Unit 6 Planar Chromatography

UNIT 6 PLANAR CHROMATOGRAPHY

Planar Chromatography

Structure 6.1

Introduction Objectives

6.2

Paper Chromatography Chromatography Principle Stationary Support Solvent Systems Development of Chromatogram Detection Methods Applications

6.3

Thin Layer Chromatography Stationary Phases Mobile Phases Apparatus and Requirements Detections Methods Plate Concept Applied to TLC High-Performance Thin Layer Chromatography (HPTLC) Applications

6.4 6.5 6.6 6.7 6.8

6.1

Quantitative Aspects of PC and TLC Comparison of PC and TLC Summary Terminal Questions Answers

INTRODUCTION

So far you have studied about the general principles of chromatography chromatography where theoretical principles including resolution and plate concept were dealt. You now know that chromatography is now a very powerful separation technique used not only for the separation of complex mixtures but it is also used for quantification of each constituent. You have also learnt about classification of various chromatographic techniques which include a wide range of ion-exchange ion-exchange chromatography, affinity chromatography, chromatography, gel filtration, f iltration, electro chromatography, chromatography, zone electrophoresis, size exclusion chromatography etc. In this unit, you will learn about planar chromatography, chromatography, which includes paper chromatography  (PC), thin layer chromatography chromatography (TLC) and electro chromatography chromatography (EC). Each of these techniques make use of a stationary phase in the form of a sheet or flat surface of a paper or any other material such as metal, glass or plastic plate coated with suitable adsorbent with the help of a binder. The mobile phase moves through through the stationary phase by capillary action, assisted by gravity or an electrical potential. Here, we shall discuss only two techniques i.e. PC and TLC and a comparative study of their principles, methodology and applications will be undertaken. Once upon a time, term planar chromatography included two-dimensional chromatography chromatography though it has now come to signify the coupling of two chromatographic techniques with different mechanisms of separation. It is called ‘planar’ because the stationary support consists of the plane surface of a paper or smooth glass plate. It chromatography (PC) and thin layer chromatography chromatography (TLC) which are includes paper chromatography the simplest of all o ther forms of chromatographic chromatographic techniques. It also includes electrophoresis or electro chromatography chromatography where the movement of mobile phase is assisted by electrical potential. However, it will not be discussed here. The paper chromatography (PC) and thin layer chromatography (TLC) have the advantage of being simple, fast and inexpensive. These have been widely used for the

53

Chromatographic Methods-I

qualitative identification of different constituents in a mixture though these could be also used for quantitative determination of the components. However, PC is not used so commonly these days and as of now, planar chromatography based on TLC has found widest applications in organic synthesis laboratory, drug industry, clinical research and for investigating investigating biochemical processes. In both the cases, sample is spotted with a micropipette on to a paper or a plate and then the chromatogram is developed using a suitable organic solvent. Each constituent present in the sample is identified on the basis of colour development using a suitable detection system. Different components components of a solute travel with different speeds and the distance travelled by each component with respect to that of solvent gives a parameter called ‘retardation factor’ or R f . Even though PC and TLC have many things in common but there are some basic differences in their operation. Here, we shall discuss the basic principles, apparatus required, methodology and some typical applications of both the techniques in the separation of complex mixtures mixtures of organic and inorganic inorganic compounds. We shall first describe paper chromatography, chromatography, its principle, methodology and applications. PC is th e simplest of all the chromatographic techniques but it is not so widely used these days. Later, we shall discuss thin layer chromatography, its methodology and applications including modern developments of plate concept and high-performance thin layer chromatography. chromatography. Remember that though PC and TLC remain primarily qualitative techniques but these could also be used for quantitative analysis. A comparative study of the two techniques will also be presented

Objectives After studying this Unit, you should be able to

54

•

explain the meaning of planar chromatography,

•

describe the principle of paper chromatography chromatography (PC),

•

give the meaning of R f  and factors affecting it,

•

discuss the type of paper used as support and the types of solvent mixtures used in paper chromatography, chromatography,

•

describe how to run and develop the paper chromatogram,

•

explain the methodology of separation of inorganic and organic mixtures,

•

give the potential applications of paper chromatography, chromatography,

•

describe the principle of thin layer chromatography (TLC),

•

list the types of supports and the types of mobile phases used in TLC,

•

explain how to run and develop the thin layer chromatogram, chromatogram,

•

discuss the advantages of two-dimensional paper and thin layer chromatography,

•

apply plate concept to TLC,

•

describe the basic principle of high-performance high-performance TLC (HPTLC),

•

discuss the potential applications of TLC,

•

describe the quantitative aspects of PC and TLC; and

•

compare PC and TLC.

