Organic compounds when isolated from natural sources or prepared by organic reactions are seldom pure; they are usually contaminated with small amounts of other compounds which are produced along with the desired product. Before carrying out the qualitative and quantitative_ analysis of the organic compounds, in order to characterise them, it is very important of purify them. The various steps involved in the characterisation of an organic compounds are:
(b) Qualitative analysis
(c) Quantitative analysis
(d) Determination of molecular mass
(e) Calculation of empirical and molecular formula
(f) Elucidation of the structure by various methods including chemical methods.
In this unit, we shall consider some of the methods commonly employed for the purification, qualitative analysis and quantitative analysis of an organic compound.
The common techniques used for the purification of a particular compound are based on its nature and also on the nature of the impurities present in it. The various methods used for this purpose are:
1. Crystallisation 2. Sublimation
3. Differential extraction 4. Distillation
5. Fractional distillation
6. Distillation under reduced pressure
7. Steam distillation 8. Chromatography.
Crystallisation is a process of solidification of a pure substance from its dissolved state. This is the most common way of purifying organic solids. This method is based upon differences in their solubility in a given solvent or a mixture of solvents. Since most of the organic compounds are not fairly soluble in water, other solvents such as alcohol, acetone, chloroform, ether, etc., are also commonly used for this purpose. The process involves the steps given below:
(i) Selection of the Solvent
A small quantity of the substance is heated with 2- 3 m1 of the solvent under examination. When partial or complete dissolution has taken place, the solution is cooled. If a considerable proportion of the dissolved substance separates out in the form of crystals, the solvent is considered suitable. Thus, a suitable solvent is one in which:
(a) The substance dissolves on heating readily and crystallises out on cooling.
(b) The solvent must not react chemically with the substance.
(c) Either it should not dissolve impurities or their solubility is much more than the substance to be purified so that during crystallisation, they should be left in the mother liquor.
Some examples are: ethanol can be used for purification of sugar containing impurities of common salt. Common salt does not dissolve in ethanol.
Similarly, a benzoic acid containing impurities of naphthalene can be purified by using hot water which dissolves only benzoic acid but not naphthalene.
(ii) Preparing the Solution
The impure substance is powdered and heated with the solvent in a flask. The amount of solvent should be just sufficient to dissolve the substance on heating. In the case of solvents with low boiling points, the flask is fitted with a water condenser or an air condenser to avoid the loss of sol vent on heating (Fig. 39.1).
The hot saturated solution prepared above is then filtered preferably through a fluted filter paper placed in a glass funnel (Fig. 39.2). The use of fluted filter paper makes the filtration rapid. If the organic substance crystallises during filtration, the filtration cannot be done with ordinary filter paper as the crystals will be formed on the filter paper itself and proper
filtration will not be possible. In such cases, the filtration is done with the help of hot water funnel (Fig. 39.3). The hot water that is circulated in the outer funnel keeps the solution hot and the crystals cannot be formed on the funnel itself
The hot filtrate is .allowed to cool slowly in a beaker or a crystallising dish. In this way, large and beautiful crystals are obtained. Scratching the sides of the vessel often facilitates crystallisation.
(v) Separation and Drying of Crystals
The crystals are separated from the mother liquor by filtration with the help on a Buchner funnel and a suction pump (Fig. 39.4). The crystals obtained on the filter paper
are washed two or three times with small quantities of the pure solvent. They are dried by pressing between folds of filter paper and are then left in a steam over or an air over for some time, if the substance is stable and high’ melting. The crystals are finally dried over sulphuric acid or calcium chloride in vacuum desiccator.
Sometimes the crystals obtained are slightly coloured because of the presence of traces of impurities. In such cases, they are redissolved in a small amount of the solvent and a little of animal charcoal is added. The suspension thus obtained is boiled, filtered and recrystallised as described above.
Sublimation is a process of conversion of a solid into gaseous state on heating without passing through the intermediate liquid state and vice versa.
This process is used for the separation of volatile solids, which sublime on heating from the non-volatile solids. The impure substance is heated in a dish covered with a perforated filter paper over which an inverted funnel is placed (Fig. 39.5). The stem of the funnel is plugged with a little cotton. Vapours of the solid, which sublime, pass through the holes in the filter paper and condense on the cooler walls of the funnel. The non-volatile impurities are left behind in the dish.
