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HPLC is one of the most powerful techniques for separation and quantitative determination of the constituents in a mixture. It works on the principle of Affinity chromatography having two phases viz: stationary and mobile phase. The constituent with lower affinity for stationary phase travels faster and vice-versa. A detector in HPLC is placed at the end of the system. Its work is to analyse the solution which is eluting from the column. Detectors are broadly classified into two groups: Bulk property detectors and solute property detectors. Bulk property detectors (Non-selective detectors) are those that measure the changes in solute and mobile phase in combination. Such detectors show fluctuation in readings even with slight change in mobile phase combination. Solute property detectors are also called as selective detectors because they give response for a particular physical or chemical property of the analyte, being ideally independent of the mobile phase. Other than that there are numerous newer developments made in detectors for more accurate determination. The present paper is meant on to focus on different detectors used in HPLC, there area of utilisation and the advancement made in recent time.
The most powerful technique to determine quantitatively and separate the mixture of composition in today’s modern chemistry is Chromatography especially High Performance Liquid Chromatography or High Pressure Liquid Chromatography . HPLC works on the principle of Affinity chromatography. The solution of the sample is injected into a column of a porous
material (stationary phase) and a liquid (mobile phase) is pumped at high pressure through the column. The mixture on travelling through the stationary phase splits into its constituents and the component with high affinity for stationary phase travels late whereas one with less affinity elutes fast. This is also based partition coefficient of the material .
A detector in HPLC is placed at the end of the system.
Its work is to analyse the solution which is eluting from the
column. The concentration of individual component of the
analyte is proportional to the electronic signal coming out of the
component of the mixture [3,4].
Bulk Property Detectors: Bulk property detectors are
those that measure the changes in solute and mobile phase in
combination. Such detectors show fluctuation in readings even
with slight change in mobile phase combination. Examples
are: refractive index and conductivity detectors. Due to poor
sensitivity and limited range, they are used less despite being
universally applicable. In general they are called as non selective
detectors because they react to the bulk property of the analyte.
Solute Property Detectors: Solute property detectors are
also called as selective detectors because they give response for
a particular physical or chemical property of the analyte, being
ideally independent of the mobile phase. Practically, it is not
possible to achieve complete independence from mobile phase
but the signal discrimination usually makes it sufficient to work
with solvent changes as in gradient elution [6,7].
Electrical Conductivity HPLC Detectors: These detectors
senses all the ions, whether they are from a solute, or from
the mobile phase. It measures the conductivity of mobile
phase along with the solute which needs to be backed-off by
suitable electronic adjustments. Thus it is a type of Electrical
Conductivity Detector. The measured electronic resistance is
directly proportional to the concentration of ions present in the
Refractive Index HPLC detectors: They are also one of
the bulk property detectors and are based on the change of the
refractive index of the eluent from the column with respect to pure
mobile phase . There aredifferent types of Refractive index
detectors: Christiansen effect detector, interferometer detector,
thermal lens detector and the dielectric constant detector. They
are mostly used for detection of non-ionic compounds that
neither fluoresce nor absorb in the UV region. They face the
drawback of being less sensitive, need of temperature control
and less suitability to gradient elution .
Electrochemical HPLC Detectors: They are termed as
“Electrochemical detectors” for the reason being that they
usually measure the current associated with the oxidation or
reduction of solutes. They act as amperometer or coulometer
in HPLC . They are classified as equilibrium and dynamic
detectors. The suitability of these detectors depends on the
volumetric characteristics of the solute molecules in the aqueous
or organic mobile phase. They are sensitive to changes in the flow
rate or composition of the eluent and require working electrode,
reference electrode and auxiliary electrode .
Light Scattering HPLC Detectors: Light scattering HPLC
detectors are useful for large molecular weight molecules like
surfactants, lipids and sugar. It measures the scattered light
coming out of the eluent. Low angle laser light scattering detector
and the multiple angle laser light scattering detectors are the two
types of Light scattering detectors available. They are also called
as Evaporative light scattering detector because in this the beam
of light by particles of compound remaining after evaporation
of the mobile phase. The importance of such type of detector is
growing with time because it acts as universal detector and does
not require a compound to have a chromophore for detection.
They can be used with gradient elution .
Ultraviolet/Visible Detectors: The most common HPLC
detectors used are UV detectors because of the fact that most of
the compounds absorb in UV or visible region. They give specific
response to the class of compounds or particular compounds
depending upon the functional group of eluting molecules
. The basis of working for optical detectors is the change
in intensity when a beam of electromagnetic radiation passes
through the detector flow cell. These detectors are of three
different types: Fixed wave length detectors, variable wavelength
detectors and diode array detectors.
