DETECTORS USED IN HIGH

PERFORMANCE LIQUID
CHROMATOGRAPHY

PREPARED BY:
TANIYA BISWAS
M.PHARM
(PHARMACEUTICAL ANALYSIS)
BENGAL SCHOOL OF TECHNOLOGY
DETECTORS
• A chromatography detector is a device used in high
performance liquid chromatography (HPLC) to detect
components of the mixture being eluted off the
chromatography column.

• The detector senses the presence of the individual

components as they leave (elute) the column.

• The detectors converts a change in effluents into an

electric signal that is recorded by data system.
IDEAL PROPERTIES OF A
DETECTOR
The detectors used in HPLC should have following ideal properties:

 High sensitivity.
 Good stability and reproducibility.
 A linear response to solute.
 Negligible base line noise.
 Should be inexpensive.
 Capable of providing information on the identity of the solute.
 A short response time independent of flow-rate.
 High reliability and ease of operation.
 The detector should be non-destructive.
 Responses independent of mobile phase composition.
 A temperature range from room temperature to at least 400 0 C
DETECTORS
OF
HPLC
The detectors used in HPLC are of majorly two types:
 Selective detectors (solute property) : These detectors respond to a particular
physical or chemical property of the solute, being ideally independent of the
mobile phase. They are as follow:

 Absorbance detectors
 Fluorescence detectors
 Electrochemical detectors
 Mass spectrometric detectors

 Universal detectors (bulk property): measure the difference in some physical

property of the solute in the mobile phase compared to the mobile phase alone.
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. They are as follow:

 Refractive index detectors

 Evaporating light scattering detectors
CLASSIFICATION OF HPLC DETECTORS
UV/VIS
- Fixed Wavelength
- Variable Wavelength
- Diode array (PDA)

Refractive index
-Deflection Detector
-Refractive Detector (Fresnel refractometer)
Fluorescence Detector
Electrochemical Detector
Conductivity Detector
Evaporative light scattering (ELSD)
Mass detector (LC-MS)
IR detector
Optical rotation detector (chiral detectors)
 ULTRAVIOLET/VISIBLE
SPECTROSCOPIC DETECTORS
• Measures the ability of solutes to absorb light at a particular wavelength(s)
in the ultraviolet (UV) or visible (Vis) wavelength range.

• When light of a certain wavelength is directed at a flow cell, the substance

inside the flow cell absorbs the light. As a result, the intensity of the light
that leaves the flow cell is less than that of the light that enters it. An
absorbance detector measures the extent to which the light intensity
decreases (i.e., the absorbance).
Three Common types of UV/Vis Absorbance Detectors
 Fixed wavelength detectors
 Variable wavelength detectors
 Photodiode array detectors

Fixed Wavelength Detector absorbance of only one given

wavelength is monitored by the system at all times (usually 254
nm)
Simplest and cheapest of the UV/VIS detectors
Limited in flexibility
Limited in types of compounds that can be monitored

Variable Wavelength Detector a single wavelength is monitored at any given

time, but any wavelength in a wide spectral range can be selected
Wavelengths vary from 190-900 nm.
More expensive, requires more advanced optics
More versatile, used for a wider range of compounds
More sensitive due to photomultiplier tube or amplification circuitry
Photo Diode Array Detector operates by simultaneously monitoring
absorbance of solutes at several different wavelengths.
Light from the broad emission source such as a deuterium lamp is collimated by an
achromatic lens system so that the total light passes through the detector cell onto a
holographic grating. In this way, the sample is subjected to light of all wavelengths
generated by the lamp. The dispersed light from the grating is allowed to fall on to
a diode array. The array may contain many hundreds of diodes and the output from
each diode is regularly sampled by a computer and stored on a hard disc.
 FLUORESCENCE DETECTOR
• Fluorescence detectors for HPLC are similar in design to the fluorometers and
spectro-fluorometers. The fluorescence detector is a near-ideal detector for those
solutes that exhibit molecular fluorescence.
• Their sensitivity depends on the fluorescence properties of the components in the
elute.
REFRACTIVE INDEX DETECTOR
• Measures the overall ability of the mobile phase and its solutes to refract or
bend light.
• Refractive index detector measures the molecule’s ability to deflect light in a
flowing mobile phase in a flow cell relative to a static mobile phase contained in
a reference cell.
• The amount of defection is proportional to the concentration of the solute in the
mobile phase.
The two chief types of RI detector are as follows.
The deflection refractometer, 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.

