized in a number of ways such as viewing under an ultravioletlight or spraying with one of a wide variety of reagents. The apparatus for performing TLC isdepicted 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 adeveloped 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 ofthe sample zone or spot.

According to the types of compounds being investigated, the separations on theplate may then be evaluated by a number of instrumental techniques. If the substances areradioactive or suspected to be so, the plate can be examined by a radioisotope scanner tolocate these substances. Under proper conditions, it is also possible to measure both the areaof a spot and its density, using a photo densitometer. These characteristic are related to thequantity of a given substances in the spot and can thereby be used to assess the amount of

the substance in the sample. A quantitative calibration or reference curve should beestablished beforehand using known amounts of the substance to be quantitated.

Thin layer chromatography (TLC) has found wide applicability in separation andidentification of both organic and inorganic substances.

The main advantage of TLC lies in the fact that the technique can carry outseparation and identification of unknown substances in very small amounts and in a veryshort time. None of the other methods can match it in this regard. Even paperchromatography which has earned the reputation of carrying out separation in very smallamounts has been exceeded by this method. For example, TLC can carry out separation oridentification in still smaller amounts and in much less time than is taken by paperchromatography.

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 plantextracts on alumina-coated glass plates and got a satisfactory separation. This discovery didnot attract the attention of other workers till 1949. In that year, J.E.Meinhard and N.F.Hallshowed some satisfactory separation with the new method. They were also the first todescribe an apparatus for coating glass plates with adsorbents. But the technique could notgain popularity because of the difficulties involved in the preparation of suitable adsorbentsand applying them in uniform layers on glass plates.

The method which was practically given up was reinvented by the untiring efforts ofEgan Stahl, a German worker, who not only found practical equipment for coating glassplates with adsorbents, but also demonstrated the usefulness of this technique on a widerange of materials. Since 1958, the use of TLC has been so widespread that it has enteredalmost every phase of analysis.

TLC has been used in separation and identification of a large variety of organiccompounds: alkaloids, terpenes, vitamins, hormones, dyes, drugs, sugars, fat, pigments,alcohols, aldehydes, acids, and many others. TLC has also found useful application in theanalysis 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, developingchamber, etc. play their individual roles. The efficiency of the former two plays a veryimportant 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 areavailable 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 alongwith some binding materials like plaster of Paris to fix the thin and porous layer on the glassplates. In addition to this kiesel gel has also been used for making thin layers. Cellulose

powders with and without binding materials is also in use. Various types of cellulose powder,including ionexchange 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 formhomogenous slurry. Then, immediately it is spread over well cleaned glass plates of standardsize (20 × 20 cm. Or 20 × 5 cm.) Arranged in a row on an aligning tray with the help of theapplicator. The applicator is first adjusted according to the thickness of the layer, after whichthe slurry is put inside the applicator and the latter is drawn by handover the glass platesarranged on the aligned tray. The coated glass plates are then allowed to dry at roomtemperature for 10 minutes to 2 hours during which the Linder present in the adsorbent setsthe adsorbent firmly over the glass plates.

After air drying, the coated glass plates are activated by drying in an oven at asuitable temperature depending on the adsorbent. The heating period similarly varies. Afteractivation the plates are stored in vacuum desiccators until they are used. If the plates are notstored in desiccators and exposed to air or kept for a long time without being used, theefficiency 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 amicro- pipette is applied to the starting point near one of the edges (narrow edge) of thechromatoplates in such a manner that the drop becomes as well as particable, and diameterof 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 thanthe paper chromatography chamber. The chamber should be saturated with the solventvapour sufficiently ahead of the actual operation. Its walls should be lined up with filterpaper soaked with the solvent to maintain steady saturation inside the chamber. The solventis put inside the chamber to a thickness of 1 cm. The spotted chromatoplates are developedby the ascending techniques. The plates are held vertically inside chamber. They can also bedeveloped by the descending technique with certain modifications of the chamber. Thedevelopment takes much less time than in the case of paper chromatography. When thedevelopment 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 keepingthem in an oven adjusted at specified temperature (100˚ - 250˚c).

Detection of the movement of spots and determination of Rf values are similar tothose in paper chromatography. Detection of spots can be made by spraying thechromatogram with visualizing reagents (e.g. ninhydrin) or in some by seeing thechromatogram in U.V.light.

Partition and ion exchange TLC can be performed in the same way. Two-dimensional thinlayer chromatography has also been achieved with success by developingthe single plate in two directions at right angles to each other utilizing two different solventsystems. Thin –layer electrophoresis has shown good success in separation of mixtures ofvarious types of compounds.

Separation of milk fat components by TLC:PROCEDUREPREPARATION OF TLC PLATES:

Silica gel G taken in quantity of 4 Gms, 11 ml of distilled water added and slurry wasmade with pastel and grinder. Further, the slurry was poured over glass plates of 10 × 20 cmsize uniformly and spread evenly with the help of a glass rod or special apparatus availablefor the purpose. The plates were then air dried and water evaporated. The plates, aftercompletion of drying, were activated at 110˚c for 20 minutes. During activation much of themoisture gels evaporated and silica gel gets properly charged. These plates, after activationwere 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 ofspotting is kept too large, overlapping of components occur and a poor resolution betweenmolecules which retards their migration.

Development:After spotting the standard as well as unknown, plates were put in chromatographic chambercontaining the solvent system. To aid saturation of the developing chamber atmosphere, theinner walls are normally lined with blotting paper or a thick chromatography paper such aswhat men 3 MM. This is conveniently done by taking a single large sheet of paper andplacing it into the dry tank so that the back and ends are lined. The front is left open so theplate may be observed. The paper should come to within 1 cm of the top of the tank. Thechromatography was allowed to proceed for a given period of time and then the plates wereremoved from other end, air dried and taken for color development of plates can be doneusing 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 ratioof 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 variouslipid components as long chain triglycerides, > short chain triglycerides, > free fatty acids, >diglycerides, > cholesteror > 1.2 diglycerides > 2 monoglycerides > phospholipids. (Figure25.3).

