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Chapter 3 High Performance Liquid Chromatography 3.1 INTRODUCTION Russian botanist Tswett67 is credited with the discovery of chromatography. In 1903 he succeeded in separating leaf pigments using a solid polar stationary phase. It was not until the 1930’s that this technique was followed up by Kuhn and Lederer68 as well as Reichstein and Van Euw69 for the separation of natural products. Martin and Synge70 were awarded the Nobel Prize for their work in 1941 in which they described liquid-liquid partition chromatography. Martin and Synge applied the concept of theoretical plates as a measure of chromatographic efficiency. This concept laid the foundation for gas-liquid chromatography (GLC) and high-performance liquid chromatography (HPLC). The GLC technique rapidly developed after Martin and James published the first use of GLC in 1952.

HPLC was derived from classical column chromatography and has found an important place in analytical techniques71. The major advancement in HPLC was found by the use of efficient separators. These separators used small particles and high pumping pressures.

3.2 THEORY OF CHROMATOGRAPHY71-73 Chromatography is an analytical method that finds wide application for the separation, identification and determination of chemical components in complex mixtures. This technique is based on the separation of components in a mixture (the solute) due to the difference in migration rates of the components through a stationary phase by a gaseous or liquid mobile phase. Figure 3.1 shows a typical chromatogram indicating the physical parameters that can be obtained directly from it. These parameters are used in chromatographic optimisation and will be discussed in the following text.

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Figure 3.1: Parameters involved in chromatographic separations82

3.2.1 Capacity Factor

The capacity factor, k’, of a compound indicates its retention behaviour on a column.

k'=

K d × Vs Cs × Vm tms − tm t s = = = Vm Cm × Vs tm tm

Kd: Distribution coefficient Vs: Volume of stationary phase Vm: Volume of mobile phase Cs: Solute concentration in the stationary phase Cm: Solute concentration in the mobile phase

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Small values of k’ show that the compound is poorly retained and elutes near the void volume. Large k’ values imply a good separation but downfalls of this are longer analysis times with peak broadening and decreases in sensitivity. Ideal separations occur with a capacity factor of between 1 and 5.

3.2.2 Resolution

The aim of chromatography is to separate components in a mixture into bands or peaks as they migrate through the column. Resolution, R, provides a quantitative measure of the ability of a column to separate two analytes. This measurement is obtained by the retention times and peakwidths which are easily obtained directly from the chromatogram.

R=

tms 2 − tms1 2Δt = w1 + w2 w1 + w2 2

tms1 and tms 2 : Gross retention times for peaks 1 and 2 respectively w1 and w2: Peak widths along the baseline of peak 1 and 2 respectively

For two peaks to be recognized as separate the resolution should be at least 0.5. Two peaks are seen as completely separate if R is greater than 1.5. The resolution can be improved by lengthening the column but this will also increase the analysis time.

3.2.3 Column Efficiency

A chromatographic column is divided into N theoretical plates. A thermodynamic equilibrium of the analytes between the mobile and stationary phase occurs within each plate. The efficiency of the column is thus expressed as the number of theoretical plates.

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N=

L H

L: Length of column packing (cm) H: Plate height

N is determined experimentally from a chromatogram using the equation:

⎛t ⎞ N = 16⎜ ms ⎟ ⎝W ⎠

2

Poor column efficiency results in band broadening.

3.2.4 Column Selectivity Column selectivity, α, is a measure of the relative separation of two peaks and is defined as the ratio of the net retention times of the two peaks.

α=

tms 2 − tm tms1 − tm

3.2.5 Distribution or Partition Coefficient

The distribution coefficient, Kd, indicates the distribution of analytes between the resin and the eluent. It is defined as the ratio of the molar concentrations of the solute in the stationary and mobile phase.

Kd =

mass of compound on resin ( g ) × volume of resin ( ml ) C s = mass of dry resin ( g ) × volume of eluent ( ml ) Cm

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Cs and Cm: Molar concentrations of the solute in the stationary and mobile phases respectively 3.2.6 Factors Affecting Band Broadening74;75 Various factors affect the peak variance, σ2, and thus the bandwidth. It is important to consider these processes in the correct design of any chromatographic system so that the variance and thus peak width can be minimised and the efficiency can be maximised. The extent of band broadening can be expressed in terms of plate height as follows:

