Chapter 3: Some Analytical Techniques
Towards the end of the 1890s, Marie and Pierre Curie noticed something that did not add up. A uranium ore called pitchblende was far more radioactive than its uranium content could explain. Something else had to be hiding inside it — something present in almost unimaginably small amounts.
To prove it, the Curies did not need a new theory. They needed separation. Working in a poorly equipped shed in Paris, Marie Curie processed tonnes of pitchblende residue, dissolving, boiling, filtering and crystallising the same solutions again and again. The difficulty was that the new element, radium, behaved chemically almost exactly like barium, which was present in far larger quantity. The two could not be told apart by any simple test.
Her solution was a purely physical difference: radium chloride and barium chloride are not equally soluble. By crystallising the mixture, collecting the crystals, concentrating the leftover solution and crystallising again — thousands of times over — she slowly pushed radium into one fraction and barium into another. By 1902 the entire effort had yielded about a tenth of a gram of radium chloride, pure enough for her to measure the atomic mass of a brand-new element.
Chemistry cannot study what it cannot first separate. That is exactly what this chapter is about.
Diagram
Fig. 3.0 – A single opening illustration in three panels: (1) crude, dirty-looking solid in a beaker of hot solvent on a flame, (2) hot filtration through a funnel with insoluble impurities left on the paper, (3) clean, well-formed crystals in an evaporating dish; caption arrow labelled “impure → pure” running beneath all three panels
Work through this chapter carefully, and by the end of it you will be able to reason out the answer to every one of these questions.
3.1 Introduction
Over the years there has been a systematic development in the techniques used for the analysis of chemical substances. In this chapter we look at the most basic of them — the techniques of purification and separation.
Why Purity Comes First
Chemical substances occur in nature in an impure stage. Even when a compound is synthesized in the laboratory, it is first obtained in a crude and impure form, mixed with leftover starting materials, side products and traces of solvent.
Before investigating the composition and properties of a substance, it is essential to obtain it in the pure form. This is not a matter of neatness. A melting point, a boiling point or a density belongs to a substance only when the sample tested is pure; an impure sample gives a shifted, unreliable value and can lead to a completely wrong conclusion about what the substance is.
The Basis of Every Technique
Methods of purification and separation of compounds depend on the difference in their physical properties — properties such as solubility, boiling point, and the tendency to stick to a surface.
Notice that these are physical differences, not chemical ones. Because no chemical reaction is involved, the substance we want is recovered chemically unchanged at the end of the process. That is the whole idea behind every technique in this chapter.
The technique you choose therefore depends on which physical property differs most sharply between the substance and its impurities, and on whether the sample is a solid or a liquid. That decision is mapped out in FC 3.1.
Flowchart
FC 3.1 – Choosing a purification technique: impure sample → is it a solid or a liquid? → if SOLID → impurity insoluble in the chosen solvent → filtration; impurity soluble → crystallization; two soluble solids of widely different solubility → fractional crystallization. If LIQUID → large difference in boiling points → simple distillation; small difference in boiling points → fractional distillation; very high boiling point or decomposes on heating → distillation under reduced pressure. If SOLUTE DISSOLVED IN WATER → more soluble in an immiscible organic solvent → solvent extraction. For very small quantities or closely similar components → chromatography
3.2 Purification of Solids
Two Kinds of Impurity
A solid substance may contain two types of impurities: (i) those which are soluble in the same solvent as the main substance, and (ii) those which are not soluble in the same solvent as the substance.
This distinction matters because it decides the technique. If an impurity dissolves in the same solvent as the main compound, both will be in solution together and no amount of pouring or straining will separate them. If it does not dissolve, the two are already in different physical states — one in solution, one as a suspended solid — and separating them becomes straightforward.
Separating the Insoluble Impurity
The second type of impurity can be separated easily using a suitable solvent to dissolve the main compound, while the impurities remain undissolved. They can then be separated by a simple process called filtration.
You have met this process outside the laboratory many times. It is the same idea as separating tea leaves from a decoction of tea, or separating sand from a mixture of sand and water.
Filtration is carried out with the help of a filter paper cone placed in a funnel, as shown in Fig. 3.1. A circular piece of filter paper is folded to form a cone and fitted in the funnel. The funnel is fixed on a stand and a beaker is kept below it. The paper is made moist, and the solution to be filtered is then poured on the filter paper.
Diagram
Fig. 3.1 – Separation of a mixture of water and sand by simple filtration: three-stage picture showing (1) beaker with mixture of water and sand, (2) folded filter paper in a filter funnel on a stand with beaker below, (3) residue held on the paper and clear filtrate collected in the beaker; label mixture of water and sand, filter paper, filter funnel, residue, filtrate
Moistening the paper is not a small detail. A damp cone sticks to the glass wall of the funnel, so the liquid cannot slip down the gap between paper and glass and carry unfiltered solid with it.
The insoluble part remaining on the filter paper is called residue, and the liquid collected in the beaker is called filtrate.
Filtration Under Suction
When filtration is carried out using a vacuum pump it is called filtration under suction. It is a faster and more efficient technique than simple filtration.
The assembly for filtration under suction consists of a thick wall conical flask with a side arm. The flask wall is deliberately thick because the pressure inside it is reduced, and a thin-walled flask could collapse inward.
The flask is connected to a safety bottle by a rubber tube through the side arm. The safety bottle is used to prevent sucking of the filtrate into the suction pump. A special porcelain funnel called a Buchner funnel is fitted on the conical flask with the help of a rubber cork, as shown in Fig. 3.2.
Diagram
Fig. 3.2 – Setup for filtration under suction: Büchner funnel with moistened filter paper resting on a porous plate, seated in a rubber bung on a thick-walled Büchner flask with side arm and rubber tubing to the suction line; label large solid particles held on the paper, porous plate (plate with holes in), rubber bung, filtrate collecting in the flask, suction from aspirator creating partial vacuum
The Buchner funnel has a porous circular bottom. A circular filter paper of correct size is placed on the circular porous bottom of the Buchner funnel, and the funnel is placed on the flask. It is moistened with a few drops of water or solvent. Suction is created by starting the pump and filtration is carried out. Crystals are collected on the filter paper and filtrate in the flask.
The size of the paper matters here as much as the moistening does. A disc that is too small leaves holes uncovered, and a disc that is too large curls up at the edge — in either case the mixture leaks past the paper instead of through it.