Fixed Wave Length Detectors: Such type of detectors does
not allow change in wavelength of the radiation. They are difficult
to find nowadays due to limitation of working wavelength. Low
pressure mercury lamp is used for very intense light at 253.7nm
Variable Wavelength Detectors: Variable wavelength
detectors can be adjusted to work on any wavelength over full
UV- visible region. The wavelength can be selected at 3nm or less.
Diode Array Detectors: In diode array detector, the sample
is subjected to light of all wavelengths generated by the lamp
at once. The lights from emission source when collimated by
an achromatic lens system, the total light passes through the
detector cell onto a holographic grating and then falls onto a
diode array which contains 100s of diodes. The chromatogram
can be produced using the UV wavelength that was falling on
that particular diode at the end of the run. DAD helps to see the
response of the analyte at different wavelengths in only single
run and thus saves time and energy .
Fluorescent detectors are the most selective, sensitive and
specific than all other HPLC detectors. Specific wavelength is
used to excite and then emit light signal in analyte atoms. They
intensity of light is monitored continuously to quantify the
analyte concentration. Fluorescence is 10-1000 times sensitive
than UV detector for strong UV absorbing compounds. Even
a single analyte in the cell can be detected by the fluorescence
detector. For some compounds which do not have fluorescence
absorbance or low absorbance, they can be treated with
fluorescence derivatives such as dansylchloride . The system
is easy to operate and relatively stable. There are three types of
Single Wavelength Excitation Fluorescence Detector: The
wavelength of excitation is fixed in such type of detectors and
they are made from low pressure mercury lamp.
Multi Wavelength Fluorescence Detector: It contains two
monochromators to select wavelength of excitation.
Laser Induced Fluorescence Detector (LIFDs): It shows
optical emission from molecules that have excited to higher
energy levels by absorption of electromagnetic radiations.
It is used as separating tool for polymerase chain reactions,
determination of solutes like proteins, nucleic acids, polycyclic
aromatic hydrocarbons and toxic elements like cyanide .
Mass Spectrometric HPLC Detectors: The detection by
mass spectrometers is based on molecular fragmentation by
electric fields and separation is based on the mass to charge
ration of the fragmented molecule. They show high selectivity
and sensitivity and are used in LC-MS technique for advantage
in terms of resolution and sensitivity. The analyte for MSD must
be in ionic form.
Infrared detectors are chosen on the basis of property of
compound to absorb infrared light i.e. it falls in the region of
4000-690 cm-1. It is a sophisticated instrument and requires
Cells or windows made of NaCl or CaF2 and the wavelength
scanning is provided by semicircular filter wedges. The detector
is suitable for polymer analysis as it differentiate the secondary
a) Transport Detectors: The transport detectors have a
carrier like metal chain, wire or disc which passes continually
through column eluting the analyte of interest from the
mobile phase. The solute is adhered on the surface as thin
film whereas the mobile phase evaporates. Moving wire and
Chain detectors are the two types of transport detectors
available for HPLC system.
b) Chiral Detectors: There are compounds and drugs
that exist in their enantiomeric form but may have
different pharmacological properties. The separation of
these enantiomers is possible by the detectors capable of
responding to the different chiral forms. They are used for
the optically active compounds like amino acids, terpenes
and sugars and other compounds. Polarimetry or Optical
rotatory dispersion (ORD) and Circular dichroism (CD) are
two techniques of chiral detection. ORD detectors are based
on differences in refractive index whereas CD detectors
differentiate enantiomers by measuring differences between
the absorption of right and left handed circularly polarised
c) Corona Discharge Detectors: Corona charged aerosol
detection (CAD), sometimes referred to as corona discharge
detection (CDD) is a newer and unique technique. In this
technique, the HPLC column eluent is first nebulized with a
nitrogen (or air) carrier gas to form droplets which are then
dried to remove mobile phase, producing analyte particles.
The principal of working of Corona discharge detectors is
the charge transfer diffusionally to the opposing stream of
analyte particles when primary stream of analyte particles is
met by a secondary stream of nitrogen (or air). The stream
of nitrogen is positively charged and produces required high
–voltage in the platinum corona wire for diffusion to take
place. The analytes then are further transferred to a collector
where it is measured by a highly sensitive electrometer,
generating a signal in direct proportion to the quantity of
analyte present [20,21].
The work of detector is to detect and give the information
to the recorder which shows it in a form of a chromatogram.
Every compounds has its own properties which is not completely
the same with one another, thus this arises a need to have
different detectors for different compounds. Before beginning
the separation by HPLC it is thus very important to study about
the nature of the compound and select the detector accordingly.
The selection of wrong detector misguides our journey of
separation and quantification. The paper summarizes different
types of detectors used in HPLC with their scope of utilisation in
Separation and determination of the compound.