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Liquid Column Chromatography UNIT 5 LIQUID COLUMN

CHROMATOGRAPHY Structure 5.1 Introduction Objectives 5.2 Recapitulation of Basics

Liquid-Solid Chromatography Liquid-Liquid Chromatography 5.3 Experimental Set up Equipment 5.4 Choice of Stationary and Mobile Phases Stationary Phases Used in Liquid-Solid Column Chromatography Stationary Phases Used in Liquid-Liquid Column Chromatography Mobile Phases in Liquid Column Chromatography 5.5 Development Techniques Frontal Analysis Displacement Development Elution Analysis 5.6 Basic Aspects of HPLC 5.7 Applications 5.8 Summary 5.9 Terminal Questions 5.10 Answers

5.1 INTRODUCTION In the previous unit (Unit 4), a thorough discussion on the classification and general principles of chromatography has been presented. While classifying the different chromatographic techniques, the main criteria used was the nature of the mobile phase. It was pointed out that a large number of diversifications are available in the case of liquid chromatography. These are mainly due to shape of the support (column and two dimensional), nature of support (simple and bonded) and the mechanism (adsorption, partition, ion exchange and sieving) responsible for separations. In this unit, it is proposed to discuss liquid column chromatography. Normally, in the liquid column chromatography, the different mechanisms cited above should be included but in the general parlance of chromatography, the technique includes only two mechanisms, adsorption and partition. Thus, the discussion in this unit will confine to liquid- solid adsorption and liquid- liquid partition chromatography. In this course, separate units have been assigned for ion exchange and gel sieving chromatography. Thus, we will be discussing only liquid-solid adsorption chromatography (LSC) and liquid- liquid partition chromatography (LLC). The situation is more or less similar to gas chromatography where we have GSC and GLC.

If we compare the two important types of chromatography, viz gas and liquid chromatography, some of the advantages of liquid chromatography become very apparent. The tremendous ability of gas chromatography to separate and analyze complex mixtures is widely appreciated but the drawback of this technique is that only 20% of known organic compounds can be handled satisfactorily by gas chromatography. Liquid chromatography, on the other hand, is not limited by sample volatility or thermal stability. Thus, liquid chromatography is ideally suited for the separation of macromolecules, ionic species, labile material products and a wide variety of other high molecular weight compounds. Liquid chromatography also enjoys certain other advantages over gas chromatography in view of the fact that very difficult separations are often more readily achieved by liquid chromatography than by

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Chromatographic Methods-I

gas chromatography. The other advantage is about the sample recovery. Separated fractions are easily collected and recovery is quantitative. The recovery of separated components in gas chromatography is also possible but is generally less convenient and less quantitative. It may be important here to point out that gas chromatography, in general, is faster than liquid chromatography.

In this unit, we will first recapitulate some of the basics of liquid chromatography. Some special features of liquid-solid adsorption and liquid-liquid partition column chromatography will be discussed. This will be followed by a discussion on the components of a liquid chromatography set up. The choice of stationary and mobile phases is very important and the considerations involved will be discussed. This will be followed by a discussion on the basic methods used for chromatographic column development. In order to highlight the importance of the technique, some of the applications to separate complex mixtures will be presented. Since the conventional liquid chromatography has undergone a major development in the form of high performance liquid chromatography, a brief idea about this will also be given at the end.

Objectives After studying this Unit, you should be able to

recapitulate some of the basic concepts of liquid-solid and liquid-liquid column chromatography,

understand the functioning of the components of an improved version of a liquid chromatographic set up,

appreciate the criteria used for the choice of stationary and mobile phase,

describe development techniques used,

get an idea about HPLC, and

enumerate some of the important applications of liquid column chromatography.

5.2 RECAPITULATION OF BASICS As stated earlier in this unit, we are going to confine ourselves to liquid-solid chromatography (LSC) and liquid-liquid partition chromatography (LLC); both of them being operated on a column. When a sample mixture is injected into a liquid chromatographic column, it begins to migrate down the column under the influence of the mobile phase. During this process, various components of the mixture will begin to separate depending upon their affinity for the stationary phase in the presence of the mobile phase. The components that are weakly retained by the stationary phase will pass through the column and be eluted earlier. Thus, there will be peaks in the order in the resulting chromatogram. The strongly retained components will elute later, the relative separation being dependent upon the degree of retention by the stationary phase for each sample component. Thus, the components pass down the column at different speeds which can be related to the distribution of each component in the stationary and mobile phases. The two forms of chromatography, (LSC) and (LLC), basically differ in the mechanism responsible for separation. In one case adsorption is responsible while partition is operative in the other. An idea about this is being given below.

