HPLC analysis:principle, theory-Video tutorial of hplc-chromatography

High pressure liquid chromatography is the full form for HPLC and as said there is use of high pressure in the principle of its operation. But also due to its efficiency in analysis of compounds it is regarded as High performance liquid chromatography. Some even call it as High patience liquid chromatography based on the long human time requirement and patience needed in its operation.

HPLC is one of the chromatography systems which is widely used in the fields of clinical research, biochemical research, industrial quality control etc. Applications of HPLC include detection, analysis, determination, quantification, derivation of molecules from mixtures (prep HPLC) of biological, plant and medical importance.

Before going into the details of HPLC theory, principle and further HPLC tutorial, let's see how it evolved and why?

Before the discovery of chromatography, techniques like gravimetric analysis, photomery,colourimetry (UV, visible detection), titrimetry (acid base detection) etc.. were sole methods available for analysis. Even the requirements of analysis for research was simple i.e. there was no necessity for analysis of complex molecules, similar molecules (i.e molecules with same chemical and physical properties).

But as research advanced there was requirement to analyse all the molecules in a given sample for better detection of problem (in clinic), impurities and also deficiencies in industry and research.

This was not possible by single technique like photometric, titrimetric etc due to greater physical and chemical similarity in molecules of a sample like phytoconstituents, amino acids, neurochemicals etc..

This posed a problem to analysis so a combined technique whose estimation is based on physical and chemical properties together was discovered in the from of chromatography. This new method then had a great disadvantage of time required in its process. Some times a single sample separation took a couple of days.

With this limitation in mind, further efforts by scientists led to the development of HPLC chromatography with further improvements in speed and efficiency of analysis.

Thus HPLC principle was discovered to analyse like compounds or similar compounds at a faster rate with better efficiency.

HPLC theory & principle

The principle involved in HPLC testing is separation of compounds in a mixture more efficiently and also quickly than that of traditional column chromatography.

The separation of compounds is due to their relative differences in travel through the column on application of pressure exerted through mobile phase or carrying liquid.

The compounds of the mixture travel with different rates due to their relative affinities with the solvent and stationary phase.

Compound with higher affinity towards stationary phase of the column travels slowly and vice-versa.

The above principle is similar to that of column chromatography but in HPLC,

The separation is more effective due to greater surface area achieved due to very small particle size of stationary phase in comparison to that used in column chromatography.

This decrease in particle size increases has disadvantage that it proportionately enhances the flow time and run time due to increased surface area. To minimize this obstacle the high pressure is applied to the flow of hplc mobile phase through the column by use of pumps.

♦ The mixture required to be evaluated is injected into a stream of mobile phase which is flowing at a defined pressure.

♦ The injected mixture now does flow over the stationary phase inside the column under the influence of pressure along with the mobile phase.

♦ During this flow based on the affinity of individual compounds in the mixture towards stationary and mobile phase, some compounds get eluted first out of the column and others latter.

♦ Outside the column they are sent into a detector where individual compounds are detected and recorded in a computer installed chromatography software.

♦ The recordings (preferably in the form of quantitative peaks) are compared with those of standard compound's HPLC values and the individual compounds are identified. So the over all theory of HPLC is relative separation and detection of compounds.

For a overview of HPLC system and operation see the video-tutorial below

HPLC analysis is of differentiated based on

a) The stationary phase in the column used: Based on the nature of stationary phase used it can be either normal phase or reverse phase hplc.

1. Normal phase chromatography: Here the column stationary phase is made of polar compounds like silica gel, alumina etc.. The polar compounds or molecules in the sample under analysis have higher affinity to the stationary phase and so they are retained longer in the column than non-polar ones. Hence non-polar compounds are eluted first under the affinity to non-polar mobile phase while polar ones are eluted later.

2. Reverse phase hplc: Here exactly the opposite of normal phase happens. The stationary phase is made of non-polar compounds like C₁₈, C₈ type of organic compounds. The mobile phase used is polar. So compounds of high polarity or eluted first while those of low polarity or no-polarity are eluted last.

Most of the applications in HPLC require evaluation of drugs, biochemical molecules and other substances used by humans and they are polar (water soluble) in nature. So, reverse phase hplc is widely used.

b) Based on purpose of use: Here HPLC is used for either

1. Analysis mode: The procedure is done to estimate different types of molecules and their individual quantities in the mixture using the help of a detector.

2. Preparative mode: Here the intention of process is to separate large amounts of specific molecule from a mixture. The molecule or substance eluted is of highest purity. The column size, sample size is comparatively large than that of analytical mode.

It will be interesting to know HPLC is one of the few appliances in research which is used at the expense of its considerable disadvantages. This is because of the advantages which only it provides and not replaceable by other equipments.

♣ Advantages of HPLC:

• It includes both aspects of analysis i.e qualitative and quantitative analysis.

• HPLC method evaluates almost all the molecules of same family.

• For example in one single run all the mono-amines like dopamine, epinephrine, serotonin can be estimated. Single run for steroids in one sample gives the data of all the steroids in that sample.

• Molecules with small differences in absorption wavelengths can be detected well due to their differences in separation time. i.e one which travels faster is measured prior to the other which is measured later. This is the prime advantages if HPLC which makes it non-replaceable.

• Substances in very low concentration like nano and picograms can be detected due to the sensitivity of HPLC detectors used like electrochemical detector, fluorescence detector etc.

• Due to its high separation efficiency, the quality of substance obtained by preparative mode or technique (prep hplc) is of high purity.

♣ Disadvantages of HPLC:

• It's an expensive technique as it requires costly HPLC instrumentation, columns and also use of highest grade of purity solvents, buffers, chemicals etc termed as HPLC grade.

• Working on HPLC requires heavy processing before estimation like mixing, homogenization, filtration, degassing, derivatization etc. These techniques are also to be performed with proper care to avoid problems in estimation.

• The systems operation requires prior hplc training and effective hplc troubleshooting skills. So prior practice is essential to run this chromatography systems.

• HPLC Data obtained is non-homogenous and is never without any noise (fluctuation) and errors during estimation.

• Its time consuming and you must have good amount of patience.

• Alteration in temperature and presence of dust in chromatography lab can greatly vary the result out put. So strict maintenance of experimental conditions is required through out the process.

This is a brief description of hplc theory, principle & hplc tutorial, for more information regarding its instrumentation, detectors, etc.. check the articles linked above. You are also welcome to leave any questions or comments for further clarification.

Thank you :-)