– K. P. Muzumdar.
aboratory should be air-conditioned. However in many cases it is not possible to avail of this facility. In that case, it must be built in such a way as to have a constant circulation of fresh air and with a good arrangement for exhaust. There should be no strong, direct sunlight not constant darkness – The place should be free from dust, moisture, heat, etc.
Furniture used inside should be of seasoned wood and all table tops should be marble tops or stainless steel tops.
Working space per worker should be quite enough so that there can be free movement without disturbing the working of the next worker. Each worker should be provided with a water tap connection and a skin. There must be smokeless heating burners or filaments.
Storage of all drugs should be in closed cupboards away from direct heat. Each drug must be well-stoppered and labelled.
Strong odorous drugs should be always kept away from the other drugs.
Mother tinctures should be stored away separately from the potentised drugs.
The important function of this homoeopathic pharmacy laboratory is to identify and to test the purity of homoeopathic drugs. This is very important factor. For the sake of experimentation it becomes necessary even to prepare the drug from the raw material. Such experimentation can further determine the standards for the drugs and later on it can be utilized on a larger scale to prepare commercial quantity of drugs.
Method of Sampling for Analysis :
Sampling from original containers:- It is recommended that gross samples of vegetables or animal drugs which which have the component parts smaller than one centimeter, in any one dimension, and all powdered or ground drugs, be taken by means of a sampler which removes a core from the top to the bottom of the container. Not less than 2 cores should be takes in the opposite direction; when the total weight of the drug to be sampled is less than 100 kilos (220 pounds), at least 250 gm. shall constitute an official sample. When the total weight of the drug to be sampled is in excess of 100 kilos, take repeated samples by the above method and according to the schedule given below, mix and quarter, reject 2 of the diagonal quarters, combine and carefully mix the remaining 2 quarters and again subject the drug to a quartering process in the same manner, until 2 of the quarters weigh at least 250 gm., which latter quarter constitute an official sample.
It is recommended that gross samples of vegetable drugs, in which the component parts are over 1 cm. in any dimension, to be taken by had, When the total weight of the drug to be sampled is less than 100 kilos, at least 500 gm. constitutes an official sample, this being taken from different parts of the container or containers. When the total weight of the drug to be sampled is in excess of 100 kilos, take repeated samples by the above-mentioned method and according to the schedule, mix and quarter, reject 2 of the diagonal quarters, combine and carefully mix the remaining 2 quarters and again subject the drug to a quartering process in the same manner, until 2 of the quarters weigh not less than 500 gm., which latter quarters constitute an official sample.
When over 100 kilos, the number sampled should not be less than 10 kilos.
When the total weight of a drug to be sampled is less than 10 kilos, it is recommended that the above methods be followed but that somewhat smaller quantities be withdrawn. In no case should the final official sample weigh less than 125 gm.
Foreign Organic Matter in Whole Vegetable Drug:
Withdraw 25 to 500 gm. from the official sample spread out n a thin layer, and separate the foreign organic matter by hand a s completely as possible.
Weigh it to determine the percentage of foreign organic matter, calculated upon the weight of drugs taken. Use the maximum quantity of sample for coarse or bulky drugs.
Preparation of Vegetable of Animal Drugs for Analysis:
The following method is of to be used where specific directions are not otherwise given in the text: Withdraw as much as may be necessary for the official sample by quartering, taking care to see that the portion is representative of the gross sample. In the case of unground or unpowdered drugs, grind the withdrawn sample, so that it will pass through a No.20 standard mesh sieve. If the sample it out in a thin layer and withdraw the portion for analysis. The disintegration of semi-solid drugs may be facilitated by the use of a meat grinder or a similar apparatus.
Cutting, Slicing and Chopping :
The process is usually employed when roots, barks, herbs, leaves, etc. are to be employed in the preparation of mother tincture. A pruning knife is quite sufficient for the work of a small quantity of substances. Knives, which have guides arranged, so that the blade has a horizontal as well as vertical movement, are preferable.
Powder-Sieve-Fineness of Powder
The fineness of powders is related to the number assigned to a certain standard sieve.
Sieves for pharmacopoeia testing are of wire cloth woven from brass, bronze or other suitable wire and are not coated or plated. The table gives the nominal dimensions, permissible variations and limits for woven wire cloth of standard sieves.
