{1 | (separation of th 3 Sep ‘ARATION METHODS: CHROMATOGRAPHY 3.1 INTRODUCTION Organic or inorga '¢ Compounds, whether obtained by synthesis or from natural sources such as plants enerally in the form of a nts o ey N ores, are gen 1 tion c thromatog mae ure compounds is essential if they are to be of any (the compounds tr Physical method of separating a mixture of compounds, during a chromatogranhae cite thixture donot undergo any chemical change as rap lc separation, Properties such as adsorption or partition of P between two phases, form the basis for the separation of mixtures.\ ee ing chromatography, it is possible to separate a mixture of organic compounds, amino acids, sugars, cations and anions] Generally, it is possible to obtain all the compounds in a mixture in a pure form. (A chromatography experiment is carried out in order to 1) know the number of compounds in a sample 2) identify the compounds 3) know the relative quantities of the compounds in a sample, and 4) effect quantitative separation of the compounds. | The common chromatographic techniques are: 1) Column chromatography (CC) 2) Thin-layer chromatography (TLC) 3) lon-exchange chromatography (IEC) 4) Gas chromatography (GC) 5) Paper chromatography (PC) 6) Gas-liquid chromatography (GLC) and 7) High-performance liquid chromatography (HPLC). in all these chromatographic methods, two phases are necessary to effect the overall fe compounds in the mixture analyzed. These two phases are: (1) the stationary phase—a phase that does not move. The stationary phase may be a52 Analytical Chemistry solid or liquid, and (2) the mobile phase—a phase that moves. ‘A mobile phase may be a liquid or gas} Chromatography Chromatography is a physical method of separating enitures of organic compounds, biomolecules, organic and inorganic salts, et» by distri é ae or partition between two phases. One of the phases is the stationary phase ant other is the mobile phase. These two phases are in contact with each oth ae mobile phase moves through the stationary phase. {The compounds in the mix : have different values of partition or distribution coefficients, and the separation based on this difference in partition coefficients. | 3.1.1 CLASSIFICATION OF CHROMATOGRAPHIC METHODS: Chromatographic methods are classified either on the basis of the stationary phase used or the mobile phase used. n based on the stationary phase Classifics Chromatographic Methods Liquid Stationary Phase (Liquid supported on an inert material) (PARTITION CHROMATOGRAPHY) rd [71 Liquid Mobile Phase Gas Mobile Phase Liquid Mobile Phase Solid Stationary Phase (ADSORPTION (CHROMATOGRAPHY) Gas Mobile Phase Eg, 1. Gas-chromatography Eg, 1. Column chromato- Eg. 1. Gas-liquid Eg. 1. Paper chromato- GO) graphy Chromato- graphy 2. Thin-layer sraphy GLC) 2. High performance ae liquid chromato graphy (HPLC) chromatography ‘The techniques that use a solid as the stationary phase are called adsorpti chromatography” Adsorption chromatography is based on the Bie eueraneen Sompounds in a mixture have different strengths of adsorption t Tene phase. ‘0 the stationary ‘The techniques that use a liquid as the stationary ph: art 28 , chromatography, Partition chromatography is based Se the reened, Pawtition Ent compounds in the mixture have different values of partiiga or ag cee r distribution coefficients in a two-phase (liquid-liquid or liquid-gas) system,Separation Methods: Chromatography 53 Classification based on the mobile ph ile phase Chromatographic Methods Liquid Mobile Phase (ued Ginoneseans —— Solid Stationary "(Adsorption ne Liquid Stationary Solid Stationary Phase - Chromatography) Partition (Adsorption) Liquid ee Eg. Column chromato. Chromatography) Eg. Gas chromatography —_Eg. Gas-liquid graphy EgPaper chromatography (GC) chromato Thin-layer High-performance graphy (GLC) chromatography eae lon-exchange e ) chromatography The classification based on the stationary phase is generally followed in the study of chromatography. Choice of the chromatographic method The selection of a chromatographic technique depends on the quantity of the sample available and the type of information desired. CC: Quantitative separations (gin-kg scale) of many classes of organic compounds TLC : Purity determinations Tentative identification of unknown compounds To monitor column chromatography Monitoring the progress of organic reactions rations of cations, anions, and amino acids, IEC :; Qualitative separatr ions i n peptides and proteins. Quantitative separations of ions Qualitative and quantitative separation of mixtures of gases Monitoring gaseous pollutants GLC :; Qualitative separation of volatile, but thermally stable compounds Purity determinations in analytical and R & D laboratories PC: Qualitative separation of water-soluble organic compounds such as amino acids, sugars, and separation of ions (cations, anions) GC$4 Anolytical Chemistry nic compounds, 10 HPLC : Qualitative separation of the majority no organic walls, ete cluding thermally unstable, non-volalle Fe and dup Purity determinations in organic, TUB manufacturing units Monitoring the progre Identification of unknown con s of reactions pounds 3.2 COLUMN CHROMATOGRAPHY . - ati eparauior \Column chromatography is a widely-used technique for the Seana fal of complex mixtures of organic compounds in the gm-kB see. ish of reaction in organic research laboratories for the separation and purificalion © Tt ; productsi\In the drug industry, two anti-cancer drugs ~ taxol and vinealeuco® lore co isolated in a pure form from their plant extracts by column chromatography. | such as silica gel In this type of chromatography, finely-powdered, porous solids such as sil 7 Rae eiaeae are packed into a burette-like or alumina, which constitute the stationary pha F f glass tube, commonly called the column. The column is clamped vertically. The mixture of compounds to be separated are dissolved in a very small volume of organic solvent and applied as a narrow band at the front end of the column. A liquid which is generally an organic solvent such as n-hexane, benzene, chloroform, constituting the mobile phase, is allowed to flow through the stationary phase by gravity. The compounds in the mixture have different adsorption strengths towards the material of the stationary phase. Therefore, the compounds in the mixture, when allowed to move down the column, move with different speeds. Weakly- adsorbed compounds move faster than the strongly adsorbed ones. The different speeds of movement of compounds in the column is known as the differential migration. By this process, the compounds get separated one from another. Figure 3.1 shows the stages in % separation of a mixture of compounds A + B, by column chromatography. In chromatography techniques that use solid material as the stationary phase, e.g., column chromatography and thin-layer chromatography, the compounds in the mixture get separated due to their differences in adsorption strength towards the stationary phase. These techniques are therefore called adsorption chromatography and the stationary phase materials (silica gel, alumina) are called adsorbent The chromatography techniques that use liquids as the mobile phase, column, thin-layer, ion-exchange, paper and high-performance liquid Dea atoge raphy are all called liquid chromatography. Thus, column chrométy graphy is a liquid chromatography technique. Tt is appropriately called solid-li wid. adsorption siaetpatography~—the solid refers (0 the stationary phase, the liquid to the mobile phase.Separation Methods: Chromatography 35 Tre Mocure A+8 PEE pieced in column MANOS VANGALAGANGOTHR!. 