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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HPLC

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.

m

s

m

mms

sm

ms

m

sd

tt

ttt

VCVC

VVKk =

−=

××

=×

='

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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HPLC

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.

2121

2

2

12

wwt

wwtt

R msms

+Δ

=+−

=

21 msms tandt : 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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HPLC

HLN =

L: Length of column packing (cm)

H: Plate height

N is determined experimentally from a chromatogram using the equation:

2

16 ⎟⎠⎞

⎜⎝⎛=

WtN ms

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.

mms

mms

tttt

−

−=

1

2α

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.

m

sd C

Cmleluentofvolumegsinredryofmass

mlsinreofvolumegsinreoncompoundofmassK =×

×=

)()()()(

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HPLC

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:

uCBAH mobilestationary Cuu

+++=

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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HPLC

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 non-

volatile samples with a molecular weight smaller than 2000.

Gas-Solid Gas-Liquid

GasChromatography

Filtration Permeation

GelChromatography

Ion-Interaction Ion-Exclusion

Ion-Exchange Ion Chrom.

Paper Chrom. Reverse-Phase

Adsorption Chrom.

TLC

Partition Chrom.

LiquidChromatography

CHROMATOGRAPHY

Figure 3.2: Types of Chromatography

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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.

39

HPLC

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.

40

HPLC

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.

41

HPLC

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

42

HPLC

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.

43

HPLC

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.

44

HPLC

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.

45

HPLC

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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HPLC

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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HPLC

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.

48

HPLC

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 ion-

exchangers used in chromatography. There are two main groups of silica-

based 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.

49

HPLC

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.

Conductivity AmperometryCoulometry

Potentiometry

ELECTROCHEMICAL

UV/VISAbsorption

RefractiveIndex

Fluorescence

Molecular SpectroscopicTechniques

AtomicAbsorption

Spectrometry

AtomicEmission

Spectrometry

Atomic SpectroscopicTechniques

SPECTROSCOPIC

Detection Methods

Figure 3.10: Summary of Detection Methods for HPLC

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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

( )K

CG ss

310−

−− −=Δ ε

λλ

ΔG: Conductance signal

−− ελλ and

s : 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 cation-

exchange 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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HPLC

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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HPLC

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

53

HPLC

techniques is atomization, a process in which the sample is volatilised in such

a manner as to produce an atomic gas.

54