5.2.1 Liquid - Solid Chromatography The liquid - solid chromatographic technique is based on adsorption phenomenon. Consider a liquid solution of two compounds which has been brought into contact with a porous adsorbent. The molecules from the solution enter the pores of adsorbent and

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Liquid Column Chromatography

become attached to its surface. These molecules are held rather loosely by van der Waals forces. Some pass back into the body of the solution, and their places in the pores of the adsorbent are taken by other molecules. Thus, there is a continual interchange between the molecules in the body of the solution and those in the pores of the adsorbent. Usually, one component tends to be more firmly held on the surface than the other, so that, when equilibrium is established, the concentration of this component in the pores will be higher than its concentration in the surrounding liquid. Assuming that the phases can be perfectly separated, the separation factor is defined by the equation

= ( X / Y )a / ( X/ Y)l i.e., , the separation factor, is equal to the ratio of the mole fractions of the two components, X over Y, in the adsorbed phase, a, divided by their ratio in the liquid phase, l.

5.2.2 Liquid-liquid Chromatography Liquid-liquid chromatography is sometimes called liquid partition chromatography. Liquid-liquid chromatography is based on the separation of the solutes by their differential partitioning between two immiscible phases. This usually involves a stationary phase coated on an inert solid support, normally silica gel and an immiscible mobile phase. Most commonly the stationary phase is more polar than the mobile phase. In some circumstances, however, it is advantageous to reverse the roles so that the stationary phase is less polar. This variation is known as reversed phase partition chromatography. The process of liquid-liquid chromatography is similar to simple batch extraction between two immiscible liquids in a separatory funnel. A successive series of such extractions forms the basis of countercurrent distribution, which is more efficient than simple one stage extraction. However, liquid-liquid chromatography is many times faster and more efficient than countercurrent extraction. This is the result of the large interface between moving and stationary phases.

In liquid-liquid chromatography, equilibrium distribution of the solutes between the mobile phase and the stationary phase takes place rapidly, and the separation of the components of a mixture results from the resulting distributions of the various solute molecules in the two immiscible phases. The distribution equilibria are described by the distribution coefficient, often called partition coefficient K. For practical chromatography, it is necessary to be able to predict a particular solvent-solute relationship in order to obtain the required separation of a mixture. The distribution of a solute between two phases is also defined in terms of capacity or retention factor, k. The different terms and concept of theoretical plates and the rate theory have already been explained in Unit 4. All these are applicable for both of these forms of column chromatography.

SAQ 1 What is the basic difference in LSC and LLC? Which one will be generally faster?

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Chromatographic Methods-I 5.3 EXPERIMENTAL SET UP

At this point, with the background that we already have, it may be important to discuss the experimental set up of liquid column chromatography. Conventionally, in the classical set up there is a simple column, packed with an adsorbent or a support material coated with a stationary phase. The mixture is fed to the column which is then irrigated with the mobile phase. The eluant is collected in small increments and put to analysis. However, with time, several improvements have taken place.

In classical liquid chromatography, the column is used only once and is then discarded. Therefore, the packing in a column has to be refilled for each separation and this amounts to a significant expense of both manpower and material. In classical liquid chromatography, the sample application requires some skill and time on the part of the operator. Solvent flow is achieved by gravity feeding of the column. Separations require several hours. The detection and quantitation are done by the manual analysis of individual fractions. Many fractions are collected normally and their processing requires much time and effort. On the other hand, in modern liquid chromatography, reusable columns are used so that a number of individual separations can be carried out on a given column. Since the cost of an individual column can now be prorated over a large number of samples, it is possible to use more expensive column packing. Precise sample injection is achieved easily and rapidly in modern liquid chromatography. Solvent flow is achieved by means of high pressure pumps with controlled flow rate which results in more reproducible operations and better and faster separations. The detection and quantitation are done with continuous detectors of various types which yield a continuous chromatogram without intervention by the operator.

Fig. 5.1(a) shows an improved version of a liquid chromatographic set up while a more sophisticated set up is shown in Fig. 5.1 (b).