H = A+

B + Cstationary u + Cmobile u u

a. Eddy Diffusion

The A term in the above equation is called eddy diffusion. This term describes the multitude of pathways that the solute molecules can follow in a packed column. Each of the paths is a different length and molecules will pass through at various rates thus leading to band broadening. Eddy diffusion can be minimised with a column that is uniformly packed with particles of constant size. Band broadening is independent of the flow rate of the eluent.

b. Longitudinal Diffusion

Molecules in a sample band tend to diffuse out of this band during it passage through the chromatographic column. This process is known as longitudinal diffusion and is the B term in the above equation. Longitudinal diffusion occurs in both the direction of flow of the mobile phase as well as in the opposite direction. Longitudinal diffusion increases at low eluent flow rates, as diffusion is a time dependant process.

c. Mass Transfer

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The C term is due to mass transfer, which is the interchange of solute molecules between the mobile and stationary phases. In an ideal chromatographic system this process would occur instantaneously but this is not the case in practice. Hence different molecules spend varying amounts of time in the stationary and mobile phases, which leads to band broadening. Band broadening increases as the eluent flow rate increases although it can be minimised by using a stationary phase with a small diameter or one, which has an active layer that is confined to the outer surface of the particle.

3.3 TYPES OF LIQUID CHROMATOGRAPHY72;73 Chromatography can be divided into three subsections namely gas, gel and liquid chromatography (Figure 3.2). Gas chromatography is used for the analysis of volatile samples, gel chromatography for non-volatile samples with a molecular weight larger than 2000 and liquid chromatography for nonvolatile samples with a molecular weight smaller than 2000.

CHROMATOGRAPHY

Gas Chromatography Gas-Solid

Gas-Liquid

Gel Chromatography Filtration

Liquid Chromatography

Permeation

Ion-Interaction

Ion-Exclusion

Ion-Exchange

Ion Chrom.

Adsorption Chrom.

Partition Chrom.

Paper Chrom.

Figure 3.2: Types of Chromatography

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Reverse-Phase

TLC

HPLC

Some of the different types of chromatography are discussed below:

3.3.1 Adsorption Chromatography

Adsorption chromatography is used for the separation of non-polar or fairly polar organic molecules. In this technique the stationary phase is the surface of a finely divided polar solid and the analyte competes with the mobile phase for sites on the surface of the packing. Retention of the analyte occurs as a result of adsorption forces. Finely divided silica and alumina are used as stationary phases with organic solvents such as hexane acting as the mobile phase. The only variable that can be altered to affect the partition coefficient of the analytes is the composition of the mobile phase. A particular advantage of adsorption chromatography is its ability to resolve isomeric mixtures.

3.3.2 Liquid - Liquid Partition Chromatography

In liquid – liquid partition chromatography an inert support is coated with a polymeric layer or with a liquid that is insoluble in the mobile phase. This separation is based on the relative solubility’s of the solutes in the mobile and stationary phases.

There are two types of partition chromatography namely normal-phase and reverse phase chromatography. Normal-phase chromatography makes use of highly polar stationary phases such as hexane for the mobile phase. Here the least polar component is eluted first where an increase in the polarity of the mobile

phase

will

decrease

the

retention

times.

Reverse-phase

chromatography is used to separate highly polar analytes, which give problems of long retention times and peak tailing with conventional absorption chromatography. In this case a nonpolar stationary phase such as a hydrocarbon is used in conjunction with a relatively polar mobile phase.

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3.3.3 Exclusion or Gel Permeation Chromatography

This technique separates analytes according to their molecular size and shape. Resins for exclusion chromatography include silica or polymer particles, which contain a network of uniform pores into which the solute and solvent molecules diffuse. As a sample moves through the column the analytes are separated as the lower molecular weight species are held back due to permeation of the particle pore whereas the higher molecular weight species are larger than the average size of the pore and are excluded. Thus the larger species move through the column faster. Exclusion chromatography differs from conventional chromatography, as there are no chemical or physical interactions between the analytes and the stationary phase.

3.3.4 Ion Exchange Chromatography

Ion exchange chromatography makes use of a resin containing a bound quaternary ammonium group for the separation of anions and a bound sulphonic acid group for the separation of cationic species. Elution is carried out with a mobile phase that contains ions, which compete with the analyte ions for the charged groups on the surface of the stationary phase. Analyte separation occurs as a result of differences in effective charge, solvated ionic radius and complex formation.

In this technique the sample migrates through the column under the influence of gravity, individual fractions are then collected and analysed.

3.3.5 Ion Chromatography

Ion chromatography is a result of the pioneering of Small, Stevens and Baumann and is the trade name for the Dionex system, which was designed for the separation of ionic species. This process consists of the separation of cations or anions by eluents on cation- or anion-exchange columns.

