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
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8. Type of detectors
Bulk property detectors
Solute property detectors
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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
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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.
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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.
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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
15
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.
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21. Contd………
In a more sophisticated detector, the
fluorescence spectrometer detector, a
combination of
Ellipsoidal mirror
Toroidal mirror
Gratings
Beam splitter
Photocell
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22. excitation
Mobile phase
Excited state
emission
(S1)
(S2)
(S3)
Ground state（S0）
Hν (fluorescence)
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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
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24. Contd…….
Respond to the deflection of light
beam caused by differing R.I.
24
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.
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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
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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.
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32. Electrochemical Detector
Most sensitive detector
Respond to substances that are
oxidisable or reductable.
3 electrodes are employed
Working electrode
Auxiliary electrode
Reference electrode
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