Separation of pigments:PROCEDURE

In a mortar place 5 ml of petroleum ether + ether and a few green leaves, crush theleaves with pastel and transfer the extract to a separating funnel by means of a pipette. Swirlthe extract with equal volume of water. Discard the lower aqueous layer. Repeat washing twotimes with water. Discard the lower aqueous layer. Take petroleum ether layer to a small

aluminums flask, dry over 2 gm of and sodium sulphate or if concentrated gel is used, by astream of dry nitrogen. Place a small spot with the help of a capillary tube on a 10 cm TLCplate. Allow the spot to dry. Use acetone as developing solvent. Develop the chromatogramusing a wide mouth bottle or a conical flask. A folded piece of filter paper is placed intobottle to moisten/ saturate the atmosphere with water. Dry the plates again ad it will bepossible to observe as many as eight colored spots. In order of decreasing Rf value, thefollowing 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 mmthickness.

EXERCISE 26

GEL FILTRATION

The technique of separating molecules of different size by passage through a gelcolumn is called gel filtration. Small molecules can enter the gel but larger molecules areexcluded from the cross- linked network (figure 26.1). This means that the accessible volumeof solvent is very much less for molecules totally excluded from the gel than for smallmolecules which are free to penetrate. The separation of molecules by gel filtration isillustrated in the diagram. First the mixture of large and small molecules is placed on top ofthe column. (figure 26.2(a)). As they pass down the column, the small molecules diffuse intothe gel ad follow a longer path than the large molecules, which are completely excluded fromthe gel particles (figure 26.2 (b)) eventually, complete separation occurs, with the largemolecules 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 orcofactors, detergents, urea, at high or low ionic strength, at 37˚ c or in the cold roomaccording 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 makepolyacrylamide (biogel p), agarose (biogel A), porous glass (bio-glas), and polystyrene (bioleads). The degree of cross- linking etc. is carefully controlled to give a range of productsable 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. Apolysaccharide, 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 bythe action of a bacterium on sucrose. Each glucose residue contains 3 hydroxyl groupsgiving the dextran a polar character. The cross linking reaction (figure 26.3). Is accompaniedwith epichlorohydrine.

CH2 CH CH2Cl O

Epoxy group

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

These gels are classified by the amount of “water regained” which is a function ofthe looseness of the structure or the extent of cross linking. The classification of sephadexgels is given in table 26.1. By sephedex gel, the useful fractionation range of molecules isonly approximate since separation depends upon the shape, charge to a minor extent andsize of the molecules. Sephadex G- 25 and G-50 are made in several particle sizes and wefind coarse, medium and fine grades. Fine grades are suitable for most laboratory workrequiring high resolution, while the coarse material is convenient for preparative work withlarge 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 temperaturerange 0-30˚c. Three different types of sepharose are available which have differentconcentrations of agarose. (Table 26.2) sepharose CL is stable over pH 3.0 to 14.0 and canbe used upto temperatures of upto 70˚c. These are some porens gels and are used tofractionate very large molecules such as nucleic acids and viruses. After use, the columns canbe washed in water and stored in the cold room for quite a time provided traces ofpreservative such as phenol or chloroform is added to prevent bacterial growth. Anothermaterial used for column chromatography is ‘sephacryl’. It is a polymer of allyldextrancovalently crossed lambda linked with N – N’ methylene –bisacrylamide (figure 26.4) andcan be obtained in a preswollen form. The material gives much faster flow rates thansephadex or biogel and 5200 can be used to separate proteins with a molecular weightbetween 5000 and 2, 50,000. The molecular weight of proteins can be determined fromelution volume after calibration of column with known molecular weight markers.

Another gel chromatography material that is used for molecular extrusionchromatography is Biogel-P of different types (table 26.3). They are made frompolyacryamide.

Principle of separation:Theory of gel filtration:

The total volume of the column (Vt) is the sum of volume of the gel matrix (Vg), thevolume of water inside the gel particle (Vi) and the volume of the water outside the gelgrains (Vo), as illustrated in figure 26.5.

I.e. VT = Vo + Vg + ViVo 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 theknowledge of dry weight of the gel (a) and the water regained (Wr) e.g. Vi = a × Wr. Theelution volume Ve of a compound is the volume required to elute that compound from acolumn i.e. Ve = Vi + Kd Vi where Kd indicates the fraction of the inner volume accessibleto a particular compound and is independent of the geometry of the column. I.e. Kd = Ve -Vo

ViSubstituting the value of Vi in the above equationKd = Ve- Vo

aWrIf Kd = 0, then Ve = Vo i.e. the elution volume would be the void volume.If Kd = 1Ve –Vo = ViOr Ve = Vi + Vo

If a molecule is completely eluded from the gel, then Kd = 0 and Ve= Vo, while if amolecule has complete accessibility to the gel, then Kd = 1.

Molecules therefore have a Kd value between 0 and 1. If Kd >1, then adsorption ofthe compound on the gel occurs.