57. Plesk Step 1 + Poles compound 8 ‘column pecking ea, Nom-nolar comaound A mode of compounds «4 117635 Flask 5 compound 8 (pure) collected Flask 2 Step 7 step 4 Fig. 3.1. Column chromatographic seperation The solvent (mobile phase) introduced at the front end of the column stationary ‘ase is called the eluent and that which leaves the column with or without theleiisseecannern emer 36 Analytical Chemistry f is te or column fraction, is called eluat® * ationary phase by the through ve column Separated compounds, collected in conical flasks, carried ion The process by which the compounds are a the mobile phase is called the development oF elutiCh BENTS 3.2.1 ADSORPTION PHENOMENON : NATURE OF ADSOR i sites used in column Table 3.1 gives the adsorbents and the nature of their active chromatography. Table 3.1 Common Adsorbents for Column Chromatography [SINo. | Adsorbent___] Nature of the surface active |. _T Silica Get Acidic 2 | Alumina Acidic and basic sites | Magnesium Silicate | Acidic 4. Kieselguhr Neutral 3. Charcoal Neutral and Acidic 6. Sucrose Neutral 7__| Starch Neutral These adsorbents are finely-divided, porous particles with large surface areas ~50m?/g. The adsorbent may be directly taken into the column as the dry powder (dry-packing method), or it may be made into a slurry in an organic solvent and | then poured into the column and the solvent drained off (wet-packing method) Silica gel and alumina are the two most common chromatographic adsorbents in use. They are cheap and readily available commercially. ilica gel Adsorbents with the general formula SiQz+H2O are called silica;-silica gel, or silicic acid. The surface of the SiO. particle is covered by hydroxyl groups (Fig. 3.2). H. HH A al F i i I 7m STITT ms IST Freehydroxys Bound and reactive hydroxyl Fig. 3.2. The surface of a silica gel particle It is the presence of these surface hydroxyl groups that is responsible for th selective adsorption properties of silica gel. Silica gel is therefore a highly pola) Solid, stationary phase. The silica gel surface is weakly acidic (pH = 3-5).Separation Methods: Chromotography 57 Alumina, acidic (A103) On heating hydrated alumina Al,Os2H:0 to 300-400°C, most of the adsorbed water is drawn off, with the remainder of the water reacting with the surface Al,O, to form hydroxyl groups. This type of alumina, used for column chromatography, is known as activated alumina, The surface of the particle of AlOs:has hydroxy groups, which are acidic in nature (pH-4), and hence it is nown as acidic alumina. Like silica gel, acidic alumina is also a highly polar, solid stationary phase, Alumina, basic (Al,0,) Heating hydrated alumina to Al,O3rH2O to 800-1000°C removes the water molecules totally to give hydroxyl-free Al;Os, the oxide ions now ae cS basic properties to alumina. Basic alumina is also a polar, solid stationary The choice of a proper adsorbent (silica gel, acidic or basic alumina) for column chromatography depends on the nature of the compounds in the mixture. The acidic type of adsorbents, ice., silica gel and acidic alumina, are used for the separation of acidic or slightly acidic compound mixtures (e.g,, carboxylic acids, phenols, etc.), while basic adsorbents, i.c., basic alumina, is used for the separation of basic compound mixtures (e.g., amines). Nature of the adsorption forces The forces that bind the compounds to the stationary phase are adsorption. The adsorption forces are purely physical in nature and are a sum of van der Waals’ forces, inductive effects and hydrogen bonding. Polar compounds bind strongly to a polar stationary phase, and less polar or non-polar compounds bind weakly to a polar stationary phase. Compound Reet ar ‘© 9 9 ce Siiee 981 az777 & rama Si Arm Site Fig. 3.3 Adsorption forces—hydrogen bonding, inductive effects and van der Waals’ forces a) van der Waals’ forces These are intermolecular forces which hold non- polar molecules together in the liquid or solid state. They are purely physical in character, and do not involve the formation of any chemical bonds. They are very weak adsorption forces. Consider a mixture of CH3(CH2)io-CH2OH (designated as A) and CHa(CHz):s-CH2OH (designated as B) to be separated by column chromatography using silica gel. B has a larger alkyl surface area than A; therefore, the van der Waals’ attraction and interaction between B and the silica gel surface is greater than that between A and the silica gel surface. B is relatively more strongly adsorbed than A on silica gel.58 Analytical Chemistry h onal groups suc functit Be trong permanent |< which Bi b) Inductive (dipole) forces In compounds which havr ps as if e C-heteroatom PO tationary phase surface as C=O, C-O, C-Cl, C-NOp, etc, th s : , CCl, ete, ; t! dipole. Compounds possessing such groups bind we inee on the strength of the groups, using their dipoles. The binding strength 4@P°™™ ¢ crional groups have (dipole. Thus, two compounds in the mixture with differ different binding strengths to the stationary phase- functional groups such as face groups by hydrogen ipole) and van der ©) Hydrogen bonding forces ‘The compounds with polar OH, NH;, COOH, etc,, bind with the stationary phase SUP ive (di bonding. These binding forces are stronger than the inducth Waals’ forces. In general, an organic compound which is b van der Waals’, dipole and H-bonding forces. r rat the polarity of the eompoute. Thus, wo or more organic compounds in See differing in molecular weight, size, or functional groups or isomer’ (sue arity trans, ortho, meta, para, structural, functional, positional), have different po Silty and therefore different adsorption strengths towards a particular stationary phase. | nary phase, such as silica gel or Polar compounds bind strongly