Fig. 5.1(a): An improved version of liquid chromatographic set up

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Liquid Column Chromatography

Fig. 5.1(b): A more sophisticated liquid chromatographic set up 1. Mobile phase reservoir 2. Slurring device 3. Lamp (Heating device) 4. Pump 5. Pressure monitoring device 6. Pump 7. Filter 8. Precolumn 9. Column 10. Pump 11. Injection port 12. Column thermostat 13. Detector 14. Recorder

5.3.1 Equipment The equipment needed to carry out modern liquid chromatography is very different from the relatively simple and unsophisticated equipment used for classical liquid chromatography separations. The schematic of equipment used for modern liquid chromatography is shown in Fig. 5.1 ( b). The components of this sophisticated set up are discussed below.

i) Mobile phase reservoir It holds one litre of the mobile phase. This reservoir is made up of stainless steel which is inert to most mobile phases and is not subject to breakage. Many different forms of reservoirs have been used, and simple units may be constructed from glass flasks or bottles of an appropriate size. Some reservoirs are designed so that the mobile phase may be degassed in situ. Degassing is required to eliminate dissolved gases, particularly oxygen. To permit in situ degassing, reservoirs are sometimes equipped with a heater, a stirring mechanism and inlets for applying vacuum and a nitrogen purge.

ii) Pumps One of the most important parts of modern liquid chromatography instrument is the pumping system. In modern liquid chromatography, the resistance to flow of the long, narrow columns packed with small particles is relatively high, and high pressures are required. Pumps are grouped into two major categories: mechanical pumps which deliver the mobile phase at a constant flow rate, and pneumatic pumps which deliver the mobile phase with a constant pressure.

iii) Filter A filter is normally placed in the line following the pump to remove fine particles of particulate material which can clog the inlet of the column. Generally a 2 sintered stainless steel filter is adequate for this purpose.

iv) Pressure monitoring device A device for monitoring the column input pressure should be inserted in the line between the pump and the chromatographic column. This pressure monitoring device indicates if there has been a plugging of the column or a failure of the pumping system.

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Chromatographic Methods-I

v) Sample introduction device Sample introduction into a liquid chromatography column is a very important factor in obtaining high column performance. The sample should be introduced as an infinitely narrow band on to the chromatographic bed. The more defused is the plug of sample in the mobile phase introduced into the column, the wider is the separated component bands at the end of the column. The sample is injected with a micro-syringe through a septum contained in a low volume inlet system. Septum materials are generally made from silicone or Neoprene. It is generally not feasible to make syringe injections above about 1500 psi through sampling ports. Therefore, at high pressure, a stop flow injection technique is normally used with syringe.

vi) Column The unpacked column must be constructed of materials that will withstand both the pressures to be used and chemical action of the mobile phase. Most columns are made up of stainless steel tubing. However heavy wall glass columns are sometimes used. Columns that will withstand pressures up to 600 psi are commercially available. For operation at high pressures, glass lined metal columns also can be used. Column end fittings should be designed with minimum dead volume. Porous plugs are used in the ends of columns to retain the packing. Straight sections of liquid chromatography columns in lengths of 25-150 cm are normally preferred. Some columns may also be bent into a U shape. Coiled columns are some times used, but are often less efficient than columns prepared in straight sections. Precolumns generally are desirable. The precolumn ensures that the mobile phase is completely saturated with the stationary phase before it passes in to the carefully prepared analytical column. The internal diameter of the column has a significant effect on the efficiency of liquid chromatography columns. For analytical studies, columns 1-4 mm internal diameter (i.d.) are normally used. Columns of larger internal diameter are used for preparative work.

vii) Column thermostat It is important to control the column temperature in liquid chromatography. The temperature variations within the column should be maintained within 0.2 0C. The larger changes in column temperature can result in significant variations in retention time.

viii) Detectors In liquid chromatography, the ideal detector should have high sensitivity, good precision and predictable response to all solutes. It should be unaffected by changes in temperature and carrier flow. It should not contribute to extra column band broadening. It must be nondestructive of the solute. Two types of detectors are in use in liquid chromatography, the bulk property or general detectors and solute property or selective detectors. Bulk property detectors measure a change in some overall physical property of the mobile phase plus that of the solute. The solute property detectors are sensitive only to the solute.

ix) Column packings The packing of columns in liquid chromatography are described in terms of adsorbent or stationary phase, the type of particle and particle size. Each of these particle characteristics has an important effect on the performance and use of a given packing material. The column are packed by various techniques such as :

Dry packing of rigid solid material using tap-fill procedure.

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Liquid Column Chromatography

Slurry packing technique for columns of hard gel. Slurry sedimentation method for soft gels.

SAQ 2 Why should the column temperature be maintained in a chromatographic set up?

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SAQ 3 What are the various techniques for column packing?

