Thakkar Arti et al.

IRJP 2 (5) 2011 1-7

INTERNATIONAL RESEARCH JOURNAL OF PHARMACY
Available online http://www.irjponline.com
Review Article

ISSN 2230 8407

HIGH PERFORMANCE LIQUID CHROMATOGRAPHY DETECTORS A REVIEW

Kaushal Ramni, Kaur Navneet, Upadhyay Ashutosh, Suri O. P, Thakkar Arti *
I. S. F. College of Pharmacy, Ferozepur Road, Ghal Kalan, Moga, Punjab, India
Article Received on: 15/03/2011 Revised on: 17/04/2011 Approved for publication: 05/05/2011
*I. S. F. College of Pharmacy, Ferozepur Road, Ghal Kalan, Moga 142 001, Punjab, India
Email: artirthakkar@gmail.com
ABSTRACT
HPLC is the most versatile and widely used elution chromatography. The technique is used to resolve and determine species in a variety of
organic, inorganic, biological, ionic and polymeric materials. Detector is the heart of an instrument and efficiency of system is dependent
upon detecting techniques. Many types of HPLC detectors exist, each of which has some valuable performance feature such as refractive
index detector, ultraviolet detector, fluorescent detector, electrochemical detector, electric conductivity detector, liquid light scattering
detector, evaporative light scattering detector. Due to strong requirement for improvements in sensitivity, selectivity and other performance
characteristics of the detector recent developments in conventional techniques and some other new technologies have been adopted such as
laser light scattering detector, charged aerosol detector, nano quantity aerosol detector, chiral detector and pulsed amperometric detector.
These detectors provide accurate concentration analysis, excellent sensitivity, wide dynamic range, consistent response and broad
applicability of the drug components. Working of these detectors involve different principles such as optical techniques, aerosol based
techniques, refractive methods, light scattering principle, amperometric and fluorescence. The present review enlightens both conventional
and advanced techniques and compares their capabilities of analyzing drug components and need for new techniques for better and wide
range of applicability.
KEYWORDS: Refractive index detector, Charged aerosol detector, Nano quantity aerosol detector, Chiral detector, Pulsed amperometric
detector.

INTRODUCTION
HPLC (High Performance Liquid Chromatography) is
the most versatile and widely used elution
chromatography. The technique is used to separate and
determine species in a variety of organic, inorganic,
biological, ionic and polymeric materials1. A standard
instrument of HPLC incorporates following components:
Solvent reservoir, Mobile phase, Pump, Loop injector,
Guard column or an in-line filter, Analytical Column,
Temperature control, Detector, HPLC Data Acquisition.
Column is connected to the injector and detector with
tubing of narrow internal diameter (0.2 mm) 2, 3.
DETECTORS
Detector in HPLC is placed at the end of analytical
column. Function of detector is to examine the solution
which is eluting from the column. An electronic signal
(output from detector) is proportional to the
concentration of individual components of analyte.
Detectors are classified as bulk property detectors and
solute property detectors. Bulk property detectors
measure the changes in the property of combined eluting
mobile phase and eluting solute. Solute property
detectors detect the changes in physical and chemical
property of eluting component of the mobile phase.

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HPLC detector should have particular characteristics

such as excellent linear response as a function of
concentration of the solute, wide linear dynamic range,
high signal to noise ratio. A robust detector with good
sensitivity works approximately in range of 0.01-100 g
of compound in elutes4. Various types of HPLC detectors
are mentioned in Tab. 1.
REFRACTIVE INDEX DETECTORS
Refractive index is a bulk property of column eluent. In
this detector, detection depends on solute modifying
overall refractive index of the mobile phase. Bulk
property detectors have an inherently limited sensitivity.
These were one of the first on-line detectors to be
developed by Tiselius and Claesson5 in 1942. This
detector can be very useful for detection of non-ionic
compounds that neither absorb in UV region nor
fluoresce. Methods employed for detection using
refractive index detector are such as angle of deviation
method and Fresnel method. Various types of RI
detectors are mentioned here.
Christiansen effect detector
This detector was developed by Christiansen on crystal
filters6,7. Liquid and particulate matter have indices of
refraction those are substantially equal to one another at