The Fresnel refractometer which measures the change in the fractions of

reflected and transmitted light at a glass-liquid interface as the refractive index of
the liquid changes.

A Simple Detector Based on the Fresnel Method of Refractive Index Measurement

ELECTROCHEMICAL DETECTORS
• It is based on the measurement of the current resulting from an oxidation/
reduction reaction of the analyte at a suitable electrode.
• The level of current is directly proportional to the analyte concentration.
• Three electrodes are employed which are:
 Working electrode
 Auxiliary electrode
 Reference electrode
ELECTRICAL CONDUCTIVITY DETECTOR
• Used in analytical applications of ion-exchange chromatography for the
detection of ionic compounds.
• Detector measures the ability of the mobile phase to conduct a current when
placed in a flow-cell between two electrodes.
• Conductivity detectors measures electronic resistance and measured value is
directly proportional to the concentration of ions present in the solution.

Two electrodes placed in mobile phase each

corresponding to one arm of a Wheatstone Bridge Typical Wheatstone Bridge
EVAPORATIVE LIGHT SCATTERING
DETECTOR (ELSD)
Detection is based on the scattering of a beam of light by particles of compound
remaining after evaporation of the mobile phase. It is a universal detector and does
not required a compound to have a chromophore for detection.
• There are three steps involved in detection:
 Nebulization
 Mobile phase evaporation
 Detection

The flow from the column is nebulized with a stream of inert gas (Nebulize
means to convert a liquid into a fine spray or mist). The mobile phase, which
must be volatile, is evaporated, leaving tiny particles of the analytes (you can
see why this detection method will not work with volatile compounds). The
particles are passed through a laser beam and they scatter the laser light. The
scattered light is measured at right angles to the laser beam by a photodiode
detector.
ELSD
 MASS SPECTROMETER AS AN
HPLC DETECTOR
• Liquid chromatography–mass spectrometry (LC-MS, or HPLC-MS) is an
analytical chemistry technique that combines the physical separation
capabilities of liquid chromatography (or HPLC) with the mass analysis
capabilities of mass spectrometry (MS).
• It is a method that combines separation power of HPLC with detection power
of Mass spectrometry.
• Mass spectrometry (MS) is a powerful analytical tool that can supply both
structural information about compounds and quantitative data relating to mass.
 INFRARED SPECTROPHOTOMETRIC
DETECTOR
Infrared (IR) detectors have been used to a limited extent only for the analysis of
non-polar lipids, with the specific absorbance for the carbonyl function between
1650 and 1860 cm-1 (or at about 5.75 microns) being the spectral region of value.

• The wavelength scanning is provided by semicircular filter wedges, the

wavelength range being from 4000-690 cm-1 .
• It is not very sensitive and have serious drawback that most mobile phase
solvents absorb strongly in the IR region.
 CHIRAL DETECTORS
• Chiral detectors are used for detection of optically active compounds such as
amino acids, sugars, terpenes and other compounds containing an asymmetric
carbon.
• There are two chiral detection techniques, polarimetry or optical rotary
dispersion (ORD) and circular dichorism (CD).
• ORD detectors are based on differences in refractive index and CD detectors
differentiate enantiomers by measuring differences between the absorption of
light and left-handed circularly polarized light due to existence of a chiral
chromophores.
 CONCLUSION
Detector is the key element which is present in any
device that is used for the identification and
estimation of any compound. It detects in a faster rate
i.e., within some fractions of second. Hence a
detector is considered as a brain of an instrument.
Without the help of an detector, no one would be
able to analyze any compound.
 REFERENCES
• Handbook of pharmaceutical analysis- by Lena Ohannesian and
Antony J. Streeter.

• Liquid chromatography detectors – by Raymond P. W. Scott.

• Chiral chromatography- by T. E. Beesley and R. P. W. Scott.

• High performance liquid chromatography detectors- Review.

International research journal of pharmacy.

• Detectors for high-performance liquid chromatography of lipids

with special reference to evaporative light-scattering detection.
By- William W. Christie