By plotting a graph of elution volume against molecular weight taking differentmarkers 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 polymerchemists to characterize molecular weight distribution in polymeric mixtures. The otherapplications 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 simplecolumns with high flow rates.e.g. With sephadex G-25, solute molecules having molecularweights over 5000 are eluted in a volume Vo, smaller molecules with molecular weightsbelow 1000 will be eluted following the macromolecules inversely in order of their size. Oneof the advantages of this method of desalting is that the macromolecules are eluted withessentially no dilution.2. Concentrating:

The diluted solution of macromolecules with molecular weights higher than theexclusion 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 preferredbecause of its more rapid action. Since salt molecules are imbibed to the same extent aswater, their concentration in supernatant solvent remains essentially unchanged and ionicstrength and pH of macromolecular solution remains essentially unaltered. This is animportant advantage in separation of proteins which are easily denatured by change intemperature.3. Fractionation:

Components separation of mixture of closely related materials having smalldifference in K- values will require long columns, slow flow rates and long times. Thedifference of molecular weights in a complex mixture is often sufficient information tocheck the solution or to give a rough idea of its composition. It has been observed and canbe 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 determinedfrom a plot of V versus log molecular weight for several known compounds. The equation isvalid over a considerable range of molecular weights, provided all compounds are closelyrelated. Values for A and B must be redetermined for each column in each set of specificconditions and for different classes of compounds. Table 26.4 lists the applications of gelfiltration.

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 aftercalibrating 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 54. Fraction collectors 55. Molecular weight standards (5 mg of each protein per ml of elution buffer)

Standard Mol.wt. Log mol.wt.Cytochrome C 12400 4.09Ovalbumin bovine serum 45000 4.65Y- Globulin 2, 10,000 5.32Blue dextran 2,000,000 -

6. Lactate dehydrogenises 1 ml7. Spectrophotometer 58. 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 justabove the top of the gel. Mix 0.1 ml of lactate dehydrogenises solution and 0.4 ml of themarkers and place this on top of the column. Allow the mixture to enter the gel, add a smallvolume of the elution buffer until the markers are clear of the top then connect the bufferreservoir. Adjust the height of the reservoir to give a flow of about 50 ml/h and collect 3 mlfractions using the fraction collector. Locate the position of the blue dextran and the glucoseand 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 standardmethod 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 ofKd against log10 mol.wt. For each marker and calculate the apparent molecular weight ofthe LDH.

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 resinpolymers, such as the strongly acidic cation exchanger Dowex -50, a polystyrene sulfonicacid, and a strongly basic anion exchanger Dowex -1, a polystyrene quaternary ammoniumsalt. Cellulose derivates such as carboxymethyl cellulose (CMC) and diethyl amino ethylcellulose (DEAE) exchangers have been very successfully used in protein purification.

The basic principle involves an electrostatic interaction with the exchanging ion andthe normal charge on the surface of the resin. These reactions are considered to beequilibrium processed and involve diffusion of a given ion to the resin surface and then tothe exchange site, the actual exchange, and finally diffusion away from the resin. The rate ofmovement of a given ionizable compound down the column is a function of its degree ofionization, the concentration of other ions, and the relative affinities of the various ionspresent in the solution for charged sites on the resin. By adjusting the pH of the elutingsolvent and the ionic strength, the electro statically held ions are eluted differently to yieldthe 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 sampleapplication and adsorption. Unbound substances can be washed out from the exchanger bedusing a column volume of starting buffer. In the second stage, substances are eluted fromthe column, separated from each other. The separation is obtained since different substanceshave different affinities for the ion-exchanger the to differences in their charge. Theseaffinities can be controlled by varying. Conditions such as ionic strength and pH. (Table27.1). Ion- exchange chromatography is capable of separating species with very minordifferences in properties, such as two proteins differing by only one amino acid- it is mostwidely used technique for separating, identifying, and quantitating the amounts of eachamino 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 performedautomatically in an amino acid analyzer.

In ion exchange chromatography one can choose whether to, bind the substances ofinterest, or to adsorb out contaminants and allow the substance of interest, to pass throughthe column. Generally it is more useful to adsorb the substance of interest, since this allowsa greater degree of fractionation.

Ion exchange may be carried out in a column or by a batch procedure. Both methodsare performed in definite stages. These stages are: equilibration of the ion exchanger,addition and binding of sample substances, change of conditions to produce selectivedesorption, and regeneration of the ion exchanger. The step- by- step procedure for ionexchange chromatography is given in figure 27.1.

Theory:The matrix many commercial ion- exchangers which have been successfully

employed for separation of biological materials are made by c0-polymerizing styrene withdivinglbenzone, however, cross- linking of molecules occurs and this produces an insolubleresin. Various degrees of cross linkage may be obtained by co-polymerizing varyingproportions 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 weightcompounds than are highly cross-linked ones, but they are also less rigid and swell morewhen paced in a buffer. These swelling characteristics must be taken into account when acolumn is prepared. Sulphonation of cross- linked polystyrene resin such as dowex 50 whichis strong acidic exchangers. The SO3H group is ionized at all except very low pH values. Ananalogous basic exchanger may be prepared by reacting cross-linked polystyrene withchloromethyl 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 tothe polystyrene- based exchangers. Cellulose is a high molecular weight compound whichcan be obtained in a highly pure state (arboxymethyl cellulose (CM- CH2 OCH2 COOHcellulose), and DEAE- cellulose (-CH2OCH2CH2N(CH2CH3)2) are examples of the mainderivates 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 orcationic, according to the nature of their affinity for either negative or positive ions. Forexample, the cation exchanger materials exchange positive ions, so it is the charge carried bythe exchangeable ion which decides whether a material is anionic or cationic and not thecharge carried on the matrix. (figure 27.3) these two types can be further divided intomaterials that contain strongly ionized groups, such as –SO3H and –NR3, and the weaklyionized groups, such as –COOH, -OH and –NH2. The strong ion exchange resins arecompletely 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 isdependent on the pH, and they can only be used at maximum capacity over a narrow pHrange.-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 ionexchanger. 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 definedexperimental conditions. In the case of some materials, large molecules may be unable to

penetrate the matrix and can only react with charged groups on the surface. In this case, theavailable capacity will be considerably less than the total capacity.

The ionizable groups commonly met are given in table 27.3 together with theirabbreviations.Ion exchange equilibria. The typical way that an ion exchange material function is illustratedin the following sample, where an anion exchange resin containing amino groups is used toseparate two negatively charged ions X- and Y- (figure 27.4).