with the polar statio1 i alumina. Non-polar compounds adsorb weakly with the polar stationary phase. For typical organic compounds, the strength of the binding increases in the following order: Hydrocarbons < aromatics < ketones < aldehydes < esters < alcohols < amines, mercaptans < acids. The differences in their adsorption strength is the basis for their chromatographic separation when a column containing the mixture, applied as a band at the front end, is eluted and developed with the mobile phase solvent. A less strongly adsorbed compound in the mixture moves faster relative to the more strongly adsorbed compound/s, thereby getting separated. Thus, in a mixture of two compounds A and B, where B is more strongly adsorbed than A to the stationary phase (B is more polar than A) on column chromatography, A is eluted first (as it moves faster) followed by B (Fig. 3.1). ationary phase uses the ind to the st These forces together contribute to 3.2.2 SOLVENT SYSTEMS Mobile phase solvent systems—elutotropic series The compounds in the mixture have different adsorption strengths towards the stationary phase. Their actual separation can be done only when a mobile phase moves through the station!) phase. The mobile phase is a pure organic solvent or a solvent mixture and is called the eluent. The common organic solvents, arranged in the increasing o de r of their polarity (dielectric constant), used as the mobile phase, are shown an Fi This arrangement of solvents in the increasing order of olari in a i ee in Table 3.2, is known as the elutotropic series. Any of these ae eae i aSeporotion Methods: Chromatography 59 as an eluent, In general, however, the elution of the column is started with the lower polarity solvent and gradually the solvent polarity is increased. For example, to begin with, benzene may be used as the eluent and later benzene : ethyl acetate 9:1 vWv can be used as the eluent. When a less polar solvent is used as the eluent, the less polar compound/s of the mixture are cluted first, as these are less strongly adsorbed to the stationary phase/ That is, the less polar solvent displaces or desorbs the less polar compounds from their adsorption. The more polar compounds of the mixture are more strongly adsorbed, and more polar solvents are needed to desorb them from their adsorption and they elute later with a relatively more polar solvent. Thus, in column chromatography where the stationary phase is polar, the starting eluent is less polar and the eluent polarity is gradually increased. The less polar compounds are eluted first, followed by the more polar compounds. Table 3.2. Solvent Systems for Column Chromatography Elutotropic Series Hexane Solvents arranged Cyclohexane | in the increasing order Benzene CHCl, increasing solvent power CHCls to desorb or dissolve EtOAc the compounds adsorbed Acetone to the stationary phase EtOH MeOH Water AcOH Choice of the starting eluent for column chromatography When the mixture of compounds to be chromatographed have different polarity, one should strictly follow the elutotropic series for the eluents. The starting eluent is n-hexane. After the collection of some fractions of the eluate, the eluent polarity is increased. Benzene is the next eluent and still later the eluent can be more polar such as benzene : ethyl acetate 9 : 1 v/v. This increase in polarity of the eluent is continued till all the compounds in the mixture are eluted. Non-polar solvents elute non-polar compounds; polar solvents elute polar compounds. The elution sequence for typical organic compounds is shown in Table 3.3. Before beginning the column chromatographic separation of a mixture, one should first study the TLC of this mixture (Section 3.3). The TLC indicates which solvent gives the best separation of the compounds in the mixture, as well as the number of compounds in the mixture. The solvent which gives the best separation in the TLC can be used as the eluent (mobile phase) for column chromatography.60 Analytical Chemistry atography Table 3.3 Elution Sequence of Compounds in Colum? Cn ‘Strong bases Slowest (need = potar sowvent o Acids Amines Alcohols Esters Aldehydes Ketones Aromatics Halocarbons Ethers fastest (will elut Olefins Hydrocarbons fe with a non-polar solvent) 3.2.3 DIFFERENTIAL MIGRATION Principle of column chromatographic separation: Partition or distribution coefficients: Differential migration (Fig. 3-1) In all types of chromatography separations, including column chromatography, two phases — the stationary phase and the mobile phase ~ are needed. In column chromatography, the stationary phase is a finely-divided, porous solid, while the mobile phase is an organic solvent or solvent mixture. First, the mixture to be separated into its constituents is applied as a small band at the front end of the column stationary phase. The compounds in the mixture get adsorbed to the stationary phase particle surface, and the different compounds adsorb with differing strengths. Now, the mobile phase is introduced into the column which moves through the stationary phase and is in contact with it. Let us consider the mixture to be separated is or two different compounds A and B. B is more polar than A, the molecules of B are strongly adsorbed to the stationary phase than A. The molecules of B get distributed or partitioned between the stationary phase and mobile phase, ie, some molecules of B are adsorbed on the stationary phase and some of B are ‘soluble’ in the mobile phase. Similarly, the molecules of A are partitioned between the stationary phase and mobile phase. B being relatively more polar than A, prefers the polar stationary phase to the mobile phase, while A prefers the polar stationary phase less than B. Thus, the concentration of B is more than A in the stationary phase. The ratio of the concentration of A in the stationary phase to that in the mobile phase is the partition coefficient of A. Similarly, the partition coefficient of the B is the ratio of the concentration of B in the stationary phase to that in the mobile phase, its value is independent of A. This is the operating principle in colum®, thin-layer, and ion-exchange chromatography as well as in normal phase HPLC. In all these types of chromatography, a polar solid acts as the stationary phase.Separation Methods: Chromatogrophy 61 Partition coefficient of a = Coue-of A in the stationary phase _ Conc. of A in the mobile phase Partition coefficient of = C2He-0f Bin the stationary phase _ Cone. of Bin the mobile phase 7 The partition coefficients are also known as distribution coefficients and are represented by kp or ky. The partition coefficients of A and B are different. No two organic compounds