Iodine, camphor, naphthalene, benzoic acid, etc., are purified by this method.
3. DIFFERENTIAL EXTRACTION
Organic compounds, whether solids or liquids, can be recovered from aqueous solutions by shaking the solution in a separating funnel with a suitable organic solvent which is immiscible with water but in which the organic compound is highly soluble. This process is known as extraction or solvent extraction. Some commonly employed solvents are, ether, benzene and chloroform. The process is carried out as follows: The aqueous solution is mixed with a small quantity of the organic solvent in a separating funnel (Fig. 39.6). The funnel is stoppered and its contents shaken for some time
when the organic solvent dissolves out the solute. The mixture is now allowed to settle and in this way solvent and water from two separate layers. The lower aqueous layer is run out by opening the tap and the solvent layer is collected separately. The whole process may be repeated to remove the solute completely from the aqueous solution. The solute is finally recovered from the organic solvent by distilling off the latter.
Distillation is a process of conversion of a liquid into vapours by heating followed by condensation of vapours so produced by cooling. The method is used for the purification of liquids which boil without decomposition and are associated with non-volatile impurities. The impure liquid is boiled and the vapours, thus, formed are condensed to get the pure liquid.
The apparatus employed for the process consists of a distillation flask having a side tube connected with a condenser. An air condenser which is a long glass tube is used for liquids with boiling points above 380 K and a water condenser in the case of other liquids. The neck of the flask is closed with a cork through which passes a thermometer and the bulb of the thermometer is kept just below the opening of the side tube. A receiver is attached to the lower end of the condenser (Fig. 39.7). The substance is placed in the distillation flask and one or two pieces of unglazed porcelain or glass bends are added to prevent bumping of the liquid .during distillation. The flask is heated on a water bath or sand
bath in the case of highly volatile and inflammable liquids, while it is heated directly with the flame when distilling high boiling liquids. The liquid changes into vapours which pass through the condenser and are condensed back into the liquid form. The liquid distillate is collected in the receiver while the impurities are left behind in the distillation flask.
5. FRACTIONAL DISTILLATION
This process is used to separate mixture of two or more miscible liquids having different boiling points. It can be carried in two ways as under.
(a) By using an apparatus of simple distillation
When the two liquids have their boiling points wide apart, say about 40° or more, the mixture may be separated by using simple distillation apparatus. The more volatile liquid (low b.p.) distils over first and is collected in a receiver. When the temperature begins to rise again, the receiver is disconnected. A new receiver is attached when the temperature again becomes constant. At this stage, the less volatile liquid distils over and is collected. The two fractions thus obtained are redistilled separately, a number of times, to ensure complete purity of the liquids.
(b) By using a fractionating column
When the boiling points of the two liquids are quite close(difference less than 30°) the separation is effected by fitting the distillation flask with a fractionating column (Fig. 39.8).
A fractionating column is a long tube provided with obstructions to the passage of the vapours upwards and that to liquid downwards. Various types of fractionating columns commonly used are shown in Fig. 39.9.
The fractionating column is fitted into the neck of a round-bottomed flask containing the mixture of liquids (say A with b.p: 350 K and B with b.p. 360 K). The column itself is fitted with a thermometer and is attached to a condenser. On heating the flask the vapours obtained consist of more of A and less of B. As the vapours rise up the fractionating column, they condense partially· and the condensed liquid flows down. Naturally, the vapours of B (having lower boiling point) condense more readily than the vapours of A (having lower boiling point). As a result, the vapours moving up get richer in A. The condensed liquid flowing down meets the ascending stream of vapours and in the process takes away more of B. This process is repeated throughout the length of fractionating column. Consequently, the vapours which escape from the top of the column into the condenser consist almost of A. Thus, the distillate received is almost pure A whereas the liquid left behind in the flask is very rich in B. This process may be repeated to achieve the complete separation of liquids.
Fractional distillation has found remarkable application in modem industry especially in the distillation of petroleum, coaltar and crude alcohol.