Although ion exchange materials are claimed to be monofunctional, in that only theion exchange process is used in separation, in practice some molecular sieving andadsorption can occur. The adsorption is small, but it can sometimes be used to separateclosely 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 chargedecreases and the macro-molecule is no longer bound. Separation is achieved as othercharged proteins remain on the column alternatively, ions can be removed by increasing theionic strength, when high concentrations of ions in the solvent displace the bound ions byincreasing the competition for the charged groups of the ion exchange material. The pH orionic strength can be altered sharply, by changing the eluting buffer, or, gradually, by meansof a gradient.Preparation of material. Ion exchange materials are first allowed to swell in the buffer andthe fines removed.The ion exchange material is then obtained in the required ionic form by washing with theappropriate solution. For example, the H+ form of a cation exchange resin is obtained bywashing 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 sodiumhydroxide then water as above.

The final stage before preparing the column is to equilibrate the material by stirringwith the eluting buffer.

The separation of amino acids by ion exchange chromatography:MATERIALS1. Chromatography column (20 cm × 1.5 cm) 52. Strongly acidic resin 30 g.3. Hydrochloric acid (4 ml/ litre) 1 litre.4. Hydrochloric acid (0.1 ml/ litre) 4 litres5. Glass wool –6. Amino acid mixture 10 mg(Dissolve aspartic acid, histidine of each and lysine in 0.1 mol/ litre HCl to a finalconcentration of 2mg/ ml)7. tris-HCl buffer (0.2 mol/ litre, pH 8.5) 3 litres8. Sodium hydroxide (0.1 mol/litre) 2 litres9. Separating funnels (500 ml) 1010. Acetate buffer (4 mol/ litre, pH 5.5) 250 ml11. Ninhydrin reagent. (Dissolve 20 g of ninhydrin and 3 g of 1 litre hydrindation in 750 mlof methyl cello solve and add 250 ml of acetate buffer). Prepare fresh and store in a brownbottle.12. Methyl cello solve (ethylene glycol monomethyl ether) 1 litre.

13. Ethanol (50 percent v/v) 1 litre.14. Ninhyrin (2 g/litre in acetone). (Care: carcinogenic!) 100 ml15. Oven at 105˚c 1.

Method:PREPARATION OF THE COLUMN- gently stirs the resin with 4 mol/ litre HCl untilfully 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 thecolumn as previously described.Elutions of amino acids carefully apply 0.2 ml of the amino acid mixture to the top of thecolumn, 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 areservoir containing 500 ml of 0.1 mol/ litre HCl. Adjust the height of the reservoir to give aflow rate of about 1 ml/ min and collect a total of forty 2 ml fractions. Test five of the tubesat a time for the presence of amino acids by spotting a sample from each tube on to a filterpaper: dip this in the acetone solution of ninhydrin and heat in an oven at 105˚c. If aminoacids are present, they will show up as blue spots on the filter paper. When the first aminoacid has been eluted, remove the reservoir of 0.1 mol/ litre HCl and allow the level of acidto fall to just above the resin. Run 2 ml of 0.2 mol/ litre tris- HCl buffer (pH 8.5) on to thecolumn, then connect to a reservoir of this buffer and continue with the elution until thethird 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 ofacid or alkali. Add 2ml of the buffered ninhydrin reagent and heat in a boiling water bath for15 min. cool the tubes to room temperature, add 3 ml of 50 percent v/v ethanol and readthe extinction at 5570 nm after allowing the tubes to stand for 10 min. set up the appropriateblanks and standards, and plot the amount of amino acid in each fraction against the volumeeluted.

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, enzymepurification, hormone preparation, studies, carbohydrate and lipids separations and vitaminand co – enzyme work etc. the special characteristic of ion exchange is that the separation isbased on charge, so that when it is combined with techniques which use other criteria forseparation, such as gel filtration (size) or affinity chromatography (bio selectivity), it is a verypowerful analytical and preparative tool.

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 exploitsthe unique property of extremely specific biological interactions to achieve separation andpurification. As a consequence, affinity chromatography is theoretically capable of givingabsolute purification, even from complex mixtures, in a single process. The technique wasoriginally 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 reversiblebinding to a specific ligand which is attached to an insoluble matrix.

M + L k + 1 MLMacro – ligand complexMolecule (attached to k - 1

Matrix)

Under the correct experimental conditions when a complex mixture containing the specificcompound to be purified is added to the insolubilized ligand, generally contained in aconventional chromatography column, only that compound will bind to the ligand. All othercompounds can therefore be washed away and the compound subsequently recovered bydisplacement 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 wayfor the particular type of support. A buffer is used which will encourage the bindingmacromolecule to be strongly bound to the ligand. The buffer generally has a high ionicstrength to minimize non – specific adsorption of polyelectrolyte ligand. The buffer mustalso contain any co – factors such as metal ions necessary for ligand – macromoleculeinteraction. Once the sample has been applied and the macromolecule bound, the column iseluted with more buffers to remove non – specifically bound contaminants. The purifiedcompound is finally recovered by either specific or non – specific elution. Non – specificelution may be achieved by a change in either pH or ionic strength. pH shift elution usingdilute acetic acid or ammonium hydroxide results from a change in the DNA by a similarprocedure. The method has also been used to isolate whole genes by hybridizing practicallysingle stranded DNA with the complementary mercurated mRNA which is then reactedwith a thioleted matrix.