3.2.1 Crystallization
When a crude solid is made of mainly one substance and has some impurities, it is purified by the process of crystallization.
It is done in four steps, and the logic of the whole method is easier to hold on to if you see the sequence together, as set out in FC 3.2.
Flowchart
FC 3.2 – Crystallization in four steps: crude solid → (i) boil with a small but sufficient quantity of a suitable solvent → hot saturated solution → (ii) hot filtration (under suction) → insoluble impurities removed as residue; hot filtrate collected → (iii) cool the filtrate → solubility falls → solution becomes supersaturated in the main solute → pure crystals separate; soluble impurities stay dissolved → (iv) filtration → pure crystals on the paper + mother liquor as filtrate
Step (i): Preparation of a Saturated Solution
A saturated solution is a solution which cannot dissolve additional quantity of solute.
A saturated solution of the crude solid is prepared by boiling it in a small but sufficient quantity of a suitable solvent. The quantity is kept small on purpose: the less solvent used, the closer the solution is to saturation, and the more solid will be forced out again when it cools.
On doing so the main solute forms an almost saturated solution, but the solution is not saturated with respect to the soluble impurities, as they are in small proportion. This single sentence is the heart of the method — the main substance is present in bulk and is close to its limit, while the impurity is present in traces and is nowhere near its limit.
Step (ii): Hot Filtration
The above solution is quickly filtered while hot. Filtration under suction allows rapid filtration. Undissolved impurities get removed in this process as residue.
Speed matters at this stage. If the solution is allowed to cool on the funnel, the dissolved substance begins to crystallize inside the filter paper, and the crystals you were trying to collect are lost along with the residue.
Step (iii): Cooling of the Filtrate
The hot filtrate is allowed to cool. Solubility of a substance decreases with lowering of temperature.
As a result, the filtrate becomes supersaturated with respect to the main dissolved solute. In other words, the cooled solution is now holding more of that solute than it can properly keep in solution at the lower temperature.
The excess quantity of the dissolved solute comes out of the solution in the form of crystals. The dissolved impurities, however, do not supersaturate the solution, as their quantity is small. These continue to stay in the solution in dissolved state even on cooling. The separated crystals are, therefore, free from soluble impurities as well.
Diagram
Fig. 3.3a – Solubility curve graph: x-axis temperature 0–100 °C, y-axis solubility in g per 100 g water (0–250); one steeply rising curve labelled KNO₃, one almost flat curve labelled NaCl; dashed vertical guide lines at 20 °C and 80 °C
Step (iv): Filtration
The crystals of the pure substance are separated by filtration. The filtrate obtained is called mother liquor. The crystals so formed are free from soluble as well as insoluble impurities.
Choice of the Solvent
The solvent to be used for crystallization must have the following properties:
- The compound to be crystallized should be least or sparingly soluble in the solvent at room temperature but highly soluble at high temperature.
- Solvent should not react chemically with the compound to be purified.
- Solvent should be volatile so that it can be removed easily.
Each condition earns its place. A solvent that dissolves the compound just as well when cold gives you almost no crystals on cooling. A solvent that reacts with the compound destroys the very substance you are purifying. And a solvent that will not evaporate cleanly leaves the dried crystals damp with it.
Water, ethyl alcohol, methyl alcohol, acetone, ether or their combinations are generally used as solvent for crystallization. The choice is done by trial and error method.
Activity: Crystallization of Common Salt from an Impure Sample
Many times the common salt obtained from the market may contain some siliceous matter and other impurities. These can be removed and larger crystals of pure NaCl can be obtained.
Apparatus: Beaker, glass rod, funnel, filter paper and stand, evaporating dish, etc.
Materials: Impure market salt or a mixture of salt and fine sand, etc.
Procedure: Arrange apparatus as shown in Fig. 3.3. Take some water in a beaker. Add salt to the beaker and stir it with a glass rod. Add more salt and stir till no more salt dissolves — this is the point at which the solution has become saturated. Heat the solution. Filter the hot solution; insoluble impurities will remain on the filter paper. Collect the filtrate in an evaporating dish and allow to cool. Crystals of pure salt NaCl will separate, leaving soluble impurities in the mother liquor. Filter the solution and collect the crystals on the filter paper and dry them.
Diagram
Fig. 3.3 – Process of crystallization in three stages linked by arrows: (1) evaporation — beaker of soluble solid in solution heated over a burner, (2) filtration — hot saturated solution poured through filter paper in a funnel on a stand, filtrate collecting in a beaker below, (3) crystallisation — crystals of the soluble solid in an evaporating dish; label soluble solid in solution, filter paper, hot saturated solution, crystals of soluble solid
Impure copper sulphate can be purified by crystallization using water as solvent. Similarly, benzoic acid can also be purified by crystallization using water as solvent.
3.2.2 Fractional Crystallization
Two or more substances in a mixture can be separated by fractional crystallisation process.
Note the condition carefully: both substances are soluble, so filtration alone is useless. What separates them is that their solubilities differ widely, so on cooling they reach their limits at different stages.
Mixture of two solutes A and B are dissolved in a suitable hot solvent to prepare a saturated solution. The saturated solution is filtered to remove dust particles and then allowed to cool.
As the solution cools, the solute which is less soluble crystallizes out first. This is the key step: the less soluble solute reaches its saturation limit earlier, while the more soluble one is still comfortably in solution.
The crystals are filtered, washed with solvent and dried. Washing removes the film of mother liquor clinging to the crystal surfaces, which would otherwise dry on them and contaminate the product.
The mother liquor is concentrated by evaporating the solvent. The second solute crystallizes from the mother liquor. These crystals are filtered to obtain the separated and purified second component. The whole sequence is set out in FC 3.3.
Flowchart
FC 3.3 – Fractional crystallization: mixture of solutes A and B → dissolve in hot solvent → saturated solution → filter to remove dust particles → cool → less soluble solute crystallises first → filter → crystals washed with solvent and dried (component 1 pure) → mother liquor → concentrate by evaporating solvent → second solute crystallises → filter → dried crystals (component 2 pure)
3.3 Distillation
Distillation is an important method used to separate (i) volatile liquids from non-volatile impurities, and (ii) liquids having sufficient difference in their boiling point.