Powdered Vegetable or Animal Drugs:
The fineness of powdered vegetable and animal drugs is expressed in the pharmacopoeia by the following terms:-
A very coarse powder (No.8): all of the particles will pass through a No.8 sieve.
A coarse powder (No.20): all particles will pass through a No.20 sieve and not more than 40 per cent through a No.60 sieve.
A moderately coarse powder (No.40): all particles will, pass through a No.40 sieve and not more than 40 per cent through a No.80 sieve.
A fine powder (No.60): all particles will pass through a No.60 sieve and not more than 40 per cent through a No.100 sieve/
For Chemicals: The specifications for chemicals, differ from those established for vegetable drugs. The terms expressing the fineness of powder chemicals are defined as follows:
A coarse powder (No.20): all particles will pass through na No.20 sieve, and no more than 60 per cent will pass through a No.40 sieve.
A moderately coarse powder (No.40): all particles pass through a No.40 sieve, and not more than 60 per cent will pass through a No.60 sieve.
A fine powder (No.80): all particles pass through a No.80 sieve.
A very fine powder (No.120): all particles pass through a No.120 sieve. For very fine powders, boiling cloth is often used for the sifting medium, although sieve cloth No.300 size or even smaller is now available. This can also be obtained, made from monel metal or from stainless steel on various compositions. When acid substances are to be sifted, horsehair sieve may be used.
Whenever the fresh sample is not available for the preparation of the mother tincture it is necessary to use the dried material after proper identification and powdering it to the desired fineness in accordance with the instructions given in the individual monographs in the pharmacopoeia.
Determination of Alcohol Content:
Measure a definite quantity of the test liquid in a round- bottomed 200 to 250 ml. flask. If the liquid contains up to 20 Percent alcohol, take for determination 75 ml. If the liquid contains from 20 Percent to 50 Percent alcohol take 50 ml., and if the liquid contains alcohol 50 Percent and above take 25 ml. for determination of alcohol content.
In cases the test liquid contains volatile matter it should undergo preliminary treatment viz. if the liquid contains volatile acid, neutralize them with alkali solution; if it contains volatile bases, neutralize them with phosphoric acid or sulphuric acid.
Liquids containing free iodine are treated before distillation with zinc in the form of powder or with a small quantity of dry sodium thiosulphate until decolourisation of the liquid. To bind the volatile sulphurous compounds add some drops of sodium hydroxide.
In case, the test liquid contains ether, essential oils chloroform, camphor, etc., add in a separating funnel an equal volume of sodium chloride solution and an equal volume of petroleum ether. Shake the mixture for two to three minutes. Wait until the layers are separated. Run the aqueous alcohol layer into another separating funnel and treat once more with half the quantity of petroleum ether. Run the alcohol water layer into a distillation flask and shake the combined petroleum ether liquids with half the quantity of saturated sodium chloride solution, which is then added to the liquid in the distillation flask. Draw in air through the liquid for half a minute to remove the last traces of petroleum ether.
If the liquid contains less than 30 Percent of alcohol, the salting out should be done with dry sodium chloride instead of its solution.
Before distillation make the distilling liquid up to 75 ml. Use tightly fitting rubber stoppers for the distillation flask and the condensers. The receive should be immersed in a vessel with cold water. To ensure uniform boiling, place some places of porcelain in the flask.
Collect 48 ml. of distillate in the receiver, bring its temperature to 20* C and make up with water to its mark (50 ml. volumetric flask). The distillate should be clear or slightly turbid. The specific gravity of the distillate is determined, pycnometrically, and the corresponding alcohol contents in percents by volume read off in the alcoholometric table.
Calculate the alcohol content of the preparation in percents by volume from the formula.
X = 5-0. a/b.
Where X is the alcohol per cent of preparation, 50 is the volume of the distillate in milliliters, a is the alcohol content of the distillate and b is the volume of the test liquid taken for distillation.
Determination of Specific Gravity :
Weight per milliliter (of a liquid) is determined by dividing the weight in air, expressed in grammes, of the quantity of the liquid which fills a pycnometer at 20* or 25* by the capacity of the pycnometer at 20* respectively, expressed in milliliters. The capacity of the pycnometer at these temperatures is ascertained from the weight in grammes of the quantity of water required to fill the pycnometer. The following data are assumed :-
The weight of 1 ml. of water in air at 20* is 0.99719 g. and at 25* is 0.99602 g.
