What is the principle of HPLC?

¿What is the principle of HPLC?

HPLC is an analytical and as well a preparative technique where a liquid is pumped through a bed of very finely packed particles. The analytes in the mobile phase are interacting with the chemical groups on the particles. Some analytes will have a higher affinity than others for the stationary particles and thus will permit the separation of the various analytes. The beginning of HPLC goes back in the 60’s where Jim Waters designed a refractive index detector for Dow Chemicals permirring to work on the separation of polymers. These separation were using gels in order to separate the molecular sizes of various polymer.

Through the years, many new modes of chromatography were invented namely:

Normal phase chromatography

Reverse phase chromatography

ion exchange

CPC (Centrifugal partition chromatography).

Typical HPLC simple isocratic setup.

Normal phase chromatography uses polar particles usually silica as a stationary phase and non polar solvants like Hexane, ethyl acetate, n-butanol and methanol in order of ascending strength. The main more of separation here is mainly the polarity of certain groups on the molecules.

Reverse phase chromatography occured in the 70’s were chemist were able to install some hydrophobic chains initially C8 and then C18 (a chain of 18 Carbons long) on the silica particles. If the carbon load is sufficient, the properties are completely changed, the column becoming a non-polar one or hydrophobic. The mobile phase used is now a very polar one like water, methanol, acetonitrile Isopropanol. This is called reverse phase because it is exactly the reverse of normal phase chromatography. Most of the HPLC separations (over 90%) are RP-HPLC today.

Typical separation of complex molecules.

ION exchange chromatography is used to separate analytes on the basis mainly of their charge, we can have anionic of cationic exchanger columns it has been used for ion chromatography.

Certains impotant organic molecules like Amino acid are often measured using this technique. Hitachi is still the main supplier of this instrument packaged as a complete system.

CPC (Centrifugal Partition Chromatography)

It is another form of chromatography were the stationary phase is also a liquid but held in place using centrifugal force. The principle stays the same, it is still and exchange of analytes according to their partition coefficient in the 2 immiscible liquids.

This form of chromatography is mainly used by people wanting to perform purification of bio-molecules ( antioxydants, flavenoids, anthocyanins etc)

The beauty is that multi grams separations can be accomplished in less than one hour and very reproducibly without the need of expensive preparative columns. Some industrial unit permit the continual purification of kilos of substances.

It has been used with great success to separate the cannabinoids content of cannabis namely the 2 major forms THC and CBD.

Usual detector that were used was mainly Simple UV detector. We now have many detectors with greatly enhanced sensitivities and selectivities like:

RI Refractive Index detector used for non chromopheric substances

ELSD (Evaporative Light Scattering Detector) more usefull than an RI detector but needs to use solvent tha can be nebulised easily and use of no salt or volatile salts only otherwise, a heavy build-up of salt will occur in the detector.

DAD Diode Array Detector, permitting to look at all the analyte at a pretty wide range of wavelength from Low UV to visible.

EC Electrochemical detectors highly sensitive but tough to use.

Fluorescence detection where the molecule are excited at one wavelength and detected at a higher one, very sensitive and specific detector.

MS of Mass spectrocopy where the cost of this detector can be several times the cost of the rest of the instrumentation. It is a very sensitive and complex instrument which needs a highly trained technician and a big research group to provide the necessary funding to keep it in good working condition. Its speed of use however permits to analyse hundreds of analysis in a day.

This is just a very brief introduction, this subject has thousands of books. RP-phase column are produced by many Corporation leading to a few thousands of models. Banks of separation do exist to facilitate the work of beginners.

Please bear in mind that the particles are so fine and the diameter of tubings as well that the operator has to filter through 0.45microns and preferably 0.22microns all the eluants and samples to be analysed. Failure to do so will result is blockages of the equipment with expensive repairs. You have been warned!


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