Detectors hplc (1)

Detectors hplc

1. A Seminar on HPLC Detectors 1

2. index Introduction Types Characterization Absorbance detector Fluorescence detector Refractive index ELSD Electrical conductivity detector Electrochemical detector 2

3. H P 3 igh erformance L iquid C hromatography

4. Instrumentation 4 Pump Injector Column Detector Mobile Phases Gradient Controller •

5. What is detector An instrument use to detect or observe Sensitive universal detector for HPLC has not devised yet Selection of detector is on the basis of analyte or sample under detection 5

6. Detection in HPLC HPLC Detectors were common laboratory instrument Usually a narrow linear range Must be solvent -compatible, stable etc. Universal--Respond to all analytes Analyte Specific Respond to specific properties of analytes Non-destructive & Destructive 6

7. 1982 study 365 Papers UV absorbance detector------71% Fluorescence detector------- 15% Refractive Index---------------- 5.4% Electrochemical ----------------4.3% Other measurements----------4.3% 7

8. Type of detectors Bulk property detectors Solute property detectors 8

9. Characterization of detector Adequate sensitivity Stability and reproducibility Wide linear dynamic range Short response time Minimum volume detected easily to reduce zero Similarity in response towards all analytes Non-destructive 9

10. Absorbance detectors UV/Visible detectors Solute property detector Three types Fixed wavelength detector Variable wavelength detector Diode array detector 10

11. Properties of Absorbance Detector Typical Z shaped. Flow through cell for absorbance measurements on eluents. Minimize extra column band broadening Pressure less than 600psi. Matched photoelectric detector. 11

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13. Fixed W avelength detector 254nm Higher detection capacity. Hg vapour lamp(discharge lamp) Focus of light through two absorption cells. Volume of cell is kept constant. 13

14. Variable Wavelength Detectors Relatively wide band pass UV-Visible spectrophotometer coupled to a chromatographic system. Offers a wide selection of UV & Visible wavelengths with increased cost. For complete spectrum, eluent flow must be stopped to trap the component of interest in the detector cell. Use of phosphorus convertor 14

15. Diode Array Detector Scanning W avelength Detector Required to obtain a real time spectrum of each solute as it elutes Work in parallel, monitoring all wavelength Xenon lamp Complete development of chromatogram 15

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17. Diode array Instrument 17 cuvette Tungsten Filament (vis) slit Diode array detector 328 individual detectors monochromator Deuterium lamp Filament (UV) slit mirror

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19. HPLC-UV Mobile phase HPLC Pump syringe 6-port valve Sample loop HPLC column UV detector Solvent waste 19

20. Fluorescence Detector Detect those materials which will fluoresce or appropriate derivatisation made to fluorescence. UV lamp provides the excitation radiation. Light is focused by the quartz lens through the cell and another lens situated normal to incident light and focuses the fluorescent light on the photo cell. 20

21. Contd……… In a more sophisticated detector, the fluorescence spectrometer detector, a combination of Ellipsoidal mirror Toroidal mirror Gratings Beam splitter Photocell 21

22. excitation Mobile phase emission (S 1 ) (S 2 ) (S 3 ) （ S ） Ground state Excited state Hν (fluorescence) 22

23. Refractive Index Detector Universal detector Least sensitive Non-ionic compounds monitoring Responds to analytes changing the RI of the mobile phase requires a separate reference flow of mobile phase Absorbance detectors are relatively cheap 23

24. Contd……. Respond to the deflection of light beam caused by differing R.I. 24

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26. UV detector RI detector 26 UV detector and RI detector

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28. ELSD (Evaporative Light Scattering Detector) Universal, destructive Useful for large molecules and wide linear range. Analytes are de-solvated in the detector. Molecules pass through a large cuvette for a UV-VIS instrument. The reduction in light intensity detected (due to scattering by the analytes) is measured. 28

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30. Detectors UV Fluorescence RI Sensitivity ng pg μg Detection selective highly selective Universal Temperature small small large Influence Gradient possible possible impossible elution 30

31. Electrical Conductivity Detector Used in ion-exchange chromatography. Consists of two electrode. bulk property detector, respond to electrolyte in the mobile phase. 31

32. Electrochemical Detector Most sensitive detector Respond to substances that are oxidisable or reductable. 3 electrodes are employed Working electrode Auxiliary electrode Reference electrode 32

33. REFERENCES Chatwal R.Gurdeep, Anand K.Sham, Instrumental method of analysis 2.625-2.626 Williards,Merritt,Dean,Settle Instrumental method of analysis 600-608 http://www.cem.msu.edu/~cem333/Week16.pdf Beckett A.H., Stenlake J.B. Practical Pharmaceutical chemistry 4 th ed.,157& 247 33

34. Contd…. Munson W.james Part-B vol.-2. Modern method of Pharmaceutical analysis Pg-73-77/ WWW.Chromatography%20-%20Introductory%20theory.htm Practical HPLC methods vol.2 Harvey David ,Modern analytical Chemistry pg-584-586 34

35. Contd ……. 35 Levin S. Dr. Detection Qualification and types of detector used in HPLC

36. 36 THANK YOU