The Idea Behind the Method
Both uses rest on the same physical fact. A liquid that boils easily can be turned into vapour and led away, while anything that will not vaporise at that temperature is left behind. If two liquids in a mixture boil at sufficiently different temperatures, the one with the lower boiling point vaporises in far greater proportion and can be collected first.
Because the substance is only vaporised and condensed, no chemical change takes place — the liquid collected is chemically the same liquid that was heated.
3.3.1 Simple Distillation
Liquids which boil without decomposition at atmospheric pressure are purified by the process of simple distillation. In this process the liquid is first converted into its vapour by boiling, and then the vapour is condensed back into liquid by cooling, and the pure liquid trickles down in the receiver.
The Apparatus
The apparatus used for simple distillation is shown in Fig. 3.4. It consists of a round bottom flask fitted with a cork having a thermometer. The flask has a side arm through which it is connected to a condenser.
Diagram
Fig. 3.4 – Simple distillation assembly: round bottom flask on a wire gauze over a burner, cork carrying a thermometer with its bulb just below the side arm, side arm leading to a water-jacketed condenser clamped on a stand, water inlet at the lower end and water outlet at the upper end, receiver flask collecting the distilled liquid; label thermometer, RB flask, condenser, water inlet, water outlet, liquid substance
The condenser has a jacket with two outlets through which water is circulated. The vapour travels through the inner tube while cold water flows through the outer jacket, so the vapour loses heat continuously along the whole length of the tube.
The liquid to be distilled is taken in the round bottom flask fixed by a clamp. The flask is placed in a water bath or oil bath, or sometimes wire gauze is kept on a stand as shown in Fig. 3.4. A bath heats the flask evenly and gently, which prevents the liquid from bumping — that is, from boiling in sudden violent bursts.
The condenser is connected to a receiver to collect the purified liquid. Care is taken that the bulb of the thermometer is just below the side arm of the round bottom flask.
How the Distillation Proceeds
The flask is heated. As the boiling point of the liquid is reached it starts boiling, and the vapours rise to the neck of the flask and pass through the side arm into the cooler parts of the condenser, which is kept cool by circulating water through its jacket. The vapours condense and the liquid is collected in the receiver.
Watch the thermometer while this happens: the temperature climbs, then holds steady as long as one component is distilling over, because the heat supplied is being used to vaporise that liquid rather than to raise the temperature. The whole sequence is summarised in FC 3.4.
Flowchart
FC 3.4 – Simple distillation: mixture in round bottom flask → heat on a water/oil bath → liquid reaches its boiling point → vapour rises to the neck → passes through side arm → enters water-cooled condenser → vapour loses heat and condenses → pure liquid trickles into the receiver; non-volatile impurity remains behind in the flask
Diagram
Fig. 3.4a – Graph of vapour temperature (y-axis, °C) against volume of distillate collected (x-axis, mL): a rise to a plateau at 56 °C, a short steep climb, a second plateau at 100 °C; the two plateaus labelled “first component distilling” and “second component distilling”
Activity: To Separate the Components of a Liquid Mixture Containing Acetone and Water
Activity: To separate the components of a liquid mixture containing acetone (b. p. \(56\ ^\circ\text{C}\)) and water (b. p. \(100\ ^\circ\text{C}\)).
Apparatus: Distillation flask with condenser, two receivers, thermometer, etc.
Chemicals: Mixture of acetone and water.
Principle: Acetone and water are two miscible liquids having a wide difference in their boiling points. Acetone boils at \(56\ ^\circ\text{C}\) while the boiling point of water is \(100\ ^\circ\text{C}\). When the mixture of acetone and water is heated and the temperature of the mixture reaches \(56\ ^\circ\text{C}\), acetone would distil off. When all the acetone distils out and when the temperature rises to \(100\ ^\circ\text{C}\), water would distil out.
Procedure: Take the mixture of water and acetone in the distillation flask and arrange the apparatus. Heat the flask on a water bath carefully. At \(56\ ^\circ\text{C}\) acetone will distil out; collect it in receiver number 1. After all the acetone has distilled, change the receiver. Discard a few mL of the liquid. As the temperature reaches \(100\ ^\circ\text{C}\), water will begin to distil. Collect this in receiver number 2.
The few millilitres discarded in between are worth understanding rather than simply following. That portion comes over while the thermometer reading is climbing from one boiling point to the other, so it is a mixture of the tail of the first liquid and the beginning of the second. Discarding it keeps both collected fractions clean.
3.3.2 Fractional Distillation
If in a mixture the difference in boiling points of two liquids is not appreciable, they cannot be separated from each other using the simple distillation assembly.
The reason is that when the boiling points are close, both liquids vaporise appreciably at the same temperature. The vapour reaching the condenser is therefore a mixture, and the “distillate” collected is only slightly richer in the more volatile liquid than the mixture you started with.
To separate such liquids, the process called fractional distillation is employed, in which a special assembly is used (see Fig. 3.5 (a)). In this assembly the distillation flask is fitted with a fractionating column (Fig. 3.5 (b)). Hence, the vapours first pass through the fractionating column. Vapours of the more volatile liquid with lower boiling point rise up more than the vapours of the liquid having higher boiling point.
Diagram
Fig. 3.5 (a) – Fractional distillation assembly: round bottom flask of liquid to be distilled in an oil bath over a burner, a packed fractionating column mounted vertically above the flask, thermometer at the top of the column, side arm to a water-cooled condenser with water inlet and outlet, adapter, and receiver collecting distilled liquid; label fractionating column with packing, oil bath, condenser, adapter, distilled liquid
Diagram
Fig. 3.5 (b) – Two types of fractionating column shown side by side: a simple packed column filled with glass beads, and a bubble plate column with a series of horizontal plates; label simple packed column and bubble plate column, and mark the large internal surface area in each
Inside the Fractionating Column
Suppose we have a mixture of two liquids (A) and (B) having boiling points \(363\ \text{K}\) and \(373\ \text{K}\) respectively. A is more volatile and B is less volatile.
As the mixture is heated, vapours of (A) along with a little of (B) rise up and come in contact with the large surface of the fractionating column. Vapours of (B) condense rapidly into the distillation flask, because B is the less volatile of the two and gives up its vapour state more readily on cooling.
While passing through the fractionating column there is an exchange between the ascending vapours and the descending liquid. The vapours of B are scrubbed off by the descending liquid; this makes the vapours richer in (A).
