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
Liquid
Chromatograph
y

4. Instrumentation
4
Pum
p
Injector
Colum
n Detector
Gradient
Controller
Mobile
Phases
•

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%
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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

12. 12

13. Fixed Wavelength 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 Wavelength 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
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16. 16

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

18. 18

19. HPLC-UV
Mobile
phase
HPLC
Pump
6-port
valve
syringe
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
Excited state
emission
(S1)
(S2)
(S3)
Ground state（S0）
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

25. 25

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

27. 27

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

29. 29

30. Detectors
UV Fluorescence RI
 Sensitivity ng pg μg
 Detection selective highly selective
Universal
Temperature small small large
Influence
 Gradient possible possible
impossible
elution
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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/We
ek16.pdf
 Beckett A.H., Stenlake J.B. Practical
Pharmaceutical chemistry 4th 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
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