detectors used In HPLC

1. Presented by Muhammad Aashir
Roll # CY-03

2. DETECTORS
• A chromatography detector is a device used
in high-performance liquid chromatography
(HPLC) to separate the components of a
mixture from a chromatography column.
• The detector senses the presence of
individual components as they leave the
column.
• Detectors convert changes in waste into an
electrical signal that is recorded by a data
system.

3. IDEAL PROPERTIES OF A DETECTOR
 The detectors used in HPLC should have following
ideal properties
 Good stability and reproducibility.
 A linear response to solute.
 Negligible base line noise.
 Should be inexpensive.
 Capable of providing information on the identity of the
solute.
 A short response time independent of flow-rate.
 High reliability and ease of operation.
 The detector should be non-destructive.

4. HPLC Detectors

5. THE DETECTORS USED IN HPLC ARE OF
MAJORLY TWO TYPES:
1. Selective detectors (solute property): These detectors
respond to a particular physical or chemical property
of the solute, being ideally independent of the mobile
phase. They are as follow:
 ✔Absorbance detectors
 ✔ Fluorescence detectors
 ✔ Electrochemical detectors Mass spectrometric
2. Detectors Universal detectors (bulk property):
measure the difference in some physical property of the
solute in the mobile phase compared to the mobile phase
alone. They are generally universal in application but tend
to have poor sensitivity and limited range. Such detectors
are usually affected by even small changes in the mobile-
phase composition which precludes the use of techniques
such as gradient elution. They are as follow:

6. CLASSIFICATION OF HPLC
DETECTORS
 QUV/VIS-
 Fixed Wavelength-
 Variable Wavelength-
 Diode array (PDA)
 Refractive index
 Deflection Detector
 Refractive Detector (Fresnel refractometer)
 Fluorescence Detector
 Electrochemical Detector
 Conductivity Detector
 Evaporative light scattering (ELSD)
 Mass detector (LC-MS)
 IR detector
 Optical rotation detector (chiral detectors)

7. ULTRAVIOLET/VISIBLE SPECTROSCOPIC
DETECTORS
 Measures the ability of solutes to absorb light at a particular
wavelength(s) in the ultraviolet (UV) or visible (Vis) wavelength
range.
 When light of a certain wavelength is directed at a flow cell,
the substance inside the flow cell absorbs the light. As a result,
the intensity of the light that leaves the flow cell is less than
that of the light that enters it. An absorbance detector
measures the extent to which the light intensity decreases
(i.e., the absorbance).

8. THREE COMMON TYPES OF UV/VIS
ABSORBANCE DETECTORS
 Fixed Wavelength Detector
 absorbance of only one given wavelength is
monitored by the system at all times (usually
254nm)
 Simplest and cheapest of the UV/VIS detectors
 Limited in flexibility
 Limited in types of compounds that can be
monitored
 Variable Wavelength Detector
 A single wavelength is monitored at given time, but
any wavelength in a wide spectral range can be
selected
 Wavelength vary from 190-900nm
 More expensive, require more advanced optics.
 More versatile, use for wider range of compound
 More sensitive due to photomultiplier tube.

9. PHOTO DIODE ARRAY DETECTOR
 operates by simultaneously monitoring absorbance of solutes at several
different wavelengths. Light from the broad emission source such as a
deuterium lamp is collimated by an achromatic lens system so that the total
light passes through the detector cell onto a holographic grating. In this way,
the sample is subjected to light of all wavelengths generated by the lamp.
The dispersed light from the grating is allowed to fall on to a diode array. The
array may contain many hundreds of diodes and the output from each diode
is regularly sampled by a computer and stored on a hard disc.

10. FLUORESCENCE DETECTOR
 Fluorescence detectors for HPLC are similar in design to
the fluorometers and spectro-fluorometers. The
fluorescence detector is a near-ideal detector for those
solutes that exhibit molecular fluorescence. Their
sensitivity depends on the fluorescence properties of the
components in the elute.