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In this technique the analyte ions are separated on a low capacity ion exchange column, followed by a micromembrane suppressor, which removes the ionic background to facilitate the conductometric detection of the analyte ions. The stationary phases used in these columns are discussed later in section 3.5.1. 3.3.6 Ion Interaction Chromatography74

Hydrophilic ionic solutes are not efficiently retained on lipophilic stationary phases when typical reversed-phase eluents are used. Ionic solutes can however be separated by the addition of a lipophilic reagent ion with the opposite charge to the eluent. This process is known as ion-interaction chromatography. A mechanism known as the ion-interaction model has been suggested for ion-interaction chromatography. This model is an intermediate between electrostatic and adsorptive effects. The lipophilic ion interaction reagent (IIR) is said to form a dynamic equilibrium between the eluent and stationary phases such that it forms an electric double layer at the stationary phase surface. The adsorbed IIR ions form a primary layer of charge to which a secondary layer of counter ions of the IRR is formed. A solute with an opposite charge to the IRR competes for a position in the secondary charged layer and subsequently moves into the primary layer. This causes a decrease in the total charge of the layer thus another IIIR ion enters the primary layer to maintain charge balance.

3.4 HPLC INSTRUMENTATION72;73;75 The main components of an HPLC system are a high-pressure pump, a column and an injector system as well as a detector (Figure 3.3). The system works as follows: eluent is filtered and pumped through a chromatographic column, the sample is loaded and injected onto the column and the effluent is monitored using a detector and recorded as peaks.

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Figure 3.3: HPLC Instrumentation

3.4.1 Analytical Pumps (Solvent Delivery System)

The requirements for HPLC pumps are as follows: they must be able to generate high pressures, have a pulse-free output, deliver flow rates ranging from 0.1 to 10 ml/min, have flow reproducibility’s of 0.5 % relative or better and they must be resistant to corrosion by a variety of solvents. Various types of pumping systems exist. These include:

a. Direct Gas-pressure Systems

This system consists of a cylinder gas pressure, which is applied directly to the eluent in a holding coil. Advantages of this pump are that it is reliable and economical although solvent changing is found to be tedious.

b. Syringe-type Pumps

In these pumps an electrically driven lead-screw moves a piston, which is able to pressurise a finite volume of solvent, and thus delivers a pulseless constant flow of solvent to the system (Figure 3.4). These pumps are found to be

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reliable although they are expensive, solvent changing is tedious and they have a finite capacity (~250 ml).

Figure 3.4: Syringe-type pump75

c. Pneumatic Intensifier (Constant Pressure) Pumps

Pneumatic intensifier pumps are operated via gas pressure. A large area piston drives a small area piston when acted on by pressure from a gas line. The gas pressure is thus amplified in the ratio of the areas of the forces of the pistons and a high-pressure liquid at constant pressure is introduced into the system (Figure3.5). If a partial blockage occurs in this system a drop in flow rate occurs but the pressure remains constant. The flow sensitivity of the detector cell will determine how much pulse damping is required in the system to suppress the detector signal caused when the flow stops during the return stroke.

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Figure 3.5: Constant-pressure pump75

d. Reciprocating (Constant Flow) Pumps

A reciprocating pump is the most generally used, as it is economical and allows a wide range of flow rates. With this pump there is no limit on the reservoir size or operating time as is commonly found with other pumps. This type of pump is electrically driven by a motor, which moves back and forth within a hydraulic chamber. On the backward stroke the piston sucks in eluent from the reservoir and due to check valves the outlet to the separation column is closed. During the forward stroke the eluent is pushed onto the column and the inlet from the reservoir is closed (Figure 3.6). The pumping motion of the piston produces a pulsed flow that requires dampening. These pumps include a high output pressure with constant flow rates and the ability to be used for gradient elution.

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Figure 3.6: Reciprocating pump75

3.4.2 Sample Introduction

The ideal method for sample introduction should enable the sample to be injected as a narrow plug onto the column so that peak broadening is negligible. The injection system should contain no void volume, as this would cause a loss of resolution.

Syringe injection through an elastomeric septum is often used although it is not very reproducible and is constricted to low pressures.

The most widely used methods are those based on sampling valves and loops. Here the sample loop is filled with sample by means of a syringe. A rotation of the valve rotor causes the eluent stream to pass through the sample loop thus injecting the sample onto the column without a noticeable change in flow (Figure 3.7). These valves have interchangeable loops and reproducibility is a few tenths of a percent relative.