6.2

Planar Chromatography

PAPER CHROMATOGRAPHY

It is one of the oldest and simplest techniques for qualitative analysis though it can also be used for quantitative determination. During later part of nineteenth century Runge, Schnbein and Goppelsroeder separated coloured dyes and other chemicals on paper or cloth which is considered as old cousin of paper chromatography. chromatography. In 1944, R. Consden and A. H. Goron, two coworkers of A. J. P. Martin, the Nobel Laureate, first reported the separation of a mixture of amino acids and laid the foundation of  paper chromatography chromatography. The technique is based on the movement of solvent phase in upward or downward direction by gravity and accordingly, it can be categorized as ascending or descending PC. There is another version of it, called circular paper chromatography  where a circular paper is taken instead of strip and the solvent phase moves in circular direction. However, it is not very commonly used.

6.2.1

Principle

The technique of paper chromatography consists of a sheet of cellulose filter paper which serves as a stationary phase or separation medium . A small amount (usually a few micrograms) of solute is placed in a small area near the end of strip. A solvent is allowed to move from the end of the paper by capillary action and after equilibration for some fixed period, the solute migrates from its initial point of application. The components of mixture are separated completely or partially in distinct coloured  zones   zones or are located by the application of different reagents or b y applying ultra-violet fluorescence. At first, PC was considered as s imply a form of liquid-liquid partition. The hydrophilic fibers of paper can hold (or b ind) water in humid atmosphere such that a large percentage of water, say > 20% by weight, may be held in filter paper. Thus, paper was considered to be the analog of a column containing a stationary aqueous aqueous phase whence solute molecules get partitioned between this water and the mobile immiscible organic solvent. Later, this model was considered to be too simple because separations were also obtained where mobile phase was miscible with water or in other cases where it was just aqueous phase. Thus, it cannot be considered as simply liquid-liquid partition mechanism. Instead, besides adsorption and hydrogen bonding, interactions between solutes and the cellulose support are involved. During the pulping and bleaching operations of paper, carboxylate and other ionizable groups are introduced into cellulose which makes the paper as ion exchanger.  R f value: It is a characteristic parameter called retardation factor and abbreviated as  R f . It represents the position of an ion or a substance with respect to solvent phase. R f o f movement of the solute to the rate of of a solute is defined as the ratio of the rate of movement of the solvent . R f is most commonly used in paper and thin layer chromatography chromatography and is considered as a characteristic of na ture of the solute sample which may, of course, change with the solvent phase. It describes relative migration of the solute with respect to the solvent and may be represented as

 R f 

=

Distance traveled by the solute Distance traveled by the solvent

=

d s d m

… (6.1)

where, d s and d m are linear distances measured from the line of origin where spots are put as illustrated in Fig. 6.1. By definition, R f value cannot exceed 1.0. Ideally, R f values must be in the range of 0.1 to 0.9 with a minimum separation of 0.05. In order to have better separation, two spots must not overlap with each other and these must be symmetric without any tailing. In order to avoid tailing, different solvent mixtures, mixed in proper ratio, must be tried. If the spot of the solute is not symmetric, then d s is measured from the position of maximum intensity or the centre of the spot.

55

Chromatographic Methods-I

Fig. 6.1: Procedure for calculation of R of R f value in paper chromatography

It has been observed that R f values are influenced by the impurities in the paper and solvent, temperature and saturation of the atmosphere. Following factors may be considered; i)

Presence of other ions e.g. presence of chloride is carried out with nitrate solutions.

ii)

 Acidity of the original solution solution-This may be needed to avoid hydrolysis and its need in the formation of soluble complex.

iii)

 Development time- Sometimes it increases with the running time. Therefore optimum time may be determined.

iv)

Presence of other cations or anions as impurities.

v)

 Ambient temperature temperature.

