The Theory of HPLC

Introduction

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Aims and Objectives

Aims and Objectives

Aims

To give a brief History of Liquid Chromatography

To compare and contrast High Performance Liquid Chromatography (HPLC) with
Gas Chromatography (GC)
To introduce the Liquid Chromatography (LC) and explain the function of each
component
To introduce the Chromatogram and explain the information it gives

Objectives

At the end of this Section you should be able to:

To outline the main separation mechanisms used in HPLC

To explain the fundamental principles of separation
To highlight the different modes of chromatography and explain their uses and
applications
Content

Origins of Liquid Chromatography 3

Why Chose Liquid Chromatography 4
Suitable Samples for HPLC 5
Comparison with Gas Chromatography 6
Typical HPLC Data 7
Chromatographic Separation Mechanisms 8
The Liquid Chromatograph 9
The Liquid Chromatographic Process 10
The Chromatogram 11
Modes of Chromatography 12
Glossary 13

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Origins of Liquid Chromatography

The Russian botanist Mikhail Tswett first used the term Chromatography (Latin for
coloured drawing) in 1906, to describe the separation that occurred when solutions of
plant pigments were passed through columns of calcium carbonate or alumina, using
petroleum ether.

Russian Botanist
Mikhail Tswett (1872-1919)

From Tswetts notebook (1910) on early

chromatographic experiments

As the mixture seeped downwards, each pigment showed a different degree of readiness
to attach itself to the absorbent, and in this way the pigments became separated as
different-coloured layers in the tube. Tswett called the new technique chromatography
because the result of the analysis was literally 'written in colour' along the length of the
adsorbent column. Eventually he found six different pigments.

Just as Tswett had discovered, modern Liquid Chromatography (LC), uses a liquid mobile
phase to transport the sample components through a column packed with a solid material
-the stationary phase.

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Why Chose Liquid Chromatography

The two main chromatographic techniques used in modern analytical chemistry are Gas
Chromatography (GC) and High Performance Liquid Chromatography (HPLC).

Gas Chromatograph
High Performance
Liquid
HPLC uses a liquid mobile phase to transport the analytes (sample)Chromatograph
through the column,
which is packed with a stationary phase material.

In contrast, Gas Chromatography uses a gaseous mobile phase to transport sample

components through either packed columns or hollow capillary columns. In most cases,
GC columns have smaller internal diameter and are longer than HPLC columns.

Typical HPLC Column

15cm x 4.6mm x 5m

Typical GC Column
30m x 0.25mm i.d.

GC has developed into a sophisticated technique since the pioneering work of Martin and
James in 1952, and is capable of separating some very complex mixtures. However, due
to limitations of volatility and thermal stability, it is only capable of separating around 23%
of known substances.

So under what circumstances would we chose HPLC to separate our sample

components?

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Suitable Samples for HPLC

The following information gives an indication of the type of sample (analyte) commonly
analysed by both HPLC and GC and relative strengths and limitations of each technique.

GC
z Samples analysed by GC must be volatile (significant vapour pressure below 250oC)
z Derivatisation to increase analyte volatility is possible but cumbersome and
introduces possible quantitative errors
z Most GC analytes are under 500 Da Molecular Weight for volatility purposes

GC is amenable to smaller more volatile analytes

such as Propanone (Acetone) (58 Da).

Regions in red on the ball and stick representation

show negative polar functional groups. The
topographical representation shows red as negative
charge and blue as positive charge.
Propanone (acetone) (58 Da)

HPLC

HPLC analysis has no volatility issues,

however the analyte must be soluble in
the mobile phase
HPLC can analyse samples over a wide
polarity range and is able to analyse
ionic samples. Mobile phase
components are selected to ensure
sample solubility
HPLC has no real upper molecular
weight limit and large proteins of many
thousands of Daltons may be analysed.
Solubility in the mobile phase may
preclude the analysis of very large
molecules
Adenosine Triphosphate (507 Da)

Important:

Large proteins and peptides can be analysed by HPLC!!!