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5.4 CHOICE OF STATIONARY AND MOBILE PHASES The success of a separation by liquid column chromatography depends upon a proper choice of stationary and mobile phases. In LSC, the stationary phase is an adsorbent. And once we talk about the adsorbent, some of its properties, viz adsorbent type, surface area, particle size and activation and regeneration become relevant. In LLC, the stationary phase is a liquid immobilized on an inert support. While considering the liquid, its polarity and tendency to leach out become important. To counter act the leaching, bonded phases have been developed. Moreover, the requirements of inert supports on which the liquid is immobilized are equally important to know. Finally, the requirements of mobile phase are to be properly understood. This section deals with of the above mentioned different aspects.

5.4.1 Stationary Phases Used in Liquid-Solid Column Chromatography Stationary phases of a great variety have been used in liquid chromatography. Presumably any finely divided or porous solid which has the adsorption capacity and which is not too soluble in the mobile phase may be used. Many different substances have been used as adsorbents including activated alumina, silica gel, carbon, magnesium oxide, magnesium carbonate, hydrated calcium silicate, talc, silver sulphide, bauxite, activated clay from bentonite, fullers earth, sucrose, and powdered cellulose.

i) Adsorbent type Various adsorbent types exhibit different selectivities towards different types of compound. Polar adsorbents such as metal oxides, magnesium silicate etc. selectively adsorb unsaturated, aromatics and polar molecules such as alcohols, amines and acids. Polar adsorbents may be further sub-divided as acidic, basic or neutral, according to the pH of the surface. Silica, magnesium silicate are acidic and thus, they chemisorb bases. The alumina surface contains both acidic and basic sites. Non-polar adsorbents such as graphitized carbon which is a strong adsorbent and kieselguhr which is a weak adsorbent show no selectivity for the adsorption of polar molecules.

ii) Surface area The surface area and pore diameter of a given adsorbent vary widely with the method of manufacture. In adsorption chromatography, the separation depends on the transport of the molecules through the system and on the interchange of

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the molecules between an adsorbed phase and a liquid phase. If the volume of the adsorbed phase per unit quantity of the adsorbent is low, both the amount of interchange between the phases and the amount of separation will be small. For this reason, when dealing with large samples, it is important to select an adsorbent with a large surface area.

iii) Particle size The effect of particle size of the adsorbent on the sharpness of chromatographic separations has been noted by many investigators. For sharp separations, it is recognized that a finely divided material is necessary. An adsorbent in the range from 100 to 200 mesh (149 to 74 ) in particle size is specified. Some authors recommend the use of more finely divided materials. However, it is more difficult to pack columns uniformly if the adsorbent is very finely divided i.e., below 50 in particle size, and columns that are poorly packed give rise to zones that are irregular in shape. Some adsorbents are available only as finely divided powders with particles below 10 in diameter, e.g. magnesium oxide. It is necessary to mix these adsorbents with filter aids such as Celite or Hyflo Super-Gel to obtain a practical rate of flow.

iv) Activation and regeneration of adsorbent The term activation refers to those processes which are used to enhance the effectiveness of an adsorbent by improving the pore structure and increasing the surface area. In general, with carbon, high temperature activation produces an organophilic adsorbent, and low temperature activation, a hydrophilic adsorbent. During adsorption, the pores of the adsorbent become filled with adsorptive molecules. The term regeneration refers to the removal of the adsorbed molecules and the return of the adsorbent to its original state. The regeneration of the adsorbent can be carried out by gentle heating if the adsorbed molecules are volatile. If they are non-volatile, then they may sometimes be removed by elution or desorption with volatile solvent, which in turn, may be removed by gentle heating. With some adsorbents overheating will destroy the pore structure e.g. silica gel, should not be heated above 200oC. On the other hand, the rugged adsorbents, fullers earth and bauxite, may be heated in an oxidizing atmosphere to temperatures sufficient to burn off the adsorbed material near 540oC.

5.4.2 Stationary Phases Used in Liquid-Liquid Column Chromatography

In liquid-liquid column chromatography, the stationary phase is a liquid that is immobilized on a inert support. The stationary phases fall into two classes: the more usual hydrophilic ones and the reversed phase. The stationary or supported liquid phases have been of many kinds varying in polarity from water to paraffin hydrocarbons. As a large number of liquid stationary phases can be held mechanically on an inert support, such stationary phases have some disadvantages like leaching out of the liquid stationary phase from the inert support. In order to eliminate such disadvantages, surface-reacted or bonded stationary phases have been developed. The advantages of these materials is that pre-columns and or presaturation of the two phases is not required. In addition, packing with bonded stationary phases are quite stable because there is no opportunity for the chemically bound stationary phase to be eluted during use. A disadvantage of bonded-phase packing is a lack of systemic information regarding the mode of retention for solutes. There are two types of surface-reacted or bonded stationary phases that are now commercially available. The first one is an esterified siliceous material e.g. Durapak and the second type is surface reacted packing e.g. Bondpack, Vydac (organic coating is a monomolecular), Permaphase ( organic coating is of many layers). Columns of bonded phase packings have been used continuously for many months without changes in chromatographic