will have the same value of partition coefficients in a two- Phase system, as in this case. Since the concentration of A is less in the stationary Phase than that of B, the partition coefficient of A is less than that of B. The concentration of A is more (relative to B) in the mobile phase, which is the moving Phase, therefore A moves faster than B, and A is eluted first. Thus, in a mixture of compounds chromatographed, the compound with the lowest value of partition Coefficient elutes first (moves faster), followed by compounds of increasing value of Partition coefficient. Thus, in this chromatography (and also in thin-layer and ion- exchange chromatography in which the solid is the stationary phase), the difference in the adsorption strengths of the compounds in the mixture to the stationary phase results in their having different partition coefficients which leads to their different rates of movement (differential migration), resulting in their separation. When a solid is the stationary phase, the compounds physically adsorb to it with different strengths, and these chromatographic methods are classified as adsorption chromatography. Thus, column chromatography and TLC and ion-exchange are adsorption chromatography. It should be noted that in column chromatography the compounds are not permanently adsorbed to the stationary phase particles. There is a continuous adsorption and desorption. There are two opposing forces on the compounds in the column. The stationary phase tends to ‘retain’ and ‘retard’ the movement of the compounds by adsorption, while the mobile phase ‘moves’ the compounds down by displacing them from their adsorption sites (desorption). The mobile phase thus desorbs the compounds from the stationary phase, dissolves them in itself and moves them down. The partition coefficients of the compounds, that is their distribution equilibrium, is dynamic with molecules constantly adsorbing from the solution and desorbing into it. ; The mobile phase solvent is continuously fed into the column until all the compounds in the mixture moving at different speeds have moved out of the column. (Fig. 3.1). The column eluate is collected in several serially-labelled conical flasks. The eluates are separately concentrated and their thin-layer chromatography (see next Section) is carried out to determine the purity of the separated compounds. In an ideal case, in the column chromatographic separation of A and B, the column is eluted with about 1 litre of benzene and the eluate is collected in 5 flasks of 200 ml each. The flasks 1 and 2 contain pure benzene only, flask 3 contains$2 Analytical Chemistry dB. k 5 contains compoun compound A only, flask 4, again pure benzene and eee B is achieved by column Thus, a quantitative separation of A + B into pure chromatography, INES 3.2.4 SEPARATION OF A MIXTURE OF 0-/p-NITROANIL (A DEMONSTRATION EXPERIMENT) ribed here for are desc ‘Two commonly used methods of column chromatography the separation of a mixture of o- and p-nitroanilines. i i 30 cm Materials needed : Chromatography column (3 em internal a cae length), Silica gel (column grade, 200 mesh, » flasks 12 No.(250 ml capacity), o-nitroaniline + p-nitroaniline (100 mg each), Cotton, TLC microslides - 12 No. (for monitoring the column chromatography separation). Before starting the experiment, check the TLC of the mixture (see Section 3.3) using benzene as the solvent. One observes two clearly separated spots of the two compounds. Wet-packing technique The chromatography column (thoroughly cleaned with chromic acid, washed with water, rinsed with methanol and dried) whose stopcock is without lubricating grease is clamped vertically, using two clamps. Using a glass rod the bottom end of the column is loosely plugged with a small piece of cotton and the column is filled with benzene to half level. 75 gm of silica gel is made into a slurry in about 400 mi of benzene in a 500 ml conical flask. It is shaken well to obtain a uniform slurry. This slurry is carefully transferred into the column with the stopcock slightly open. The slurry slowly settles down. Any excess benzene is drained out. When all the benzene is drained out and the slurry level in the column is unchanged, close the stopcock. The slurry height is about 23) of the length of the column. In a beaker, dissolve the mixture of o-nitroaniline and p-nitroaniline in a minimum volume of benzene (about 5 ml is needed for this). Using a pipette, carefully transfer this solution onto the top of the silica gel slurry in the column and slightly open the stopcock. The’ compound solution percolates and spreads as a small band at the top. Close the stopcock and insert a piece of cotton above the slurry, fill the column with benzene (mobile phase) and immediately open the stock, diet the eluate flow-rate to 10 ml/min. Feed the benzene continuously fom the top. Collect the column eluate in 10 sequentially-labelled 250 ml conical flasks, In each conical flask, collect about 150 m! of eluate. Initially, an orange band ic soem at theSeparation Methods: Chromatography 63 top of the column. After about 30 minutes one observes two bands, one a clear bright orange band moving faster than the pale yellow band, with a white gap in between them. The eluate in the flasks labelled 1, 2, 3 is colourless and is only pure benzene. The flasks 4, 5, 6 contain the orange compound (o0-nitroaniline). The eluate in flask 7 and 8 is colourless and is pure benzene. The eluate in flasks 9 and 10 is pale yellow and contains the compound p-nitroaniline. The solutions in all the flasks are concentrated to a small volume (10-15 ml) and their TLC is examined. TLC (as described in the next Section) gives information about the purity of the compounds in the 10 flasks. From the TLC information, the solutions in flasks 4, 5, 6 are combined, and the solvent is evaporated to give pure o-nitroaniline ~ 90 mg. Similarly, the solutions in flasks 9 and 10 are concentrated and the solvent is evaporated to give pure p-nitroaniline ~ 90 mg. Dry-packing technique The above column chromatographic separation of o-and p-nitroanilines can also be performed using the dry-packing technique. In this method, fill the column with silica gel directly to about 3/4 level of the column (no solvent is used for this purpose). Now, disconnect the column, and withdraw about 1/5 of the silica gel from the column and transfer it into a beaker containing the mixture of o- and p- nitroanilines and a few millilitres of CHCls. With continuous stirring on a hot-water bath, evaporate the CHCl. The mixture of compounds, o- and p-nitroanilines are now adsorbed to the silica gel. It appears as a yellow powder. Now, clamp the column vertically and carefully pour this yellow powder at the top, place a piece of cotton, open the stopper and fill the column with benzene. Adjust the eluate flow rate to 10 ml/min. Feed benzene continuously from the top. Collect the column eluate in 10 serially-labelled 250 ml conical flasks. Concentrate the eluate fractions on a water bath and check the TLC. As in the wet-packing technique, the 1 3 fractions do not contain any compound, 4, 5, 6 fractions contain orange-yellow o-nitroaniline (~ 90 mg), and fractions 9 and 10 contain pale yellow p-nitroaniline (~ 90 mg). 