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Comparison with Gas Chromatography

GC
z Temperatures in GC can exceed 350oC and samples that are thermally unstable
(labile) may decompose
z Many GC detectors such as the Flame Ionisation Detector (FID) are destructive and
the analyte does not survive analysis in-tact and therefore cannot be recovered
z GC samples are prepared in organic solvents and extraction of analytes from aqueous
samples will be necessary
z Sample size is usually between 1 and 5L with typical detector sensitivity between
nanograms (ng) and picograms (pg) on column

Operates at high temperatures and is

destructive toward the analyte.

The Flame Ionisation Detector

HPLC
z HPLC is usually carried out at (or around) room temperature and most HPLC
detectors apart from the Mass Spectrometer are non-destructive
z HPLC samples are prepared in a solvent system that has the same or less organic
solvent than the mobile phase and volumes of 1 to 50 L are common (1-10g of
analyte per 1g packing material)

HPLC UltraViolet (UV) Detectors operate at room temperature, are non-destructive (the
analyte may be recovered) and typical sensitivity is in the order of nanograms.

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Typical HPLC Data

HPLC - Even though the Flame ionisation detector may be more universal and sensitive,
the UV detector is non-destructive, relatively sensitive (nanograms of analyte on column),
and also has the capability of producing spectra associated with sample components. This
can aid qualitative analysis and assist with identification of sample components.

Typical Mass Spectrum from GC-MS Equipment

HPLC and GC - can both use Mass Spectrometers (MS) as detection systems to assist
with analyte identification although MS detectors are destructive. HPLC-MS is a less
mature technique and there are no spectral libraries available for compound identification
as there are with GC instruments. However, HPLC-MS (LC-MS) is a burgeoning
technique that can assist in the characterisation of sample components in a wide variety
of application types.

Summary - If the sample cannot be analysed by Gas Chromatography without lengthy

sample preparation (indicating issues with volatility), then HPLC should be the technique
of choice. HPLC is the best choice for higher molecular weight analytes and analytes
which may potentially degrade when heated.

Mass Spectrometers: produce spectra that can assist with the identification
(qualitative analysis) of sample components for both GC and LC

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Chromatographic Separation Mechanisms

HPLC separations involve both the mobile phase (a liquid) and the stationary phase
(usually materials of varying hydrophobicity chemically bonded to a solid support). In
contrast, GC uses the mobile phase only to carry the analyte through the column.

As an illustration - the amount of water in an HPLC mobile phase will determine how
strongly a hydrophobic analyte is repelled into the stationary phase and how well
retained it is. The chemical nature of the stationary phase will also govern how strongly
the analyte is retained. For this reason, HPLC is a technique that is driven by the
selectivity achieved using two interacting phases.

Typical Liquid Chromatographic Separation Mechanism

In HPLC the analyte is able to interact with the mobile and stationary phases brining a
high degree of SELECTIVITY.

In contrast, analytes in a capillary GC column will only be retained due to their interaction
with the stationary phase (usually an immobilized liquid of varying hydrophobicity) coated
onto the inner walls of the GC column. There are less options for improving selectivity in
GC, however, as it is very highly efficient this is often enough to achieve the desired
separation.

Typical Gas Chromatographic Separation Mechanism

In GC the analyte interacts with the stationary phase only. The technique shows less
selectivity but is highly efficient.

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The Liquid Chromatograph

In HPLC, several instrument and column chemistry parameters need to be optimised in

order to generate a satisfactory separation.