6. DISTILLATION UNDER REDUCED PRESSURE
Principle. A liquid boils when its vapour pressure becomes equal to the applied pressure or external pressure. If applied pressure is decreased, the same liquid will now, boil at a lower temperature. Thus, this process is employed if the liquid has a tendency to decompose near its boiling point. Under reduced pressure, the liquid will boil at a low temperature and the temperature of decomposition will not be reached. Glycerol, for example, boils with decomposition at 563 K but if the pressure it reduced to 12 mm, it boils at 453 K without decomposition.
The apparatus used for distillation under reduced pressure is shown in Fig. 39.10. The liquid to be distilled is taken in a two necked flask called “Claisen’s flask”. It is fitted with a long capillary tube in the main neck and a thermometer in the side neck. The side tube of the second neck is connected to a condenser carrying a receiver at the other end. The receiver is further connected to a vacuum pump and a manometer which indicates the pressure. The whole apparatus is tested to be air-tight.
The flask is usually heated on an oil bath or sand both. During the distillation a stream of air bubbles is allowed to pass through the capillary tube to prevent bumping by ensuring steady pressure. The desired pressure is maintained by working the pump.
Distillation under reduced pressure finds a number of applications in various industries. For example, in sugar
7. STEAM DISTILLATION
The process of steam distillation is employed in the purification of a substance from non-volatile impurities provided the substance itself is volatile in steam and insoluble in water. This method is based on the facts that:
(i) A liquid boils at a temperature when its vapour pressure becomes ·equal to the atmospheric pressure.
(ii) The vapour pressure of a mixture of two immiscible liquids is equal to the sum of the vapour pressures of the individual liquids.
In the actual process, steam is continuously passed through the impure organic liquid. Steam heats the liquid but itself gets practically condensed to water. After some time, the mixture of the liquid and water begins to boil because the vapour pressure of the mixture becomes equal to the atmospheric pressure. Obviously, this happens at a temperature which is lower than the boiling point of the substance or that of water. For instance, a mixture of aniline (b.p. 453 K, with decomposition), and water (b.p. 373 K), under normal atmospheric pressure, boils at 371 K. At this temperature the vapour pressure of water is 717 mm and that of aniline is 43 mm and, therefore, the total vapour pressure is equal to 760 mm. Thus in steam distillation the liquid gets distilled at a temperature lower than its boiling point and any chances of decomposition are avoided.
The proportion of water and liquid in the mixture that distils over is given by the relation
w1 / w2 = p1 xl8
w2 P2 x M
where w 1 and w2 stand for the masses of water and the organic liquid that distils over; p 1 and p2 represent the vapour pressures of water and the liquid at the distillation temperature and M is molecular mass of the liquid (molecular mass of water being 18).
The apparatus set up for steam distillation is shown in Fig. 39 .11. The impure liquid (or the solid, along with some water) is taken in a round-bottomed flask which is kept in a slightly slanting position. It is attached to a steam generator and steam is passed into the flask. The flask may be gently heated on a sand bath to prevent undue condensation of steam in it. The distillate consisting of water and the organic substance is collected in the receiver. The pure substance is separated from water with the help of a separating funnel or by extraction with a suitable solvent
It is most useful and modem technique of separation and purification of organic compounds. The method was first developed by Tswett in 1903.for the separation of coloured substances into individual components. Since then the method has undergone tremendous modifications. Nowadays various types of chromatographic methods are in use to separate any mixture, into its constituents irrespective of whether it is coloured or colourless.
Chromatography in general, may be described as the technique of separating the constituents of a mixture by the differential movement of individual components through the stationary phase under the influence of mobile phase.
Different types of chromatographic techniques are employed depending upon the nature of stationary and mobile phases.
Two of the important categories of chromatography are:
(A) Adsorption chromatography
(B) Partition chromatography
A. ADSORPTION CHROMATOGRAPHY
This category of chromatography is based on the principal of differential adsorption of various components of the mixture on a suitable adsorbent. Some components are more strongly absorbed than others. When the mobile phase is allowed move over the stationary phase (adsorbent), the components of the mixture move by varying distances over the stationary phase. Two main types of chromatographic techniques based on the principal of differential adsorption are:
• Column chromatography
• Thin layer chromatography.
This is the simplest chromatographic method. In this technique, the mixture of substances or the substance to be purified is passed through the column of suitable adsorbent. The constituents get adsorbed to the different extent. In general the following steps are involved (Fig. 39.12).
