This document discusses different types of detectors used in high performance liquid chromatography (HPLC). It describes that detectors sense the individual components as they leave the chromatography column. There are two main types of detectors: selective detectors which respond to a particular property of the solute, and universal detectors which measure differences in a bulk property between the solute and mobile phase. Examples of common detectors discussed are UV-visible, fluorescence, refractive index, light scattering, conductivity, electrochemical, mass spectrometry, and infrared detectors. Each has distinct operating principles and applications for qualitative and quantitative analysis in HPLC.
3. A chromatoghraphy is a device used in high performance liquid chromatography
(HPLC) to detect component of the mixure being eluted off the chromatography
column.
The detector sense the presence of the individual components as they leave to column.
4. Ideal properties of a detector
High sensitivity and reproducible, predictable response
.Respond to all solutes, or have predictable specificity .
Wide linear dynamic range; Response that increases linearly with the amount of solute .
Response unaffected by changes in temperature and mobile phase flow .
Respond independently of the mobile phase .
Reliable and convenient to use .
Nondestructive of the solute .
Provide qualitative and quantitative information on the detected peak .
Fast response
5. The detectors used in HPLC are of majorly two types:
Selective detectors (solute property) : respond to a particular physical or chemical property of
the solute
Absorbance detectors
Fluorenscence detectors
Electrochemical detectors
Mass spectrometric detectors
6. The detectors used in HPLC are of majorly two
types:
Universal detectors(bulk property):measure the difference in some physical property of
the solute in the mobile phase compare to the mobile phase alone.
Refractive index detectors
Evaporating light scattering detectors
8. Classification of HPLC detectors
Conductivity detector
Evaporative light scattering detector (ELSD)
IR detector
Mass detector ( LC-MS)
Optical rotation detector (Chiral detector)
9. Uv detectors
The UV-visible absorbance detector is the most common HPLC detector
in use today since many compounds of interest absorb in the UV
(or visible) region (from 190–600 nm). Sample concentration, output as
absorbance, is determined by the fraction of light transmitted through
the detector cell by Beer’s Law:
A= logً I0/I=Ebc
10. Fixed wavelength detector
Absorbance of only one given wavelength is monitored by system at all times(254nm)
12. DIOD array detector
Operates by simultaneously monitoring absorbance of solutes at several different
wavelengths.
13. Fluorescence detector
Fluorescence detectors (FL) measure the optical emission of light by solute molecules
after they have been excited at a higher energy wavelength and can be very sensitive for
compounds that have native fluorescence or that can be made to fluoresce through
derivatization.
15. REFRACTIVE INDEX DETECTION
RI detectors measure the difference in optical refractive index between mobile phase
and the sample; no chromophore on the solute molecule is required.
The most common RI detector design is the deflection refractometer where the light
from a tungsten source lamp is directed through a pair of wedge-shaped flow cells,
(reference and sample).
17. LIGHT SCATTERING DETECTORS
The ELSD works on the principle of evaporation (nebulization) of the mobile phase
followed by measurement of the light scattered by the resulting particles. The column
effluent is nebulized in a stream of nitrogen or air carrier gas in a heated drift tube and
any nonvolatile particles are left suspended in the gas stream.
The particles are passed through a laser beam and they scatter the laser light.
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19. CONDUCTIVITY DETECTION
Conductivity detectors are the detector of choice for ion chromatography or ion
exchange separations when the analyte does not have a UV chromophore. In a
conductivity detector, the resistance (or strictly the impedance) between two electrodes
in the flow cell is measured.
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21. Optical rotation detector (Chiral detector)
Chiral detector are used for detection of optically active compounds such as amino
acid,sugars,terpenes and another compounds containing asymmetric carbon.
There are two chiral detection techniquse,polarimetry or optical rotary dispersion(ORD) and
circular dichorism(CD).
ORD detector are based on differences in refactive index and CD detectors differentiate
enantiomers by measuring difference between and absorption of light –handed circularly
polarized light due to existence a chiral chromophores.
22. ELECTROCHEMICAL DETECTORS
(EC) detector is one of the most sensitive and selective HPLC detectors available. EC detectors
require the use of electrically conductive HPLC mobile phases (buffers suffice) and, when
properly used and maintained, are the standard bearer when it comes to response levels for the
HPLC analysis of compounds such as catecholamines and neurotransmitters.
Three electrod are employed are which :
working electrods
Auxiliary electrods
reference electrods
24. MASS SPECTROMETER
Liquid chromatoghraphy-mass spectrometry(LC-MS) is as analytical technique that
combines the physical separation capabilities of liquid chromatograghy with the mass
analysis capabilities of mass spectrometry.
Mass spectrometry is a powerful analytical tool that can supply both structural
information about compounds and quantitative data relating to mass.
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26. INFERAED DETECTOR
IR detector have been used to a limited extent only for the analysis of
non-polar lipids,with specific absorbance for the carbonyl function between 1650 and
1860 being the spectral region of value.