This process is repeated each time the vapours and liquid come in contact with the surface in the fractionating column. Each contact is, in effect, a miniature distillation — and the column provides a great many of them, one above the other, which is exactly what a single side arm cannot provide.
Rising vapours become richer in (A) and escape through the fractionating column and reach the condenser, while the liquid in the distillation flask is richer in B. The separated components are further purified by repeating the process. The exchange inside the column is set out step by step in FC 3.5.
Flowchart
FC 3.5 – Exchange inside a fractionating column: mixture heated → vapour of A + a little B rises → meets cool packing surface → less volatile B condenses and runs back down as descending liquid → descending liquid scrubs more B out of the rising vapour → vapour now richer in A rises higher → contact repeated at every level of the column → vapour leaving the top is almost pure A → condenser → receiver; branch: liquid returning to the flask becomes progressively richer in B
Mixtures of acetone (b.p. \(329\ \text{K}\)) and methyl alcohol (b.p. \(337.7\ \text{K}\)); acetone and benzene (\(353\ \text{K}\)) can be separated by fractional distillation.
This process is used in the petroleum industry to separate different fractions of crude oil.
Flowchart
FC 3.6 – Fractional distillation of crude oil: crude oil → heated in a furnace → vapour enters the base of the fractionating tower → rises past bubble plates, cooling as it goes → highest plates (lowest boiling range): refinery gas and petrol → middle plates: naphtha and kerosene → lower plates: diesel oil → base of tower (highest boiling range, does not vaporise): lubricating oil and residue
3.3.3 Distillation Under Reduced Pressure
Liquids having a very high boiling point, or those which decompose on heating, are purified by carrying out distillation under reduced pressure.
In this method the liquid is made to boil at a temperature which is below its normal boiling point by reducing the pressure on the surface of the liquid. Pressure is reduced using a water pump or vacuum pump.
Note the term “normal boiling point”: it means the boiling point measured at atmospheric pressure. A boiling point is not a fixed property of a liquid on its own — it is a property of the liquid at a stated pressure.
In soap industry, glycerol is separated from soap by using this technique.
3.4 Solvent Extraction
When an organic substance is present in an aqueous solution, it can be extracted from that solution by shaking it with an organic solvent in which the substance is more soluble.
The organic liquid should be immiscible with water and be able to form two layers. Two liquids are said to be immiscible when they do not mix into a single solution but settle into separate layers, the denser one below — as oil does over water. This is essential, because if the two liquids mixed, there would be no second layer to draw off.
How the Extraction Is Carried Out
In this process the solute distributes itself between two immiscible liquids. From the aqueous phase the solute gets extracted in the organic phase. Extraction of compound takes place based on the difference in solubility of compound in two liquids, as seen in Fig. 3.6.
Diagram
Fig. 3.6 – Solvent extraction in a separating funnel, drawn as two side-by-side funnels: “before extraction” showing an upper solvent layer and a lower aqueous layer containing the organic compound, and “after extraction” showing the organic compound now present in the upper solvent layer with a clear aqueous layer below; label solvent layer, aqueous layer, organic compound in aqueous layer, organic compound in solvent layer, stopcock
Shaking is what makes the process work quickly. It breaks one liquid into countless small droplets within the other, creating an enormous contact area, so the solute can pass from one phase into the other in seconds rather than hours.
On shaking for a few times with small volumes of organic phase, most of the solute gets extracted into the organic phase. Notice that the text says a few small volumes rather than one large volume — each fresh portion of solvent starts out completely free of the solute and so pulls across another share of what is left behind.
The mixture is then allowed to stand until the two layers separate cleanly, and the lower layer is run out through the stopcock. The organic solvent is then removed by distillation and the solute is collected. The whole sequence is set out in FC 3.7.
Flowchart
FC 3.7 – Solvent extraction: aqueous solution of the organic compound → transfer to separating funnel → add a small volume of immiscible organic solvent → shake → allow layers to settle → run off the lower layer through the stopcock → repeat the shaking with fresh small volumes of solvent → combine the organic extracts → remove the solvent by distillation → pure solute collected
The solvent extraction process is important as it helps clean separations in a short time span.
Continuous Extraction
If the solute is less soluble in the organic phase, then a technique called continuous extraction is used, where the same amount of organic solvent is used repeatedly for extraction.
This technique involves continuous distillation of the solvent within the same assembly. The solvent is boiled, its vapour is condensed, and the fresh condensed solvent falls back on the material being extracted; the extract then drains back into the boiling flask, and the cycle repeats. Thus use of a large quantity of organic solvent is avoided. A well-known assembly of this kind, the Soxhlet extractor, is shown in Fig. 3.6a, and its cycle in FC 3.8.
Diagram
Fig. 3.6a – Soxhlet extraction assembly: round bottom flask of solvent heated below, extraction chamber above it holding a porous thimble containing the sample, side tube for rising vapour and a siphon tube on the other side, reflux condenser on top; label solvent flask, thimble with sample, vapour arm, siphon arm, condenser
Flowchart
FC 3.8 – Continuous (Soxhlet) extraction cycle: solvent boiled in the flask → vapour rises through the side arm → condensed by the condenser above → hot pure solvent drips onto the sample in the thimble → solvent dissolves out the solute → chamber fills to the top of the siphon → extract siphons back into the flask → solute accumulates in the flask (it is non-volatile) → solvent boils again; returns to the start and repeats until extraction is complete
Because the solute is left behind in the boiling flask each time while only the solvent evaporates, every drop reaching the sample is fresh, pure solvent — which is exactly why a small quantity can do the work of a very large one.
3.5 Chromatographic Techniques
Chromatography is a technique used to separate components of a mixture, and also to purify compounds. The name of the technique comes from the Greek word Chroma, meaning Colour.
In 1903, Tswett discovered this technique for separating the coloured components found in plants. The name made obvious sense then, because the separated substances announced themselves as bands of colour — although today the same technique is used far more often on colourless substances than on coloured ones.
The Principle and the Two Phases
The principle of separation of substances in this technique is similar to solvent extraction, i.e. distribution of the solutes in two phases. You have already met that idea in the previous section, where a solute distributed itself between water and an immiscible organic liquid. Chromatography uses the same idea, but with one of the two phases held still.
In chromatography we use two phases for separation: (a) stationary phase and (b) mobile phase.
