A Seminar on

HPLC Detectors

1

INDEX

Introduction

Types

Characterization

Absorbance detector

Fluorescence detector

Refractive index

ELSD

Electrical conductivity detector

Electrochemical detector

2

H

P

3

igh

erformance

L iquid

Chromatograph

y

Instrumentation

4

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

5

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%

7

Type of detectors

Bulk property detectors

Solute property detectors

8

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

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 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

15

16

Diode array Instrument

17

cuvette

Tungsten

Filament (vis)

slit

Diode array detector

328 individual detectors

monochromator

Deuterium lamp

Filament (UV)

slit

mirror

18

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

21

excitation

Mobile phase

emission

(S1)

(S2)

(S3)

（S0）Ground state

Excited state

Hν (fluorescence)

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

RI detector

26

UV detector and RI detector

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 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.

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/Week16.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-73-77/

WWW.Chromatography%20-

%20Introductory%20theory.htm

Practical HPLC methods vol.2

Harvey David ,Modern analytical

Chemistry pg-584-586

34

Contd…….

35

Levin S. Dr. Detection Qualification

and types of detector used in HPLC

36

THANK YOU