Ordinary deviations in the density of air do not affect the result of determination significantly.
The specific gravity of a substance is the weight of a given volume of that substance at a stated temperature, as compared with the weight of an equal volume of water at the same temperature, all weighings being taken in air. A suitable pycnometer is used for the determination of the Specific Gravity.
Determination of pH Value:
The pH value (Hydrogen ion concentration) of an aqueous liquid may be defined as the common logarithm of the reciprocal of the hydrogen ion concentration expressed in grammes per litre. This helps to qualitatively determine the acidity or the alkalinity of a solution. This is determined potentiometrically by means of a glass electrode and a suitable pH meter. The actual instructions to use the instrument are always given with the instrument by the manufacturer.
The acid base balance in a liquid is important from the point of view of stability of a liquid in terms of its properties. Storage, exposure to temperature and pressure tend to alter the pH, thus making the liquid unsuitable from its indicated use. It also helps to detect any adulteration in the liquid under question.
Determination of Optical Rotation and of Specific Rotation:
Certain substances, in a pure state or in solution, possess the property of rotating the plane of polarised light i.e. the incident light possess the property of rotating the plane of polarised light i.e. the incident light emerges in a plane forming an angle with the plane of the incident light.
These substances are said to be optically active and the property of rotating the place of polarised light is known as Optical Rotation. This property may be utilized either for assaying or identifying a substances. The place of polarised light may be rotated to the right or the left when a plus sign (+) or a minus sign (-) respectively is placed before the number indicating the degrees of rotation.
Optical rotation is defined as the angle through which the plane of polarised light is rotated when a layer of a liquid or solution of substance, one decimetre in thickness and at a temperature of 25*, unless otherwise stated, is examined by sodium light.
Apparatus – A polarimeter on which angular rotation, accurate to 0.05* can be read, may be used for general purposes.
Procedure – For liquid substances, take a minimum of five readings of the rotation of the liquid and also for the empty tube at the specified temperature. For solid substances, dissolve in a suitable solvent and take five readings of the rotation of the solution and the solvent used. Calculate the average of each set of the five readings and find out the correct optical rotation from the observed rotation and the reading with the blank (average).
Determination of Ash:
Take about 2 or 3 g. accurately weighed, of the pulverised drug in a tarred platinum or silica dish previously ignited and weighted. Scatter the powdered drug in a fine even layer on the bottom of the dish. Incinerate by gradually increasing the heat, not exceeding dull red heat, until free from carbon. Cool and weigh. If a carbon-free ash cannot be obtained in this way, exhaust the charred mass with hot water, collect the residue on an ashless filtrate, evaporate to dryness land ignite at a low temperature. Calculate the percentage of ash with reference to the air-dried drug. This helps to detect low-grade, exhausted or adulterated drugs.
Determination of Total Solids:
The term `total solids’ is applied to the residue obtained when the prescribed amount of the preparation is dried to constant weigh under the conditions specified below :-
Apparatus : Shallow, flat-bottomed flanged dishes, about 75 mm. in diameter and about 25 mm. deep made of nickel or other suitable metal of high heat conductivity which is not affected by boiling water.
Method: Weight accurately or measure an accurate quantity of the preparation and place in a tarred dish, evaporate at as low a temperature as possible, until the alcohol is removed, and heat on a water-bath until the residue is apparently day. Transfer to an over and dry to constant weight at 105* C. Owing to the hygroscopic nature of certain residues, it may be necessary to use dishes provided with well-fitting covers and to cool in an efficient desiccator.
Determination of Alcohol-Soluble Extractive:
Macerate 5 g. of the air-dried drug, coarsely powdered with 100 ml. of alcohol of the specific strength in a closed flask for twenty-four hours, shaking frequently during six hours and allowing it to stand for eighteen hours. Filter rapidly, taking precautions against loss of alcohol, evaporate 25 ml. of the filtrate to dryness in a tarred flat-bottomed shallow dish, dry at 105* and weigh. Calculate the percentage of alcohol-soluble extractive with reference to the air dried drug.
Determination of Water-Soluble Extractive:
Method 1 : Proceed as directed for the determination of alcohol- soluble extractive, using chloroform water instead of alcohol.
