1. RIPER
AUTONOMOUS
NAAC &
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SIRO- DSIR
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
2. RIPER
AUTONOMOUS
NAAC &
NBA (UG)
SIRO- DSIR
Raghavendra Institute of Pharmaceutical Education and Research - Autonomous
K.R.Palli Cross, Chiyyedu, Anantapuramu, A. P- 515721 2
Introduction
Principle
Types of detectors
List of Detectors
Conclusion
Reference
Contents:
3. RIPER
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Raghavendra Institute of Pharmaceutical Education and Research - Autonomous
K.R.Palli Cross, Chiyyedu, Anantapuramu, A. P- 515721 3
• 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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Raghavendra Institute of Pharmaceutical Education and Research - Autonomous
K.R.Palli Cross, Chiyyedu, Anantapuramu, A. P- 515721 4
• 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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Raghavendra Institute of Pharmaceutical Education and Research - Autonomous
K.R.Palli Cross, Chiyyedu, Anantapuramu, A. P- 515721 5
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
6. RIPER
AUTONOMOUS
NAAC &
NBA (UG)
SIRO- DSIR
Raghavendra Institute of Pharmaceutical Education and Research - Autonomous
K.R.Palli Cross, Chiyyedu, Anantapuramu, A. P- 515721 6
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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Raghavendra Institute of Pharmaceutical Education and Research - Autonomous
K.R.Palli Cross, Chiyyedu, Anantapuramu, A. P- 515721 7
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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Raghavendra Institute of Pharmaceutical Education and Research - Autonomous
K.R.Palli Cross, Chiyyedu, Anantapuramu, A. P- 515721 8
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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Raghavendra Institute of Pharmaceutical Education and Research - Autonomous
K.R.Palli Cross, Chiyyedu, Anantapuramu, A. P- 515721 9
• 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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Raghavendra Institute of Pharmaceutical Education and Research - Autonomous
K.R.Palli Cross, Chiyyedu, Anantapuramu, A. P- 515721 10
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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K.R.Palli Cross, Chiyyedu, Anantapuramu, A. P- 515721 11
• 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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Raghavendra Institute of Pharmaceutical Education and Research - Autonomous
K.R.Palli Cross, Chiyyedu, Anantapuramu, A. P- 515721 12
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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Raghavendra Institute of Pharmaceutical Education and Research - Autonomous
K.R.Palli Cross, Chiyyedu, Anantapuramu, A. P- 515721 13
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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K.R.Palli Cross, Chiyyedu, Anantapuramu, A. P- 515721 14
Fluorescence Detector
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K.R.Palli Cross, Chiyyedu, Anantapuramu, A. P- 515721 15
• 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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Raghavendra Institute of Pharmaceutical Education and Research - Autonomous
K.R.Palli Cross, Chiyyedu, Anantapuramu, A. P- 515721 17
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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Raghavendra Institute of Pharmaceutical Education and Research - Autonomous
K.R.Palli Cross, Chiyyedu, Anantapuramu, A. P- 515721 18
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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Raghavendra Institute of Pharmaceutical Education and Research - Autonomous
K.R.Palli Cross, Chiyyedu, Anantapuramu, A. P- 515721 19
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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Raghavendra Institute of Pharmaceutical Education and Research - Autonomous
K.R.Palli Cross, Chiyyedu, Anantapuramu, A. P- 515721 20
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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Raghavendra Institute of Pharmaceutical Education and Research - Autonomous
K.R.Palli Cross, Chiyyedu, Anantapuramu, A. P- 515721 21
Mass Spectrometer
22. RIPER
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Raghavendra Institute of Pharmaceutical Education and Research - Autonomous
K.R.Palli Cross, Chiyyedu, Anantapuramu, A. P- 515721 22
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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Raghavendra Institute of Pharmaceutical Education and Research - Autonomous
K.R.Palli Cross, Chiyyedu, Anantapuramu, A. P- 515721 23
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:
24. RIPER
AUTONOMOUS
NAAC &
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SIRO- DSIR
Raghavendra Institute of Pharmaceutical Education and Research - Autonomous
K.R.Palli Cross, Chiyyedu, Anantapuramu, A. P- 515721 24
Optical Rotation Detector
25. RIPER
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Raghavendra Institute of Pharmaceutical Education and Research - Autonomous
K.R.Palli Cross, Chiyyedu, Anantapuramu, A. P- 515721 25
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:
26. RIPER
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Raghavendra Institute of Pharmaceutical Education and Research - Autonomous
K.R.Palli Cross, Chiyyedu, Anantapuramu, A. P- 515721 26
Charged Aerosol Detection
27. RIPER
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SIRO- DSIR
Raghavendra Institute of Pharmaceutical Education and Research - Autonomous
K.R.Palli Cross, Chiyyedu, Anantapuramu, A. P- 515721 27
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:
28. RIPER
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NAAC &
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SIRO- DSIR
Raghavendra Institute of Pharmaceutical Education and Research - Autonomous
K.R.Palli Cross, Chiyyedu, Anantapuramu, A. P- 515721 28
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:
29. RIPER
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NAAC &
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SIRO- DSIR
Raghavendra Institute of Pharmaceutical Education and Research - Autonomous
K.R.Palli Cross, Chiyyedu, Anantapuramu, A. P- 515721 29