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Thakkar Arti et al. IRJP 2 (5) 2011 1-7

a specific wavelength. Variations of transmitted light can
determine changes of refractive index of liquid.
Differences in the intensity of light transmitted through
sample cell and reference device can be determined.
Interferometer detector
Detector response depends on change in effective path
length of light beam passing through cell. Detector
consists of carrier such as metal chain, wire or disc that
passes continually through column, extracting a sample
of mobile phase containing solute. Mobile phase is
eliminated by evaporation leaving solute as a coating on
carrier wire. Scanning is done with specific sensing
technique8.
Thermal lens detector (TL)
TL effect is induced when a laser beam passes through a
material and the absorbed energy is converted into heat.
Change in the optical path length induced by a
temperature rise produce a lens like optical element at
sample. This affects the propagation of a probe beam
through TL. Measurement of the beam centre describes
thermo-optical properties of sample9,10.
Dielectric constant detector
Refractive index of substance is complementary property
to dielectric constant and in some circumstances; it is
direct function of dielectric constant. Relationship
between dielectric constant (e) and refractive index (n)
for non polar substances is given by equation11 (i)
e = n2
(i)
Dielectric constant detector provides an electrical signal
that is proportional to concentration of a component
being passed through the detector.
ULTRAVIOLET DETECTORS
As most organic compounds absorb light in the UV
region (190400 nm) and visible region (400750 nm),
UVVIS spectophotometric detectors are most
commonly used detectors in HPLC. Fixed wavelength,
variable wavelength and diode array detectors are
available for detection in these regions. All the UV-VIS
detectors are based on Beer-Lambert law of ability of
solute to absorb light at defined wavelengths based on
chemical structure and functional groups present in the
solute molecule. Sensitivity of the detector depends on A
(1%, 1 cm) value of analyte. Source of UV light is a
deuterium or high pressure xenon lamp while for visible
range tungsten lamp is preferable. A beam of light is
allowed to pass through a flow cell mounted at the end of
column. Schematic diagram of UV detector is described
in Fig. 112.
Fixed wavelength UV detector
Fixed wavelength detectors operate at a single
wavelength either at 254 or 280 nm in the UV region.
Most popular lamp is low pressure mercury vapour lamp

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which generates light at wavelength of 254 nm.

Variable wavelength UV detector
These detectors can be adjusted to operate at any
wavelength over full UV-Visible range. The wavelengths
can be selected with difference of 3 nm or less in this
detector.
Multi wavelength UV detector
This detector selects a single or narrow range of
wavelengths. There are two types of multi wavelength
detector, dispersion detector that monitors eluent at one
wavelength, used for separation of some carboxylic
acids, a series of common fatty acids13 and the diode
array detector (DAD) that monitors solute absorption
simultaneously over a range of wavelengths. DAD is
used for detection and identification of poison in
traditional medicines14,15. The ideal multi wavelength
detector is combination of dispersive system and diode
array detector.
FLUORESCENCE DETECTOR
Fluorescence detectors are most sensitive, specific and
selective among existing modern HPLC detectors. It is
possible to detect presence of a single analyte molecule
in the flow cell. Fluorescence sensitivity is 10-1000
times higher than UV detector for strong UV absorbing
materials16. Schematic diagram of simple form of
fluorescence detector17 is shown in Fig. 2.
Single wavelength excitation fluorescence detector
The excitation light is normally provided by a low
pressure mercury lamp. Fixed wavelength fluorescence
detector have sensitivity of about 1 x 10-9 g/ml and linear
dynamic range of about 500 with a response index of
0.96 < r <1.0418.
Multi wavelength fluorescence detector
It contains two monochromators, one to select the
excitation wavelength and second to select the
fluorescence wavelength.
Laser induced fluorescence detector (LIFD)
It is optical emission from molecules that have been
excited to higher energy levels by absorption of
electromagnetic radiations. LIFD is most sensitive
optical detection technique. LIFD is usually used with
capillary electrophoresis technique19. LIFD is applied as
separating tool for analysis of polymerase chain reaction
products, determination of solutes like proteins, nucleic
acids, polycyclic aromatic hydrocarbons, toxic elements
like cyanide20.
TRANSPORT DETECTORS
A transport detector consists of a carrier such as metal
chain, wire or disc that passes continually through
column eluent extracting sample of mobile phase
containing solute as a thin film adhering to its surface.
Mobile phase is eliminated by evaporation leaving solute