Method 2 : Add 5 g. to 50 ml. of water at 80* in a stoppered flask. Shake well and allow to stand for ten minutes; cool to 15* and add 2 g. of kieselgur filter. Transfer 5 ml. of the filtrates to a tarred evaporating basin 7.5 cm. in diameter, evaporate the solvent on a water-bath, continue drying for half an hour; finally dry in a steam oven for two hours and weigh the residue. Calculate the percentage of water-soluble extractive with reference to the air-dried drug.
Determination of Melting Range:
The melting-range of a substance is the range between the corrected temperature at which the substance begins to form droplets and the corrected temperature at which it completely melts, as shown by the formation of a miniscus.
Definition of Melting Point :
When a change of state from solid to liquid takes place the temperature at which it takes place is the Melting Point.
It is constant for a pure chemical at a given pressure. However, the effect of pressure is quite negligible and can be neglected in the practical work. If there is any impurity the melting point is lower than that of the pure substance.
Determination of Boiling Range:
The boiling range of a substance is the range of temperature within the whole, or a specified portion of the substance distills.
Definition of boiling point:
Boiling proceeds at a definite temperature, for any single substance at a fixed pressure. This temperature is called the Boiling Point. This is denoted usually at one atmosphere pressure.
Procedure – 100 ml. of liquid to be examined is placed in the distillation flask, and a few glass beads or other suitable substances are added. The bulb of the flask is placed centrally over a circular hole varying from 3 to 5 cm. in diameter (according to the boiling range of the substance under examination) in a suitable asbestos board. The thermometer is held concentrically in the neck of the flask by means of a well- fitting cork in such a manner that the bulb of the thermometer remains just below the level of the opening of the side-tube. Heat the flask slowly in the beginning and when distillation starts, adjust heating in such as starts, adjust heating in such a manner the the liquid distills at a constant rate of 4 to 5 ml. per minute. The temperature is read when the first drop runs from the condenser and again when the last quantity of liquid in the flask is evaporated.
The boiling ranges indicated apply at a barometric pressure of 760 mm. of mercury.
Determination of Congealing Temperature :
The congealing temperature of a liquid or of a melted solid is the highest temperature at which it solidifies.
The congealing temperature of the liquid is the same as the melting temperature of the solid but since the liquid may be cooled to a temperature below its congealing temperature without assuming the solid form the following method is employed to determine the congealing temperature of a liquid or a melted solid.
Apparatus : A test tube about 2 cm. internal diameter and about 10 cm. long, suspended by means of a bored cork inside a larger tube, about 3 cm. in diameter and 12 cm. in length, nd a standardized thermometer. Unless otherwise directed, place about 10 ml. of the liquid, or 10 g. of the melted solid, to be tested in the inner test tube previously dried; suspend the inner tube inside the outer tube and cool them together in water or in a suitable freezing mixture in a temperature about 5* below the expected congealing temperature. The temperature of water or freezing mixture should be adjusted according to the congealing liquid with a standardized thermometer until it begins to solidify. Congealing may be induced by adding a small crystal of the substance to the liquid or by rubbing the inner walls of the test tube with the thermometer. The highest temperature reached during solidification is regarded as the congealing temperature.
Determination of Refractive Index :
The refractive index (n) of a transparent substance is the ratio of her velocity of light in air to its velocity in that material under like conditions. It is equal to the ratio of the sine of the angle of incidence made by a ray in air to the sine of the angle of refraction made by the ray in the material being tested.
The refractive index (n) is, therefore, a physical property which is characteristic of a particular substance and is, therefore, useful as a means of identifying substances and establishing their purity. In many cases refractive index provides the basis for the quantitative analysis of simple mixture as of alcohol and water, if the relationship of refractive index and concentration is know. It is a simple matter to determine the refractive index of a liquid. The latter lends itself especially well to the examination of the more volatile liquids. Both instruments are provided with a polished prism with prism with which the liquid to be examined is in contact and through which light that has been refracted through the liquid is also refracted through the prism. The fast ray of light entering the prism provides a sharp demarcation between light and dark areas and is determined by the refractive index of the liquid. The index may be read directly, as in the able instrument, or in terms of an arbitrary, scale, the reading old which may be converted to refractive index by referring to tables supplied with the instrument.
Chromatography has opened new dimensions to the study of phytochemistry. It is a process for separating the components of a mixture by producing different rates of flow or movement for each component in a counter current system. In this a liquid flows over a stationary solid phase, carrying with it solutes which have varying degrees of affinity for the stationary phase. Different rates of flow are thus produced for each solute and physical separation is achieved. Paper Chromatography is easier to do than Thin Layer (Silica Gel) Chromatography.
