8

COLUMN AND THlrHAYER LIQUID CHROMATOGRAPHY

packing techniques are used in which the particles are suspended in a suitable
solvent and the suspension (or slurry) driven into the column under pressure.
The essential features for successful slurry packing of columns have been
summarised.P" Many analysts will, however, prefer to purchase the commercially
available HPLC columns, for which the appropriate manufacturer's catalogues
should be consulted.
Finally, the useful life of an analytical column is increased by introducing a
guard column. This is a short column which is placed between the injector and
the HPLC column to protect the latter from damage or loss of efficiency caused
by particulate matter or strongly adsorbed substances in samples or solvents. It
may also be used to saturate the eluting solvent with soluble stationary phase
[see Section 8.2(2)J. Guard columns may be packed with microparticulate
stationary phases or with porous-layer beads; the latter are cheaper and easier
to pack than the microparticulates, but have lower capacities and therefore
require changing more frequently.
Detectors. The function of the detector in HPLC is to monitor the mobile phase
as it emerges from the column. The detection process in liquid chromatography
has presented more problems than in gas chromatography; there is, for example
no equivalent to the universal flame ionisation detector of gas chromatography
for use in liquid chromatography. Suitable detectors can be broadly divided
into the following two classes:
(a) Bulk property detectors which measure the difference in some physical

property of the solute in the mobile phase compared to the mobile phase
alone, e.g. refractive index and conductivity* detectors. They are generally
universal in application but tend to have poor sensitivity and limited range.
Such detectors are usually affected by even small changes in the mobile-phase
composition which precludes the use of techniques such as gradient elution.
(b) Solute property detectors, e.g. spectrophotometric, fluorescence and electrochemical detectors. These respond to a particular physical or chemical
property of the solute, being ideally independent of the mobile phase. In
practice, however, complete independence of the mobile phase is rarely
achieved, but the signal discrimination is usually sufficient to permit
operation with solvent changes, e.g. gradient elution. They generally provide
high sensitivity (about 1 in 109 being attainable with UV and fluorescence
detectors) and a wide linear response range but, as a consequence of their
more selective natures, more than one detector may be required to meet
the demands of an analytical problem. Some commercially available
detectors have a number of different detection modes built into a single
unit, e.g. the Perkin-Elmer '3D' system which combines UV absorption,
fluorescence and conductimetric detection.
Some of the important characteristics required of a detector are the following.
(a) Sensitivity, which is often expressed as the noise equivalent concentration,

i.e. the solute concentration, Cn, which produces a signal equal to the
detector noise level. The lower the value of C n for a particular solute, the
more sensitive is the detector for that solute.

* The conductance detector is a universal detector for ionic species and is widely used in ion
chromatography (see Section 7.4).
224

EQUIPMENT FOR HPLC

8.3

(b) A linear response. The linear range of a detector is the concentration range

over which its response is directly proportional to the concentration of

solute. Quantitative analysis is more difficult outside the linear range of
concentration.
(c) Type ofresponse, i.e. whether the detector is universal or selective. A universal
detector will sense all the constituents of the sample, whereas a selective
one will only respond to certain components. Although the response of the
detector will not be independent of the operating conditions, e.g. column
temperature or flow rate, it is advantageous if the response does not change
too much when there are small changes of these conditions.
A summary of these characteristics for different types of detectors is given
in Table 8.2.
Table 8.2 Typical detector characteristics in HPLC
Type

Response

Amperometric
Conductimetric
Fluorescence
UV /visible absorption
Refractive index

Selective
Selective
Selective
Selective
Universal

Linear range'"

10- 1 0
10- 7
1O~

10- 8
10- 6

104-10 5
103-10 4
103-104
104-10 5
103-10 4

'"The range over which the response is essentially linear is expressed

as the factor by which the lowest concentration (en) must be
multiplied to obtain the highest concentration.

A detailed description of the various detectors available for use in HPLC is

beyond the scope of the present text and the reader is recommended to consult
the monograph by Scott. 55 A brief account of the principal types of detectors
is given below.