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Liquid Column Chromatography

characteristics. These bonded phase materials are available with several types of functional groups and used for separations of many types of solutes. There are four different techniques now in use for preparing liquid coated packing for liquid-liquid column chromatography:

1. Solvent evaporation technique.

2. In situ coating procedure.

3. The solvent filtration technique.

4. The equilibration technique.

Supports for liquid-liquid partition chromatography In liquid-liquid partition chromatography, the stationary liquid phase is supported on an inert support. An ideal support material has to meet the following requirements.

It should be chemically inert and it must not dissolve or swell in the stationary phase.

It should display good wetability by the stationary phase and it should neither dissolve nor react with the mobile phase.

It should consist of particles as identical as possible which allow the most uniform and reproducible packing.

It should have large enough surface to retain the stationary phase as a thin uniform film. Porous supports generally meet this requirement.

It should allow the columns to have an acceptable pressure drop as regards the mobile phase.

It should have sufficient mechanical stability. It must not grind during column packing, impregnation of stationary phase or regeneration of support material.

When applied for routine analysis or for preparative purposes, it must be relatively cheap, easily available and permit regeneration.

5.4.3 Mobile Phases in Liquid Column Chromatography Various physical and chemical properties govern the choice of mobile phases. The most important factor is the influence of the mobile phase on the selectivity of the system. The solubility of the samples and influence of such properties as surface tension and viscosity are also important. Solubility of some samples, especially polymers, limits the choice of the mobile phase and the use of certain detectors imposes constrains.

The choice of mobile phase in liquid column chromatography is all important. If water-deactivated silica is used as an adsorbent, the solvent is then varied to give k values in the optimum range (1< k

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Binary solvent mixtures are often used in liquid solid column chromatography. There are several advantages in the use of binary solvent mixtures e.g. solvent strength changes continuously with composition. Another advantage of binary solvents is that solvent viscosity can be kept low.

While considering the choice of mobile phases in liquid-liquid column chromatography, some basic characteristics of mobile phases must be considered. The mobile phase must be immiscible with the stationary phase. It should have viscosity as low as possible for higher column permeability and or efficiency. The detector can also limit the phases which can be used. e.g. strongly UV-absorbing solvents should be avoided with an ultraviolet photometric detector. The cost, toxicity, purity and stability of a solvent also should be taken into account. Most important is the selectivity of a liquid-liquid system for a given sample. In liquid-liquid column chromatography, the k values of solutes are generally controlled by changing the mobile phase. The scale of solvent polarity is defined by the Hildebrand solubility parameter, . The parameter, , is a good measure of what is commonly called polarity. Non-polar solvents have low values of , while polar solvents have large values. The values of for different solvents are shown in Table 5.1.

Table 5.1: Solvent Strength and Polarity Data Solvent Hildebrand

solubility parameter

Solvent strength

o

Solvent Hildebrand solubility

parameter

Solvent strength

o

n-Pentane 7.1 0.00 Ethylene dichloride

9.7 0.44

Isooctane 7.0 0.01 Triethyl amine 7.5 0.54 Petroleum ether 0.01 Acetone 9.4 0.56 Cyclohexane 8.2 0.04 Dioxane 9.8 0.56 Cyclopentane 8.1 0.05 Tetrahydrofuran 9.1 0.57 Carbon tetrachloride

8.6 0.18 Ethyl acetate 8.6 0.58

Xylene 8.8 0.26 Methyl acetate 9.2 0.60 i-Propyl ether 7.0 0.28 Nitromethane 11.0 0.64 Toluene 8.9 0.29 Acetonitrile 11.8 0.65 Benzene 9.2 0.32 Dimethyl

sulfoxide 11.5 0.75

Ethyl bromide 8.8 0.35 n-Propanol 10.2 0.82 Ethyl sulfide 8.6 0.38 Ethanol 11.2 0.88 Chloroform 9.1 0.40 Methanol 12.9 0.95 Methylene chloride

11.9 0.42 Ethylene glycol 14.7 1.1

SAQ 4 What are the different techniques to prepare liquid coated support?

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Liquid Column Chromatography

SAQ 5 What is meant by solvent strength of a mobile phase?

