HPLC Analysis| Principle, Method & Applications of this Chromatography

HPLC analysis is one of the types of chromatography used to isolate and analyze mixtures.

HPLC in full form is “High-pressure liquid chromatography.”

Unlike column chromatography, here high pressure is employed in the process. Hence the name.

But also due to its efficiency in the analysis of compounds, it is regarded as High-performance liquid chromatography.

On the humorous side, some even call it as High patience liquid chromatography based on long time duration needed to run it.

HPLC chromatography is one of the widely used techniques in the fields of;
a. Clinical research,
b. Biochemical research,
c. 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?

HPLC analysis

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Before the discovery of chromatography, techniques like gravimetric analysis, photometry, colorimetry (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 the same chemical and physical properties).

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

This was not possible with a single technique like photometric, titrimetric, etc. due to greater physical and chemical similarity in molecules of a sample like phytoconstituents, amino acids, neurotransmitters, 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 form of chromatography. This new method then had a significant disadvantage of time required in its process. Sometimes 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 analyze like compounds or similar compounds at a faster rate with better efficiency.

HPLC Analysis Principle

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

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

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

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

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

The separation is more efficient due to greater surface area achieved due to a tiny particle size of stationary phase in comparison to that used in column chromatography.

This decrease in particle size increases has the 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 HPLC method or process steps include

♦ 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 later.

♦ 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 overall theory of HPLC is relative separation and detection of compounds.

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

Types of HPLC

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 stationary phase of the column 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 the 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 the process is to separate large amounts of a particular molecule from a mixture. The molecule or substance eluted is of the highest purity. The column size, the sample size is comparatively large than that of analytical mode.
HPLC advantages and disadvantages

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 equipment.

♣ Advantages of HPLC:

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

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

• For example, in one single run, all the monoamines like dopamine, epinephrine, serotonin can be estimated. A 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 before 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 the 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 method as it requires costly HPLC instrumentation, columns and also uses of the 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 active HPLC troubleshooting skills. So prior practice is essential to run these chromatography systems.

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

• It’s time-consuming, and you must have a good amount of patience.

• Alteration in temperature and presence of dust in chromatography lab can greatly vary the result output. So strict maintenance of experimental conditions is required throughout 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 🙂

Also see Column chromatography

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