HPLC – Back to Basics

Colin Stone
Clinical Biochemistry Dept.
Kings College Hospital NHS Trust
Definitions
 Chromatography is a separation process that is
achieved by distributing the components of a
mixture between two phases, a stationary phase
and a mobile phase. Those components held
preferentially in the stationary phase are
retained longer in the system than those that
are distributed selectively in the mobile phase.
As a consequence, solutes are eluted from the
system as local concentrations in the mobile
phase in the order of their increasing distribution
coefficients with respect to the stationary phase;
therefore, a separation is achieved.
History
 The first scientist to recognize chromatography
as an efficient method of separation was the
Russian botanist Tswett, who used a simple form
of liquid-solid chromatography to separate a
number of plant pigments.

 Early 1940’s - Martin and Synge introduced

liquid-liquid chromatography to separate acetyl
amino acids
History
 Nearly four decades before high efficiency liquid
chromatography columns became a reality.

 By the mid 1960s the development of all aspects

of chromatography were virtually complete.

 Today, HPLC is an extremely versatile technique

with many applications & systems available.
Definitions

 Chromatography works by exploiting

differences in molecular characteristics eg.
structure, size, charge, hydrophobicity

 Utilising the diversity of molecules and by

changing HPLC operating conditions, a
wide variety of separations, even of very
similar substances, can be carried out.
Terminology
 HPLC first described as:
High Pressure Liquid Chromatograpy

 When marketed, known as:

High Performance Liquid Chromatography

 Known now by most users as:

High Price Liquid Chromatography
Types of HPLC

 Normal phase
 Reversed phase
 Ion exchange
 Affinity
 Size exclusion
Normal Phase HPLC
 Adsorption of analytes on the polar, weakly
acidic surface of silica gel (eg. TLC)

 Stationary Phase.: Silica (pH 2-8), Alumina (pH

2 - 12), Bonded Diol, and NH2

 Mobile Phase: Non-polar solvents (Hexane,

CHCl3)

 Applications: Non-polar and semi-polar samples

Reverse Phase HPLC

 Polar mobile phase & non-polar stationary

phase
 Highly polished uniform sized spheres of
silica particles with pores
Reverse Phase HPLC
 Partition of analytes between mobile phase and
stationary phase - adsorption on the surface of
bonded phase and inside pore spaces
 Stationary Phase: Hydrophobic surfaces of
moieties bonded on silica (C18, C8, C5, Phenyl,
CN)
 Mobile phase: Mostly buffer with small % of
organic solvent
 Applications: ~80% of all separations done on
RP HPLC.
Reverse Phase HPLC
 Different sorption affinities between analytes
results in their separation

 More polar analytes retained less

 Analytes with larger hydrophobic part are

retained longer
Ion exchange HPLC
 The stationary phase contains ionic groups
which interact with the ionic groups of the
sample molecules.

 The method is suitable for separating ionic

metabolic products and organic ions.
Affinity HPLC
 Highly specific biochemical interactions provide
the means of separation.
 The stationary phase contains specific groups of
molecules which can only adsorb the sample if
certain steric and charge-related conditions are
satisfied
 Affinity chromatography can be used to isolate
proteins (enzymes as well as structural
proteins), lipids, etc., from complex mixtures
Size exclusion HPLC
 Used for large mw compounds - proteins and
polymers

 Separation mechanism is sieving not partitioning

 Stationary phase - porous silica or polymer

particles (polystyrene, polyacrylamide)

 Well-defined pore sizes (40-2500 Å)

HPLC hardware
 HPLC systems are comprised of individual
modules
Definitions
 Mobile phase:
Solvent flowing through the column
 Stationary phase:
Particulate which mobile phase flows through
 Column:
Tube containing tightly packed particulate
 Chromatogram:
Resulting separation graph from chromatography
Mobile phase reservoir

 Module to hold upto 4 1L flasks containing

mobile phase(s), other solvents
Degasser

 Essential that all solvents entering HPLC

system are degassed
 Can either degas by:
 Vacuum chamber
 Sparging (helium)
Pump
 Reciprocating Pump
 Up to 10,000 psi, small internal volumes
 Produces pulsation
Autosampler
 Temperature control
 Variety of vial size / shapes
 Ability to sample from 96 well plates
Sample injection
Introduce small sample (0.1-100 µL) without
depressurization of system
Sample injection
 Need to load sample into loop, then wash out
into mobile phase flow
Columns
 In-line filters / guard columns necessary
 Analytical column usually a stainless steel tube,
but can also be made of PEEK
Columns
 Column production & silca surface bonding can
vary from manufacturer

Mobile Phase: Sample:

60% CH3CN 1. Uracil
40% 50mM KH2PO4, pH 3.2 2. Pyridine
3. Phenol
4. Dimethyl phthalate
5. N,N-Dimethylaniline
6. 4-Butylbenzoic acid
7. Toluene
Detectors
 Variety of detectors that can be attached to
HPLC systems
 Common detectors:
UV Fluorescence
UV PDA Mass spec
Electrochemical / RI CE
 Recent development:
Corona CAD
UV detector

 Most common detector

 Absorption of UV wavelengths to produce
signal
Chromatogram

•The baseline is any part of the chromatogram

where only mobile phase is emerging from the
column.
•The peak maximum is the highest point of the
peak.
•The injection point is that point in
time/position time when/where the sample is
placed on the column.
•The retention time is the time elapsed
between the injection point and the peak
maximum. Each solute has a characteristic
retention time.
•The relative retention time is calculated as a
fraction of retention time to an internal standard.
Varying HPLC chromatography

 Three simple ways to change pattern of

chromatography on HPLC:

 Temperature of column
 pH of mobile phase
 Concentration of organic solvent / counterion
Isocratic elution
 Use of one mobile phase ie. uniform polarity of
solvent

 Comprised mostly of aq. buffer and some

organic solvent

 Analytes retarded as they travel down column

 Easiest method for elution of the column

Isocratic elution
Gradient elution
 Use of two or more mobile phases ie. solvent
polarity continuously varied

 First phase usually comprised of aq. buffer, other

phases mostly organic solvent

 Analytes travel down the column at the same

speed, but at different time intervals

 More complex method for elution of the column

Gradient elution
Isocratic vs. Gradient elution
Solvent selection

 Choice & strength of organic solvent can

effect chromatography
HPLC sample preparartion
 Usually, a degree of sample ‘clean up’ is required for
HPLC analysis.

 Solvent extraction

 Solid phase extraction (SPE)

 Microdiffusion

 In line SPE
Modern HPLC systems

 Tower design
Future developments at Kings

 Replacement HPLC to be purchased 2006

 UV
 UV PDA
 EC
 Fluorescence
 LCMS
Questions ?