A Seminar on

HPLC Detectors
1

INDEX
Introduction
Types
Characterization
Absorbance

detector
Fluorescence detector
Refractive index
ELSD
Electrical conductivity detector
Electrochemical detector
2

High

Performanc

eiquid

Chromatogra
phy

Instrumentation
Gradient
Controll
er

Pum
p
Mobile
Phases

Inject
or

Colum
n

Detector

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

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

1982 study
365

Papers
UV absorbance detector------71%
Fluorescence detector------- 15%
Refractive Index---------------- 5.4%
Electrochemical ----------------4.3%
Other measurements----------4.3%

Type of detectors
Bulk

property detectors
Solute property detectors

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

Absorbance detectors
UV/Visible detectors
Solute

property detector
Three types
Fixed wavelength detector
Variable wavelength detector
Diode array detector

10

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

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

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

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

16

Diode array Instrument

mirror

Diode array detector

328 individual detectors

Tungsten
Filament (vis)
slit
slit

cuvette
Deuterium lamp
Filament (UV)

monochromator

17

18

HPLC-UV
HPLC
Pump

Mobile
phase

Sample
loop

6-port
valve

HPLC
column
UV
detector

syringe
Solvent
waste
19

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
20

Contd
In

a more sophisticated detector,

the fluorescence spectrometer
detector, a combination of

Ellipsoidal mirror

Toroidal mirror
Gratings
Beam splitter
Photocell
21

(S1)
Excited state
(S2)
(S3)

excitation

emission

H (fluorescence)

S0
Ground state
Mobile phase

22

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

Contd.
Respond

to the deflection of light

beam caused by differing R.I.

24

25

UV detector and RI detector

RI detector

UV detector

26

27

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

Detectors
UV

Fluorescence

Sensitivity

ng

pg

Detection

selective

RI
g

highly selective

Universal
Temperature
Influence
Gradient

small small

possible

large

possible

30

Electrical Conductivity
Detector
Used

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

31

Electrochemical Detector
Most

sensitive detector
Respond to substances that are
oxidisable or reductable.
3 electrodes are employed

Working electrode

Auxiliary electrode

Reference electrode

32

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/W
eek16.pdf
Beckett A.H., Stenlake J.B. Practical
Pharmaceutical chemistry 4th
ed.,157& 247
33

Contd.
Munson

W.james Part-B vol.-2.

Modern method of
Pharmaceutical analysis Pg-7377/
WWW.Chromatography%20%20Introductory%20theory.htm
Practical HPLC methods vol.2
Harvey David ,Modern analytical
Chemistry pg-584-586
34

Contd.
Levin

S. Dr. Detection
Qualification and types of
detector used in HPLC

35

THANK
YOU
36