3.3 THIN-LAYER CHROMATOGRAPHY (TLC) Thin-layer chromatography (TLC) is the most extensively used of all chromato- graphic methods in an organic laboratory. The theoretical basis of TLC is exactly the same as the column chromatography described earlier. Both these chromatographic techniques are adsorption chromatography. While column chromatography is used ‘to effect large-scale quantitative separations to give pure organic compounds, TLC “needs an extremely small quantity of the sample (less than a milligram), and in64 Anolytical Chemistry qualitative SeP2r ns. It is used to in) effects an extremely 10 10 mi A ly short time (say 5 i iat compounds in the sample. TLC has sever: determi IDetermine the number of compounds in a sample (purity Monitor the progress of organic reactions: ‘ith suspected 1 3+ Identify unknown compounds by a comparison wit standards. 4. Monitor the progress of column chrom “ification process such as crystallization, ~5Select the starting eluent for column chi 6. Effect small-scale (10-100 mg) quantitat nique is known as preparative TLC. separation OF any hic ata on, solvent extraction. distillation. romatography- a ive separation of mixtures. This asists of: (1) preparation of fate as a thin layer). (2) @PP) TLC plate using o: A thin-layer chromatography experiment co’ plates (coating the stationary phase on a glass pl of the sample solution as a spot, (3) development of the solvents (mobile phase), and (4) detection of the compounds. 3.3.1 COATING MATERIALS AND PREPARATION OF TLC PLATES Stationary phase materials: Adsorbents : Preparation of TLC plates fn thin-layer chromatography, as in the case of column chromatograp! Stationary phase materials are silica gel and alumina. These are the adsorb for TLC. For TLC, a special grade of silica gel and alumina known as sil G and alumina G, where G stands for calcined gypsum\CaSO, $H20, are us Silica gél G and alumina G contain 10-13% by weight of gypsum which acts a binder The silica gel G or alumina G is finely-powdered and fiour-like. (T column ¢hromatography-grade silica gel or alumina have 60-200 mesh size do not contain gypsum).(The silica gel G or alumina G is made into a si (CHCly:MeOH - 2:1 v/v or water and applied as a thin layer (about 250um thick: on a microscope slide or glass plate of 20 x 5 cm size} Due to the water MeOH gypsum becomes CgSO,.2H,0 and binds the adsorbent silica gel or alumina firm to the glass plate. (The water used for coating the adsorbent to the glass pla. removed by drying the TLC plate in a hot-air overt When CHCl;:MeOH is usea the TLC plate is dried just by exposing it to the air for 5 minutes. The thin layer of silica gel G or alumina G coated on the glass plate is the stationary phase for this chromatography, the glass plate is only acting as a support for this coatin; This solid stationary phase is also called an adsorbent. {In addition to silica gel i and alumina G, materials such as finely-powdered cellulose and polyamide are also” dsorbents for TLC. The choice of a proper adsorbent depends on the used as a Raid ig tre oteSeparation Methods: Chromatography 65 type of compounds to be separated or analyzed) Silica gel G is widely used for routine TLC work. Silica gel G : Majority of organic compounds, in particular, phenols, esters, acids Alumina G Basic compounds Cellulose and: Polyhydroxy and highly polar organic compounds Polyamide Sample application : Spotting : Adsorption To know the number of compounds in a sample using the TLC method, a dilute solution of the sample (about 1 mg/1 ml) is made in a volatile organic solvent such as CHCI;, CH;COCHs, McOH, etc. Using a microcapillary tube, a few microlitres of the sample solution is applied as a spot on the adsorbent-coated TLC plate at a distance of about 1 cm from the end of the plate (see Fig. 3.4). In this process, a Lid Spotted TLc plete }=-Glass chamber Silica get a coated el TLC plete {solution Eluent solvent Tem | point si (mobile phase) Step 1 Step2 Step3 Preparation of Spotting Development of TLC plate TLC plate: start A (less poler) B (more poler) Step 4 "Step 5 Developed plate Detection oe in-layer chromatography66 Analytical Chemistry rent. This step is know, few micrograms of the sample is delivered onto the arse compounds in the sample as the spotting of the sample or sample applicatio®. 1. pifferent compounds in are adsorbed onto the surface of the adsorbent Particle™. 4 described in deta the sample are adsorbed with different adsorption SUC" P7. trent of adsorption earlier, under the column chromatography technidh® "' giooje interactions, ang or binding is due to a combination of van der Waals» “1 ciohe, size, shape H bonding. These adsorption forces depend on the mo” mpounds have the same and/or functional groups in the compound. No two aia the sample solution strength of binding towards a given adsorber Assume thet © a 4 an. spotted contains two compounds A and B, where B is more BOM a \dsorbents, silica gel G and alumina G, are highly polar an rage a 7 gel more polar B is strongly ; , the stationary phases. rate 2 polar stationary phase, the aoe a bind strony adsorbed relative to fo a polar stationary phase, P* and less polar compounds bind weakly.) 