Each of the following items needs to be optimised in order to generate a chromatogram

that is suitable for qualitative or quantitative purposes.

z Mobile Phase Composition

z Bonded Phase Chemistry
z Column and Packing Dimension
z Injection Volume
z Sample Pre-treatment and concentration
z Mobile phase Flow Rate
z Column Temperature
z Detector Parameters

A typical Liquid Chromatograph

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Where:

1: The mobile phase composition (usually water and an organic solvent plus other
additives) needs to be optimised in order to gain good separation.
2: Degassers are often used to remove air from the mobile phase leading to better
chromatographic baselines.
3: The detector conditions are chosen to give the best response to the analytes of interest
and achieving good sensitivity.
4: The column dimensions and stationary phase chemistry are chosen and optimised to
give separations of the quality required.
5: The autosampler introduces a plug of sample into the mobile phase flow which is then
swept onto the column.
6: Dual reciprocating pumps are used to deliver the mobile phase at back pressures of up
to 400Bar. As steady stream of liquid delivered at constant flow rate is important.

The Liquid Chromatographic Process

The mobile phase is continuously pumped at a fixed flow rate through the system and
mixed (if required) by the pump. The injector is used to introduce a plug of sample into the
mobile phase without having to stop the mobile phase flow, and without introducing air
into the system.

The mixture of components is carried in a narrow band to the top of the column. Some
compounds in the sample mixture will have a greater preference for the stationary phase
than the mobile phase and will be retained in the column longer. Those components that
are not retained as strongly are carried by the mobile phase down the column. The longer
the column, the more opportunities for interaction with the stationary phase and the
greater the separation.

Liquid Chromatographic Process

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The detector is then used to respond to a physico-chemical property of the analyte
(perhaps a UV Chromophore consisting of a set of conjugated double bonds or an
aromatic group). This response is digitally amplified and sent to a data system where it is
recorded as the chromatogram.

The Chromatogram

As the components elute from the column they pass into a detector where some
physico-chemical property of the analyte produces a response from the detector. This
response is amplified and plotted against time giving rise to a chromatogram.

The High Performance Liquid Chromatogram

Components (such as the injection solvent) that are not retained within the column elute
at the dead time or hold up time t0. There are various ways of measuring this parameter
using unretained compounds such as Uracil. Some workers use the first baseline
disturbance caused by the passage of the sample solvent to estimate the value of t0.

Those compounds (analytes and sample components) that are retained, elute as
approximately Gaussian shaped peaks later in the chromatogram. Retention times
provide the qualitative aspect of the chromatogram and the retention time of a compound
should be the same under identical chromatographic conditions. The chromatographic
peak height or peak area is related to the quantity of analyte. For determination of the
actual amount of the compound, the area or height is compared against standards of
known concentration.

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Modes of Chromatography

The mode of chromatography is usually defined by a combination of a certain stationary

phase type with a certain mobile phase type.

For example, Normal Phase chromatography has a non-polar mobile phase with a more
polar stationary phase. Reverse phase HPLC (as the name implies) uses a system in
which the mobile phase is more polar that the stationary phase.

The main modes of chromatography and their uses are described in the table.

Table 1. The Main Modes of Liquid Chromatography

Types of compounds Mode Stationary phase Mobile phase
Neutrals Reverse phase C18, C8, C4, Water/Organic Modfiers
Weak acids cyano, amino
Weak bases
Ionics, Bases, Acids Ion Pair C18, C8 Water/Organic
Ion-Pair reagent
Compounds not Normal Phase Silica, Amino, Organics
soluble in water Cyano, Diol
Ionics/Inorganic ions Ion exchange Anion or Cation Aqueous/Buffer
Exchange Resin Counter Ion
High Molecular Weight Size Exclusion Polystyrene Silica Gel Filtration Aqueous
Compounds, Polymers Gel Permeation Organic

The ability of HPLC to separate such a wide diversity of species leads to its popularity in
a wide range of industries. It is important to note that HPLC is capable of separating
analytes in the following categories:

z High MWt (>2000)

z Lower MWt (<2000)
z Polar
z Non-polar
z Ionisable
z Cationic
z Anionic

Application Areas of HPLC

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Glossary

Analyte - The compound of interest to be analyzed by injection into and elution from an
HPLC or GC column.