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Figure 3.7: Flow paths of the load and inject positions of an injection valve76

In stopped-flow injection, the eluent flow is stopped and the sample is injected directly onto the head of the column by means of a syringe. The pump is then switched on again.

3.4.3 Separation Columns

Heavy-wall glass, stainless steel and plastic are among materials that can withstand high pressures and are thus used to construct HPLC columns. They must also be able to resist the chemical action of the mobile phase. Wall irregularities will cause a well-packed column to channel near the wall or packing interface thus the tubing must have a smooth, precision bore internal diameter. Channels would cause peak broadening and a decrease in efficiency.

Column connections are made with low dead-volume fittings, which prevent stagnant pockets of eluent.

Usually a short guard column is placed in front of the analytical column. This serves to increase the life of the analytical column by removing particulate matter and contaminants from the solvents.

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3.5 TYPES OF STATIONARY PHASES Various stationary phases are available for HPLC and are discussed below. 3.5.1 Polystyrene/Divinylbenzene – Based Resins71

In ion chromatography, the support material is a polystyrene/divinylbenzene (PS/DVB) based resin that is relatively stable with respect to pH. The copolymerisation of PS with DVB is used to give the resin mechanical stability. The amount of DVB in the resin is denoted as “percent crosslinking”. The percentage of crosslinking is directly related to the extent to which PS/DVB resin shrinks or swells in an aqueous media or in the presence of organic solvents. If the resin shrinks a loss in column efficiency occurs as a dead volume occurs at the beginning of the column. Swelling of the resin leads to higher column backpressures. The optimum degree of crosslinking is said to be 2-5%.

a. Anion - Exchange Resin

The anion-exchange resins used by Dionex are composed of a surface sulphonated PS/DVB core (10-25 μm) and a totally porous latex particle (0.1 μm), which is completely aminated (Figure 3.8). Electrostatic and van der Waals interactions are used to agglomerate the latex particles onto the core particles. It is the latex particles that carry the actual ion exchange function.

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Latex Particle

Surface Sulphonated Substrate

Figure 3.8: The structure of a latex anion exchange particle71

The length of the diffusion path as well as the rate of diffusion is determined by the particle size of the latex material.

Advantages of this stationary phase include mechanical stability of the resin due to the inner core, which also ensures moderate backpressures. Rapid exchange processes and thus high efficiencies occur due to the small, totally porous, latex particles. Surface sulphonation greatly reduces swelling and shrinking of the material. The selectivity of the resin can be varied by making use of various quaternary ammonium bases.

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b. Cation – Exchange Resins

The stationary phase of a cation exchange column is based on inert, surface sulphonated, crosslinked polystyrene (Figure 3.9).

Figure 3.9: The structure of a latex cation exchange particle71

The exchange process for a cation, M+, occurs as follows:

~SO3-H+

+

M+A-

~SO3-M+

+

H+A-

3.5.2 Silica – Based Resins74

Silica-based resins make up one of the most important classes of ionexchangers used in chromatography. There are two main groups of silicabased materials namely polymer-coated and functionalised silica materials. Polymer-coated materials consist of silica particles, which are coated, with a layer of polymer such as polystyrene, silicone or fluorocarbon and then derivitised to introduce functional groups. The advantage of polymer-coated materials is that diffusion in the thin layer of the polymer occurs more rapidly than it would in totally polymeric particles. Functionalised silica materials comprise a functional group, which is chemically bonded directly to a silica particle. The silica particles used in both groups can be either pellicular or microparticulate. A disadvantage of silica-based resins is that they can only be operated over a limited pH range. At a pH below 2 the covalent bond linking the ion-exchange functionality to the silica substrate becomes unstable. At high pH values dissolution of the silica matrix itself occurs.

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3.5.3 Chelating Resins74

Chelating resins, which are able to separate metal ions, are made up of a suitable ligand immobilized onto a stationary phase. Many chelating resins have been synthesised using styrene-divinylbenzene polymers or silica as the support material. The ligands are chemically bound to the stationary phase by an appropriate reaction. Solute retention is altered by manipulating the eluent pH or by adding a competing ligand to the eluent. The success of using chelating resins is dependant on the rate at which the metal-ligand complex is formed and dissociated. Broad peaks are characteristic of slow formation and dissociation rates.

3.6 Detection Methods71;72;74 There is no one highly sensitive, universal detector system used for HPLC. The system used is thus based on the requirements which need to be met such as detection limits, expense etc. A summary of detection methods, which are used with HPLC separation, can be seen in Figure 3.10 and some methods are discussed below.