332 SOLVENTS FOR DEVELOPMENT Development of the TLC plate : Mobile phase solvents : Chromatographic separation Figure 3.4, shows the various stages in the TLC. To separate A from B, the spotted TLC plate is to be developed or run. This is done by taking a few millilitres of organic solvent such as n-hexane, benzene, CHCls, EtOAc, or MeOH (the same as those used in column chromatography) in a capped glass bottle of appropriate dimensions. This is called the TLC chamber. The organic solvents taken in the TLC chamber constitute the mobile phase for this chromatography; they are called eluents. The sample spotted TLC plate is carefully dipped into the solvent and supported vertically. The solvent level in the chamber must be slightly below the level of the spot on the TLC plate. The solvent moves up the plate (ascends) through the stationary phase adsorbent by capillary action. (In this respect, TLC differs from column chromatography, where the solvent descends through the adsorbent). As the solvent moves up, the compounds A and B also move up, but at different speeds and thereby get separated. Compound B being more polar is more strongly adsorbed and moves slower, relative to the less polar A. The moving solvent edge, called the solvent front, is ahead of thé compounds A and B. The movement of the solvent on the TLC, leading to the separation of A and B, is known as running or development. When the solvent Tront Teaches about 4/5 of the TLC plate (it nseds 5 ini Tor microscope slides, and 30 min for 20 x 5'ém plates), the plate is taken out of the chamber, the solvent front marked with a pin or pencil and tH! eis air-dried to allow the solvent to evaporate. Pp she plat Bi ce Th val ph duiSeparation Methods: Chromatography 67 3.3.3 DETECTION OF COMPOUNDS IN TLC The separated compounds, A and B, are seen directly if they are coloured. If the compounds are colourless, the developed plates are kept in a stoppered bottle containing a few crystals of iodine. Within a few minutes, brown spots appear “wherever the compounds are present. Principle of TLC Since a solid is used as the stationary phase and a liquid as the mobile phase, the TLC is a solidUtiquid chromatography. The physical principle involved in the separation of compounds in a mixture is adsorption. Thus, TLC is an adsorption chromatography. The separation of compounds is due to their different adsorption strengths to the adsorbent. The role of the mobile phase solvent is to desor the compounds from their binding to the adsorbent, dissolve the compounds in themselves and carry the compounds upwards, in the direction of the movement of the solvent. The compounds move due to dynamic adsorption and desorption. Thus, in the case of the sample containing A and B, where B is more polar than A and the stationary phase is polar, A is partitioned or distributed between the stationary phase (adsorbent) and mobile phase (solvent). Similarly, B is partitioned between the adsorbent and the solvent. a Cone. of A in the stationary phase Partition coefficient of A = = ven Conc. of A in the mobile phase ~ "A Cone. of B in the stationary phase _ ition coeffici = S aa ica Concor lini hel mobil phase pia The partition coefficient of B is greater than A, because B being more polar than A has greater affinity to the stationary phase than A. Compound A with a lower value of partition coefficient is present more in the mobile phase, the moving phase, and therefore moves faster than B. Thus, the compounds get separated due to differences in their adsorption strengths which results in different values of Partition coefficients. Choice of the Solvents for TLC The common organic solvents used as the mobile phase or eluents for TLC are n-hexane, CCl,, benzene, CHCls, CH2Ch, ethylacetate, acetone, methanol, water, acetic acid and pyridine. To move and resolve non- polar compounds on TLC, non-polar solvents are used. To move and resolve polar compounds, polar solvents are used. The role of the solvent is to desorb and move the compound. : To know the number of compounds in a sample, the TLC experiment must be68 Analytical Chemistry solvents. In ONE Of th, ity . ifferent polar single spot done 3-4 times on different plates using diMerer” apie shows 8 cae pl solvent systems, the resolution will be good. ve: uch 4 1 a. When ding, « TLC experiment, the sample is @ pure compount a ular speed depending Dn on the plate (there is no separation), it moves vent. whens Ore than the polarity of the compound and the polarily ©. impure and the numbe: one spot is seen in the TLC of a sample, that sf P ‘ow, it is possible tc of spots indicate the number of compou' nds in the samP nds from the arcs assess the approximate relative concentra tions of the comp oemation, very little and intensity of the spots. TLC is mainly used ee ei y of the sample needed, and also less time is neede partic ¢ $0) for analysis. 3.3.4 Ry VaLues IN TLC Ry, (retardation factor) values and its applications Solvent front A 4, & O-p8 | i | point of sample in as Fig. 3.5. Ry valves in thin-loyer chromatography ‘The thin-layer chromatography particulars (data) of a compound are expressed in terms of retardation factor (R,) values. The stationary phase “retards” the motion of the compounds by adsorption. Different Compounds are retarded to different extents, depending on their adsorption strength. In TLC, the solvent moves ahead of the compounds and the edge of the solvent is called the solvent front. The ratio of the distance that the compound travels to the distance the solvent front travels is called the Ry value. The Ry value of a compound can be calculated by measuring the distances in centimetres, between the solvent front and the point of sample application (d,) and the distance from the middle of the spot to hte joint of sample application (ds, dz) (Fig. 3.5), for compounds A and B p distance moved by compound A (ds) Ry of compound A = = y of compt distance moved by solvent front(d,) distance moved by compound B (do) 2 Ry of compound B= y distance moved by solvent front{d;Separation Methods: Chromatography 69 than 1. The Ry value depends on Jumina), (2) nature of the solvent These parameters should be lue of a compound remains The Ry value of a compound is always less the: (1) nature of the adsorbent (silica gel or al system, and (3) thickness of the adsorbent coating. mentioned while reporting the Ry values. The Ry val the same whether it is in its pure form or if it is present in a mixture. The R, value is an important physical property of a compound, and is useful to identify an unknown compound by its TLC comparison with a suspected reference or standard, Solutions of the unknown and of the suspected reference compounds are spotted on the same TLC plate, developed and the spots detected. If the Ry values of the unknown and the reference compound are the same, then they are identical. 