DETECTORS IN HPLC

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Raghavendra Institute of Pharmaceutical Education and Research - Autonomous
K.R.Palli Cross, Chiyyedu, Anantapuramu, A. P- 515721 1
DETECTORS IN HPLC
A seminar as a part of curricular
requirement for I year M. Pharm I semester
Presented by
K. Tarun (20L81S0805)
Dept. of Industrial Pharmacy
Under the guidance/Mentorship of
Dr. Hindustan Abdul Ahad M. Pharm, Ph.D, FAGE
Life member of IPA & APTI
Professor & Head, Dept. of Industrial Pharmacy

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Introduction
Principle
Types of detectors
List of Detectors
Conclusion
Reference
Contents:

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• A chromatography detector is a device to detect components of the mixture being
eluted off the chromatography column.
Properties:
1. Low drift and noise level.
2. High sensitivity.
3. Fast response.
4. Wide linear dynamic range.
5. Low dead volume (minimal peak broadening).
6. It should be non-destructive.
Introduction:

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• HPLC is based on adsorption as well as partition chromatography.
• It is depends on the nature of stationary phase.
• Determination of volatile and non-volatile compounds.
Principle:
Stationary
phase
Solid Adsorption
Stationary
phase
Liquid Partition

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Types of detectors
Bulk Property
Detectors:
Refractive
Index
Detector
Evaporative
Light
Scattering
Detector
Solute
Property
Detectors
Electrochemi
cal Detector
UV-
visible
Detector
Mass
Detector
(LC-MS)
Fluoresc
ence
Detector

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1. UV-visible Detector
a. Fixed / Single Wavelength Detector
b. Variable Wavelength Detector
c. Photodiode Array Detector / DAD
2. Refractive Index Detector
3. Fluorescence Detector
4. Electrochemical Detector
5. Conductivity Detector / Potentiometry
6. Evaporative Light Scattering Detector (ELSD)
7. Mass Detector (LC-MS)
8. IR Detector
9. Optical Rotation Detector (ORD) / Chiral Detectors
10.Corona Discharge Detectors
List of detectors:

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1. UV-Visible Detector:
• They give specific response to the class of compounds or particular compounds
depending upon the functional group of eluting molecules.
• When the light of a certain wavelength is directed at a flow cell, the substance
inside the flow cell absorbs light.
• These detectors are different types:

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 Such type of detectors does not allow change in wavelength of the radiation.
 Absorbance of only one given wavelength is monitored by the system at all times
(usually 254nm).
 Limited in flexibility.
 Simplest & Cheapest of the UV/Visible.
a. Fixed Wavelength Detector:

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• These can be adjusted to work on any wavelength over full UV-Visible region.
• The wavelength can be selected at 3nm or less.
• More expensive.
• More versatile.
b. Variable Wavelength Detector:

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Light from the broad emission source such as deuterium lamp.
It is collimated by an achromatic lens system.
So that the total light passes through the detector cell onto a holographic grating.
The dispersed light from the grating is allowed to fall on to a diode array.
c. Photodiode Array Detector:

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• 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 detection is proportional to the concentration of the solute in the
mobile phase.
2. Refractive Index Detector:

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o The are different types are:
Deflection refractometer
Fresnel refractometer
Interferometer detectors
o Mostly used for detection of non-ionic compounds that neither fluorescence nor
absorb in the UV region.
o Draw backs:
Less sensitive
Temperature control
Less suitability to gradient elution.

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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.
The system is easy to operate and relatively stable.
Sensitivity depends on the fluorescence properties of the components in the elute.
3. Fluorescence Detector:

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Fluorescence Detector

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• There are 3 types of fluorescence detectors:
Fluorescence
Detector Single Wavelength Excitation
Detector
Multi Wavelength Fluorescence
Detector
Laser Induced Fluorescence
Detector (LIFD)

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• Usually measure the current associated with the oxidation or reduction of solutes.
• They are sensitive to changes in the flow rate or composition of the eluent.
• The level of current is directly proportional to the analyte concentration.
• Three electrodes are employed which are:
Working electrode
Auxiliary electrode
Reference electrode
4. Electrochemical Detector:

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It is used in analytical applications for the detection of ionic compounds.
Measures the ability of the mobile phase to conduct a current when placed in a
flow-cell between 2 electrodes.
Measured Value is directly proportional to the concentration of ions present in the
solution.
5. Conductivity Detector:

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It is useful for large molecular weight molecules like surfactants, lipids and sugar.
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
Does not required a compound to have a chromophore for detection.
There are 3 steps involved in detection:
o Nebulization
o Mobile phase evaporation
o Detection
6. Evaporative Light Scattering Detector (ELSD):

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The mobile phase which must be volatile.
The flow from the column is nebulized with a stream of inert gas.
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.

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The detection by mass spectrometers is based on molecular fragmentation by
electric fields.
Separation is based on the mass to charge ration of the fragmented molecule.
It is a method that combines separation power of HPLC with detection power of
mass spectrometry.
They show high selectivity & sensitivity.
In LC-MS technique advantage of resolution and sensitivity.
The analyte for mass spectrometer must be in ionic form.
7. Mass Spectrometer:

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Mass Spectrometer

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Infrared detectors are chosen on the basis of property of compound to absorb
infrared light.
It is a sophisticated instrument and requires Cells or windows made of NaCl or
CaF2.
The wavelength scanning is provided by semicircular filter wedges, the wavelength
range from 4000-690 cm-1.
The detector is suitable for polymer analysis.
Not very sensitive
Mobile phase solvents absorb strongly in the IR region.
8. IR Detector:

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o Used for detection of optically active compounds such as amino acids, sugars &
terpenes containing an asymmetric carbon.
o There are 2 chiral detection techniques:
Polarimetry / optical rotary dispersion (ORD)
Circular dichroism (CD)
o ORD detectors are based on differences in refractive index.
o CD differentiate enantiomers by measuring differences between the absorption of
light & left handed circularly polarized light due to existence of a chiral
chromophore.
9. Optical Rotation Detector (ORD) / Chiral
Detectors:

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Optical Rotation Detector

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Other name for this is Charged Aerosol Detection (CAD).
It is a newer and unique 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 analytes then are further transferred to a collector where it is measured by a
highly sensitive electrometer.
10. Corona Discharge Detector:

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Charged Aerosol Detection

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The work of the detector is to detect and give the evidence to the
recorder which shows it in a form of a chromatogram. It senses at a more rapidly.
Each compound has its properties that are not the same as one another, thus arises a
need to have different detectors for different compounds.
Conclusion:

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1. Swartz M. HPLC detectors: a brief review. Journal of Liquid Chromatography &
Related Technologies. 2010 Jul 13;33(9-12):1130-50.
2. Sunil A, Anju G, Rajat V. HPLC Detectors, Their Types and Use: A Review.
Organic & Medicinal Chemistry International Journal. 2018;6(5):143-6.
3. Sims JL, Carreira JA, Bragg RA. Optical rotation detection for reversed phase
HPLC: Investigation of solvent effects. Chirality. 1997;9(2):122-5.
4. Sagliano Jr N, Hartwick RA. Micro-HPLC detectors: a review. Journal of
chromatographic science. 1986 Nov 1;24(11):506-12.
References:

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DETECTORS IN HPLC

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