(i) Preparation-of Column
A column is prepared in a long burette like glass tube having a stopcock near the bottom. A plug of cotton or glass wool is placed at the bottom of the column to support the adsorbent. The tube is packed uniformly with suitable adsorbent which is usually taken in the form of a slurry in petroleum ether. This constitutes the stationary phase. The commonly employed adsorbents are: activated aluminium oxide (alumina), magnesium oxide, silica gel, starch and Fuller’s earth. Nearly one-fourth of the tube is left empty. A loose plug of cotton or glass wool is then placed at the top of the adsorbent column.
(ii) Adsorption Process
The substance to be purified is added, (as such if it is a liquid or in the form of its solution in some suitable solvent if it is a solid) at the top of the column and allowed to pass
slowly through it. As it passes through the column, the different components of the mixture get adsorbed. Different constituents of mixture get adsorbed to the different extent and forms bands at different parts of column.
It is a process of extraction of the adsorbed components from the adsorbent with the help of suitable solvent called eluent. Eluent is added as soon as the last portion of the mixture to be separated enters the column. This acts as a moving phase. The eluents usually employed are petroleum ether, carbon tetrachloride, benzene, alcohol, etc., and their selection depends upon the relative solubilities of the components of the mixture in them. More than one eluent may be used in certain cases. The eluents dissolve out the different components selectively. The weakly adsorbed component is eluted more rapidly than a strongly adsorbed component. Such a progressive separation of a mixture is shown in Fig. 39.13. Different components of the mixture are collected in the form of different fractions in separate conical flasks. The eluent from each fraction is then distilled off to get the various components in pure form.
This technique is now widely used in research laboratories for the purification of different substances and for the separation of mixtures. For example, a mixture of an alcohol and a liquid hydrocarbon may be separated by using alumina as adsorbent and petroleum ether as eluent.
Thin Layer Chromatography (TLC)
Thin layer chromatography is another type of adsorption chromatography, which involves separation of the substances of a mixture over a thin layer of an adsorbent. Various steps involved are (i) Preparation of TLC plate two pencil lines are draw across the width of the plate about 1 em each from the top and bottom end. The lower pencil mark is called the starting line and the upper line is called the finish line or solvent line. A thin layer (about 0.2 mm thick) of an adsorbent (silica gel or alumina) is spread over a glass plate of suitable size.
The plate is known as thin layer chromatography plate (TLC plate). (ii) Separation process the solution of the mixture to be separated is applied as a small spot about 2 cm from one end of the TLC plate. The glass plate is then placed in a closed jar containing the solvent (Fig. 39.14). As the solvent in the jar moves up, the components of the mixture move up along the plate to different distances depending on their degree of adsorption and separation takes place. The relative adsorption of each component of the mixture is expressed in terms of its retention factor i.e., R, value (Fig. 39.15).
R= Distance moved by the substance from base line /
Distance moved by the solvent from base line
For or example, R t for component A= x A / y ; R f for compound B= x B / y
The spots of coloured compounds are visible on TLC plate due to their original colour. The spots of colourless compounds which are invisible to the eye can be detected by any of the following techniques.
(a) u .v light. This method is used for certain organic compounds which produce fluorescence effect in u .v light. The spots of such substance can be detected by placing under u .v lamp.
(b) Use of iodine vapours. In this method, the developed TLC plate is placed in a covered jar containing a crystals of iodine. Spots of the compounds which adsorb iodine will become brown.
(c) Chemical methods. This method involves spraying of suitable chemical reagents on the TLC plate. · For example, amino acids may be detected by spraying the plate with ninhydrin solution. Similarly, aldehyde/ketone can be detected by spraying the plate with 2, 4-dinitropheny hydrazine.
B. PARTITION CHROMATOGRAPHY
Partition chromatography is liquid-liquid graphy unlike TLC and column chromatography represents solid-liquid chromatography. In chromatography, the stationary phase as well as mobile are both liquids.