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 liquidand a gas phase, is a widely used method for the qualitative and quantitave analysis of a largenumber of compounds size it has high sensitivity, reproducibility and speed of resolution. Ithas proved to be most valuable for the separation of compounds of relatively low polarity. Astationary phase of ‘liquid’ material such as silicone grease is supported on an inert granularsolid. 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 asnitrogen or argon. The column is maintained in an oven at an elevated temperature whichvolatilizes the compounds to be analyzed. The basis for the separation of compound beinganalyzed is the difference in the partition coefficient of the volatilized compounds as they arecarried through the column by the gas. As the compounds leave the column they passthrough a detector. This is linked via an amplifier to a chart recorder, which records a peakas 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 columnSP (stationary phase) filled with silicon or other non-volatile liquid, then through lower partof 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 thecolumn with STD. The detector (d) is a thermal conductivity cell called a doublekapharometer. The principle of this detector is Wheatstone bridge. Heat is condensed awayfrom a hot body by passing a gas over hot body. The rate of heat removal will depend on thenature of the gas, other factors being constant. Thus 1/ 5th the heat conductivity of airwhereas H2 gas is seven times more effective than air.

In figure 29.2 the identical gas chromato C1 and C2 contains identical pieces ofplatinum wire. They are heated by identical quantity of electric current. The resistance of twowires will be identical if the same gas passes through C1 and C2 at a constant rate, however ifinert gas such as helium passes over C1 and methyl ester of fatty acids is mixed with SP, andthis mixture passes over wire C2 , C2 will be surrounded by a gas with different thermalconductivity (probably a low conductivity in this case). The C2 wire will not lose as muchheat as the C1 wire, and so will have higher resistance. If the resistance of C1 and C2 are firstjust 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 wasintroduced. This can be measured and taped with an electrical tracing paper recorder.

An example of such a tracing is shown below

Twenty ml of mixture of methyl esters of C10- C20 fatty acids dissolved in methylcorporate 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, afilament current of 260 mA and charge speed of 15 inches/ hr. this apparatus used was anaerograph 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 mostcommonly used support is celite (diatomaceous silica) which because of the problem ofsupport/ sample interaction, is often treated so that hydroxyl groups which occur in celiteare modified. The support particles have an even size which, for the majority of practicalapplications, 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 thepolyethylene 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 generallyretained on the column for excessive periods of time if they are applied directly. Thisexcessive is inevitably accompanied by poor resolution and peak tailing. This problem can beovercome by derivatisation of these polar groups. Methylation, silanisation andtrifluoromethyl- 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-syringethrough a septum in the injection port which is attached to the top of the column. Normallybetween 0.1 and 10 mm3 of solution is injected. It is a common practice to maintain theinjection region of the column at a slightly higher temperature than the column itself. Thishelps to ensure rapid and complete volatilization of the sample. Sample injection isautomated 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 temperaturemust be within the working range of the particular stationary phase and is chosen to give abalance between peak retention time and resolution. In isothermal analysis a constanttemperature is employed. In the separation of compounds of weight it may be advantageousto 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

response range (106). A mixture of hydrogen and air is introduced into the detector to give aflame, the jet of which forms one electrode, whilst the other electrode is brass or platinumwire mounted near the tip of the flame. When the sample components emerge from thecolumn they are ionized in the flame, resulting in an increased signal being passed to therecorder. The carrier gas passing through the column and the detector gives a smallbackground signal, which can be offset electronically to give a baseline.

The nitrogen- phosphorous detector (NPD), which is also called a thermionicdetector, is similar in design to a FID, but has a sodium salt fused onto the electrode systemor a burner tip embedded in a ceramic tube containing a sodium salt or a rubidium chloridetip

The electron capture detector (CED) responds only to substances which captureelectrons, 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 atthe beginning of the chromatogram.

The three main forms of detector respond to this solvent with varying sensitivitythereby affecting the detection and resolution of rapidly eluting solutes. In cases whereauthentic samples of the test compounds are not available for calibration purposes or incases where the identity of the compounds is not known, the detector may be replaced by amass spectrometer. More recently, GLC has been linked to other types of detector includingan infer-red spectrophometer and to a nuclear magnetic resonance Spectrometer, theresulting 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-polarcompounds which do not need derivatisation; compounds are characterized by theirretention time or preferably by their relative retention time to a standard referencecompound. In the analysis of compounds which form a homologous series, for example themethyl esters of the saturated fatty acids, there is a linear relationship between the logarithmof 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 forquantitative analysis is the Retention Index (RI) which is based on the retention of acompound 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 capacityto calculate RI values automatically.

EXERCISE 30

HIGH PERFORMANCE (PRESSURE) LIQUIDCHROMATIGRAPHY (HPLC)

Principle:Originally, HPLC was referred to as high pressure liquid chromatography but

nowadays the term high performance liquid chromatography is preferred since it betterdescribes the characteristics of the chromatography and avoids creating the impression thathigh pressure are an inevitable pre- requisite for high performance. This is new known notto be the case and the term medium pressure liquid chromatography (MPLC) has beencoined 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 andthe number of theoretical plates per unit length, although there are limits to the length of acolumn due to the problem of peak broadening. As the number of theoretical plates isrelated to the surface area of the stationary phase it follows that the smaller the particle sizeof 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 fardiscussed rely on gravity or low pressure pumping systems for the supply of eluant to thecolumn. The consequences of this is that the flow rates achieved are relatively low and thisgives greater time for band broadening by simple diffusion phenomena. The use of fasterflow rates is not possible because it creates a back- pressure which is sufficient to damagethe matrix structure of the stationary phase, thereby actually reducing eluant flow andimpairing resolution. In the past decade there has been a dramatic development in columnchromatography technology which has resulted in the availability of new smaller particlessize stationary phases which can withstand these pressures and of pumping systems whichcan give reliable flow rates. These developments, which have occurred in adsorption,partition, ionexchange, exclusion and affinity chromatography, have resulted in faster andbetter resolution and explain why HPLC has emerged as the most popular, powerful andversatile form of chromatography.