This technique is based on the difference in rates at which components in the mixture move through the stationary phase under the influence of the mobile phase.
First the mixture of components is loaded at one end of the stationary phase, and then the mobile phase, which is a pure solvent or a mixture of solvents, is allowed to move over the stationary phase.
Depending on the relative affinity of the components toward the stationary phase and mobile phase, they remain on the surface of the stationary phase or move along with the mobile phase, and gradually get separated.
Read that sentence as a tug of war. Every component is pulled two ways at once: the stationary phase holds it back, the mobile phase carries it forward. A component held strongly moves slowly; one held weakly is swept along. Since no two substances are held with exactly the same strength, the components fall behind one another and separate.
Classification by the Stationary Phase
The stationary phase can be a solid or a liquid. Depending on the stationary phase, chromatography is classified into Adsorption Chromatography and Partition Chromatography.
This one difference — solid or liquid — decides how the components are held. On a solid, they stick to the surface. In a liquid, they dissolve throughout. The two situations are given different names, and they form the two subsections that follow.
3.5.1 Adsorption Chromatography
This type of chromatography is based on the principle of differential adsorption.
Adsorption means the sticking of a substance to the surface of a solid, as distinct from absorption, in which it would be taken up throughout the bulk. In adsorption chromatography, the solid stationary phase offers an enormous surface, and each solute clings to that surface with its own characteristic strength.
Adsorption chromatography is of the following two types.
i. Column Chromatography
This type involves the separation of components over a column of stationary phase. The stationary phase material can be alumina or silica gel — both are finely divided solids offering a very large surface area for adsorption.
A slurry of the stationary phase material is filled in a long glass tube provided with a stopcock at the bottom and a glass wool plug at the lower end. The plug is what holds the powder back while allowing liquid to drain, and the stopcock lets the operator control how fast the liquid runs out.
The mixture to be separated is dissolved in a small amount of appropriate solvent and is then loaded on top of the adsorbent column. Keeping this volume small matters: a concentrated, narrow band at the top gives sharp, well-separated bands later, while a dilute, spread-out loading gives smeared ones.
A suitable mobile phase, which could be a single solvent or a mixture of solvents, is then poured over the adsorbent column. The mixture along with the mobile phase slowly moves down the column.
The solutes get adsorbed on the stationary phase, and depending on the degree to which they are adsorbed, the solutes get separated from each other. The most strongly adsorbed component is retained on the column and others move down the column to various distances, forming bands as seen in Fig. 3.7.
Diagram
Fig. 3.7 – Column chromatography, three stages of separation drawn side by side: (1) glass column with stopcock and glass wool plug, packed with adsorbent, mixture of compounds (a+b+c) as a narrow band at the top under a layer of solvent, (2) partly developed column showing band a separated above band b+c, (3) fully developed column showing three distinct bands a, b and c at different heights; label solvent, mixture of compound (a+b+c), adsorbent (stationary phase), glass wool, stopcock
The component which is less strongly adsorbed is desorbed first and leaves the column first, while the strongly adsorbed component is eluted later. Desorption is simply the reverse of adsorption — the solute letting go of the surface and returning to the flowing solvent; elution is the washing of a component out of the bottom of the column by the mobile phase.
The solutions of these components are collected separately. On evaporating the solvent, the solutes can be recovered. The complete operation is set out in FC 3.9.
Flowchart
FC 3.9 – Column chromatography: pack glass tube (stopcock + glass wool plug) with a slurry of alumina or silica gel → dissolve mixture in a small volume of solvent → load as a narrow band on top of the column → pour mobile phase over the column → mobile phase carries the mixture down → components separate into bands by degree of adsorption → least strongly adsorbed component elutes first, collect in a separate receiver → change receiver as each band emerges → most strongly adsorbed component elutes last → evaporate solvent from each fraction → pure components recovered
A recorded demonstration of a column being packed, loaded and run is well worth watching online before attempting one in the laboratory; seeing the bands move makes the description above much easier to picture.
ii. Thin Layer Chromatography
A thin layer (0.2 mm thick) of adsorbent silica gel or alumina spread over a glass plate acts as the stationary phase. The plate is called the TLC plate or chromplate.
The mixture of solutes is applied on the chromplate as a small spot about 2 cm from one end of the plate, as shown in Fig. 3.8. The spot is kept small for the same reason the band in a column is kept narrow — a broad starting spot gives broad, overlapping spots at the end.
Diagram
Fig. 3.8 (a) – Stages in thin layer chromatography, three jars in sequence with arrows between them: (1) TLC plate standing in a closed jar with the spot just above the shallow mobile phase, (2) mobile phase risen partway up the plate with the spot beginning to resolve, (3) fully developed plate with two separated spots at different heights; label chromplate, mobile phase, sample spot
The plate is then placed in a closed jar containing the mobile phase, such that the spot is well above the mobile phase. The jar is kept closed so that its air becomes saturated with solvent vapour, which stops the solvent from evaporating off the plate as it climbs.
As the mobile phase rises up, the components of the mixture move along with it. They move up to different distances depending upon their degree of adsorption and thus get separated.
Flowchart
FC 3.10 – Thin layer chromatography: coat glass plate with a 0.2 mm layer of silica gel or alumina → apply the mixture as a small spot about 2 cm from one end → place the plate in a closed jar with the spot standing above the level of the mobile phase → mobile phase rises up the layer → components travel different distances according to their degree of adsorption → remove the plate and mark the solvent front → locate the spots: coloured components seen directly; branch: colourless components → view under UV light, or expose to iodine vapour in a chamber, or spray with a suitable reagent
If the components are coloured, they appear as separated coloured spots on the plate. If the components are not coloured but have the property of fluorescence, they can be visualised under UV light. Alternatively, the plate can be kept in a chamber containing a few iodine crystals; the iodine vapours are adsorbed by the components and the spots appear brown.
Amino acids are visualised by spraying the plate with a solution of ninhydrin. This is known as a spraying agent.
Diagram
Fig. 3.8 (b) – Developed chromatogram: a closed jar with a glass plate coated with adsorbent standing in solvent, showing base line and sample dot; beside it the removed plate marked with the distance travelled by the solvent and the distances travelled by the various separated spots, with arrows for each measurement; label jar, adsorbent coated on glass plate, base line, sample dot, solvent, distance travelled by the solvent, distance travelled by the various dyes
3.5.2 Partition Chromatography
In this type of chromatography the stationary phase and mobile phase both are liquids.