Since R f  value depends on the distribution of complex species of the cations with different organic solvents, solvents should be carefully chosen. The technique of paper chromatography is primarily used for qualitative identification though it could also be used for quantitative determination but with a poor precision. Hence, it could be at best considered as semi-quantitative semi-quantitative technique.

SAQ 1 Which of the following phenomenon is responsible for the rise of solvent in p aper chromatography? i)

Capillary action

ii)

Gravity

iii)

Ion-exchange

iv)

Chemical affinity

…………………………………………………………………………………………... …………………………………………………………………………………………... …………………………………………………………………………………………...

SAQ 2 What is the ideal r ange of R f values? i) 0.0 to 0.9

ii) 0.5 to 1.0

iii) 0.05 to 0.95

iv) 0.1 to 0.9

…………………………………………………………………………………………... …………………………………………………………………………………………... …………………………………………………………………………………………...

56

6.2.2

Stationary Support

As mentioned already, the stationary support material is a highly purified c ellulose filter paper such as Whatman No. 1, 2, 31 and 3 MM or acetyl acid paper having hydrophilic affinity for water. The solvent penetrates the fiber and causes swelling of paper changing its dimensions. Polymeric cellulose structure contains several thousand anhydroglucose anhydroglucose units lin ked through oxygen atoms. Alternatively, modified forms of paper such as impregnated with alumina, silica gel, hydrous zirconium oxide, ion exchange resin may be used. Sometimes paper is coated with chelating agent solution such as dimethylglyox d imethylglyoxime, ime, 8-hydroxyquinoline 8-hydroxyquinoline etc. However, it should not 2+ 2+ have any impurities of Ca , Mg , Fe3+, Cu2+ etc so as to avoid interference. The paper is impregnated either neat or dissolved in a volatile so lvent. The solvent should evaporate slowly so that stationary phase may distribute homogeneously. In this case, coated liquid phase may interfere with the detection of separated spots. The paper shows following properties: 1.

weak ion-exchange properties

2.

adsorptive properties

3.

water holding property

4.

mild reducing agent

Planar Chromatography

The quality of paper and its porosity play an important role in paper chromatography as it determines the rate of movement of the solvent used. Thick paper with increased sample capacity may be used for preparative studies. The most suitable cardboards are Schleicher and Schull 2070, SS2171 which can take a load up to 1 g at each point of application. Generally, a paper strip is cut into 4 cm × 30 cm for one dimensional PC. For two dimensional PC, however, a 30 cm × 30 cm square sheet is commonly used.

SAQ 3 Explain why following types of paper can not be used for paper chromatography? chromatography? i)

Ordinary filter paper

…………………………………………………………………....................................... …………………………………………………………………....................................... …………………………………………………………………………………………... …………………………………………………………………....................................... ii)

Glazed paper

…………………………………………………………………………………………... …………………………………………………………………....................................... …………………………………………………………………....................................... iii)

Butter paper

…………………………………………………………………………………………... …………………………………………………………………....................................... …………………………………………………………………....................................... iv)

Bond paper

…………………………………………………………………………………………... …………………………………………………………………....................................... ………………………………………………………………….......................................

57

Chromatographic Methods-I

6.2.3

Solvent Systems

The nature of solvent plays an important role in the development of paper chromatogram. The solvent should be free from impurities and dried before use. Polar phase such as water is adsorbed by the paper and held stationary whereas the less polar solvent such as ethanol, acetone, glycol, formamide, acids, and amines flow through easily. Though pure solvent may be used but a mixture of solvents is preferred. Many solvent mixtures can be used provided these are not immiscible with one another. The following criteria may be adopted for the choice of the solvent; •

The solvent should not react chemically with any of the components of the sample mixture.

•

The composition of solvent mixture should not change with time. It means that none of its components should be volatile.

•

The solvent should not interfere with the detection of spots.

•

The distribution ratio should be independent of solute concentration.

•

The minimum difference between the Rf values of any two components components should be 0.05 or 0.1 so that they may be separated easily.