Principle. Partition chromatography is based continuous differential partitioning (distribution) components of a mixture between stationary and mob phases. One of the common example of partition chromatograph is paper chromatography. In paper chromatography, a special quality paper known as chromatography paper is used. Paper is mainly cellulose, but cellulose does not constitute stationary phase. The stationary phase in paper Chromatography is water, trapped or chemically bound to cellulose. The mobile phase is liquid mixture of two or more substances with water as one of the component.
Process. A suitable strip of chromatographic paper is taken and starting line is drawn across the width of the paper about 1 or 2 cm from the bottom. A spot of the mixture of components to be separated is applied on the starting line with the help of fine capillary or syringe. The spotted paper is then suspended in suitable solvent (or a mixture of solvents (Fig. 39.16). This solvent acts as the mobile phase. The solvent rises up the paper by capillary action and flows over the spot.
The different component of the mixture travel through different distances depending upon their solubility in (or partioning between) the stationary and mobile phases. When the solvent reaches the top end the paper is taken out and is allowed to dry. The paper strip so developed is known as a chromatogram. The spots of the separated coloured compounds are visible at different heights from the position of initial spot on the chromatogram. The spots of the separated colourless compounds may be observed either under ultraviolet light or by the use of an appropriate spray reagent as discussed under thin layer chromatography.
CRITERIA OF PURITY OF ORGANIC COMPOUNDS
After purification, the test of purity of an organic compound is the next important step. A pure organic compound possesses characteristic physical properties such as refractive index specific gravity boiling point, crystalline structure and melting point. If a purified sample shows the same properties that the pure substance is known to possess, if may be considered as pure and no further purification is required. However, for practical purposes in laboratory only melting point (in the case of solids) and boiling point (for liquids) are used as criteria for a purity. A pure organic solid has a definite and sharp (sudden, rapid and complete) melting point, while an impure substance has a lower and indefinite melting point. The melting point of a solid may be defined as the temperature at which the solid and liquid states of the compound are in equilibrium with each other at an external pressure of 1 atmosphere.
Determination of melting point of a solid A capillary tube, 5 to 6 cm long and 1 mm in diameter, is closed at one end and finally dried and powdered organic solid is inserted in it. The tube is tapped to accumulate the crystals at the bottom of the capillary tube. Filling is continued until the length of the packed solid is 3 to 4 mm.
The apparatus used for the determination of melting point is shown in Figs. 39.17(a), (b) and (c). The bulb of the thermometer is dipped in sulphuric acid bath and then taken
out. The capillary tube is then placed along with the thermometer in such a manner so that the sealed end of the capillary is near the bulb. The capillary tube sticks to the thermometer due to surface tension and viscosity of the acid. The thermometer is now lowered in the bath and held in a position so as to keep half of the capillary tube out the bath. The bath is slowly heated as to keep the temperature uniform throughout the bath. When the solid in the capillary tube just shows signs of melting, the heating is stopped. The temperature at which the substance just melts and becomes transparent is recorded. This is the melting point of the organic solid.
Thiele’s tube or round bottom flask (50 mL) or a boiling tube can be used for the determination of melting point of an organic solid.
If the determined melting point is the same as that of the pure compound, the sample in question is also said to be a pure substance.
Mixed melting point: The melting point of two thoroughly mixed substances is called mixed melting point. This can also be used for ascertaining the purity of a compound.
The substance, whose purity is to be tested, is mixed with a pure sample of the same compound. The melting point of the mixture is determined. If the melting point of the mixture is sharp and comes out to be the same as that of pure compound, it is sure that the compound under test is pure. On the other hand, if the melting point of the mixture is Jess than the melting point of the pure compound, the compound in question is not pure.
Boiling point: The boiling point of a liquid is defined as the temperature at which the vapour pressure of a liquid is equal to external pressure. Boiling points are also normally quoted for standard atmospheric pressure. A pure organic liquid boils at a fixed temperature which is characteristic of that substance.
If enough liquid is available, it can be distilled in a distillation apparatus and the constant temperature recorded by the thermometer is the boiling point (Fig. 39.18).
Boiling point is not as reliable a test of purity as is the melting point for the solids. There are many liquids which are miscible with other liquids and mixtures have fixed boiling points (azeotrope). Thus, other physical properties are being used for deciding the purity.
Fig. 39.18 Determination of boiling point.
When the amount the liquid is small, Siwoloff’s method (capillary tube method) is used.