There are various method of chromatography study viz. paper, than layer, columnar and gas chromatography. Each method has it special advantage. However, paper chromatography is much more advantageous to homoeopathic tincture study, as the separation is more marked.
The mechanics of operating paper chromatography is quite simple. The paper specially designed for this purpose is kept in a closed chamber where the atmosphere is saturated with the solvent vapour. If the developing solvent is water-saturated n-butanol, equal volumes of water and butanol are shaken together until they are mutually saturated. The butanol layer is then separated and placed in a reservoir which will be used to irrigated the paper strips. The aqueous layer is transferred to a second container. Both the aqueous and organic phases are then placed in a large covered chamber and allowed to stand until the atmosphere is saturated with the solvent vapours. The water which is absorbed by the paper is regarded as the stationary phase. A drop of solution containing the unknown mixture is applied a few inches from one end of the strip and allowed to dry. The paper is placed in the chromatography chamber and allowed to equilibrate with the vapour for about an hour. This is done to obtain reproducible Rf value. However, a know substance can be run side by side – Co TLC – to avoid the waiting period. The development is carried out by ascending or descending method.
It will be noticed that in the ascending method the movement of the solvent is somewhat slower than in the descending method. The distances travelled by the solvent and the solute are recorded. They are interpreted in terms of Rf values :-
Rf = Distance travelled by solute
Distance travelled by solvent
Rf is defined as the fractional distance travelled by a given solute in relation to the total distance travelled by the solvent front. Rf stands for Relative Flow.
Standard Drop Measure :
This was recommended at the Brussels Conference in 1902. The specifications are that the drop measure is 3 mm. in external diameter at the delivery end and, when held vertically, delivers 20 drops water, the total weight of which is between 0.9 g. and 1.1 g. at 15* C.
It must be borne in mind that one drop is not one minim, and 60 drops are not equal to 1 fluid dram.
100 ordinary drops or 60 minims of water are equal to one fluid dram.
But the volume of a drop of fluid depends on many factors such as density, temperature, viscocity, surface tension, etc. Thick substances like gums or syrups produce large drops, while chloroform and bromine form very small drops.
Tea Spoon :
No definite standardisation of a tea-spoon has yet been determined. Spoons are of many sizes. But owing to common and constant use of tea spoon in homes and other places, it may be said that 5 ml. can be taken as an average measure of a tea spoon.
The simplest and the most commonly used instrument for measuring temperature is a liquid thermometer. But the so accurate ones are the gas thermometers, which work on the principle of the relation of pressure and temperature. However, these cannot be used because of the difficulty in using glass apparatus that is used in a pharmacy laboratory.
Mercury is the most common liquid that is used in the thermometers.
There are three scales in vogue, but internationally only one is used now and that is Centigrade. The other two which were also used were Fahrenheit and Reaumur. Conversion of Scales :
(1) *C to *F – Multiply by 1.8 and add 32
(2) *F to *C – Subtract 32 and divide by 1.8
To maintain, raise or lower a temperature, heat may be applied directly to the point or conveyed through the medium of water, steam or air by pipes directly. Lately, heating devices have been introduced by the use of the property of electricity.
In many processes it is essential to regulate the temperature, the commonest form of contrivance used in the laboratory is water-bath. Heat is applied through gas, fuel or electricity. By using liquid inside this bath, a constant boiling temperature can be maintained. Thus boiling water will provide a constant temperature of 100*C (at NAP). Saline bath or oil bath is also used if the temperature regulation is required at more than 100* C. Glycerin bath is, however, used if 250* C is to be maintained. Sand bath is used to regulate and apply uniform heat to avoid any breakage and sudden heating. A very thin firm of sand is sufficient.
There are new temperature-regulating devices electrically operated and automatic temperature controls based on thermostatic principles, but such instruments are costly.
Molecules are composed of elements which may have translational, rotational or vibrational modes. Molecules as result of these movements have energy. The energy from ground level gets excited if light or electro-magnetic waves are passed through it. It is possible to effect a change in a particular type of molecular energy using appropriate frequency and wavelength of the incident radiation and measure them. This is the basis of spectroscopy.