Some of the solvents commonly employed employed for the separation of cations are mentioned below; Typical Mobile Phases for P C          

Isopropanol,-ammonia -water (9:1:2) n-Butanol-acetic acid -water (4:1:5) Water-phenol Formamide-chloroform Formamide-chloroform -benzene Formamide-benzene Formamide-benzene -cyclohexane Dimethylformamide -cyclohexane Kerosene-(7:3)-isopropanol Paraffin oil-dimethylform oil-dimethylform amide -methanol-water

i)

n-Butanol saturated with 3M hydrochloric acid: equal volumes of alcohol and acid are shaken together and the upper layer is used.

ii)

Acetylacetone saturated with water: 7.5 mL acetylacetone is mixed with 0.05 mL hydrochloric acid and 2.5 mL dried acetone.

iii)

Acetone containing 5% (v/v) water and 8% (v/v) hydrochloric hydrochloric acid

iv)

Glacial acetic acid containing 25% (v/v) dried methanol methanol

v)

Methanol

vi)

Methyl ethyl ketone containing 30% (v/v) water and 1% (w/v) potassium thiocyanate

vii)

Methyl acetate containing 3% (v/v) methanol and 10% (v/v) water

viii) Pyridine containing 10% (v/v) water ix)

Dried n-butanol containing 40% (v/v) dry methanol

The solvents must be refluxed over suitable drying agent such as potassium hydroxide for acetone and ethyl methyl ketone, or anhydrous calcium sulphate for n-butanol or as prescribed in literature.

6.2.4

Development of Chromatogram

Paper strips are cut in appropriate size (usually 4-5 cm × 35-40 cm for a single spot but breadth may be changed for multiple spots) and stored under controlled conditions of humidity. A thin pencil line is drawn across the paper 2-3 cm from the edge and a circle is put at its centre. The sample is dissolved in a volatile solvent and it is spotted in the centre of line using a lambda (or micro) pipette of usually 5-10 micro liters or even with a capillary in case of qualitative analysis. The spot must be as small as possible without any spread for better separation and symmetric spots offer separation. It is best done by placing the sample drop wise, drying it with hot air blower (or hair drier) to evaporate the solvent as illustrated in Fig. 6.2. Sample spot may also be dried using infrafil lamp in a chamber. After drying, another drop may be put and dried. Several spots of different samples or a sample and the standard can be made across the line with a minimum separation of 1- 2 cm. The paper is now ready for development.

58

Planar Chromatography

Fig. 6.2: Illustration of (a) spotting of solute sample on paper using a capillary tube and (b) drying process using hair dryer

Important equipment used in paper chromatography is a development chamber where chromatogram is developed in a controlled environment. Some commercially available development chambers chambers used in ascending and descending descending PC are shown in Fig. 6.3. In ascending chromatography, chromatography, the paper is supported by means of a clip as shown in Fig. 6.3 (a).

Fig. 6.3: Some typical Developing Developing chambers for paper chromatography: (a) Ascending and (b) Descending

In descending PC, the top edge of the paper is h eld down by a glass rod or strip as shown in Fig. 6.3 (b). The development chamber is presaturated with the solvent

59

Chromatographic Methods-I

system and then paper is hung with its end dipping in the solvent system as shown in the figure. The solvent starts rising slowly and then it stops a fter some time. It may take an hour or even longer. The development time will depend on the complexity of the mixture of solutes being separated, solvent system and the quality of paper and the ambient temperature. The diffusion of the solvent and the resulting separation into spots is termed as development of the chromatogram. It is essential that paper should be equilibrated with the solvent vapours in the chamber. For good resolution, reasonable  R f values must be in the range of 0.4 to 0.8 with typical separation time being 1 to 2 hours. Following are the main sources of error: •

Lateral diffusion of the solutes.

•

Variation in the structure of paper.

•

These become important especially for the concentrated solutes.

•

Variation in the geometry of the assembly for the standard and unknown samples. However, this error can be easily eliminated by using the same tank for the two samples where similar si milar experimental conditions are maintained.

