HIGH PERFORMANCE LIQUID CHROMATOGRAPHY (HPLC): RATIONALE, EQUIPMENT, TYPES - CHEMISTRY - 2024

The high-performance liquid chromatography is an instrumental technique used in chemical analyzes which separate mixtures are allowed, purify and quantify its components, and further studies. It is known by the abbreviation HPLC, derived from English: High Performance Liquid Chromatography.

Thus, as its name indicates, it works by manipulating liquids. These consist of a mixture composed of the analyte or sample of interest, and one or more solvents that act as the mobile phase; that is, the one that drags the analyte through all the HPLC equipment and the column.

HPLC equipment. Source: Dqwyy

HPLC is widely used by quality analysis laboratories in many companies; such as pharmaceuticals and food. The analyst in question must prepare the sample, the mobile phase, check the temperature and other parameters, and place the vials inside the wheel or carousel for the equipment to perform the injections automatically.

The HPLC equipment is coupled to a computer through which the generated chromatograms can be observed, as well as to start the analyzes, control the flow rate of the mobile phase, program the type of elution (isocratic or gradient), and turn on the detectors (UV -Vis or mass spectrophotometer).

Basis

Unlike conventional liquid chromatography, such as paper or silica gel filled column chromatography, HPLC does not depend on gravity for the liquid to wet the stationary phase. Instead, it works with high pressure pumps, which irrigate the mobile phase or eluent through the column with greater intensity.

In this way, it is not necessary to pour the mobile phase every so often through the column, but rather the system does it continuously and with higher flow rates.

But the efficiency of this technique is not due exclusively to this detail, but also to the tiny filler particles that make up the stationary phase. Being smaller, its contact area with the mobile phase is greater, so it will interact better with the analyte and its molecules will separate more.

These two characteristics, plus the fact that the technique allows the coupling of detectors, make HPLC far superior to thin-layer or paper chromatography. Separations are more efficient, the mobile phase travels better through the stationary phase, and chromatograms can detect any failure in the analysis.

Equipment

Simplified diagram of the operation of an HPLC equipment. Source: Gabriel Bolívar.

Above is a simplified diagram of how an HPLC equipment works. The solvents are in their respective containers, arranged with hoses so that the pump takes a small volume of them into the equipment; we thus have the mobile phase.

The mobile phase or eluent must be degassed first, in such a way that the bubbles do not affect the separation of the analyte molecules, which is mixed with the mobile phase once the equipment has made the injections.

The chromatographic column is located inside an oven that allows to regulate the temperature. Thus, for different samples there are adequate temperatures to achieve high-performance separations, as well as a wide catalog of columns and types of fillings or stationary phases for specific analysis.

The mobile phase with the dissolved analyte enters the column, and from it the molecules that "feel" less affinity for the stationary phase elute first, while those that are more retained by it elute later. Each eluted molecule generates a signal displayed on the chromatogram, where the retention times of the separated molecules are observed.

And on the other hand, the mobile phase after passing through the detector ends up in a waste container.

HPLC types

There are many types of HPLC, but among all of them the most outstanding are the following four.

Normal phase chromatography

Normal phase chromatography refers to one where the stationary phase is polar in nature, while the mobile phase is nonpolar. Although it is called normal, in fact it is the least used, with the reverse phase being the most widespread and efficient.

Reverse phase chromatography

Being an inverse phase, now the stationary phase is apolar and the mobile phase polar. This is especially useful in biochemical analysis, as many biomolecules dissolve better in water and in polar solvents.

Ion exchange chromatography

In this type of chromatography, the analyte, with a positive or negative charge, moves through the column, replacing the ions it houses. The higher the charge, the higher its retention, which is why it is widely used to separate ionic complexes of transition metals.

Size Exclusion Chromatography

This chromatography, rather than separating, is in charge of purifying the resulting mixture. As its name indicates, the analyte is separated no longer depending on how closely related it is to the stationary phase, but according to its size and molecular masses.

Smaller molecules will be more retained than large molecules, since the latter are not trapped between the pores of the polymeric column fillings.

Applications

HPLC allows for both qualitative and quantitative analysis. On the qualitative side, by comparing the chromatogram retention times under certain conditions, the presence of a particular compound can be detected. Such presence may be indicative of disease, adulteration, or drug use.

Therefore, it is a computer part of diagnostic laboratories. Likewise, it is found within the pharmaceutical industries, as it allows to check the purity of the product, as well as its quality with regard to its dissolution in the gastric environment. Starting materials are also subjected to HPLC to purify them and ensure better performance in drug synthesis.

HPLC allows the analysis and separation of complex mixtures of proteins, amino acids, carbohydrates, lipids, porphyrins, terpenoids, and is essentially an excellent option for working with plant extracts.

And finally, molecular exclusion chromatography allows you to select polymers of different sizes, since some may be smaller or larger than others. In this way, products with low or high average molecular masses are obtained, this being a determining factor in their properties and future applications or synthesis.

References

1. Day, R., & Underwood, A. (1989). Quantitative Analytical Chemistry. (fifth ed.). PEARSON Prentice Hall.
2. Bussi Juan. (2007). High performance liquid chromatography.. Recovered from: fing.edu.uy
3. Wikipedia. (2019). High-performance liquid chromatography. Recovered from: en.wikipedia.org
4. Clark Jim. (2007). High Performance Liquid Chromatography. Recovered from: chemguide.co.uk
5. Matthew Barkovich. (December 05, 2019). High Performance Liquid Chromatography. Chemistry LibreTexts. Recovered from: chem.libretexts.org
6. GP Thomas. (April 15th of 2013). High Performance Liquid Chromatography (HPLC) - Methods, Benefits and Applications. Recovered from: azom.com