Separation of components takes place by continuous differential partitioning of the components between the stationary and mobile phases. Partitioning means dividing: as the mobile liquid flows past the stationary liquid, each component keeps sharing itself between the two, again and again along the whole path. A component that favours the stationary liquid lags behind; one that favours the mobile liquid moves ahead.
Paper Chromatography
For example, paper chromatography: in this technique a special quality paper, Whatmann paper number 1, is used. The water trapped in the fibres of the paper acts as the stationary phase.
This point is easy to misread. The paper itself is not the stationary phase — it is only the support. The stationary phase is the water held in its fibres, which is why paper chromatography belongs with partition and not with adsorption.
The solution of the mixture is spotted on the strip of the chromatography paper at about 2 cm from one end of the paper using a glass capillary. The paper is then suspended in a chamber containing the mobile phase, taking care that the spot does not dip in the mobile phase (Fig. 3.9).
Diagram
Fig. 3.9 – Stages in paper chromatography, three photographs or drawings in sequence: (1) paper strip clipped above a dish of solvent with a row of small coloured spots on the base line, (2) solvent risen partway with the spots beginning to stretch and resolve, (3) fully developed strip with each original spot separated into several coloured spots at different heights; label base line, solvent level, separated spots
The mobile phase rises up by capillary action and flows over the spot. Capillary action is the same effect that draws water up a narrow tube or into blotting paper — the liquid creeps through the fine spaces between the paper fibres without any pump or pressure.
Partitioning of the components takes place between the stationary phase (water) and the mobile phase.
Different solutes are retained differently on the paper depending on their selective partitioning between the two phases. This developed paper strip is the chromatogram.
Similar to TLC (thin layer chromatography), the coloured components are visible as coloured spots, and the colourless components are observed under UV light or by using a spraying agent.
Retention Factor (\(R_f\))
Migration of the solute relative to the solvent front gives an idea about the relative retention of the solutes on the stationary phase. This is termed as the \(R_f\) of the solute.
\[R_f = \frac{\text{distance travelled by the solute from the base line}}{\text{distance travelled by the solvent from the base line}}\]
Both distances are measured from the base line — the pencil line on which the original spot was applied — as shown in Fig. 3.10. The solvent front is simply the highest level the mobile phase reached, and it must be marked immediately after the plate or paper is taken out, before the solvent evaporates and the mark is lost.
Diagram
Fig. 3.10 – Retention factor: a developed strip showing the base line at the bottom, a single spot part way up, and the solvent front near the top; arrow x marking the distance travelled by the solute from the base line and arrow y marking the distance travelled by the solvent from the base line; label solvent front, spot, base line
Diagram
Fig. 3.10a – A developed paper chromatogram of an ink sample shown against a vertical centimetre scale: pencil base line at 0 cm, four separated spots at roughly 2.0, 3.6, 3.9 and 7.2 cm, solvent front marked at 8.0 cm; label base line, spots, solvent front, scale in cm
3.6 Summary
Chemical substances occur in nature in an impure stage, and substances synthesized in the laboratory are obtained in a crude and impure form. They must be purified before their composition and properties can be investigated.
Methods of purification and separation depend on the difference in the physical properties of the substances involved — chiefly solubility, boiling point, and the tendency to be held on a surface. Since no chemical reaction is used, the purified substance is recovered chemically unchanged.
A solid may contain impurities that are soluble in the same solvent as the main substance, and impurities that are not soluble in it. The insoluble type is removed by filtration, in which the insoluble solid left on the filter paper is the residue and the liquid collected below is the filtrate.
Filtration under suction uses a vacuum pump, a thick-walled conical flask with a side arm, a safety bottle, and a porcelain Buchner funnel with a porous bottom. It is faster and more efficient than simple filtration.
Crystallization purifies a crude solid that is made mainly of one substance. It is done in four steps: preparation of a saturated solution by boiling the solid in a small but sufficient quantity of a suitable solvent; hot filtration to remove undissolved impurities as residue; cooling of the filtrate, during which the solution becomes supersaturated in the main solute and pure crystals separate while the small quantity of soluble impurity stays dissolved; and filtration of the crystals, leaving behind the mother liquor.
A saturated solution is one that cannot dissolve an additional quantity of solute at that temperature. Solubility decreases with lowering of temperature, and this is what forces the crystals out on cooling.
The solvent chosen for crystallization must dissolve the compound only sparingly at room temperature but highly at high temperature, must not react chemically with the compound, and must be volatile so that it can be removed easily. Water, ethyl alcohol, methyl alcohol, acetone, ether or their combinations are generally used, the choice being made by trial and error. Impure copper sulphate and benzoic acid are both purified by crystallization using water as solvent.
Fractional crystallization separates two or more soluble substances having widely different solubilities in the same solvent at room temperature. On cooling the hot saturated solution, the less soluble solute crystallizes out first; the crystals are filtered, washed and dried, and the mother liquor is then concentrated to crystallize the second component.
Distillation separates volatile liquids from non-volatile impurities, and liquids having a sufficient difference in their boiling points. The liquid is converted to vapour by boiling and the vapour is condensed back to liquid in a receiver.
Simple distillation is used for liquids which boil without decomposition at atmospheric pressure. The assembly uses a round bottom flask with a thermometer whose bulb is kept just below the side arm, a water-jacketed condenser, and a receiver. A mixture of acetone (b. p. \(56\ ^\circ\text{C}\)) and water (b. p. \(100\ ^\circ\text{C}\)) can be separated this way, acetone distilling first.
Fractional distillation is used when the difference in boiling points is not appreciable. The distillation flask is fitted with a fractionating column, in which ascending vapours and descending liquid exchange repeatedly, so the rising vapour becomes progressively richer in the more volatile component while the liquid in the flask becomes richer in the less volatile one. Acetone (b.p. \(329\ \text{K}\)) and methyl alcohol (b.p. \(337.7\ \text{K}\)), and acetone and benzene (\(353\ \text{K}\)), are separated in this way, and the method is used in the petroleum industry to separate the fractions of crude oil.
Distillation under reduced pressure is used for liquids with a very high boiling point or those which decompose on heating. Reducing the pressure on the surface of the liquid with a water pump or vacuum pump makes it boil below its normal boiling point. Glycerol is separated from soap in the soap industry by this technique.