6.2.5

Detection Methods

After development of chromatogram, the solvent front is marked and the solvent is dried. The spots of the separated compounds are then detected in a variety of ways by using any one of the following methods. i)

Reactions with colouring reagents such as d imethylglyoxime imethylglyoxime for Ni and hydrogen sulphide gas for any of Gr II elements

ii)

Fluorescence producing reagents

iii)

Inherent visible colours of the components such as sulphides and oxides

iv)

Radioactivity measurement of radiotracers

v)

Electrochemical methods such as potentiometry, conductance measurements and polarography polarography have been successfully used for the detection and quantitative analysis in paper chromatography. However, these methods are of limited importance and not commonly used.

The reagents employed include diphenylthiocarbazone diphenylthiocarbazone (dithizone), rubeanic acid, diphenyl dithiocarbazide, alizarin, salicylaldoxime, morin, potassium ferrocyanide, ferrocyanide, potassium chromate, ammonium sulphide and hydrogen sulphide gas. In many cases, two or more of these reagents are advantageous. The spots are characterized by their characteristic R f values. Identical R f values for a known and an unknown compound using several different solvent systems provide a good evidence that two are same especially if they run si de by side along the same strip of paper. Example

60

i)

In a paper chromatographic chromatographic separation of cations- Ag +, Pb+ and Hg+, solvent front rises to 18.4 cm while cationic spots were observed at 15.8, 12.1 and 5.9 cm, respectively. Calculate R f values of the metal ions.

ii)

Emission gases of a two wheeler were tested for pollutant metals by paper chromatography. chromatography. A spot corresponding corresponding to R f value of 0.65 was observed. What is the possible pollutant metal ion in the emission gases?

Solution: 

Here,

 R f value for Ag+ = 15.8/18.4 15.8/18.4 = 0.86  R f value for Pb+ = 12.1/18.4 = 0.66 +

 R f value for Hg =

Planar Chromatography

5.9 /18.4 = 0.32

As R f value of 0.65 is comparable to 0.66 corresponding to Pb + , it can be concluded that the pollutant in the emission gases is likely to be lead.

6.2.6

Applications

Paper chromatography chromatography has been widely used for separation and identification of cations in inorganic mixtures, organic functional groups, proteins and enzymes in biochemical work. It is particularly useful in the separation of closely related compounds such as isomers, homologues, isotopes and species having different valency or oxidation states. It has been found especially useful in the following: •

identification of trace metals in ores,

•

checking purity of pharmaceuticals and fermentation,

•

ripening of fruits and fermentation products,

•

detection of adulterants and contaminants in foods and drinks by comparing chromatogram of pure compound with that of the adulterant.

Sometimes, it happens that all the components of a mixture can not be separated using a single solvent system; some components separate better in one solvent and some in another. In such cases, two-dimensional paper chromatography  may be employed. In this technique, a square paper of 30 cm x 30 cm is taken and the sample is spotted at one corner of the sheet. Chromatogram is developed using one solvent whence solute migrates parallel to one edge of the paper. Next time, the paper is turned at 90o and developed in a second solvent system which carries the solutes into the unused portion of the paper. A typical chromatogram of a protein hydrolysate using ninhydrin-stained spots is shown in Fig. 6.4.

Fig. 6.4: Illustration of two-dimensional paper chromatogram chromatogram of a protein hydrolysate where solute is placed in corner at H and it is first run towards the right with an acetic acid-butanol solvent and then at perpendicular using phenol + resol/water solvent

Some typical applications are described below:

61

Chromatographic Methods-I

i)

Separation and Identification of Mn, Ni, Co and Zn

A combination of Mn, Ni, Co and Zn may be separated by paper chromatography chromatography using Whatman No. 1 filter paper strip by developing with a mixture of acetone, water-hydrochloric acid system. The spots are located by the colouring reagents. These elements are identified by comparing with their characteristic R f values of known with those in unknown mixture. The characteristic R f values are as follows: Ni Mn Co Cu ii)

- 0.09 - 0.21 - 0.43 - 0.61

Separation of a mixture of pesticides

A combination of pesticides such as aldrin, endrin, lindane, heptachlor, DDT, BHC etc. may be separated on Whatman No. 1 filter paper treated with refined mineral oil in diethyl ether (5% v/v) and washed with 75% aqueous acetone. The mobile phase used is 3:1 methanol-water (v/v). The R f values for aldrin, heptachlor, DDT, endrin and lindane were found to be 0.37, 0.48, 0.60, 0.62 and 0.89, respectively. iii)

