Unit 5 Liquid Column Chromatography

UNIT 5 LIQUID COLUMN CHROMATOGRAPHY

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 C hromatography Stationary Phases Used in Liquid-Liquid Column C hromatography Mobile Phases in Liquid Column Chromatography

5.5

Development Techniques Frontal Analysis Displacement Development Elution Analysis

5.6 5.7 5.8 5.9 5.10

5.1

Basic Aspects of HPLC Applications Summary Terminal Questions Answers

INTRODUCTION

In the previous unit (Unit 4), a thorough discussion discussion on the classification and general principles of chromatography has been presented. While classifying the different chromatographic 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  (column and two dimensional), nature of support  (simple  (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, chromatography, the technique includes only two mechanisms, adsorption  and partition. Thus, the discussion in this unit will adsorption and liquid- liquid partition chromatography. In this confine to liquid- solid adsorption course, separate units have been assigned for ion exchange and gel sieving chromatography . Thus, we will be discussing only liquid-solid adsorption chromatography chromatography (LSC) and liquid- liquid partition chromatography 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, chromatography, some of the advantages of liquid chromatography become very apparent. The tremendous ability of gas chromatography 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. 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 chromatography also enjoys certain other advantages over gas chromatography in view of the fact that very difficult separations are often more readily a chieved by liquid chromatography than by

35

Chromatographic Methods-I

gas chromatography. 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, 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 adsorption and liquid-liquid partition column chromatography 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 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 conventional liquid chromatography has undergone undergone a major development in the form of high performance liquid chromatography, 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 chromatographic set up,

•

appreciate the criteria used for the choice of st ationary 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 RECAPITULATION OF BASICS

As stated earlier in this unit, we are going to confine ourselves to liquid-solid chromatography chromatography (LSC) and liquid-liquid partition chromatography chromatography (LLC); both of them being operated on a column. When a sample mixture is injected into a liquid chromatographic 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 la ter, the relative separation being dependent dependent upon the degree of retention 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 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

36

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

Liquid Column Chromatography

α = ( X / Y )a / ( X/ Y)l i.e., α , 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 chromatography is sometimes called liquid partition chromatography chromatography. Liquid-liquid chromatography 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 chromatography. The process of liquid-liquid chromatography 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 one stage extraction. However, liquid-liquid chromatography 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, chromatography, equilibrium distribution of the solutes b etween 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, 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 di fference 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, 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, 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. packing. Precise sample injection is achieved easily and rapidly in modern liquid chromatography. chromatography. Solvent flow is achieved by means of high pressure pumps with controlled flow rate which results in more reproducible operations operations and better and faster separations. The detection and quantitation a re done with continuous detectors of various types which yield a continuous chromatogram chromatogram without intervention by the operator. Fig. 5.1(a) shows an improved version of a liquid chromatographic 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 up 1. Mobile phase phase reservoir reservoir 2. Slurring device 3. Lamp (Heating device) device) 4. Pump 5. Pressure monitoring device 6. Pump Pump 7. Filter 8. Precolumn 9. Column 10. Pump 11. Injection port 12. Column thermostat 13. Detect Detector or 14. Recorder

5.3.1

Equipment

The equipment needed to carry out modern liquid chromatography chromatography is very different from the relatively simple and unsophisticated equipment used for classical liquid chromatography chromatography separations. The schematic of equipment used for modern liquid chromatography 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 i s 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 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 2µ sintered stainless steel filter is adequate for this purpose.

iv)

Pressure monitoring device

A device for monitoring the column input pressure should 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.

39

Chromatographic Methods-I

v)

Sample introduction device

Sample introduction into a liquid chromatography 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 t hrough 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 chromatography columns in lengths of 25-150 cm are normally preferred. Some columns columns may also be bent into a “U” shape. Coiled columns are are some times used, but are 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 analytical column. The internal diameter of the column has a si gnificant effect on the efficiency of liquid chromatography columns. 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 i mportant to control the column temperature in liquid chromatography. The 0 temperature variations within the column should be maintained within ± 0.2 C. 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 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 l iquid chromatography, 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 :

•

40

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

• •

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

Liquid Column Chromatography

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

…………………………………………………………………………………... …………………………………………………………………………………... …………………………………………………………………………………... 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 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 l iquid 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, fuller’s 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 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, chromatography, the separation depends on the transport of the molecules through the system and on the interchange of 41

Chromatographic Methods-I

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 µ) 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 µ 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 µ 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 st ructure 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 a dsorbents overheating will destroy the pore structure o e.g. silica gel, should not be heated above 200 C. On the other hand, the rugged adsorbents, fuller’s 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 Phases Used in Liquid-Liquid Column Chromatography

In liquid-liquid column chromatography, 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. 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 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 opportunity for the chemically bound bound stationary stationary phase to be eluted during use. A disadvantage 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

42

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: chromatography: 1.

Solvent evaporation technique.

2.

In situ coating procedure.

3.

The solvent filtration technique.

4.

The equilibration t echnique.

Liquid Column Chromatography

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. 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 a lso 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 ´