High-performance liquid chromatography (HPLC) uses high pressure to
force the mobile phase through a closed column packed with micro metresized
particles. This allows rapid separation of complex mixtures. Several
operating modes of HPLC are possible. These are:
- Normal phase (NPHPLC): the sample should be soluble in a
hydrophobic solvent, e.g. hexane, and should be non-ionic. The mobile
phase is non-polar while the stationary phase is polar, e.g. silica, cyano,
- Reversed phase (RPHPLC): the sample should be soluble in water or a
polar organic solvent, e.g. methanol, and should be non-ionic. The mobile
phase is polar while the stationary phase is non-polar, e.g. C18 (ODS), C8 (octyl), phenyl.
- Size exclusion chromotography (SEC): this is used when the major
difference between compounds in a mixture is their molecular weight. It is
normally used for compounds with molecular weights greater than 2000.
The mobile phase should be a strong solvent for the sample. Aqueous
SEC is called gel filtration chromatography (GFC) and is used for
separation of proteins and other biomolecules, while organic SEC is
called gel permeation chromatography (GPC) and is used for the
separation of polymers.
- Ion exchange chromotography (1EC): it is used when compounds are
ionic, or potentially ionic, e.g. anions, cations, organic acids and bases,
amino acids, catecholamines, peptides. The mobile phase is typically a
buffer and the choice of pH is critical. Two types can be differentiated:
SAX (strong-anion exchange) and SCX (strong-cation exchange).
The essential components of an HPLC system are a solvent delivery system, a
method of sample introduction, a column, a detector and an associated
readout device (Fig. 32.16).
Solvent delivery system
|Fig. 32.16 Components of an HPLC system.
This should fulfil certain requirements:
- It should be chemically inert.
- It should be capable of delivering a wide flow-rate range.
- It should be able to withstand high pressures.
- It should be able to deliver high flow-rate precision.
- It should have a low internal volume.
- It should provide minimum flow pulsation.
Although several systems are available that meet these requirements, the most
common is the reciprocating or piston pump. The choice of solvent delivery
system depends on the type of separation to be performed:
- Isocratic separation: a single solvent (or solvent mixture) is used
throughout the analysis.
- Gradient elution separation: the composition of the mobile phase is
altered using a microprocessor-controlled gradient programmer, which
mixes appropriate amounts of two different solvents to produce the
The main advantages of gradient HPLC are that you can control mobilephase
composition. This allows you to resolve closely related compounds and
provide faster elution of strongly retained compounds thereby producing
reduced analysis times and faster method development time. However, these
advantages have to be compared with some disadvantages, such as the initial
higher cost of the equipment compared with an isocratic system. Also, after
each gradient run, a re-equilibration of the system is required to return to the
initial mobile-phase conditions.
The most common method of sample introduction in HPLC is via a rotary
valve, e.g. a Rheodyne® valve. A schematic diagram of a rotary valve is
shown in Fig. 32.17. In the load position, the sample is introduced via a
syringe to fill an external loop of volume 5, 10 or 20 µL. While this occurs,
the mobile phase passes through the valve to the column. In the inject
position, the valve is rotated so that the mobile phase is diverted through the sample loop, thereby introducing a reproducible volume of the sample into
the mobile phase. The procedure for injection of a sample is shown in Fig.
32.18. In Fig. 32.18a the syringe is filled with the sample/standard
solution (typically 1mL). Then the outside of the syringe is wiped clean with
a tissue (Fig. 32.18b). The syringe is placed into the Rheodyne® injector of
the chromatograph while in the 'load' position (Fig. 32.l8c) and the plunger
on the syringe is depressed to fill the sample loop. Finally, the position of the
Rheodyne'f valve is switched to the 'inject' position to introduce the sample
into the chromatograph (Fig. 32.l8d) and then the syringe is removed from
the injection valve. The procedure for the preparation of a series of
calibration solutions is shown in Box 32.1.
|Fig. 32.17 Schematic diagram of a rotary valve.
|Fig. 32.18 Sample injection in HPLC.
This is usually made of stainless steel, and all components, valves, etc., are
manufactured from materials which can withstand the high pressures
involved. The most common form of liquid chromatography is reversed
phase HPLC. In RPHPLC the most common column packing material
consists of C18 or octadecylsilane (ODS). A chemically bonded stationary
phase is shown in Fig. 32.19. However, some of the surface silanol groups
remain unaffected. These unreacted groups lead to undesirable chromatographic
effects, such as peak tailing. One approach to remove the
unreacted silanol groups is end capping. In this way, the silanol group is
reacted with a small silylating group, e.g. trimethylchlorosilane. An
alternative approach to nullify the action of the silanol groups is to add
triethylamine to the mobile phase, which modifies the silica surface while in
|Fig. 32.19 A C18 stationary phase.
Most HPLC systems are linked to a continuous monitoring detector of high
sensitivity, e.g. phenols may be detected spectrophotometrically by monitoring the absorbance of the eluent at 280 nm as it passes through a flow
cell. Other detectors can be used to measure changes in fluorescence, current
or potential, as described below. Most detection systems are non-destructive,
which means that you can collect eluent with an automatic fraction collector
for further study.
