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Detector of HPLC
- 1. Detector of HPLC PRESENT BY: Khatereh zarkesh Ph.D student of pharmaceutics at SUMS
- 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
- 7. Classification of HPLC detectors UV/VIS Fixed wavelength Variable wavelength Diod array (PDA) Refractive index Reflection detectors Refractive detectors (Fresnel Refractometer) Fluorescence detector Electrochemical detector
- 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)
- 11. Variable wavelength detector Wavelength vary from 190-900
- 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.
- 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.
- 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.
- 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.