HPLC detector types: Principle in their working
HPLC detectors constitute an important accessories of HPLC instrument.
This part of HPLC helps in detection and identification of compounds in the sample injected.
These detectors are designed to have certain properties like
♦ Being inert (non-reactive) to the samples injected and the mobile phases passing through.
♦ Preferably non-destructive to the sample.
♦ Produce quick and quantitative response.
♦ Produce uniform, reliable and reproducible detection and analytic data.
♦ Compatible for all types of compounds under testing.
♦ Should have good sensitivity .i.e able to detect compounds at very low concentration in the ranges below micro-grams, nano-grams etc. as the sample quantity may be lower in many cases.
All types of HPLC detectors comply with most of the above properties.
UV detectors: These detector systems work on the spectroscopy principle. The sample detection depends on absorption of UV ray energy by the sample.The equipment comprises of accessories in order as UV source, grating (for light defraction),sample passing through a tubing exposed to rays, photo cell, charge conductor etc.
When the UV rays emitted by lamp pass through gratings, rays split into different wavelengths. One specific wavelength rays are passed through sample. Some amount of light is absorbed by sample and the unabsorbed rays which fall on photo cell.
These rays on collision on photo cell produces electrons whose current is recorded. This is indicative of nature and quantity of sample. This UV wavelength range of absorption is specific for sample .
These are the HPLC detectors used in general, unless there is requirement for analysis of special compunds.
They are capable to detect very wide range of compounds.
The sensitivity ranges till microgram quantity of estimation.
PDA detectors: These are detectors which follow principle similar to UV detectors but the range of detection extends from UV, visible and to some extent to IR region. Thus the advantages are higher sensitivity and measures the entire absorption range i.e it gives scan of entire spectrum.
HPLC Detector types
Based on the method or principle used in detection the detectors available are UV detectors, Fluorescent detectors, Electrochemical detectors and Photo diode-array detectors (PDA).
Refractive index detectors; These are detectors which measure the change of refractive index of the eluant from the column with respect to pure mobile phase. They have several disadvantages like lack of high sensitivity, non suitability for gradient elution, and also require strict temperature control ±0.001oC to operate at their highest sensitivity.
Principle in UV Floroscent detectors
HPLC Fluorescence detector
In this detector the fluorescence rays emitted by sample after absorbing incident light is measured as a function of quality and quantity of the sample.
The equipment comprises of accessories in order as light source, sample passing through a tubing exposed to rays, grating (for light defraction), photo cell, charge conductor etc.
Xenon arc lamp is used to produce light for excitation of sample molecules. These light rays excite the sample molecules. These excited molecules emit florescence, which pass through gratings. These gratings pass the florescence at specific wavelength to photo cell which is recorded.
The detector is suitable for compounds which can produce florescence.
Some compounds are chemically altered to produce fluorescence by chemical derivatization to estimate by this detector.
These detectors have high precision and sensitivity (with less noise in data).
Compounds are measurable till nanogram quantities.
HPLC Electrochemical detector
This detector is specially suitable to estimate oxidisable & reducible compounds.
The principle is that when compound is either oxidized or reduced, the chemical reaction produces electron flow. This flow is measured as current which is the function of type and quantity of compound.
The system has electrodes like working electrode where oxidation or reduction takes place and reference electrode which acts to subtract conductivity of mobile phase to that of sample.
This electrode is suitable for compounds which can't be assayed by UV detector especially due to their similarities in light absorption properties ex: monoamines.
This detector has super sensitivity which ranges till picograms measurement. So very minute quantity of compounds present in the samples can be measured.
This electrochemical detector produces severe noise or fluctuations in peaks. So it is tedious to work with when compared to other detectors.
As such all types of HPLC detectors are used based on the requirement of labs.
Precautions in selection and use of detector
1. Always select a suitable detector which is well tested and prescribed after proper validation of the said compound by HPLC analysis. This can be known by standard books of reference like Pharmacopoeias of U.S. India, British etc.
The detectors should be checked for their response to commands by the operating software for any external connection problems.
2. The calibration of detectors is to be performed for any new experiment to acheive stability in data and responses.
3. The mobile phase used should be of hplc grade and free from air bubbles, this may give errors in data.
4. The room temperature be preferably constant & low.
The technology of detectors is evolving further by combination of other analytical methods.
For any queries use the comments section to get solutions.
More by this Author
HPLC relies on pressure application on the mobile phase. This pressure should be uniform. Presence of gases can fluctuate the pressure
TLC chromatography or thin layer chromatography is an analytical technique for easy identification of photo-constituents.
Paper chromatography is a type of planar chromatography which can be performed faster and in an easy way. It is useful for detection of components in a mixture.