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SAQ 6 What is the advantage of using a binary solvent mixture as a mobile phase in LSC?

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SAQ 7 What does Hildebrand solubility parameter signify?

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5.5 DEVELOPMENT TECHNIQUES After having learnt about the stationary and mobile phase, it is important to know as to how the columns are developed. There are three basic methods of chromatographic developments. 1. Frontal analysis.

2. Displacement development. 3. Elution analysis.

Let us know them in a little more detail.

5.5.1 Frontal Analysis In frontal analysis, a large sample in a suitable solvent is passed through a short adsorption column previously saturated with solvent and the effluent is analyzed continuously until its composition is identical with that of the original sample. Consider a three component mixture containing equal quantities of each component fed continuously onto a column. Because of the forces between solute and stationary phase, each solute will be retained to a different extent as it comes into equilibrium with the stationary phase while passing through the column. The first component to elute will be that which is held least strongly in the stationary phase, then the second

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component will elute but in conjunction with the first component, and finally, the most strongly held of the three will elute in conjunction with the first and second components. Subsequently, there will be no change in concentration of solute in the mobile phase and the concentration of the respective solutes will be the same as the feed mixture. The concentration profile resulting from frontal analysis is shown in Fig. 5.2 (a). The continuous curve shows the total concentration of solutes in the eluent, plotted against volume of mobile phase passed through the column, and the dotted curves represent a similar concentration profile but for each individual component. Frontal analysis was employed as a development procedure in the early stages of chromatography and before detection procedures were fully effective. It is not often used today, and certainly not for quantitative analysis. The reason for this is that no individual component is completely separated from the others in the mixture.

5.5.2 Displacement Development Displacement development has the advantages of being able to accommodate large samples and giving sharp separations. It depends on the competition between solutes for the active sites of the adsorbent and is only really effective in separating very strongly adsorbed materials. In displacement development, all the substances in the sample will be held on the stationary phase so strongly that they cannot be eluted by the mobile phase; they can, nevertheless, be displaced by substances that are held on the surface by stronger forces. However, there will be competition between individual solutes and, when the sample is placed on the column, all the immediately available active sites of the adsorbent will be occupied by the most strongly held component.

As the band of the sample moves down the column, the next available sites will be occupied by the next strongly retained component. Thus, all the components array themselves along the column in order of their adsorption strength. To develop the chromatogram, another substance called the displacer is introduced into the mobile phase stream, the displacer has an even higher affinity for the adsorbent than any of the components to be separated.

Thus, on coming into contact with the sites occupied by the most strongly adsorbed component, it will displace this component into the mobile phase and thus, move onto the next group of sites occupied by the next component which will then itself be displaced. Thus, the displacer drives the adsorbed components progressively along the column, each component displacing the one in front, until they are eluted in the same order in which they were adsorbed on the column. The least strongly held being eluted first. The concentration profile of displacement development is shown in Fig. 5.2 (b). Displacement development has very limited applications as a separation technique and is only very rarely used in quantitative analysis.

5.5.3 Elution Analysis The elution analysis is the most common technique in chromatography. Employing elution analysis, complete separation can be achieved. This technique is being widely used for quantitative analysis. A relatively inert solvent is used in large quantities to transport the components down the column.

The nature of the solvent influences the equilibrium between stationary and mobile phases. The nature of the solvent can also affect the shape of the distribution isotherms. Bands that might travel down the column with a symmetrical concentration profile (Fig. 5.2 (c)) with one solvent might be quite unsymmetrical with another. When elution analysis is used to separate materials with widely differing distribution coefficients, the use of single elution solvent is not practical. If the eluent chosen is strong enough to remove the most strongly held materials in a reasonable time, it will carry the more weakly held materials through the column too rapidly. On the other hand, if the eluent is weak, it will be difficult to elute more strongly held materials.

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Therefore, when the sample components have a wide range of distribution coefficients, graded eluents are used. When utilizing graded eluents, the usual procedure is to begin with the weakest solvent capable of eluting one of the sample components and to change increasingly stronger solvents until all of the sample has been eluted. The change from one member of the series to the next is made by a stepwise increase in concentration of the stronger solvent. This technique is called as gradient elution analysis. The gradient elution technique can be used not only with mixed solvents but also with any phenomena that will give a solvent gradient in the column, e.g. pH or ionic strength.

Fig. 5.2: (a) Frontal analysis; (b) Displacement development; and (c) Elution analysis

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SAQ 8 What is meant by development of column? What are the different ways of achieving it? ...