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as a coating on carrier. Carrier is then scanned by an
appropriate sensing technique to monitor the residual
solute. Moving wire, chain and disc detector are type of
transport detectors available for HPLC system
(schematic diagram in Fig. 3).
Moving wire detector
In 1960 decade Karmen21; Scott and Lawrence developed
a modified form of original moving wire detector.
Carrier wire from a spool passes through an oven and
any residual lubricants remaining on wire after drawing
are burnt off. Wire was passed through an evaporator at
about 105 C. In evaporation chamber a nitrogen stream
is allowed to flow counter-current to wire movement to
improve rate of solvent evaporation. The wire carrying
solvent-free solute enters (via a restriction) a pyrolysis
tube that is maintained at about 500 C.
Chain detector
Haahti and Nikkari22 described a device that employed
chain loops in place of wires as carrier (Fig. 3 B).
Carrier, a gold chain driven by a synchronous motor,
passes through a coating block where it is wetted with
column eluent. Chain enters an evaporator tunnel, is
heated, and solvent volatilized leaving solute deposited
on chain. Chain exits tunnel into actual flame of a flame
ionization detector.
ELECTROCHEMICAL DETECTOR
Electrochemical detector responds to those substances
that are oxidizable or reducible. A reaction takes place at
the surface of electrode, due to which generation of
electron leads to production of electrical signals23.
Suitability of electrochemical detector depends upon
voltammetric characteristics of solute molecules in
aqueous
or
aqueous-organic
mobile
phase.
Electrochemical detector requires three electrodes,
working electrode (oxidation or reduction takes place),
auxiliary electrode and reference electrode (which
compensate any changes in background conductivity of
mobile phase). This detector can be grouped into two
types, the dynamic detector which involves electron
transfer and the equilibrium detector which do not
promote oxidation or reduction of analyte24.
Dynamic detector
Dynamic or amperometric detection relies on oxidation
or reduction of analyte at a potential corresponding to
energy required to free or add electron from molecule.
Dynamic type of detection system is involved in multi
electrode array detector which allows detection of
several analyte at a time by operating each electrode in
array at different potential25. Neuro-active substances are
identified and separated using this detector.
Equilibrium detector
This detector measures conductance of flowing solvent

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stream. Changes in conductance induced by solute are

recorded by the detector.
ELECTRICAL CONDUCTIVITY DETECTOR
Electrical conductivity detector provides universal,
reproducible, high-sensitivity detection of all charged
species such as anions, cations, metals, organic acids,
and surfactants. These detectors measure conductivity of
the total mobile phase hence categorized in bulk density
detectors. Sensors of electrical conductivity detector
consist of a flow-through cell which has few l of
volume containing two electrodes. Electrodes are usually
made up of platinum, stainless steel or some other noble
metal. It is used for determination of alkali and alkaline
earth cations26.
LIQUID LIGHT SCATTERING DETECTOR
These detectors are based on measurement of scattered
light. When light is scattered by a polymer or large
molecular weight substance present in the column eluent,
it is examined by passing through an appropriate sensor
cell. High intensity beam of light is used for illuminating
the scattered light which is achieved by the use of a laser.
There are two types of detectors based on laser principle
such as low angle laser light scattering (LALLS) detector
and multiple angle laser light scattering (MALLS)
detector.
Low angle laser light scattering detector
It is also known as low angle light scattering (LALLS),
Fraunhofer diffraction or Mie Scattering detector. It
applies to those particles which are larger than
wavelength (1) of light. Angle of scattering is
dependent on size of the particles and thus for large
particles, light scatters from edge of particle. Thus
intensity of light scattered at different angles can help to
determine relative amount of different size particles. In
LALLS detector scattered light is measured at very small
angle to incident light (virtually 0). This detector
provides sensitive, simple and accurate molecular weight
measurement for large molecules. Schematic diagram of
LALLS detector is shown in Fig. 4 (A)27.
Multiple angle laser light scattering (malls) detector
In MALLS detector scattering measurements are made at
a number of different angles, none of which are close to
incident light. Variation of scattered intensity depends
upon size, shape, material, orientation, and internal
structure of the particle. Schematic diagram of detector is
shown in Fig. 4 (B)28.
AEROSOL BASED DETECTORS
These are generally classified into evaporative light
scattering detector (ELSD), charged aerosol detector
(CAD) and nano quantity aerosol detector (NQAD).
ELSD is based on light scattering principle while CAD
detects charged aerosols and NQAD count particles