3.3.5 APPLICATIONS OF TLC IN CHEMISTRY 1. Monitoring a chemical reaction In several organic reactions, the reactant is slowly transformed into the product. The Ry values of the reactant and product are generally different. As the chemical reaction progresses, at different time intervals, a small quantity of the reaction mixture is withdrawn using a microcapillary tube and spotted on the TLC plate. Alongside this spot, the ‘reactant solution is spotted, the TLC plate developed, and spots detected. In the earlier stages of the reaction, ‘one observes spots in the reaction mixture which are due to the reactant and the product, while in the later stages of the reaction the TLC of the reaction mixture shows only the product spot. (Fig. 3.6) (a) Fig. 3.6 Monitoring a chemical reaction by TLC: (a) early stages of reaction, and (b) later stages of reaction. y It is essential that all 2. Monitoring the progress of column chromatograph: TLC. The column eluates column chromatographic separations are monitored by ; are collected in 100-200 ml fractions, in sequentially-numbered, 250 ml conical70 Analytical Chemistry ately concentrated 4, ions are separa’ , e. tractictions that show the an Tle ‘ jumn chromatographic he co ‘ : behaviour are combined, and the solvent evaporated. ee aa th ae separation of the mixture A + B is good, the first oving compound in pure form, contain no compound, flasks 3, 4, 5 contain the fast-m oo omar 0 cont: ‘ flasks 6 and 7 contain no compound and flasks 8: 9» 10 omavographic separation compound in the pure form. If, however, the column © moving compound, the of A + B is poor, the initial fractions contain the pure fasi-™ tast eee anae tons middle fractions contain a mixture of A and B, a while OT aby monitoring the pure slow-moving compound. All this information '§ the column chromatography by TLC. flasks (Flasks numbered 1 to 10). Thes 4 small volume and their TLC examines 3.3.6 PREPARATIVE TLC Preparative thin-layer chromatography TLC can be used for relatively small e ehel preparative separations, that is, quantitative separations in the-10-100, mg scale. TLC plates 6f 20 x 20 cm size, with a thick layer (0.5-2 mm) of adsorbent, are used. The samiplé Solution, containing about 10-100 mg of compounds, is applied as a band, instead of spots, at 1 cm from one end of the TLC plate, The plate ‘is developed and the separated compounds form bands (Fig. 3.7). To locate the position of the bands, a small portion of the-plate is visualized by iodine vapour or UV light, and the rest of the plate is covered with a filter paper. The bands are then scraped from the plate and collected in separate flasks, and the compound is leached from the adsorbent using an organic solvent. Filtration and evaporation of the solvent yields the pure compound (Fig. 3.7). Check area Solvent front A+B+C * 20 x 20cm TLC plete coating thickness (2mm) Fig. 3.7 Preparative thin-layer chromatography: (a) e sample Be 1 {b) developed TLC plate with separated bands imple application as a band,Separation Methods: Chromatography 71 3.3.7 DEMONSTRATION EXPERIMENT IN TLC Experimental procedure of thin-layer chromatography Step 1(a) Preparation of the TLC plates of 20 x 5 em size Glass plates of 20 » 5 cm (25 nos.) are thoroughly cleaned with chromic acid, rinsed with distilled water and wiped dry. These are arranged lengthwise, in rows of four on a thick plastic sheet (This is part of the TLC kit supplied by several manufacturers). About 50 gms of silica gel G is weighed into a 500 mi conical flask and 100 ml of distilled water is added to it and shaken well for 1-2 minutes. The resulting slurry is transferred into a TLC applicator or hopper (which is again part of the TLC kit), which is mounted on the glass plates at one end of the plastic sheet. The applicator is slowly and steadily moved over the glass plates (Fig. 3.8). A uniform thin coating of silica gel G on the glass plates is thus obtained. The coated plates are allowed to air dry for about 15 minutes and then transferred to the drying rack (also part of the TLC kit) and kept in a hot oven at 100° for 2 hrs. This is done to remove water from the adsorbent. The TLC plates are cooled and stored in a dust-free place These plates are used for analyzing complex mixtures of organic compounds, and the developed chromatograms can be preserved for longer periods. Direction of 8 - Hopper containing slurry b- Adjustable trailing face c- “Bed' with raised edges 4d - Spread plates 2 Glass plates Fig. 3.8 Moving spreader opparatus b) Preparation of TLC microslides The microscope slides are thoroughly washed with chromic acid, rinsed with distilled water and wiped dry. About 250 ml of the solvent mixture of CHCl3:MeOH - 2:1 v/v is transferred into a wide-mouthed, glass-stoppered bottle of 10 cm x 6 cm dimensions, silica gel G (about 35 g) is slowly added with continuous stirring till a slurry is obtained. The bottle is stoppered and shaken well. Two clean, dry microscope slides held back-to-back are dipped into the slurry (Fig. 3.9). These slides are slowly removed and the excess slurry is allowed to drain out of the edge of the container (If the coating is thin and grainy, the slurry should be thickened by the addition of more silica gel G). The two slides are separated and allowed to air dry on a clean paper for 5 minutes. Any excess adsorbent is removed from the edges of the slides, and the TLC plates are ready for use.72° Analytical Chemistry plate: Spotting AbOU 1 me oy the TLC ple into a dilute solutic, Step 2 Application of the sample onto tRE |g is made im uti -tube acetone, M the comple poe analyzed is taken in a t8-1UPE Toy (CHChs, seetone, MeOx by adding a few drops of any volatile organ sample solution Is applieg ete). Using a fine ca rilary tube, a few microlitres © tg end of the plate. Whi or pottednat a distance of about 1 em from ie °C" tog adsorbent at the poin: spotting, care should be taken not to scrape oes small as possible (3-4 mm) of application, and the size of the spot show ts can be applied, while on q On a 20 x 5 cm plate about 5 different sample SPO microslide 3 sample spots can be app! lied (Fig. 3-4)- Fig. 3.9 Preparation of TLC microslides Step 3. Development of the TLC plate A few millilitres of one of the orga solvents or solvent mixture (hexane, benzene, CHCl;, EtOAc MeOH) is takel! in the TLC development chamber (for a trial tun, benzene i CHCI, which 2 of medium poleriy an be used). The spotted TLC plate is placed vertically "Separation Methods: Chromatography 73 thé development chamber and quickly covered with a lid. The solvent level in the chamber should be below the level of the spots on the plate. The solvent runs up the plate by capillary action. This slow movement of the solvent, carrying the compounds with it, is known as development. The development is continued till the solvent runs up to about 4/5 of the coated plate (the longer the solvent run, the better the resolution). The time needed for this is about 30 mts for the 20 x 5 cm plates and about 5 minutes for the microslides (Fig. 3.4). Step 4 Visualization/detection of the compound spots in the developed plate The developed TLC plate is removed from the TLC development chamber, the solvent front is immediately marked (when the plate is still wet with solvent) using a pin or pencil (for Ry value calculation purposes), and the plate is kept aside for a few minutes to allow the solvent