Detectors of HPLC

Detectors Used in HPLC, desirable features, detector performance criteria, uv-visible detector, photo diode array detector, fluorescence detector, refractive index detector, elsd detector, ms detector

1. DETECTORS OF HPLC Presented by: Aditya Sharma M.S. (Pharm) Pharmaceutical Analysis NIPER Guwahati

2. DETECTORS Purpose : The purpose of the detector in an HPLC system is to identify the presence of certain compounds of interest in the eluent from the HPLC column The analyte undergoes physico -chemical interaction. The detector provides the electrical output signal which is proportional to the quantity of analyte within detector at a given moment. Integrator quantifies the amount of analyte present. Two broad classes of Detectors:- Bulk property detectors : Respond to any change in certain physical property of the HPLC eluent which is common to both analyte and mobile phase and its magnitude is altered by the presence of analyte. Example: RI, ELSD Solute property detector : They respond to certain unique properties of the analyte such as fluorescence or electrochemical property

3. Desirable features  Should be equally sensitive to all eluted peaks Should not be affected by changes in temperature Should not be affected by changes in mobile phase composition Should be able to monitor small amounts of analytes i.e. highly sensitive Should not contribute to band broadening, hence the flow cell should be small Easy to operate and cheap

4. Detector Performance Criteria Detector performance characterized by sensitivity, noise, limit of detection, linear and dynamic range and detection volume. Noise : is a random short-term variation in the output signal. Limits the size of signal and thus amount of analyte. Short term noise : occurs at sub second level, much more rapidly than variations in response to elution of an analyte peak. Fuzzy baseline. Long term noise : comprises random variations in the output signal with a time scale similar to peaks due to elution of compounds from HPLC column and leads to confusion as to the identity of the peak.

5.  Sources of Noise: Shot noise: electrical optical devices. Flicker noise: lamps and lasers Pulsation of the pump pistons Dirty flow cell Detector noise increases with the age of the instrument. Drift: Drift is very slow variation in the detector output signal, occurring on a longer time scale of minutes or hours. Makes integration difficult.

6. Sensitivity: LOD: The signal should be 3 to 5 times of the noise Sensitivity: LOQ: The signal should be 10 to 12 times of the noise Linearity and dynamic range: Linear range is the range of analyte concentrations over which the detector output signal varies in linear proportions to the analyte concentration. The region where a change in analyte concentration produces a change in output signal is the dynamic range, although without this necessarily being a linear relationship. Band broadening : It is due to two main detector parameters Cell volume: Too small a cell volume impairs the sensitivity (as a certain amount of product is needed to produce any signal). A standard cell volume is 8 – 10µl. Detection time constant: Time constant can be defined as the minimum time required by a system to reach 98% of its full scale value.

7. Types of Detectors:

8. UV / VISIBLE DETECTOR  T hey are the work horses of HPLC and constitute about 70% of all detection system Sensitive, wide linear range, relatively unaffected by temperature fluctuations and is also suitable for gradient elution and relatively inexpensive. It records compounds that absorb UV or visible light. Absorption occurs above 200 nm if the molecule has at least:. A double bond adjacent to an atom with lone pair of electron (X=Y-Z:) Bromine, Iodine or sulphur Ketone group or a nitro group Two conjugated double bonds (X=Y-Z=A) An aromatic ring

9.  Lamps or Light source : Fixed wavelength or variable wavelength Fixed wavelength : Mercury Lamps : emit at 253.7 nm Cadmium Lamps: emit at 229 nm Zinc Lamps: emit at 214 nm Variable wavelength : Deuterium Lamp: emit continuous UV spectrum up to 340 nm Tungsten Lamp: emit in the near UV & visible ranges (340 to 800nm)

10. PHOTODIODE ARRAY DETECTOR It is UV technique but produces a 3D output i.e. On X axis time, Y-axis absorbance and Z axis wavelength Chromatogram: On X axis time and Y-axis absorbance Spectrum: On X axis wavelength and Y-axis absorbance Allows simultaneous collection of chromatograms at different wavelengths during a single run. Main application is in the field of method development Used for peak purity testing.

11. FLUORESCENCE DETECTORS When light is absorbed by a molecule and an electron is promoted to a higher energy state and while coming back to its ground state it loses energy by emission of a photon, this process being called fluorescence. Design: Light from the lamp (D2 or a xenon) passes through an excitation filter, which provides essentially monochromatic light of the desired wavelength for excitation of sample molecules. This light passes through the flow cell causing sample molecules to fluoresce at a higher wavelength than that used for excitation. Resultant light passes onto the detector for quantitation of the emission signal. As the fluorescent emission is very low, photomultiplier rather than photodiode is used.

12. ELECTROCHEMICAL DETECTORS  Electrochemical detectors are based on amperometric measurements. Also called amperometric detectors. Principle of operation is the oxidation or reduction of analyte in a flow -through electrolysis cell with a constant applied electrical potential. e.g. oxidation of hydroquinone Detectors where only a low percentage of the analyte is reacted- amperometric. All the analyte reacts- coulometric . Catecholamines , phenols,aromatic amines are easily oxidized. Quinones and some nitro-aromatics are easily reduced.

13.  The detector cell in which the electrochemical reaction takes place has three electrodes namely working, auxiliary and reference. Electrolysis of analyte occurs at working electrode and auxiliary electrode supplies the current. Reference electrode measures the solution potential. Potentiostat is used to control the voltage at auxiliary electrode in order to maintain the constant potential difference between the solution and the working electrode. Sensitivity is high. Low detection limits can be achieved.

14. REFRACTIVE INDEX DETECTOR The velocity of electromagnetic wave varies as it passes from one medium to another. The ratio of its velocity in vacuum to that in given medium is known as RI of the medium. The RI detector measures the change in RI of the mobile phase due to the presence of dissolved analyte. RI detector is very useful for analysis of sugars which have poor UV absorbance or fluorescence measurements without chemical derivatization. RI detector can not be used in case the solute and mobile phase has the same RI.

15. Disadvantages

16. EVAPORATIVE LIGHT SCATTERING DETECTOR (ELSD) A universal detector. The effluent from column is nebulized and evaporated as it passes through the drift tube. Analyte particles are detected as they pass through light scattering cell. Non-volatile analytes and volatile mobile phase. Compatibility with gradient elution possible. Useful for impurity analyses.

17. MASS SPECTROMETER (MS) DETECTOR LC-MS is a hyphenated technique, combining separation power of HPLC with the detection power of MS. For using MS there is a need for an interface that will eliminate the solvent and generate gas phase ions, for MS. MS detector has three distinct parts: Ion source Analyzer Detector For all MS techniques, an analyte is first ionized in the ion source since the MS can only detect charged species. Based on their mass to charge (m/z) ratio the ions are separated and focused in the mass-analyzer. Mass Analyzers: There are many types of mass analyzers in MS. E.g. Triple Quadrupole