Speciation of Different Anions of Sulphur

In its unique application, paper chromatography chromatography has been successfully used for speciation studies of sulphur such as S2-, SO32-, SO42-,S2O32-, S4O62- using radiotracer 35S. Whatman No. 1 filter paper was used along with solvent mixtures of dioxane, n-butyl alcohol-1N ammonia (1:1:1) and acetone- isopropanol-liquor propanol-liquor ammonia (1:1:1). Different spots corresponding corresponding to various anions can be identified by radioactivity measurements using a G. M. counter. 2222The R f values for SO 4 , S2O3  , S4O6 and S  were found to be 0.12, 0.43, 0.54 and 0.77, respectively. Similarly, R f values for S2O32- and SO32- were found to be 0.14 and 0.61, respectively. Paper treated with silicone or paraffin oil permits reversed phase paper chromatography chromatography where mobile phase is a highly polar solvent. This technique is referred to as ‘ reverse-phase ’. Commercially available papers coated with reverse-phase PC ’. adsorbent or ion-exchange resin offer additional applications of adsorption and ion-exchange ion-exchange paper chromatography. iv)

Autoradiography using Radiotracers

In this technique, a radiotracer or a radiolabelled solute sample is used. After development of chromatogram, chromatogram, it is kept i n contact with a photographic film of the same size. After some time when the film is developed then dark spots are observed in place of spots corresponding corresponding to the movement of solute components. This is called autoradiography . It has been found to be especially useful for the study of distribution and metabolism of compounds administered to the plants or animals. In a unique experiment, experiment, the Nobel Nobel Laureate Calvin and coworkers identified intermediate steps involved in the photosynthesis of carbohydrates from atmospheric CO2 in the presence of light and chlorophyll by using 14C, 32P and 3H. Plants were placed in the a tmosphere containing containing 14C labeled CO2 and irradiated with light as shown in Fig. 6.5. The plants were removed after different irradiation periods of light and the molecular components were separated using paper chromatography chromatography.. The presence of radioactive atoms in a compound by following autoradiography autoradiography was taken as a proof of the involvement of that compound in the photosynthesis.

62

Planar Chromatography

Fig. 6.5: Illustration of Autoradiography Autoradiography method used for the determination of compounds involved in photosynthesis

6.3

THIN LAYER CHROMATOGRAPHY

Thin layer chromatography is different from paper chromatography in that a flat

63

Chromatographic Methods-I

surface of glass, metal or plastic coated with adsorbent such as silica gel, alumina or any other material, is used. The technique discovered by Ismailoff and Scraiber in 1938 is faster, more sensitive and has better resolution than paper chromatography. chromatography. TLC is often used to develop optimal conditions for separation by liquid column chromatography. chromatography. The technique has become a workhorse of the drug industry for purification of products. It has also found widespread widespread use in clinical, industrial and environmental laboratories. Recent developments have elevated it from the level of semi-quantitative analytical procedure to one in which highly reliable quantitative separations can be performed.

6.3.1

Stationary Phases

The stationary phase used in TLC can be an adsorbent, an ion exchanger, a molecular sieve or it can serve as the support for a liquid film. It c onsists of a finely divided powder of particle size 5 to 50 µm. A variety of stationary supports are available for coating the smooth surface of glass, metal or plastic. Plastic sheets have the advantage that these can be e asily cut to any shape or size as required l. Silica gel G (60-120 mesh) remains the most frequently used coating materia. It contains hydroxyl hydroxyl groups on the surface which form hydrog h ydrogen en bonds with polar molecules. The adsorbed water prevents other polar molecules from reaching the surface. Hence, the gel is activated by heating to remove the adsorbed water. In modified silica gel, H and OH groups can be replaced by other functional groups similar to bonded phase. Alumina containing hydroxyl groups or oxygen atoms is another commonly used adsorbent. Other adsorbents widely used are powdered cellulose, magnesium silicate, calcium silicate, activated charcoal, natural diatomaceous earth, ion exchange resins such as Dowex50W-strong acid cation exchanger in sodium or hydrogen form and Dowex-1-strong base anion exchanger in chloride form. Kieselghur is o ften used for the separation of sugars. In fact, any adsorbent material that can be used in adsorption or column chromatography chromatography can be used u sed in TLC. Here, an aqueous slurry of the powder is prepared by mixing the adsorbent with a binder such as plaster of Paris (CaSO4) and polyvinyl alcohol to help it adhere to the backing material. It is uniformly spread over the plate manually or by using one of the commercial forms of spreader as shown in Fig. 6.6. The thickness of the layer is usually in the range 0.1 to 0.3 mm but for preparative work much thicker layers are preferred. The solvent is evaporated off and adsorbents are activated by placing in an oven at 110o C for a few hours. Precoated ready-to-use plates and sheets are also commercially available.