Column packing:Three forms of column packing material are available based on a rigid solid (as opposed togel) structure. These are:

i. Microporous supports where micropores ramify through the particles which aregenerally 5 to 10 µm in diameter;

ii. Pellicular (superficially porous) supports where porous particles are coated onto aninert 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 inertsupport.

For adsorption chromatography, adsorbents such as silica or alumina are available asmicroporous or pellicular forms with a range of particle size. Pellicular systems generallyhave a high efficiency but low sample capacity, and therefore microporous supports arepreferred where applicable. All forms of HPLC column packing are characterized by their

regular spherical shape which distinguishes them from conventional materials. These smallspheres pack most efficiently and give good flow properties.

In liquid – liquid partition systems, the stationary phase may be coated onto the inertsupport. 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 maygradually wash off the liquid phase with repeated use. To overcome this problem bondedphases have been developed where the liquid phase has been covalently bonded to thesupporting material which may be silica or a silicone polymer. The silicone polymer bondedphases have the particular advantage that as well as not being eluted by the developingsolvent, they are chemically, hydrolytically and thermally stable. In normal phase liquid –liquid chromatography, the stationary phase is a polar compound such as alloys nitride oralloylamine derivatives and the mobile phase a non – polar solvent such as hexane. Forreverse – phase chromatography, the stationary phase is non – polar compound such as a C8or C18 hydrocarbon and the mobile phase a polar solvent such as water / acetonitrile orwater / methanol mixtures.

Many different types of ion exchangers are available of which the cross – linkedmicroporous polystyrene resins are widely used. Pellicular resin forms are also available, asare bonded phase exchangers covalently bonded to a cross – linked silicone network. Theseresins 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 areavailable 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 canwithstand only low pressure. The supports for affinity separations are similar to those forexclusion separations. The spacer arm and ligand are attached to these supports by similarchemical means to those used in conventional low pressure affinity chromatography. Table30.1 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). Straightcolumn of 20 to 50 cm in length and 1 to 4 mm in diameter are generally used thoughsmaller capillary columns are available. The best columns are precision bored with aninternal mirror finish which allows efficient packing of the column. Porous plugs of stainlesssteel or Teflon are used in the ends of the columns to retain the packing material. The plugsmust be homogeneous to ensure the uniform flow of solvent through the column. It isimportant in some separations involving liquid partition and ion – exchange that the columntemperature 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 packtheir own columns since this is cheaper than purchasing pre – packed columns. Severalmethods are available for packing columns and the method used will depend on the natureof the packing material and the dimensions of the particles. The major priority in the packingof a column is to obtain a uniform bed of material with no cracks or channels. Rigid solids

and hard gels should be packed as densely as possible, but without fracturing the particlesthe packing process. The most widely used technique for column packing is the highpressure slurring technique. A suspension of the packing is made in a solvent of equaldensity to the packing material. The slurry is then rapidly pumped at high pressure into acolumn with a porous plug at its outlet. The resulting bed of packed material within thecolumn 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 isnecessary for them to be allowed to swell first in the solvent to be used in thechromatographic process before packing under pressure. Soft gels cannot be packed underpressure and have to be allowed to pack from slurry in the column under gravitationalsedimentation only, in a similar way to the packing of columns for conventional columnchromatography.

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, ortwo or more solvents mixed in fixed proportions. Alternatively a gradient elution system maybe used where the composition of the developing solvent is continuously changed by use ofsuitable gradient elution system may be used where the composition of the developingsolvent is continuously changed by use of a suitable gradient programmer. In the majority ofcases this involves the use of two pumps. All solvents for use in HPLC systems must bespecially purified since traces of impurities can affect the column and interfere with thedetection system. This is particularly the case if the detection system is measuringabsorbance at below 200 nm. Purified solvents for use in HPLC systems are availablecommercially, but even with these solvents a 1 to 5 µm micro filter is generally introducedinto the system prior to the pump. It is also essential that all solvents are degassed before useotherwise gassing tends to occur in most pumps. It tends to be particularly bad for aqueousmethanol and ethanol solvents, gassing (the presence of air bubbles in the solvent) can altercolumn 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 itvigorously with a magnetic stirrer, subjecting it to a vacuum, ultrasonic vibration, or bybubbling 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 smallparticles, and high pressures are therefore required to achieve satisfactory flow rates. Themain feature of a good pumping system is that it is capable of outputs of at least 3.4 * 1.07Pa (5000 p.s.i) and ideally there must be no pulses of flow through the system. There mustbe a flow delivery of at least 10 cm3 min-1 for normal analysis, and upto 30 cm3 min-1 forpreparative 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 pressureor constant displacement.

Constant pressure pumps produce a pulse less flow through the column, but anydecrease in the permeability of the column will result in lower flow rates for which the pumpwill not compensate. These pumps will not compensate. These pumps operate by theintroduction of high pressure gas into the pump, and the gas into the pump, and the gas in

turn forces the solvent from the pump chamber into the column. The use of an intermediatesolvent between the gas and the eluting solvent reduces the chances of dissolved gas directlyentering the eluting solvent and causing problems during the analysis.