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5.6 BASIC ASPECTS OF HPLC Possibly, the most significant advancement in chromatography since 1966 is the development of High Performance Liquid Chromatography (HPLC). The current practice in HPLC is based on the application of pressure to liquid systems.

The HPLC is characterized by a long, narrow column packed with very fine powder with spherical particles, operated at high liquid pressures, and possessing a continuous monitoring facility for the common effluent.

Such chromatographs may be quite expensive, but in turn, it is possible to achieve efficiencies comparable with those obtainable in gas chromatography, with separating times of the same order and the ability to work with non-volatile materials. Technically, the development work in HPLC has concentrated on the production of pulse-free pumps for generating the high pressures required and sensitive detectors for monitoring the column effluent.

In HPLC, the quantitative measurements are readily carried out on the chromatogram since the amount of substance present is proportional to its peak area. The analysis of standard samples is often necessary for comparative purposes. The advantages of HPLC technique are as follows:

a) Non-volatile materials can be handled. b) Ability to operate long columns at near optimum solvent velocity throughout

their length.

c) The nature of mobile phases can be varied to produce either stepwise or gradient elution or both.

The technique is discussed in detail in Unit 8.

5.7 APPLICATIONS The liquid column chromatography has been used to separate a wide range of sample types. Several interesting separations of compounds containing metal ions, isomers of cobalt complexes involved in the synthesis of vitamin B-12 and metal--diketones have been achieved. Steroids and related synthetically prepared compounds have been separated by liquid-liquid column chromatography.

Various pesticides have also been separated by liquid-liquid column chromatography. The stationary and mobile phases used for the separation of various compounds have been shown in Table 5.2. The separations listed only give a birds eye view of the variety of difficult separations that can be achieved by liquid chromatography.

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Table 5.2: Applications of Liquid Column Chromatography

Sample Separation Stationary Phase Mobile Phase Column Parameters

Steroids containing progesteron, androsteron, testosteron, 19-nortestosteron

1% --oxydipropionitrile on Zipax

Heptane Flow 1 mL/min

Pesticide residues containing methylparathion, parathion,

Diatomaceous earth coated 2,2,4-trimethylpentane

60.1 % water, 38.8 % etnanol,0.8 % acetic acid, 0.21 % NaOH,0.09 % KCl

18 0.27 cm glass column

Flow rate 3.4 mL/hr

Vitamins ( mixture ) Permphase ODS H2O to CH3OH 5 %

2 mL/min

Pesticides containing aldrin, DDT, DDD, lindane, endrin

Corasil--II n-hexane Column 20 2.3 cm

Flow rate 3.0 mL/mm

Barbiturates containing hexobarbital, phenobarbital, amobarbital, barbital

Vydac 2 % methanol in heptane

1m 2mm stainless steel column

Catecholamines from tisue, plasma, urine

Acidified alumina 1.0 M acetic acid, 0.2 M perchloric acid

Formaldehyde in air Silica coated with DNHP

Acetonitrile

Polycyclic aromatic hydrocarbons

Alumina Cyclohexane-benzene mixture

Coccidiostat in poultry feedstuffs

Silica gel Acetonitrile-chloroform (1:1)

5.8 SUMMARY This unit embodies discussion on liquid column chromatography emphasizing on the separations by adsorption and partition mechanism. To start with some of the advantages of liquid chromatography (LSC, LLC) over gas chromatography (GSC, GLC) have been highlighted. Some of the basics aspects relevant to LSC and LLC have been recapitulated. The different components of a modern liquid column chromatographic set up have been discussed. Since the success of a chromatographic separation depends on the choice of stationary (adsorbent/ liquid coated support) and mobile phases, the criteria used for their selection have been elaborated. The methods used for the development of a column with their special features have been discussed. Based on the background developed in the unit, an idea about HPLC is given. Finally, the unit ends with a few typical applications of liquid column chromatography.

5.9 TERMINAL QUESTIONS 1. In what particular respect, the liquid chromatography scores over gas

chromatography?

2. What are the requirements of a good detector for liquid chromatographic set up?

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3. What are the essential characteristics of a support material for liquid-liquid partition chromatography?

4. What is meant by activation and regeneration of an adsorbent?

5. What are important requirements of an appropriate mobile phase?

6. What is the role of displacer in the development of column by displacement technique?

7. Comment on the applications of liquid column chromatography in environmental analysis by citing two examples.

5.10 ANSWERS

Self Assessment Questions 1. The basic difference between LSC and LLC is that in LSC the separation takes

place due to difference in adsorption while in LLC the difference in partition is responsible for the separation. Generally LLC is faster.

