detectors hplc (1)
TRANSCRIPT
A Seminar on
HPLC Detectors
1
INDEX
Introduction
Types
Characterization
Absorbance detector
Fluorescence detector
Refractive index
ELSD
Electrical conductivity detector
Electrochemical detector
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H
P
3
igh
erformance
L iquid
Chromatograph
y
Instrumentation
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Pum
pInjector
Colum
n Detector
Mobile
Phases
Gradient
Controller
•
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
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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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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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Type of detectors
Bulk property detectors
Solute property detectors
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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-destructive9
Absorbance detectors
UV/Visible detectors Solute property detector
Three types
Fixed wavelength detector
Variable wavelength detector
Diode array detector
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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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12
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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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 convertor14
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
Diode array Instrument
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cuvette
Tungsten
Filament (vis)
slit
Diode array detector
328 individual detectors
monochromator
Deuterium lamp
Filament (UV)
slit
mirror
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HPLC-UV
Mobile
phase
HPLC
Pump
syringe
6-port
valveSample
loop
HPLC
column
UV
detector
Solvent
waste19
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
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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excitation
Mobile phase
emission
(S1)
(S2)
(S3)
(S0)Ground state
Excited state
Hν (fluorescence)
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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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Contd…….
Respond to the deflection of light
beam caused by differing R.I.
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25
UV detector
RI detector
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UV detector and RI detector
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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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Detectors
UV Fluorescence RI
Sensitivity ng pg μg
Detection selective highly selective
Universal
Temperature small small large
Influence
Gradient possible possible
impossible
elution30
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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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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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 4th ed.,157& 247
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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
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Contd…….
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Levin S. Dr. Detection Qualification
and types of detector used in HPLC
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