DETECTORS USED IN HIGH PERFORMANCE

LIQUID CHROMATOGRAPHY

PREPARED BY:TANIYA BISWAS

M.PHARM (PHARMACEUTICAL ANALYSIS)

BENGAL SCHOOL OF TECHNOLOGY

DETECTORS

• A chromatography detector is a device used in high performance liquid chromatography (HPLC) to detect components of the mixture being eluted off the chromatography column.

• The detector senses the presence of the individual components as they leave (elute) the column.

• The detectors converts a change in effluents into an electric signal that is recorded by data system.

IDEAL PROPERTIES OF A DETECTOR

The detectors used in HPLC should have following ideal properties:

High sensitivity. 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. Responses independent of mobile phase composition. A temperature range from room temperature to at least 400 0 C

DETECTORS

OF HPLC

The detectors used in HPLC are of majorly two types: 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 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:

Refractive index detectors Evaporating light scattering detectors

CLASSIFICATION OF HPLC DETECTORS

UV/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)

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).

Three Common types of UV/Vis Absorbance Detectors Fixed wavelength detectors Variable wavelength detectors Photodiode array detectors

Fixed Wavelength Detector absorbance of only one given wavelength is monitored by the system at all times (usually 254 nm)Simplest and cheapest of the UV/VIS detectorsLimited in flexibilityLimited in types of compounds that can be monitored

Variable Wavelength Detector a single wavelength is monitored at any given time, but any wavelength in a wide spectral range can be selectedWavelengths vary from 190-900 nm. More expensive, requires more advanced opticsMore versatile, used for a wider range of compoundsMore sensitive due to photomultiplier tube or amplification circuitry

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 wavelengthsgenerated 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.

• 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.

FLUORESCENCE DETECTOR

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.

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.

A Simple Detector Based on the Fresnel Method of Refractive Index Measurement

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

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.

Two electrodes placed in mobile phase each corresponding to one arm of a Wheatstone Bridge Typical Wheatstone Bridge

EVAPORATIVE LIGHT SCATTERING DETECTOR (ELSD)

• 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.

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.

ELSD

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.

INFRARED SPECTROPHOTOMETRIC

DETECTOR

• 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.

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.

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.

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.

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.

• Detectors for high-performance liquid chromatography of lipids with special reference to evaporative light-scattering detection. By- William W. Christie