2. The column temperatures should be controlled because large variations in temperature can result in to significant variations in retention time.

3. The various techniques used for column packing are: i) Dry packing of rigid material using tap fill procedure. ii) Slurry packing technique for columns of hard gel. iii) Slurry sedimentation method for soft gel.

4. The different techniques to prepare liquid coated support are i) Solvent evaporation technique. ii) In situ coating procedure. iii) Solvent filtration technique. iv) Equilibration technique.

5. The solvent strength, 0, controls the k values. These values are given with respect to an adsorbent. The values for solvents arranged in increasing strengths is referred as elutropic series. An elutropic series is used to find the right solvent strength by trial and error approach.

6. The advantage of using a binary solvent mixture in LSC is that solvent strength changes continuously with the composition and the appropriate composition can be selected. Moreover, the solvent viscosity can be controlled and kept low.

7. The Hildebrand solubility parameter is a measure of the polarity of the solvent. Non polar solvents have low values of while polar solvents have large values.

8. The development of a column essentially means separating the different components of mixture fed to the column by irrigating it with a suitable mobile phase. There are three basic methods for chromatographic development which are as follows: i) Frontal analysis

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Liquid Column Chromatography

ii) Displacement development iii) Elution analysis.

Terminal Questions 1. Liquid chromatography scores over gas chromatography because the later is not

applicable in cases of compounds which are thermally unstable or not easy to volatilize. In the case of GC, the quantitative recovery of the components is not easily achieved while in LC it is easy to recover the samples quantitatively.

2. A good detector should have high sensitivity, good precision and predictable responsible to all solutes. It should be unaffected by temperature changes and solvent flow. It should not contribute to extra band broadening. It should be non-destructive to the solute.

3. The essential characteristics of a support material for liquid-liquid partition chromatography are as given under. i) It should be chemically inert. It should neither dissolve nor swell in the

stationary phase. ii) It should have large enough surface to retain the stationary phase as a thin

uniform film. Porous supports generally meet this requirement. iii) It should allow the column to have an acceptable pressure drop as regards

the mobile phase. iv) It should have sufficient mechanical strength and should not grind during

column packing, impregnation or regeneration of support material. v) If being used for routine analysis and commercial purposes it should be

relatively cheap, easily available and permit regeneration.

4. Activation refers to those processes which are used to enhance the effectiveness of an adsorbent by improving the pore structure and increasing the surface area. The term regeneration refers to the removal of the adsorbed molecules and bring the adsorbent to its original state.

5. The requirements of an appropriate mobile phase are given as under: i) It should have viscosity as low as possible for higher column permeability

and or efficiency. ii) It should give a retention/ capacity factor for solutes preferably between 2

and10. iii) The cost, toxicity, purity and stability should be given due consideration. iv) The detector response to the solutes should not be interfered with.

6. To develop a chromatogram by displacement technique, a displacer is introduced in to the mobile phase stream. The displacer has even higher affinity for the adsorbent than any of the components to be separated. Thus, on coming in to contact with the sites occupied by the most strongly adsorbed component, it will displace this component in the mobile phase and thus move on to the next group of the sites occupied by the next component which will then itself be displaced. Thus, the displacer drives the adsorbed components progressively along the column, each component displacing the one in front until they are eluted in the same order in which they were adsorbed on the column. The least strongly held is eluted first.

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Chromatographic Methods-I

7. There is a great awareness about the pesticide pollution. Organophosphorus pesticides like methyl parathion and parathion are considered a little safer than organochlorine like DDT. Liquid chromatography has been successful in separating mixtures of organophosphorus and also that of organochlorine pesticides. They can be subsequently quantified individually. Another class of pollutants is polycyclic aromatic hydrocarbons. The technique has also been successful in separating them.

Further Reading 1. Chromatographic Methods, By R. Stock and C. B. F. Rice. Champman and

Hall and Science Paperback.

2. Fundamentals of Chromatography, By H. G. Cassidy, Interscience Publishers. 3. Chromatography: A Review of Principles and Applications, By E. Lederer and

M. Lederer, Elsevier Publishing Company.

4. Principles of Adsorption Chromatography, By L. R. Snyder, Marcel Dekker, Inc.

5. Quantitative Analysis Using Chromatographic Techniques, By Elena Katz (Editor), John Wiley & Sons.

6. Introduction to Modern Liquid Chromatography, By L. R. Snyder and J. J. Kirkland, A Wiley-Interscience Publication.

7. Organic Trace Analysis, By Using Liquid Chromatography, By J. F. Lawrence, Academic Press.