Constant displacement pumps maintain a constant flow rate through the columnirrespective of changing conditions within the column. One form of constant displacementpump is a motor-driven syringe type pump where a fixed volume of solvent is forced fromthe pump to the column by a piston driven by a motor. Such pumps, as well as providinguniform solvent flow rates, also yield a pulse less solvent flow which is important as certaindetectors are sensitive to changes in solvent flow of constant displacement pump. Thepiston is moved by a motorized creak and entry of solvent from the reservoir to the pumpchamber and exit of solvent to the narrow band onto the column. There are two methodswhich are generally used. The first method makes use of a micro syringe designed towithstand 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 immediatelyabove 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 pumpswitched on again. This is termed a stop flow injection. The second method of sampleintroduction is by use of a loop injector. This consists of a metal loop of small volume whichcan be filled with the sample. By means of an appropriate value, the eluant from the pump ischanneled through the loop, the outlet of which leads directly onto the column. The sampleis column is regulated by check values. On the compression stoke solvent forced from thepump chamber into the column. During the return stroke the exit check value closes andsolvent is drawn in via. The entry value to the pump chamber, ready to be pumped onto thecolumn on the next compression stoke. Such pumps produce pulses of flow and pulsedampness are usually incorporated into the system to minimize this pulsing affect. Allconstant displacement pumps have in-built safety cut-out mechanisms so that if the pressurewithin 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 beintroduced as an infinitely thus flushed onto the column by the eluant, without interruptionsolvent flow to the column. Automatic versions of loop injectors are commercially available.

Repeated application of highly impure samples such as sera, urine, plasma or wholeblood, which have preferably been deproteinated, may eventually cause the column to loseits resolving power. To prevent this occurrence a guard column is installed between theinjector and the analytical column. This guard column is a short (2 to 10 cm) column. Of thesame internal diameter, and packed with similar material to that present in the analyticalcolumn. 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 moleculeshave been purified using the approach. Reverse phase partition HPLC is particularly usefulfor 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

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 andproteins. Instruments dedicated to the separation of proteins have given rise to thetechnique of fast protein liquid chromatography (FPLC). There are no unique principlesassociated with FPLC; it is simply based on reverse phase and ion-exchange chromatographyand on chromato – focusing. Microlore glass- lined stainless steel columns 1 mm diameterand 2.5 cm long have recently been developed which enable very small amounts of sample tobe used with separation taking as little as 10 minutes.

EXERCISE 31

DETERMINATION OF FREE FATTY ACIDS IN GHEE

Milk fats contain a little concentration of free fatty acids in their composition. Uponpassage of time due to splitting of glycerides by the action of lipase the concentrations offree fatty acids increase resulting into hydrolytic rancidity.CH2OOC – R1 CH2OH1 lipase 1CHOOC – R2 CHOH + R1COOH + R2COOH1 1CH2OOC – R3 CH2OH + R3COOHTriglyceride 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 inmolecular weight, it is therefore preferable to express the free fatty acids in terms of acidsvalue. 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 gheesample.

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 acidsin the sample calculated as oleic acid.

3. Degree of acidity: it is the total titratable acidity present in the sampleexpressed as percentage.

Principle:The free fatty acids presents in the ghee are estimated by titration with alkali using

phenolphthalein as an indicatoro 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.

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 asight 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/W3. 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)

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 andnutritional characteristics and are of permanent importance from economic, commercial andfunctional view points. This deterioration in quality of ghee generally arises from two majorpathways viz hydrolytic rancidity and oxidative rancidity. The other process is ketonicrancidity.

The oxidative rancidity affects an isolated fat is caused by the action of oxygen. Airin 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 chemicalreaction occurring between atmosphere oxygen and unsaturated lipid components mediatedby a variety catalysts resulting in destruction at valuable nutrients, undesirable flavours andodors affecting the palatability of foods and even generation of toxic compounds. It exhibitsauto catalytic property, once initiated, cannot be stopped. Oxidation of unsaturated lipidsfollows 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 doublebond in unsaturated lipids.

The free radical auto oxidation is described in terms of initiation propagationtermination process. Auto oxidation may be considered as a two step process:

a. Primary auto oxidation reaction which may lead to the formation of hydroperoxide.

b. Further reaction of decomposition of hydro peroxides by cleavage of C-Cbonds 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 amountof oxygen and water (short chain fatty acids)

Peroxide value is the measure of oxidative rancidity (peroxide value) of ghee. It isexpressed as ml of 0.002 N sodium thiosulphate solutions per gm of sample, or since 2electrons are provided by the iodide to reduce one molecule of peroxide; it is expressed asm. eq. per kg fat or mill mole per kg fat. The peroxide value of normal ghee range from 1.5to 50.

The other chemical methods to measure oxidative deterioration in lipids and lipidcontaining foods products include.

1. Determination of peroxide by modified stamm method.2. Using loftus – hill method3. thio- barbituric acid (TBA) value

4. Determination of carbonyl content5. 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 isinserted 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 thendetermined 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-

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.

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. Donot add starch until the end point is almost reached.

7. Note the ml of sodium thiosulphate required to change the colour from violet tocolourless and express the result in terms of peroxide value.

EXERCISE 33

DETERMINATION OF AFLATOXINS FROM FOOD ANDFEEDS BY COLUMN AND THIN LAYERCHROMATOGRAPHY

Importance:Carcinogenic nature and involvement of aflatoxins in the disease of animals and

humans have collectively urged major public health organizations to stipulate maximumlevels of aflatoxins that could be permitted in food and feeds. Many organizations in ournation and others, including other import- export agencies, have fixed maximum permissiblelimit 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 thepoint of view of the effect on the health of humans and livestock or to make the productacceptable 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. Theirinsoluble character in diethyl ether, petroleum ether and hexane affords a method to separatethem from certain interfering pigments and fats. Characteristic fluorescence and adsorptionunder 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. Extraction3. Purification, and4. Detection or estimation.

Materials:1. glass- waves (pipettes, conical flasks, glass stoppered flask, measuring cylinder

etc.).2. Petroleum ether3. Chloroform4. Mechanical shaker5. Glass distilled water6. Rough and whatman filter paper7. Anlnydrous sodium sulphate8. Glass column, 2.5 cm internal diameter9. Silica gel G and silica gel for chromatography10. Hot air oven11. Cleaning solvent, diethyl ether: hexane (3:1 v/v)12. Eluting solvent; chloroform: ethanol (97:3 v/v)13. Flash evaporator

14. Boiling waterbath15. 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 toflask 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

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

(C). PurificationTake 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 madewith 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). estimationMake 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 havingconcentration 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 withstandards.