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through optical detection. The working of general
aerosol detector can be explained by Fig. 5 (A)29.
Evaporative light scattering detector (ELSD)
This detector is based on three steps such as nebulisation,
evaporation and detection. Eluent from HPLC column is
transformed into a fine mist of droplets, using nebulizer.
Small droplets are selected in order to minimize
background noise. These droplets are allowed to
evaporate using a hot evaporation (drift) tube at a
different temperature. Solutes molecules left as fine
particulate matter are passed through an optical head
designed to measure light scattering30. It provides
universal measurement under isocratic and gradient
conditions. In advance systems such as low temperature
ELSD, evaporation is done at very low temperature. This
unique feature minimizes the thermal decomposition of
analyte. This technology is highly sensitive, efficient and
reproducible and enables detection of semi-volatile and
thermo-sensitive compounds due to detection through
low temperature evaporation and gas supported focusing
within the optical head. Compounds such as caffeine,
Carbohydrates, lipids or triglycerides, surfactants and
polymer blends or copolymers can be analyzed.
Charged aerosol detector (CAD)
Charged aerosol detection is a unique HPLC detection
technology that delivers advanced and better capabilities
of analysis than other detection technologies such as
refractive index, UV, ELS (Evaporative Light
Scattering). It can deliver excellent sensitivity, wide
dynamic range, superior reproducibility, consistent
response and broad applicability31. In CAD technology,
eluent is nebulised with nitrogen gas and dried to
produce a stream of analyte particles which are collided
with a stream of positive ions produced by corona
discharge. This impact produces charged analyte
particles which are detected by an electrometer.
Detection is based on the particle size and mobility of
analyte. A secondary stream of nitrogen becomes
positively charged as it passes a high-voltage corona
wire32. Schematic diagram of detector is shown in Fig. 5
(B).It is applicable for virtually any non-volatile or semivolatile compounds including pharmaceuticals, lipids,
proteins, steroids, surfactants, carbohydrates, polymers,
oligosaccharides and in chemical as well as food
industries. Drugs like ibuprofen, dextrin, amitriptyline,
estradiol, propranol and salicylic acid are analysed by
CAD33.
Nano quantity aerosol detector (NQAD)
A new aerosol-based HPLC detector using condensation
nucleation has been developed to provide high sensitivity
and a wider linear range than existing aerosol-based
HPLC detectors. The NQAD utilizes a water

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condensation particle counter (WCPC) that selectively

grows particles by condensation of water vapour. The
droplets evaporate leaving particles in the form of nonvolatile residue. As level of non-volatile residue
increases the size of particles increases enough to be
detected optically. NQAD converts particle count rate
into a chromatogram output signal34. This detector offers
a wide dynamic range with superior linearity when
compared to other aerosol-based detectors. The detector
is ideal for searching drug impurities, degradation
products and excipients. NQAD is applicable in analysis
of nonvolatile and semi-volatile compounds such as
amino acids, carbohydrates, steroids, caffeine, cations,
cholesterol, amine, artemisinin and dihydroartemisinin,
sulfonic acid and sulfanilamide, saccharin, theophylline
and sucrose35.
CHIRAL DETECTOR
Chiral detector is used for detection of optically active
compounds such as amino acids, sugars, terpenes, and
other compounds containing an asymmetric carbon.
Chiral compounds have strong chirality in the near UVUV region than in the visible to near IR region. There are
two chiral detection techniques, polarimetry or optical
rotary dispersion (ORD) and circular dichroism (CD).
ORD detectors are based on differences in refractive
index and CD detectors differentiate enantiomers by
measuring differences between the absorption of right
and left-handed circularly polarized light.
Polarimetric detectors
Polarimetric detector is a useful instrument for detecting
and measuring chiral molecules in HPLC. Solutions of
such molecules have property of rotating plane-polarized
light in preferred direction and to a specific degree. This
specific property for each chiral molecule is measured by
detector. Amount of optical rotation, , depends on the
number of optically active species through which light
passes, and thus depends on both sample path length and
analyte concentration. Specific rotation, [], provides a
normalized quantity to correct for this dependence, and is
defined as:
(ii)
Where is measured optical rotation in degrees, l is
sample path length in decimeters (dm), and d is density if
the sample is pure liquid, or concentration (g/100ml) if
sample is a solution. Polarimetric detectors are useful in
isolating and identifying compounds crystallized from
various solvents or separated by HPLC. Compounds
such as amino acids, antibiotics, analgesics, diuretics,
vitamins etc and flavours like camphor, orange oil and
lemon oil are examined by this detector36.
Circular dichroism detector
Circular dichroism based detectors differentiate