UV/visible detectors are widely used and have the advantages of versatility,
sensitivity and stability. Such detectors are of two types: fixed wavelength and
variable wavelength. Fixed-wavelength detectors are simple to use, with low
operating costs. They usually contain a mercury lamp as a light source,
emitting at several wavelengths between 254 nm and 578 nm; a particular
wavelength is selected using suitable cutoff filters. The most frequently used
wavelengths for analysis of organic molecules are 254 nm and 280 nm. Variable
wavelength detectors use a deuterium lamp and a continuously adjustable
monochromator for wavelengths of 190-600 nm. For both types of detector,
sensitivity is in the absorbance range 0.00 1-1.0 (down to ≈ 1 ng), with noise
levels as low as 4 × 10−5
. Note that sensitivity is partly influenced by the path
length of the flow cell (typically 10 mm), see Fig. 32.20. Monitoring at shortwavelength
UV (e.g. below 240 nm) may give increased sensitivity but
decreased specificity, since many organic molecules absorb in this range.
Additional problems with short-wavelength UV detection include instrument
instability, giving a variable base line, and absorption by components of the
mobile phase (e.g. organic solvents, which often absorb at < 210 nm).
An important development in chromatographic monitoring is diode array
detection (DAD). The incident light comprises the whole spectrum of light
from the source, which is passed through a diffraction grating and the
diffracted light detected by an array of photodiodes. Typical DAD can
measure the absorbance of each sample component at 1-lOnm intervals over
the range 190-600nm. This gives an absorbance spectrum for each eluting
substance which may be used to identify the compound and give some
indication as to its purity. An example of a three-dimensional diode array
spectrum is shown in Fig. 32.21.
Many aromatic organic molecules, including some polycyclic aromatic
hydrocarbons, show natural fluorescence (Table 26.1), or can be made to
fluoresce by pre-colurnn or post-column derivatization with a fluorophore.
Fluorescence detection is more sensitive than UV/visible detection, and may
allow analysis in the picogram (10−12
g) range. A fluorescence detector
consists of a light source (e.g. a xenon lamp), a diffraction grating to supply
light at the excitation wavelength, and a photomultiplier to monitor the
emitted light (usually arranged to be at right angles to the excitation beam).
The use of instruments with a laser light source can give an extremely narrow
excitation waveband, and increased sensitivity and specificity.
Electrochemical detectors offer very high sensitivity and specificity, with the
possibility of detection of femtogram amounts of electroactive compounds
such as catecholamines, vitamins, thiols, purines, ascorbate and uric acid. The
two main types of detector, amperometric and coulometric, operate on similar
principles, i.e. by measuring the change in current or potential as sample
components pass between two electrodes within the flow cell. One of these
electrodes acts as a reference (or counter) electrode (e.g. calomel electrode),
while the other - the working electrode - is held at a voltage that is high
enough to cause either oxidation or reduction of sample molecules. In the
oxidative mode, the working electrode is usually glassy carbon, while in
reductive mode a mercury electrode is used. In either case, a current flow
between the electrodes is induced and detected.
Mass spectrometry used in conjunction with chromatographic
methods can provide a powerful tool for identifying the components of
complex mixtures, e.g. pharmaceuticals. One drawback is the limited capacity
of the mass spectrometer - due to its vacuum requirements - compared with
the volume of material leaving the chromatography column. Similarly, in
HPLC, devices have been developed for solving the problem of large solvent
volumes, e.g. by splitting the eluent from the column so only a small fraction
reaches the mass spectrometer.
The computer-generated outputs from the mass spectrometer are similar to
chromatograms obtained from other methods, and show peaks
corresponding to the elution of particular components. However, it is then
possible to select an individual peak and obtain a mass spectrum for the
component in that peak to aid in its identification. This has helped
to identify hundreds of components present in a single sample, including
flavour molecules in food, drug metabolites and water pollutants.
Recording and interpreting chromatograms
|Fig. 32.20 UV detector cell for HPLC.
|Fig. 32.21 Diode array detector absorption spectra of the eluent from an HPLC separation of a mixture of four steroids, taken every 15 seconds.
For analytical purposes, the detector output is usually connected to a
computer-based data acquisition and analysis system. This consists of a
personal computer (PC) with data acquisition hardware to convert an
analogue detector signal to digital format, plus software to control the data
acquisition process, store the signal information and display the resulting
chromatogram. The software will also detect peaks and calculate their
retention times and sizes (areas) for quantitative analysis. The software often
incorporates functions to control the chromatographic equipment, enabling
automatic operation. In sophisticated systems, the detector output may be
compared with that from a 'library' of chromatograms for known
compounds, to suggest possible identities of unknown sample peaks.
In simpler chromatographic systems, you may need to use a chart recorder
for detector output. Two important settings must be considered before using
a chart recorder:
- The base-line reading - this should be set only after a suitable quantity
of mobile phase has passed through the column (prior to injection of the
sample) and stability is established. The chart recorder is usually set a
little above the edge of the chart paper grid, to allow for base-line drift.
- The detector range - this must be set to ensure that the largest peaks do
not go off the top of the chart. Adjustment may be based on the
expected quantity of analyte, or by a trial-and-error process. Use the
maximum sensitivity that gives intact peaks. If peaks are still too large
on the minimum sensitivity, you may need to reduce the amount of
sample used, or prepare and analyse a diluted sample.