Solvent extraction removes an organic substance from an aqueous solution by shaking it with an organic solvent in which the substance is more soluble. The organic liquid must be immiscible with water and form two layers; the solute distributes itself between the two liquids. Shaking a few times with small volumes extracts most of the solute, and the solvent is then removed by distillation. If the solute is only slightly soluble in the organic phase, continuous extraction is used, in which the same solvent is distilled and reused within one assembly, avoiding the use of a large quantity of solvent.
Chromatography separates the components of a mixture and also purifies compounds. Its name comes from the Greek Chroma, meaning colour, and it was discovered by Tswett in 1903 for separating coloured plant components. It uses a stationary phase and a mobile phase, and works because the components move at different rates through the stationary phase depending on their relative affinity for the two phases.
Depending on whether the stationary phase is a solid or a liquid, chromatography is classified as adsorption chromatography or partition chromatography.
Adsorption chromatography is based on differential adsorption — different solutes are adsorbed to different extents on the stationary phase. In column chromatography, a slurry of alumina or silica gel is packed in a glass tube; the mixture is loaded on top and washed down by the mobile phase, forming bands. The least strongly adsorbed component is desorbed and eluted first, the most strongly adsorbed last, and the solutes are recovered by evaporating the solvent from each collected fraction.
In thin layer chromatography, a 0.2 mm layer of silica gel or alumina on a glass plate acts as the stationary phase. The mixture is applied as a small spot about 2 cm from one end, and the plate is placed in a closed jar so that the spot stays above the mobile phase. Coloured components appear as coloured spots; colourless ones are viewed under UV light, exposed to iodine vapour, which turns the spots brown, or sprayed with a reagent such as ninhydrin for amino acids.
Partition chromatography has liquids as both phases, and separation occurs by continuous differential partitioning of the components between them. In paper chromatography, the water trapped in the fibres of Whatmann paper number 1 is the stationary phase; the mobile phase rises by capillary action over the spot, and the developed strip is called the chromatogram.
The retention factor describes how far a solute has migrated relative to the solvent front: \[R_f = \frac{\text{distance travelled by the solute from the base line}}{\text{distance travelled by the solvent from the base line}}\] It has no unit, and its value indicates how strongly the solute is retained on the stationary phase.
The techniques of this chapter can be seen as one decision chain, summarised in FC 3.11.
Flowchart
FC 3.11 – Choosing and applying a purification technique: impure sample → SOLID branch: insoluble impurity → filtration (simple or under suction) → residue and filtrate; soluble impurity in small proportion → crystallization (saturated solution → hot filtration → cooling → filtration) → pure crystals + mother liquor; two soluble components of widely different solubility → fractional crystallization → successive crops. LIQUID branch: non-volatile impurity or wide difference in boiling points → simple distillation; small difference in boiling points → fractional distillation through a fractionating column; very high boiling point or decomposes on heating → distillation under reduced pressure. SOLUTE IN AQUEOUS SOLUTION branch: more soluble in an immiscible organic solvent → solvent extraction in a separating funnel; only slightly soluble in it → continuous extraction. SMALL QUANTITIES OR CLOSELY SIMILAR COMPONENTS branch: solid stationary phase → adsorption chromatography (column or thin layer); liquid stationary phase → partition chromatography (paper) → measure \(R_f\)
3.7 Check Your Concepts
Assertion: In crystallization, the soluble impurities are left behind in the mother liquor. Reason: The hot solution is almost saturated with respect to the main solute but is far from saturated with respect to the impurities. State whether the assertion and the reason are correct, and whether the reason correctly explains the assertion.
Why is the solvent used to prepare the saturated solution in crystallization described as “small but sufficient”? What would go wrong if a large excess were used?
Explain why the hot filtration step in crystallization must be carried out quickly, and state what is lost if it is done slowly.
A student chooses a solvent in which the compound dissolves equally well when hot and when cold. Predict what will happen on cooling the filtrate, and identify which condition for a good crystallization solvent has been ignored.
Why is a thick-walled conical flask, and not an ordinary thin-walled one, used for filtration under suction?
State the function of the safety bottle in the suction filtration assembly, and describe what could happen if it were left out.
Arrange the following steps of crystallization in the correct order and name each step: cooling of the filtrate; filtration of the crystals; preparation of a saturated solution; hot filtration.
Distinguish between crystallization and fractional crystallisation with respect to (i) the purpose of the technique, (ii) the quantity in which the second substance is present, and (iii) the fate of the mother liquor.
In fractional distillation, explain why the liquid remaining in the distillation flask becomes progressively richer in the less volatile component.
Assertion: Glycerol is separated from soap by distillation under reduced pressure. Reason: Lowering the pressure on the surface of a liquid raises the temperature at which it boils. State whether each statement is correct, and correct any statement that is wrong.
Why must a boiling point always be quoted along with the pressure at which it was measured? Relate your answer to the working of distillation under reduced pressure.
Match the technique in Column I with the example in Column II.
| Column I | Column II |
|---|---|
| a. Distillation under reduced pressure | i. Separating the fractions of crude oil |
| b. Fractional distillation | ii. Separating tea leaves from a decoction |
| c. Filtration | iii. Separating glycerol from soap |
| d. Crystallization | iv. Separating the coloured pigments of a leaf |
| e. Chromatography | v. Purifying impure copper sulphate using water |
A mixture of two miscible liquids whose boiling points differ by only a few kelvin is distilled in a simple distillation assembly. Describe what the thermometer will show and explain why the distillate collected is not pure.
Why is the mixture loaded as a narrow band at the top of a column in column chromatography, rather than being poured in as a dilute solution?
In column chromatography, which component leaves the column first — the one that is strongly adsorbed or the one that is weakly adsorbed? Explain your answer in terms of the time each component spends on the stationary phase.
A student applies the sample to a TLC plate as a large, broad smear instead of a small spot. Predict the appearance of the developed plate and explain the result.
In paper chromatography, the paper is only the support and not the stationary phase itself. Identify the true stationary phase and use your answer to explain why paper chromatography is classified as partition and not adsorption chromatography.
Why is the base line on a chromatography paper drawn with a pencil rather than with a pen?
A student suspends the TLC plate so deep in the jar that the sample spot lies below the level of the mobile phase. Predict what will be observed at the end of the run and explain the observation.