Refractive index detectors. These bulk property detectors are based on the
change of refractive index of the eluant from the column with respect to pure
mobile phase. Although they are widely used, the refractive index detectors
suffer from several disadvantages - lack of high sensitivity, lack of suitability
for gradient elution, and the need for strict temperature control (+ 0.001 C)
to operate at their highest sensitivity. A pulseless pump, or a reciprocating pump
equipped with a pulse dampener, must also be employed. The effect of these
limitations may to some extent be overcome by the use of differential systems
in which the column eluant is compared with a reference flow of pure mobile
phase. The two chief types of RI detector are as follows.
1. The deflection refractometer (Fig. 8.4), which measures the deflection of a
beam of monochromatic light by a double prism in which the reference and
sample cells are separated by a diagonal glass divide. When both cells contain
solvent of the same composition, no deflection of the light beam occurs; if,
however, the composition of the column mobile phase is changed because
of the presence of a solute, then the altered refractive index causes the beam
to be deflected. The magnitude of this deflection is dependent on the
concentration of the solute in the mobile phase.
2. The Fresnel refractometer which measures the change in the fractions of
reflected and transmitted light at a glass-liquid interface as the refractive
225

COLUMN AND THIN-tAYER LIQUID CHROMATOGRAPHY

__-+-------- Light beam

Mirror ------i.

--;---~------- Reflected beam

Reference
solvent

Fig. 8.4 Refractive index detector.

index of the liquid changes. In this detector both the column mobile phase
and a reference flow of solvent are passed through small cells on the back
surface of a prism. When the two liquids are identical there is no difference
between the two beams reaching the photocell, but when the mobile phase
containing solute passes through the cell there is a change in the amount of
light transmitted to the photocell, and a signal is produced. The smaller cell
volume (about 3 ,uL) in this detector makes it more suitable for high-efficiency
columns but, for sensitive operation, the cell windows must be kept
scrupulously clean.

Ultraviolet detectors. The UV absorption detector is the most widely used in

HPLC, being based on the principle of absorption of UV visible light as the
effluent from the column is passed through a small flow cell held in the radiation
beam. It is characterised by high sensitivity (detection limit of about
1 x 10- 9 g mL -1 for highly absorbing compounds) and, since it is a solute
property detector, it is relatively insensitive to changes of temperature and flow
rate. The detector is generally suitable for gradient elution work since many of
the solvents used in HPLC do not absorb to any significant extent at the
wavelengths used for monitoring the column effluent. The presence of air bubbles
in the mobile phase can greatly impair the detector signal, causing spikes on
the chromatogram; this effect can be minimised by degassing the mobile phase
prior to use, e.g. by ultrasonic vibration. Both single and double beam
(Fig. 8.5) instruments are commercially available. Although the original
detectors were single- or dual-wavelength instruments (254 and/or 280 nm),
some manufacturers now supply variable-wavelength detectors covering the
range 210~800 nm so that more selective detection is possible.
No account ofUV detectors would be complete without mention of the diode
array (multichannel) detector, in which polychromatic light is passed through
the flow cell. The emerging radiation is diffracted by a grating and then falls
on to an array of photodiodes, each photodiode receiving a different narrowwavelength band. A microprocessor scans the array of diodes many times a
226

EQUIPMENT FOR HPLC

Hg lamp
source

Quartz
lens \

t
+

Dualchannel
cell

Movable
~ calibrated
filter

Sample
~Photocell

8.3

~ Reference
\

photocell

Compound
UV filter

Fig. 8.5 Block diagram of a double-beam UV detector.

second and the spectrum so obtained may be displayed on the screen of a VDU
or stored in the instrument for subsequent print-out. An important feature of
the multichannel detector is that it can be programmed to give changes in
detection wavelength at specified points in the chromatogram; this facility can
be used to 'clean up' a chromatogram, e.g. by discriminating against interfering
peaks due to compounds in the sample which are not of interest to the analyst.