11. REFRACTIVE INDEX DETECTOR
 Measures the overall ability of the mobile phase and its solutes to
refract or bend light.
 Refractive index detector measures the molecule's ability to deflect
light in a flowing mobile phase in a flow cell relative to a static mobile
phase contained in a reference cell.
 The amount of defection is proportional to the concentration of the
solute in the mobile phase.

12.  The Two chief types of RI detector are as follows.
The deflection refractometer, which measures the deflection of a
beam of monochromatic light by a double prism in which the reference
and sample cells are separated by a diagonal glass divide.
The Fresnel refractometer which measures the change in the
fractions of reflected and transmitted light at a glass-liquid interface as
the refractive index of the liquid changes.

13. ELECTROCHEMICAL DETECTORS
 It is based on the measurement of the current resulting from an
oxidation/ reduction reaction of the analyte at a suitable electrode.
 The level of current is directly proportional to the analyte
concentration.
 Three electrodes are employed which are:
 Working electrode
 Auxiliary electrode
 Reference electrode

14. ELECTRICAL CONDUCTIVITY DETECTOR
 Used in analytical applications of ion-exchange chromatography for
the detection of ionic compounds.
 Detector measures the ability of the mobile phase to conduct a
current when placed in a flow-cell between two electrodes.
 Conductivity detectors measures electronic resistance and measured
value is directly proportional to the concentration of ions present in
the solution

15. EVAPORATIVE LIGHT SCATTERING
DETECTOR (ELSD)
Detection is based on the scattering of a beam of light by
particles of compound remaining after evaporation of the mobile
phase. It is a universal detector and does not required a
compound to have a chromophore for detection.
There are three steps involved in detection:
➤ Nebulization
➤ Mobile phase evaporation
➤ Detection
The flow from the column is nebulized with a stream of inert gas
(Nebulize means to convert a liquid into a fine spray or mist).
The mobile phase, which must be volatile, is evaporated, leaving
tiny particles of the analytes (you can see why this detection
method will not work with volatile compounds). The particles are
passed through a laser beam and they scatter the laser light.
The scattered light is measured at right angles to the laser beam
by a photodiode detector

16. ELSD

17. MASS SPECTROMETER AS AN HPLC
DETECTOR
 Liquid chromatography-mass spectrometry (LC-MS, or HPLC-MS) is
an analytical chemistry technique that combines the physical
separation capabilities of liquid chromatography (or HPLC) with the
mass analysis capabilities of mass spectrometry (MS).
 It is a method that combines separation power of HPLC with
detection power of Mass spectrometry.
 Mass spectrometry (MS) is a powerful analytical tool that can supply
both structural information about compounds and quantitative data
relating to mass.

18. INFRARED
SPECTROPHOTOMETRICDETECTOR
 Infrared (IR) detectors have been used to a limited extent only for the
analysis of non-polar lipids, with the specific absorbance for the
carbonyl function between 1650 and 1860 cm-1 (or at about 5.75
microns) being the spectral region of value.
 The wavelength scanning is provided by semicircular filter wedges,
the wavelength range being from 4000-690 cm-1.
 It is not very sensitive and have serious drawback that most mobile
phase solvents absorb strongly in the IR region.

19. CHIRAL DETECTORS
 Chiral detectors are used for detection of optically active compounds
such as amino acids, sugars, terpenes and other compounds
containing an asymmetric carbon.
 There are two chiral detection techniques, polarimetry or optical
rotary dispersion (ORD) and circular dichorism (CD).
 ORD detectors are based on differences in refractive index and CD
detectors differentiate enantiomers by measuring differences
between the absorption of light and left-handed circularly polarized
light due to existence of a chiral chromophores.

20. CONCLUSION
 Detector is the key element which is present in any
device that is used for the identification and estimation of
any compound. It detects in a faster rate i.e., within some
fractions of second. Hence a detector is considered as a
brain of an instrument. Without the help of an detector, no
one would be able to analyze any compound.

21. REFERENCES
 Handbook of pharmaceutical analysis- by Lena
Ohannesian and Antony J. Streeter.
 Liquid chromatography detectors - by Raymond P.
W. Scott.
 Chiral chromatography- by T. E. Beesley and R. P.
W. Scott.
 High performance liquid chromatography detectors-
Review. International research journal of
pharmacy."