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 PerkinElmer '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 Cn for a particular solute, the more sensitive is the detector for that solute.
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

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 theconcentration 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 glassliquid interface as the refractive 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 phasecontaining solute passes
Fig. Refractive index detector.
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
singleor
dualwavelength instruments
(254 and/or 280 nm),
some manufacturers now
supply
variablewavelength
detectors
covering the range 210~800
nm so that more selective
detection is possible. No
Fig. 8.5 Block diagram of a double-beam UV detector.
account of UV 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 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 indetection
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

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 mobile
phases, e.g. containing inorganic salts or mixtures of water with water-miscible organic
solvents, but such 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; and
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.

Effect of Temperature
EFFECT OF TEMPERATURE ON ANALYTE IONIZATION
The use of elevated temperatures for the reversed-phase HPLC separation of mixtures has
been used primarily for increasing column efficiency or shortening run time and
enhancing separation selectivity . Elevated temperatures also increase solute solubility
and diffusivity. Column efficiency is also expected to increase with temperature as
diffusion rate increases. However, temperature can also affect the dissociation constants
of the ionizable components, and this can lead to anomalous retention behavior of these
compounds as a function of temperature (i.e., increases in retention with increase in
temperature) . The pH of a phosphate buffer and acetate buffer are not significantly
affected by change in temperature . For acidic analytes, depending on the type of acid and
its intrinsic properties, the analyte pKa may not vary as a function of temperature.

Phenolic and carboxylic acids pKas do not vary significantly with a change of
temperature. However, basic analytes may experience greater changes in their ionization
constants with increase of the temperature. The weaker an acid, the greater the change in
the analyte pKa (mainly seen for basic compounds) with a change in temperature.
Essentially for basic compounds the analyte is being analyzed in its more neutral form
with an increase in the temperature and may experience increases in retention at higher
analysis temperatures. Therefore the temperature of the separation should also be taken
into consideration when performing method development, especially for basic
compounds. Basic compounds that have pKa values >6 usually experience the greatest
changes in retention with increase in temperature. The pKa values of these basic
compounds decrease with an increase in temperature, thereby making them more neutral
when analyzed at higher temperatures.
Selectivity
Although temperature has been proposed as a variable in altering selectivity, it has not
been widely used, because the majority of analytes show very similar changes on
changing temperature (especially over the limited conventional temperature range).
Significant differences may be observed if temperature can cause ionization changes or if
analytes with very different functional groups are present. However, care must be taken
in these situations because relative retention changes with column temperature could
result in a lack of method robustness, especially if caused by ionization changes.
Efficiency
One of the reported advantages of raising the temperature of a chromatographic
separation is an increase in peak efficiency. This is usually attributed to a reduction in
the viscosity of the eluent and an increase in the diffusion rate of the analyte as the
temperature is increased. A higher diffusion rate should reduces the mass transfer term
effect The improvement in efficiency is normally regarded as most significant for larger
analytes, such as biological and synthetic macromolecules, whose size reduces their
mobility. For smaller molecules the effects are relatively small, and often an increase in
efficiency can be attributed to a reduction in the retention factor on raising the
temperature

Antigens
The term antigen refers to any substance that is capable of eliciting the formation of
aspecific antibody directed against that substance. An antigen canbe a drug, peptide,
protein, carbohydrate, or lipid. Antigens can also be a combination ofthese substances,
such as a glycoprotein or lipoprotein.
The large number of substances that can act as antigens means that physicochemical
techniques like traditional chromatography or electrophoresis can be used to isolate
suchsubstances. However, the most efficient approach for this work is to use
immunoaffinity separations based on antibodies that are specific for these agents..

Affinity Methods for Antigen Isolation

As described earlier, immunoaffinity methods are
probably the most practical approaches for isolating
specific antigens. This makes use of the immunological
activity of the antigens by binding them to
immunoaffinity columns containing immobilized
antibodies. The isolation of antigens by this approach
can employ polyclonal antibodies, affinity-purified
antibodies, or specific monoclonal antibodies to isolate
a sing|le compound from essentially any matrix. An
example of such a separation is given in Figure
Monoclonal antibodies have been used to isolate a large
number of antigens and are often the reagents of choice
when performing such isolations [13., 140].
Immunoaffinity separations have been successfully
applied to many fields, including the isolation of
allergens, the purification of parasite antigens for
diagnostic purposes, and isolation of fungal or viral
components . Specific antigens associated with
receptors and other cellular components have been
successfully isolated using immobilized-antibody columns. In addition, antibody-based
systems have been used to purify recombinant proteins from expression systems such as
bacterial cultures . Along with whole antibodies, antibody fragments can be immobilized
and used as immunoadsorbents for antigen isolation. As early as 1981. Kennedy and
Barnes used immobilized F(ab)2 fragments to isolate class-specific IgG from human
serum. In the same manner, antibody Fab fragments and scFvs have been successfully
used to isolate whole bacteria and tagged recombinant proteins .
A technique that is becoming increasingly popular for immunoaffinity separations is a
method performed with antibody-coated paramagnetic panicles ( Figure). Although this
approach has been mainly used to isolate cells bearing specific antigens, there is great
potential for this to be used as a rapid technique for isolating immunologically specific
antigens . Immunomagnetic separations based on anti-immunoglobulin-coated particles
have been employed in the recovery of muscle tropomyosin, vimentin. and myosin heavy
chain . Similarly, Kausch et al. employed a procedure using streptavidin-coated particles
to isolate a number of cell organelles that had been previously labeled with biotinylated
antibodies. Angen et al. used antibody-coated Dynabeads to isolate pleuropneumoniae
serotype 2 bacteria from pure cultures and suspensions.