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enantiomers by measuring differences between the
absorption of right and left-handed circularly polarized
light due to the existence of a chiral chromophore. These
detectors provide sensitive and selective detection of
chiral compounds that have a UV absorption in the 220420 nm range. CD detector also provides a UV signal in
addition to CD signal which allows optical purity
determinations without chiral separation37. Detection is
unaffected by gradient elution or other changes in nonchiral components during elution. Polarimetric or optical
rotation based detectors remain as an alternative for
compounds that do not contain chromophore by allowing
universal detection with reduced sensitivities.
Compounds such as flavanone, sugar, triadimefon, transstilbene oxide, naproanilide , pindolol, indapamide,
ibuprofen and flubiprofen can be examined using this
technique.
PULSED AMPEROMETRIC DETECTOR (PAD)
This technique was developed to remove the poisons
from electrode surface during detection. A three step
waveform system called as pulsed amperometric
detection is applied to electrode approximately once
every second. Waveform system consists of
measurement and recording of current at electrode
followed by surface oxidation to remove foreign particles
and then oxidation stripping. Amperometric detection
utilizes a flowing current to initiate a chemical
conversion of electro-active analyte. PAD is an
important method for separation and detection of polar
aliphatic compounds which have poor detection
properties and require derivatization for optical
measurements. PAD combines Amperometric detection
at a noble metal electrode with positive and negative
potential excursions for on-line cleaning and reactivation
of the electrode. The result is direct detection of
compounds containing amine, alcohol, or sulphur
moieties and carbohydrates38.
CONCLUSION
Thus new technologies like LLSD, CAD, NQAD, PAD,
and chiral detector have been proved to be powerful new
detection methods for HPLC that perhaps comes closest
to achieving true "universality" in a single platform
detection technology. These systems deliver superior
performance than conventional detectors which include
their broad applicability which enables easy detection of
large number and variety of molecules, consistent
response and high sensitivity (nanogram level).
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26. Bruce KG, Karin D, Bruno A. Electrical Conductivity Particle
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#

Types of Detectors

Refractive index Detector

Ultraviolet Detector

Fluorescent Detector

Transport Detector

Electrochemical detector

6
7

Liquid light scattering

Detector

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Table 1. TYPE OF HPLC DETECTORS

Sub type of Detectors
a) Christiansen effect
Detector
a)Variable wavelength UV
absorption Detector

b) Interferometer
Detector
b)Fixed wavelength UV
Detector

a)Single wavelength
excitation fluorescence
Detector
a) Moving wire Detector

b)Multi wavelength
fluorescence Detector

a) Dynamic Detector or
b) Equilibrium Detector
Multi electrode array
Detector
Electric conductivity Detector
a) Low angle laser light
b) Multiple angle laser light
scattering Detector
scattering Detector
Advanced Detectors

Aerosol based Detectors

a) Evaporative light
scattering Detector

10

Chiral Detector

a) Polari metric Detector

11

Figure 1: Typical UV detector

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b) Chain Detector

c)Thermal lens
Detector
c)Multi
wavelength
dispersive UV
Detector
c)Laser induced
fluorescence
Detector
c) Disc Detector

b) Charged aerosol Detector

c) Nano
quantity
aerosol
Detector

b) Circular dichroism
Detector
Pulsed Amperometric Detector

Figure 2: Simple fluorescence detector

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Figure 3: Transport detectors (A) Moving wire detector (B) Chain detector
(A)

(B)

Figure 4: Liquid light scattering detector (A) Low angle light scattering detector (B) multiple angle laser light scattering detector.

(A)

(B)

Figure 5: (A) Aerosol based detector, (B) Corona charged aerosol detector

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