Fig. 6.6: Apparatus used for preparing TLC plates where the applicator is filled with the slurry of adsorbent and binder in a solvent.

Thin layers of Sephadex superfine superfine gel can be prepared for size exclusion. exclusion. Gel is soaked in water for a few days and then spread on the plate. These are not dried but stored wet. Capillary action through these molecular sieves is much slower, typically of the order of 1 to 2 cm/hour. Hence, it takes longer period to develop these plates.

64

The following precautions must be followed while handling TLC pl ates: 1.

The surface of the TLC plates should not be touched. The plates should be handled carefully by holding at the edges so as to avoid any contamination due to sweat.

2.

The plates should be cleaned thoroughly so as to remove any extraneous material that might contaminate the adsorbent.

Planar Chromatography

SAQ 4 Explain why the flat surfaces of the following materials cannot be used as an inert support for the coating of the adsorbent in TLC? i)

Plywood

…………………………………………………………………………………………... …………………………………………………………………………………………... …………………………………………………………………………………………... ii)

Asbestos sheet

…………………………………………………………………………………………... …………………………………………………………………………………………... …………………………………………………………………………………………... iii)

Card board

…………………………………………………………………………………………... …………………………………………………………………………………………... …………………………………………………………………………………………... iv)

Glass with granulated surface

…………………………………………………………………………………………... …………………………………………………………………………………………...

6.3.2

Mobile Phases

The choice of the mobile phase is largely empirical but some general guidelines can be formulated. A mixture of organic solvents and water with the addition of acid, base, or complexing agent to optimize the solubility of the components of a mixture can be used. It may be emphasized that a large degree of trial and error is involved in the selection of the mobile phase. The polar solvents can themselves become strongly adsorbed; thus, producing an undesirable situation of partition system. The following criteria may be adopted. i)

The developing solvent must be of the highest purity because even trace amounts of impurities may yield irreproducible results.

ii)

Good separation of polar or ionic solutes can be achieved with a mixture of water and n-butanol. The criteria used for the selection of developing solvent are the same as for column chromatography.

iii)

The eluting power of solvents increases in the order of their polarities e.g. from hexane to acetone to alcohol to water. An eluotropic series, given in Table 6.1, can be used for the selection of the best solvent or the solvent mixture for a sample.

iv)

If the stationary phase is hydrophobic, mixtures of benzene, cyclohexane, and chloroform in different ratios provide sa tisfactory mobile phase.

65

Chromatographic Methods-I

Table 6.1: Eluotropic series of mobile phase phase Solvent

Solvent strength o

ε 

6.3.3

n- Pentane Cyclohexane

0.00 0.04

Carbon tetrachloride Toluene Chloroform Methylene chloride Tetrahydrofuran Acetone Methyl acetate Acetonitrile n- and iso-propanol Ethanol Methanol Water Ethylene glycol

0.18 0.29 0.40 0.42 0.45 0.56 0.60 0.65 0.82 0.88 0.95 1.00 1.11

Apparatus and Requirements

Commonly, the plates in sizes of 2.5×10, 2.5×15, 2.5×20, 5×20, 10×20, and 20×20 in centimeters are available. These are of two types: conventional and high-performance . The former have thicker layers of about 0.25 mm with particle size of < 20 µm. On the other hand, the high performance plates usually have thickness of 0.1 mm and particle size of