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.

Waters-ECD detector
Waters-ECD detector

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.

Thanks...............

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Comments 36 comments

munirahmadmughal profile image

munirahmadmughal 5 years ago from Lahore, Pakistan.

"HPLC detector types"

The hub is informative and also thought provoking that research on rays has shown how great is the system set by Creator of all things that it is working the same way everywhere. The more the mankind is searcing and

researching the hidden treasures of the Creator are generously unfloded for the mankind. The mind that explores and re-explores the secrets of nature is also a great gift of God Almighty. The precision and accuracy kept in every particle is itself a glorifcation of the praises of the Creator of all.

Thanks giving is therefore necessary at every moment that passes.

The science and technology is the other name of reasonable and consistent awareness. Nothing is without wisdom.

May God bless all.


bheem profile image

bheem 5 years ago Author

munirahmadmughal thanks for ur comment. you are right by your view. I would further add that inspite of tremendous research and strive by human brain,for precicion and accuracy, still we are far behind in achieving them.


Tara_in_NE profile image

Tara_in_NE 5 years ago

Hi! I've worked with tons of HPLC's, but I have to say the Shimadzu Prominence HPLC with the PDA detector is by far the best HPLC I have ever worked with, in terms of being superb in terms to separation and of being user friendly.

I love THE INSTANT determination of PEAK PURITY that a PDA can do! And the ability to look at a spectrum from ALL angles. It's awesome!

Do you guys do any IQ, OQ and PQ performance checks on your detectors? If so what are your protocols?

Thanks so much in advance if you would like to share them. Great hub.


bheem profile image

bheem 5 years ago Author

Thanks for your comment Tara.........


arshita verma 5 years ago

The perfect information in few words about detectors


bheem profile image

bheem 5 years ago Author

thanks for the visit arshita......


crystolite profile image

crystolite 5 years ago from Houston TX

Thanks so much for the information. It is applauding


bheem profile image

bheem 5 years ago Author

you are welcome crystolite


farhad_pd 5 years ago

please provide me recent advancement in HPLC & LC-MS and also their impact on pharmaceutical analysis


ahmad 5 years ago

any new research related to HPLC?


Ahmad ali 5 years ago

thank you sir for giving such wonderfull informations. THANK YOU.


bheem profile image

bheem 4 years ago Author

You are Welcome Ahmad...


muslimah bestari 4 years ago

its such a really good information given here.. Thanx alot


bheem profile image

bheem 4 years ago Author

muslimah bestari! Glad that you found the article interesting. You are welcome again....


asma 4 years ago

Assalam.oalikum kindly sir,tell me about IQ,OQ and PQ performance of HPLC.i don,t have knowledge about it in this term i know the term LOQ and LOD but don,t knoe IQ,OQ and PQ. increase my knowledge

Best Regard,

Asma


Abdullah Alghamdi-Riyadh 4 years ago

Asma, the terms are as follows: (IQ)Installation Qualification, (OQ)Operational Qualification, (PQ)Performance Qualification. These terms are used for instrument validation.


bheem profile image

bheem 4 years ago Author

@Asma and Abdullah! thanks for your comments and participation.....


sathya 4 years ago

i need more information about ELSD and ECD detector function and principle and what type of analysis can use this detector


bheem profile image

bheem 4 years ago Author

@Sathya!

Evaporative Light Scattering Detector (ELSD) is one which is complementary to UV detector. It helps in detection of photons scattered by vapor form of molecules like in volatile substances...These molecules may not be detected by fixed wavelength UV detector due to differences in absorbtion wavelengths among different molecules.


chakravarthi 4 years ago

which is better detector used for HPLC?


bheem profile image

bheem 4 years ago Author

Chakravarthi! detector selection depends on the sample taken for analysis, cost factor and the level of resolution needed. I think florescent is better for most samples and to estimate even minute concentrations.


faiza 4 years ago

asallam.thanks for hplc information..i want to study furthur what are the uses of uv-visible dectector,which are used in hplc.and i want to know the what type of toxicants uv-visible dectector(hplc)detects oper method with pr and also want to know the its mobile and stationary phases?and thanks i am waiting for the answer.


V SINGH 3 years ago

CAN MS REPLACE THE FUNCTION OF PDA DETECTORS IN LC


bheem profile image

bheem 3 years ago Author

Hi V SINGH !

The use of mass spectroscopic detector has different purposes in analysis than PDA detectors. Further use of mass spectroscopy is more expensive and complex process than use of PDA detector in LC.In PDA the type and concentration of specific type of molecules is known. In MS the different aspects of molecule and its constitution is known.


tushar 3 years ago

what is difference between waters (EMPOWER) PDA and Agilent (CHRMELEON) PDA


bheem profile image

bheem 3 years ago Author

tushar! perhaps only difference may be the companies and small improvements of one over other, but the internal applications might still be the same. yet check for more info from others..


cuttler profile image

cuttler 3 years ago from HubPages

good hub...been following your work for a while and learned so much. Keep up the good work and keep em coming. voted up and useful


bheem profile image

bheem 3 years ago Author

thanks for your complement cuttler!


savani hiren 3 years ago

i like it


Vandana 3 years ago

I got the complete information about HPLC in much easy and understandable way.... thank u hub :)


Apeksha 2 years ago

Thnx sir ...


bheem profile image

bheem 2 years ago Author

Apeksha! you are welcome.


thanx sirr... 2 years ago

i got much more info about hplc detectors


bheem profile image

bheem 24 months ago Author

Welcome....


SWETHA 21 months ago

I WANT TO KNOW MUCH MORE ABOUT THE DETECTORS AND THEIR USE


SWAPNA 21 months ago

WHY WE ARE USING UV DETECTORS IN HPLC

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