Two components of a mixture give almost identical \(R_f\) values in a particular solvent. Suggest what change could be made to separate them more clearly, and explain why this should work.
Explain why the retention factor has no unit, and why its value can never be greater than 1.
Chromatography can separate quantities far too small to be seen, whereas crystallization cannot. Explain the reason for this difference, based on how each technique achieves separation.
Continuous extraction recycles one small portion of solvent instead of using many fresh portions. Explain how the assembly achieves this, and state one environmental advantage of working in this way.
Name the technique you would select in each of the following cases, and justify each choice in one sentence:
- Separating sand from a solution of common salt.
- Purifying benzoic acid contaminated with a small amount of a soluble impurity.
- Separating a liquid that decomposes below its normal boiling point from a non-volatile residue.
- Recovering an organic compound dissolved in water, using an immiscible solvent.
- Checking how many coloured dyes are present in a sample of ink.
Both solvent extraction and chromatography are said to work on the distribution of a solute between two phases. State clearly what plays the part of each phase in the two techniques, and explain the one important difference between them.
Which of the following techniques is used for purification of solid organic compounds?
- Crystallisation
- Distillation
What do you understand by the terms
- residue
- filtrate.
Why is a condenser used in distillation process?
Why is paper moistened before filtration?
What is the stationary phase in Paper Chromatography?
What will happen if the upper outlet of the condenser is connected to the tap instead of the lower outlet?
Give names of two materials used as stationary phase in chromatography.
Which properties of solvents are useful for solvent extraction?
Why should spotting of mixture be done above the level of mobile phase?
Define:
- Stationary phase
- Saturated solution
What is the difference between simple distillation and fractional distillation?
Define
- Solvent extraction
- Distillation.
List the properties of solvents which make them suitable for crystallization.
Name the different types of Chromatography and explain the principles underlying them.
Why do we see bands separating in column chromatography?
How do you visualize colourless compounds after separation in TLC and Paper Chromatography?
Compare TLC and Paper Chromatography techniques.
Label the diagram and explain the process in your words.
Diagram
Fig. 3.11 – Unlabelled line drawing for the student to label: a funnel with a porous plate seated in a rubber bung on a thick-walled conical flask with a side arm and rubber tubing leading away; blank leader lines drawn to the funnel, the porous plate, the bung, the side arm, the tubing and the liquid collecting in the flask
3.8 Practice With Numericals
The solubility of a salt X is \(110\ \text{g}\) per \(100\ \text{g}\) of water at \(60\ ^\circ\text{C}\) and \(22\ \text{g}\) per \(100\ \text{g}\) of water at \(10\ ^\circ\text{C}\). A saturated solution of X is prepared in \(200\ \text{g}\) of water at \(60\ ^\circ\text{C}\) and cooled to \(10\ ^\circ\text{C}\). Calculate the mass of crystals obtained.
For the salt in the previous question, calculate the percentage of the dissolved salt that is recovered as crystals on cooling, and state where the rest of it has gone.
A student starts with \(25.0\ \text{g}\) of a crude solid and, after crystallization, obtains \(21.5\ \text{g}\) of dry pure crystals. Calculate the percentage recovery.
The solubility of a compound is \(8\ \text{g}\) per \(100\ \text{g}\) of water at \(20\ ^\circ\text{C}\) and \(80\ \text{g}\) per \(100\ \text{g}\) of water at \(90\ ^\circ\text{C}\). What is the minimum mass of water needed to just dissolve \(40\ \text{g}\) of the compound at \(90\ ^\circ\text{C}\), and what mass of crystals would separate if that solution were cooled to \(20\ ^\circ\text{C}\)?
A liquid boils at \(78\ ^\circ\text{C}\) and a second liquid in the same mixture boils at \(351\ \text{K}\). Express both boiling points in kelvin, find the difference between them, and comment on what your answer implies about separating the two.
Two liquids have boiling points of \(329\ \text{K}\) and \(373\ \text{K}\). Express both in degrees Celsius and state, with a reason, which distillation assembly you would use for the mixture.
On a developed TLC plate the solvent front is \(7.5\ \text{cm}\) from the base line and a single component has travelled \(3.0\ \text{cm}\) from the base line. Calculate the \(R_f\) value of the component.
In a paper chromatography run the solvent front travels \(10.0\ \text{cm}\) from the base line. Two components have \(R_f\) values of \(0.35\) and \(0.80\). Calculate the distance travelled by each component, and state which of the two is held more strongly by the stationary phase.
A solute travels \(5.4\ \text{cm}\) from the base line and has an \(R_f\) value of \(0.60\). Calculate the distance travelled by the solvent front from the base line.
A TLC plate is \(12.0\ \text{cm}\) long and the sample is spotted \(2.0\ \text{cm}\) from the lower end. After development, a spot is found \(9.0\ \text{cm}\) from the lower end of the plate and the solvent front is \(11.0\ \text{cm}\) from the lower end. Calculate the \(R_f\) value of the spot, taking care to measure both distances from the base line.
\(100\ \text{mL}\) of an aqueous solution contains \(5.0\ \text{g}\) of an organic compound. After shaking with an immiscible organic solvent and separating the layers, the organic layer is found to contain \(4.1\ \text{g}\) of the compound. Calculate the percentage of the compound extracted, and the mass left behind in the aqueous layer.
A thin layer chromatography plate carries an adsorbent layer \(0.2\ \text{mm}\) thick. Express this thickness in centimetres and in metres, using scientific notation for the value in metres.
3.9 Objective Questions
- Which of the following methods can be used to seperate two compounds with different solubilities in the same solvent?
- Fractional crystallization
- Crystallization
- Distillation
- Solvent extraction
- Which of the following techniques is used for seperation of glycerol from soap in soap industry?
- Distillation under reduced pressure
- Fractional distillation
- Filtration
- Crystallization
- Which technique is widely used in industry to seperate components of mixture and also to purify them?
- Steam distillation
- Chromatography
- Solvent extraction
- Filtration
- A mixture of acetone and benzene can be seperated by the following method:
- Simple distillation
- Fractional distillation
- Distillation under reduced pressure
- Sublimation
- Colourless components on chromatogram can not be observed by the following:
- Using UV light
- Using iodine chamber
- Using the spraying reagent
- Using infrared light