Fluorescence detectors. These devices enable fluorescent compounds (solutes)

present in the mobile phase to be detected by passing the column effluent through
a cell irradiated with ultraviolet light and measuring any resultant fluorescent
radiation. Although only a small proportion of inorganic and organic compounds
are naturally fluorescent, many biologically active compounds (e.g. drugs)
and environmental contaminants (e.g. polycyclic aromatic hydrocarbons) are
fluorescent and this, together with the high sensitivity of these detectors, explains
their widespread use. Because both the excitation wavelength and the detected
wavelength can be varied, the detector can be made selective. The application
of fluorescence detectors has been extended by means of pre- and post-column
derivatisation of non-fluorescent or weakly fluorescing compounds (see
Section 8.4).
Electrochemical detectors. The term 'electrochemical detector' in HPLC
normally refers to amperometric or coulometric detectors, which measure the
current associated with the oxidation or reduction of solutes. In practice it is
difficult to use electrochemical reduction as a means of detection in HPLC
because of the serious interference (large background current) caused by
reduction of oxygen in the mobile phase. Complete removal of oxygen is difficult
so that electrochemical detection is usually based on oxidation of the solute.
Examples of compounds which can be conveniently detected in this way are
phenols, aromatic amines, heterocyclic nitrogen compounds, ketones, and
aldehydes. Since not all compounds undergo electrochemical oxidation, such
detectors are selective and selectivity may be further increased by adjusting the
potential applied to the detector to discriminate between different electroactive
species. It may be noted here that an anode becomes a stronger oxidising agent
as its electrode potential becomes more positive. Of course, electrochemical
detection req uires the use of conducting mo bile phases, e.g. containing inorganic
salts or mixtures of water with water-miscible organic solvents, but such

227

COLUMN AND THIN-LAYER L1nUID CHROMATOGRAPHY

conditions are often difficult to apply to techniques other than reverse phase
and ion exchange chromatography.
The amperometric detector is currently the most widely used electrochemical
detector, having the advantages of high sensitivity and very small internal cell
volume. Three electrodes are used:
1. the working electrode, commonly made of glassy carbon, is the electrode at
which the electroactive solute species is monitored;
2. the reference electrode, usually a silver-silver chloride electrode, gives a stable,
reproducible voltage to which the potential of the working electrode is
referred; a n d "
3. the auxiliary electrode is the current-carrying electrode and usually made of
stainless steel.
Despite their higher sensitivity and relative cheapness compared with ultraviolet
detectors, amperometric detectors have a more limited range of applications,
being often used for trace analyses where the ultraviolet detector does not have
sufficient sensitivity.

8.4 DERIVATlSATlON
In liquid chromatography, in contrast to gas chromatography [see Section 9.2(2)J,
derivatives are almost invariably prepared to enhance the response of a particular
detector to the substance of analytical interest. For example, with compounds
lacking an ultraviolet chromophore in the 254 nm region but having a reactive
functional group, derivatisation provides a means of introducing into the
molecule a chromophore suitable for its detection. Derivative preparation can
be carried out either prior to the separation (pre-column derivatisation) or
afterwards (post-column derivatisation). The most commonly used techniques
are pre-column off-line and post-column on-line derivatisation.
Pre-column off-line derivatisation requires no modification to the instrument
and, compared with the post-column techniques, imposes fewer limitations on
the reaction conditions. Disadvantages are that the presence of excess reagent
and by-products may interfere with the separation, whilst the group introduced
into the molecules may change the chromatographic properties of the sample.
Post-column on-line derivatisation is carried out in a special reactor situated
between the column and detector. A feature of this technique is that the
derivatisation reaction need not go to completion provided it can be made
reproducible. The reaction, however, needs to be fairly rapid at moderate
temperatures and there should be no detector response to any excess reagent
present. Clearly an advantage of post-column derivatisation is that ideally the
separation and detection processes can be optimised separately. A problem
which may arise, however, is that the most suitable eluant for the chromatographic
separation rarely provides an ideal reaction medium for derivatisation; this is
particularly true for electrochemical detectors which operate correctly only
within a limited range of pH, ionic strength and aqueous solvent composition.
Reagents which form a derivative that strongly absorbs UV /visible radiation
are called chromatags; an example is the reagent ninhydrin, commonly used
to obtain derivatives of amino acids which show absorption at about 570 nm.
Derivatisation for fluorescence detectors is based on the reaction of nonfluorescent reagent molecules (ftuorotags) with solutes to form fluorescent
228