Vapour pressure - the absolute pressure at which the vapour contained in a substance is
at equilibrium with its liquid or solid phase

Derivatisation - to chemically alter an analyte to impart favourable chromatographic

behaviour (e.g. increase volatility)

Quantitative - chemical analysis undertaken to determine (measure) the quantity or

amount of each analyte within a mixture

Polarity - the greater the difference in electron affinity (electronegativity) between atoms
in a covalent bond, the more polar the bond. Partial negative charges are found on the
most electronegative atoms, the others are partially positive. The molecular electrostatic
potential is the potential energy of a hydrogen ion at a particular location near a molecule.

Negative electrostatic potential corresponds to: partial negative charges.

Positive electrostatic potential corresponds to: partial positive charges.

Ethanoic (acetic) acid Ethanamide

Nanogram 1 x 10-9 g

Femtogram 1 x 10-12 g

Organic Solvent - A solvent is a substance that dissolves another substance or

substances to form a solution (a homogeneous mixture). Liquid solutions that do not have
water as a solvent are called non-aqueous solutions. A broad and common class of non-
aqueous solvents is called organic solvents. In the context of chemistry and materials,
organic refers to materials based on carbon (an element abbreviated as C). Additional
elements that are commonly found in organic materials are hydrogen (H), nitrogen (N),
oxygen (O), phosphorus (P) and sulfur (S).

Qualitative involving distinctions based on qualities; "qualitative analysis attempts to

determine the chemical identity of constituents of a substance or mixture"

MassSpectrometers a mass spectrometer is an instrument that measures the masses

of individual molecules that have been converted into ions, i.e., molecules that have been
electrically charged

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Mobile Phase A fluid that percolates through or along the stationary bed, in a definite
direction. It may be a liquid (Liquid Chromatography) or a gas (Gas Chromatography). In
gas chromatography the expression, Carrier Gas may be used for the mobile phase. In
elution (liquid) chromatography the expression Eluent is also used for the mobile phase.

Stationary Phase The stationary phase is one of the two phases forming a
chromatographic system. It may be a solid, a gel or a liquid. If a liquid, it may be
distributed on a solid. This solid may or may not contribute to the separation process. The
liquid may also be chemically bonded to the solid (Bonded Phase) The expression
Chromatographic Bed or Sorbent may be used as a general term to denote any of the
different forms in which the stationary phase is used. A stationary phase that is covalently
bonded to the support particles or to the inside wall of the column tubing is known as a
Bonded Phase.

Hydrophobic Meaning " water fearing". Hydrophobic compounds do not dissolve easily
in water, and are usually non-polar. Oils and other long hydrocarbons are hydrophobic

Selectivity also called the Separation Factor (), described the separating power of a
chromatographic system (By definition, the value of the separation factor is always greater
than one). The relative retention value calculated for two adjacent peaks (VR2' > VR1'):

= tR2'/tR1'

Efficiency also called Plate Number (N), describes the broadening of the
chromatographic band by using the chromatographic peak width. Plate number is
indicative of column performance, calculated from the following equations which depend
on the selection of the peak width expression (see Peak Width):

N = 16(tR /wb)2

TR = peak apex retention time, wb = width of the beak at the base measured using
tangents to the peak sides.

Elute To chromatograph by elution chromatography. The process of elution may be

stopped while all the sample components are still on the chromatographic bed or
continued until the components have left the chromatographic bed.

t0 (tM) The volume of the mobile phase (or the corresponding time) required to elute a
component the concentration of which in the stationary phase is negligible compared to
that in the mobile phase. In other words, this component is not retained at all by the
stationary phase. Thus, the hold-up volume (time) is equal to the Retention Volume (Time)
of an Unretained Compound. The hold-up volume (time) includes any volumes contributed
by the sample injector, the detector, and connectors.

tM = VM / Fc

Uracil a compound often used to determine the hold up time (t0) of an

analytical column (system). Whilst Uracil was not retained on traditional
HPLC phases, some modern phases are capable of retaining the
compound. Care should therefore be taken when using Uracil to ensure
that the t0 value is correct.

Gaussian following the shape of a normal distribution as described by Gauss used

to describe a symmetrical bell shaped peak.

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