Detection Methods ELECTROCHEMICAL Conductivity

SPECTROSCOPIC

Amperometry Coulometry

Molecular Spectroscopic Techniques

Potentiometry

UV/VIS Absorption

Fluorescence

Figure 3.10: Summary of Detection Methods for HPLC

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Refractive Index

Atomic Spectroscopic Techniques Atomic Absorption Spectrometry

Atomic Emission Spectrometry

HPLC

3.6.1 Electrochemical Detection Methods a. Conductivity Detection

Conductivity is frequently used for detection purposes as all ions are electrically conducting thus conductivity detection should be universal in response. Conductivity detectors are also relatively simple to construct and operate, thus they find wide applicability. Conductivity detection is based on conductance of an eluent prior to and during elution of an analyte. The detector response equation for an anion-exchange system is

ΔG =

(λ

s−

− λε − )Cs

10 − 3 K

ΔG: Conductance signal

λs and λε : Limiting equivalent ionic conductances of the analyte and eluent −

−

anions respectively

Cs: Concentration of the analyte anion K: Cell constant

The above equation shows that when conductivity detection is used to monitor the effluent from an anion-exchange column the observed signal for an eluted solute is proportional to the solute concentration as well as the difference in the limiting equivalent ionic conductances between the eluent and solute ions. A similar equation can be derived for the conductimetric detection of a cationexchange separation. It can be seen from the above equation that sensitive detection is possible as long as there is a considerable difference in the limiting equivalent ionic conductances of the solute and eluent ions. The resulting difference can be positive or negative depending on whether the eluent ion is weakly or strongly conducting resulting in direct or indirect detection respectively.

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b. Amperometric Detection

Amperometric detection is used for ions, which have a pK value of above 7 and thus cannot be detected by conductivity as the products formed are only slightly dissociated.

Amperometric detectors usually consist of a three-electrode measuring cell, which contains a working electrode, a reference electrode and a counter electrode. The potential required for oxidation, or reduction of the species being analysed is applied between the working electrode and the Ag/AgCl reference electrode. A “glassy carbon” electrode acts as the counter electrode and functions to preserve the potential during operation as well as to prevent the destruction of the reference electrode. The detector functions as follows when an electrochemically active substance flows through the measuring cell it is partially oxidised or reduced. This produces an anodic or cathodic current, which is proportional to the concentration of the analyte. This signal is subsequently converted into a chromatographic peak.

c. Potentiometric Detection

Potentiometry is the process by which potential changes at an indicator electrode are measured with respect to a reference electrode at a constant current. Potentiometry enables ion concentration determinations as the potential of the indicator electrode varies with the concentration of the ions in solution that come into contact with the electrode. Potentiometric detection has found wide applicability in aqueous solutions of which ion-selective electrodes are the most generally used. Potentiometry coupled with ion chromatography is however limited as it has moderate sensitivity as well as slow response and poor baseline stability on flowing solutions.

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3.6.2. Spectroscopic Detection Methods a. Molecular Spectroscopic Techniques i. UV Detectors UV detectors measure the change in the UV absorption as the solute passes through a flow cell. In a UV transparent solvent UV detectors are concentration sensitive. Direct detection has a flaw as not all inorganic ions have appropriate chromophores but this can be compensated for by using the method of derivitisation. This is done by mixing the effluent with a chromogenic reagent in a post column reactor. The formed chelate complex subsequently absorbs at a particular wavelength. ii. Refractive Index Detectors The refractive index of a medium is the ratio of the speed of light in a vacuum to the speed in the medium. These detectors measure the change in refractive index in the eluent as the solute passes through the sample cell. This method of detection is less sensitive than UV detection although non-chromatographic compounds can be measured directly without derivitisation. iii. Fluorometric Detection In this detection system the solute is excited by UV radiation at a particular wavelength and the emission wavelength is detected. Fluorometric detection has been used with naturally fluorescent compounds but compounds can be reacted to produce fluorescent derivatives. b. Atomic Spectroscopic Techniques Atomic spectroscopy includes atomic absorption spectroscopy as well as atomic emission spectroscopy (which will be discussed in more detail in Chapter 4). The spectroscopic determination of atomic species can only be performed on a gaseous medium in which the individual atoms are well separated from one another. Thus the first step in atomic spectroscopic

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techniques is atomization, a process in which the sample is volatilised in such a manner as to produce an atomic gas.

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