to evaporate. If all the compounds in the sample examined are coloured, a visual inspection is sufficient to locate the spots. If all or some of the compounds in the sample analyzed are colourless, the position of each of the compounds in the separated mixture can be visualized by using iodine vapour. The developed TLC plate is kept in a stoppered bottle containing a few crystals of iodine for about 5 minutes. The iodine vapour is adsorbed into the areas of the plate containing organic compounds. Brown spots, due to iodine charge transfer complexes, appear on a white background, Instead, the iodine vapour may be blown onto the TLC plate from a dropper-type glass tube containing a few crystals of iodine. Almost all organic compounds can be detected by this technique. The other methods of detection of TLC compound spots are: (1) Looking at the TLC plate under UV light. Wherever the compound spots are present, fluorescent spots are seen. (2) Spraying the plate with 5% ethanolic HaSO, and heating it in an oven at 100°C for 10-15 min., all compounds are charred and appear as dark spots, and (3) Spraying colouration reagents such as FeCl, solution (for the detection of phenols) or 2,4-DNP solution (for the detection of aldehydes and ketones), are the other methods for the detection and visualization of spots. 3.4, ION-EXCHANGE CHROMATOGRAPHY | Con-exchange chromatography is a technique for separating mixtures of charged compounds, such as cations (K+, Na*, Ca**, Cu**, Lanthanides), anions (Cl-, Br-, I), amino acids (ala, asp, orn), proteins or neutral molecules that can develop a charge in acidic or basic media such as carboxylic acids and amine: 4 (Ton-exchange chromatographic separations are done by using porous resin beads (granules) to which are bonded acidic groups such as -SO;_H* or -COO-H*, or basic groups such as -CH2N*R3X~ or -CH2NR2. ( Depending on the type of bonded group used for ion-exchange chromatography, 2 NNER: 86 Analytical Chemistry inutes, is a charag, gsed in min! . ration times PT ature of the stationary 5, ton tl juent. Retention times (Rn, ‘5 time. (Rete ography: A compe’ paper: column is its retention se ent 0 d_ It is depen of the el! property of the compoun rate of flo hromal the nature of the eluent and ee T dd paper chron hromatography hy. : of similar uty a the Ry values a8 mixtures) are SePATALC inj, in whether it is in its pure form 0} nN acids (stan! are ‘ an i amino acids ("a1 amino acids in the mn, 4 fo same value of retention time. Pure individual s in se . ted. The on with the retep, eluted and their retention ee a entiod times, bY comprded chromatogra wants ie ells oa te at oe oe ms eer times of the standards. 7 . | proportional to their concentration mixture of amino acids, the informatio, 8 If the sample analyzed is an unknown MTT |. follows: nas 2 | obtained from the automatic amino acid ana of the i different chrom: . the number of amin 1. The number of peaks in the chromatogram se 2 forces. acids. « simes in the chro i i times in the Matogra iti and their retention Table 3 2. From the positions of the peaks ei cette standards, the amino ai, be and by comparison with the retention in the mixture are identified. . | ‘onal to the concentration of the amino acids 20 3. The areas of the peaks are proporti Hl 1 3M 3.5 PAPER CHROMATOGRAPHY : 4 Paper chromatography is generally used for the separation of water-soluble organic 1 and inorganic compounds or highly polar compounds, such as amino acids ani “31 sugars. Mixtures of cations and mixtures of anions, in the solution form, are alo Th separated by paper chromatography. In paper chromatography, the stationary phase is water, which is supported ty Thus. the paper. The paper used for paper chromatography Contains/22% ‘by weight ol phas« water which is the stationary phase. The mobile phase for paper chromatograply Static is generally a polar organic solvent and water. perfec In paper chromatography, the Stationary phase is a liquid (water) and the mobie Sli phase is also a liquid (organic solvent + water). For this reason, paper chromatof raphy is also called liquid-liquid chromatography. These two teh i tituter 3.5. two phase system (and can be considered as equivalent to t Damiseile i ids taken in a separating funnel). The compounds in a mixture (oe a Tech partition or distribution between the two liquid phases, F € (e.g, A + B) underg? shap - Further, the compounds ° a lit the mixture A and B have different values of parti The differences in the partition coefficients form 1 of compounds using paper chromatography. For th, is one kind of partition chromatography. Aba OF distribution coefficien® — Con fe basis for separating mix amin Teason, paper chromatogrép”Separe "eperation Methods: Chromatography 87 Natui PAPER 3.5.1 RE OF PAPER—sUpporr, STATIONARY Pt , HASE per: Stationary phase ‘The : ~ Paper used f cotton cellulose. It is com; ‘OF Paper ch a circular or strip-form. (The paper wnufactured by the Whatman, pete of filter paper, and generally those 1 chromatograph Fann yee ted (Te 3 papel graphy work, the Whatmann No. rade of Taper is used, Ths : celulose fibres randomly matted together to has a porous. character. ie the water molecules (22% by weight te ee eae wrist molecules constitute the stationary aie of the ae Co aber ip. € water molecules are held to the cellulose by adsorption forces. incorporated into the paper at the time of manufacture.) paper consists of a mass of smal form a 3-dimensional network wit Table 3.4 Characteristics of Whatmann Chromatography Papers Grade Flow rate of Water Density gem Thickness mm in_mm/30 min ~ 20 85 0.58 0.16 -2 115 0.54 0.18 1 130 0.54 0.16 3 MM 130 0.56 0.33 3 130 0.49 0.38 4 180 0.46 0.20 7 190 0.50 0.88 31 ET 225 0.36 ues LET 225036 SB The role of the paper is that of an inert support for the water stationary phase. Thus, overall, the paper used for paper chromatography constitutes the stationary phase. iIt should be noted that in all chromatographic techniques with liquid as the stationary phase (paper chromatography, gas-liquid chromatography GLC, high- performance liquid chromatography HPLC) the liquid is supported on an inert solid support for otherwise, this liquid moves or flows out and cannot be stationary. 3.5.2 APPLICATION OF SAMPLE, SOLVENT SYSTEMS: MOBILE PHASE i \ .1 paper of a suitable size and Technique of paper chromatography (Whatman No.1 pap shape i Shown (At one end of the papers ata distance of about 3-4 cm, draw a line with a pencil. On this line the sample solution/s to be analyzed is spout Consider a sample solution containing two compounds A and B)(which ce amino acids, sugars, cations, anions, etc.) The solution is spotted at the mar!