Molecular change in Branch of Spectroscopy used
Property Energy level Based on Region of molecular instrument property used
1.Rotational Rotational Rotational Microwave
2.Vibrational Vibrational Vibrational Infra-red (IR)
3.Electronic Electronic Electronic Visible and Ultraviolet (UV)
It is the third method which finds wide application in determination (identification and estimation) of drugs.
The visible region corresponds to wave length 4000-4000A* (Angstrom 10 negative power of 8 cm; nm 10 negative power of 7 cm). UV region is 2000-4000k.
Coloured substance owe their colours due to presence of one or more unsaturated linkage (c=c, c=0 N=N etc.) which are called chromophores. A substitution on chromophores may lead to increase in colour (C-Br, C-OH, C-NH2 etc.). They are called auxochrome. Normally such substitution leads to red-shift called bathocromic effect. Result of these lead to change in absorption. Intensity of emitted light will decrease as the thickness or concentration of absorbing media (Drug solution) increase. The statement is mathematically expressed as follows (Beet Lambert Law):
Log Io/10It= Ecl, where to Io & It are intensifiers of incident and transmitted light, l is path length of absorbing solution in centimetres, is concentration of the drug in moles/litre.
log Io/10It is called absorbance or optical density
E is known as molecular extinction, coefficient.
At a particular wave length absorption of the drug become maximum which is called mad, it differs from drug to drug. There are instruments which can scan a solution, a drug at different wavelengths and automatically record absorption. Such a record gives a graph with a peak which may be very sharp, broad or very broad. These maximas lead to identification and estimation of drugs.
The Schoniger oxygen Flask method of for Sulphur and Phosphorus Apparatus : Schoniger’s oxygen flask.
Weigh accurately about 5-10 mg of sample onto a piece of ashless whatman filter paper which is folded in such a way that the tail (wick) is free. This is than placed in a platinum basket or carrier suspended from the ground glass stopper of 500 ml conical flask. The flask containing about 50 ml absorbing solution (2.5M Nitric acid for phosphorus and 30 Percent hydrogen peroxide for sulphur), is filled with oxygen and then sealed with the stopper with the platinum basket attached.
The wick of the sample paper can either be ignited before the stopper is placed in the flask, or better still ignited by remote electrical, control, or by an infra-red lamp.
After standing for a few minutes until any combustion could has disappeared, the flask is shaken for 2-3 minutes to ensure complete absorption. The solution can then be treated by a method appropriate to the element being determined.
Reagent : Ammonium vanadate solution : Dissolve 2.5g ammonium vanadate (NH4 VO3) in 500ml hot water, add 20ml concentrated nitric acid and dilute with water to 1 lit. in a graduated flask.
Ammonium molybdate solution : Dissolve 50g ammonium molybdate (NH4) 6M07 024 4h2O in warm water and dilute to 1 lit in a graduated flask. Filter the solution before use.
Procedure: Dilute 50ml solution of schoniger oxygen flask to 100ml with 2.5M Nitric acid in volumetric flask. Place a 10ml aliquot of this solution in a 100ml graduated flask add 50ml water, 10ml of ammonium vanadate solution and 10 ml of ammonium vanadate solution and 10ml water, 10ml of ammonium vanadate solution and 10ml of ammonium molybdate solution at 465nm against blank prepared in the same manner omiting sample in 1 cm cell.
Prepare a series of solutions from potassium dihydrogen phosphate covering the range 0-2mg phosphorus per 100ml and containing the same concentration of acid, ammonium vanadate and ammonium molybdate as the previous solution. Construct a calibration curve and use it to calculate the concentration of phosphorus in the sample.
Procedure: Boil 50ml solution obtained from schoniger oxygenflask to remove all hydrogen peroxide. Adjust the pH to 4 with dilute hydrochloric acid or ammonia solution. Make up the value to 1000ml. Take 50ml of this aliquot in 100ml volumetric flask, add 10ml of a buffer (pH 4; 0.05M solution of A. R. potassium hydrogen phthalate) and 50ml of 95 per cent ethanol. Dilute to the mark with distilled water, and 0.3g of barium chloranilate and shake the flask for 10 minutes.
Remove the precipitate by filtering and measure the absorbance of the filtrate with spectrophotometer at 530 nm against a blank prepared in the same manner. Construct a calibration curve using standard potassium sulphate solutions prepared from the A. R. salt.