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 lysinecomplexes. These lysine complexes can result from reactions with reducing sugars(producing maillard products), oxidized polyphenols, and oxidized lipids, glutaminyl andasparaginyl 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- aminogroup 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 bindingprocedure, furesine method, or reduction by NaBH4. Several microbiological assays havebeen developed for available lysine, as have in vitro enzymatic hydrolysis methods. In vitrodiestability assays assume that protein digestability of each amino acid, including reactivelysine. Enzymatic hydrolysis with pepsin plus pancreetin or with pronase makes use of thefact that these proteolytic enzymes release only reactive lysine.

A water- soluble reagent, trinitrobenzene sulphonic acid (TNBS) can be used for freelysine measurement. TNBS reacts with lysine derivatives formed early in maillars browningwhich 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 withsome other compound. (E.g. An aldehyde such as an aldose sugar2 in such a way that part oflysine becomes nutritionally unavailable. If the combination of the є amino group withanother compound such as an aldose sugar also prevents a chemical reaction between theamino group and an amino coupling reagent, then the extent of combination of the lysineamino group could be measured, thus giving an indication of the amount of lysine ofbiological value (figure 34.1).

Reagents:1. 0.1 % agar solution2. 1 M sodium bicarbonate3. 1 % 2, 4, 6 – trinitrobenzene sulfonic acid (freshly prepared)4. 11 M HCl5. 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:

1. for the estimation of 2,4,6- trinitrobenzene sulphonic acid (TNBS) reactive lysine, suspend250 mg biscuit powder (defatted) in 0.1 % weight/volume agar solution.2. Homogenize the suspension in a potter- elvehjem all glass homogenizer and make thevolume 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 sodiumbicarbonate 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 for2 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 excessof 2, 4, 6-TNBS and other interfering substances by shaking thrice with 5 ml of diethyl ethereach time.9. Evaporate the residual ether by placing the tubes in a boiling water bath. Make the volumeof 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 therespective blank.11. For blank, cease the reaction by adding first 3 ml of 11 M hydrochloric acid followed by1 ml of 1 percent (w/v) solution of TNBS reagents; follow all other steps in the samemanner as in the case of samples.12. Plot standard curve taking lysine (BDH) ranging in concentrations from 50 to 250mg/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 proteinin 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.

EXECRISE 35

ESTIMATION OF PHOSPHOLIPIDS BY THIN LAYERCHROMATOGRAPHY

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 paperNo. 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 anddried. Slurry was prepared by mixing 8 g of silica gel –G with 22 ml of water and shaking forabout 2 min. and used promptly. A plate was placed under the tough of an applicator withleading edge protruding about one inch beyond the gate. The slurry was poured into thetough to prepare single dimensional plate. Another glass plate was immediately pushed undergate of tough in a continuous movement. The coated plates were immediately placed on auniform surface for superficial drying. After about 30 min. the plates were transferred to anoven 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 acapillary tube at a line about 2 cm above the end of the plate. After evaporation of thesolvent, the plate was insert in a saturated chromatographic chamber containing a solventsystem consisting of chloroform: methanol: ammonia: water (65:35:5:2.5) as suggested byMorrison v/v et al. (1980). Total time taken for separation was about 2 h. thechromatograms, after drying were sprayed with Hanes reagent (prepared by mixing 5 ml of60 percent w/w perchloric acid, 10 ml of 1N hydrochloric acid and 25 ml of 4 percentammonium molyldate in 100 ml acetone) as suggested by stahl (1969),

The chromatograms were incubated at 110˚c until the developments of bluecoloured 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.

EXERCISE 36

ESTIMATION OF CHOLESTEROL BY COLORIMETRICMETHOD (from cream, butter and ghee by the method of schoenheimer, R andSperry, 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 filterpaper.

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 mixing20 ml acetic anhydride and 1 ml concentrated H2SO4 and then incubating in ice-bath for27 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 totalcholesterol 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 bythe method of Bindal and Jain (1973).

Materials:1. milk/butter/ghee2. Chloroform AR3. Acetic analydride-H2SO4 AR mixture (30:1) fresh4. Stock cholesterol solution 2mg/ml in CHCl35. working standard cholesterol dilute the above solution to 5ml with CHCl3 (114) to give0.4mg/ml6. Alcohol: acetone (1:1)

Method:1. Place 10ml alcohol – acetone in a centrifuge tube and add 0.5g milk/0.2g butter2. Immerse the tube in boiling waterbath 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 boilingwaterbath.6. Cool and dissolve the residue in 5 ml CHCl37. Set at the same time series of standard tubes containing cholesterol and blank with (3) Schloroform.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.

cal dairy chemistry.manual

Documents

dairy food compendium dairy - perkinelmer€¦ · dairy...

· monade 280/400 cal 270/400 280/400 300/440 280/400 cal...

dairy & dairy goat

cal poly slo dairy products technology center

dairy industry/ dairy science “introduction to the dairy...

chips & salsa ....

dairy microbiology - national dairy research institute

cannoli 3.99 cheesecake 3.95 cal 190 cal 610 brownie ... ·...

fall 2014 newslettercaholstein.com/2014 fall...

dairy whole dairy packet 1 worksheets

(703)...(703) appetizers taquitos (3) cal 850 calamari...

chapter 8 - dairy and non-dairy

european dairy industry the european dairy industry …...

dairy cattle selection. dairy cattle selection overview...

pasta · 2019. 10. 25. · pasta mac & cheese salad dessert...

college dairy college dairy - gdcwsopore.com

dairy microbiology and dairy products

cowbank dairy farmland fund - parliament.vic.gov.au dairy...

dairy products technology (dairy technology)

dairy knowledge fair nutrition of dairy animals -...