Introduction
Chemistry is the study of the elements that make up the universe. These building blocks are of different forms and require many methods of analysis. High-Performance Liquid Chromatography has become such an essential aspect of chemistry. It helps to identify, differentiate and understand mixtures.
What is Chromatography?
It is a technique, which is used in laboratories to separate the mixture appropriately. These mixtures are dissolved in a fluid such as water, solvent and sometimes gas, also known as the mobile phase. The mobile phase carries it through a column, plate or capillary tube and sometimes a sheet. These systems will get fixed on the material, which is known as the stationary phase.
The separation is due to the affinity of the mixture for the stationary phase. These mixtures stay shorter or longer on the stationary phase due to their interaction with the stationary phase material. According to this, the mixtures get separated and identified based on their affinity to the stationary phase.
Among all the other chromatography types, it is in use and one of the convenient chromatography techniques used by chemists across various sectors.
What is High-Performance Liquid Chromatography?
High-performance liquid chromatography (HPLC) is also high-pressure liquid chromatography. This technique is widely in use in analytical chemistry.
High-Performance Liquid Chromatography used for-.
- separation
- Identification
- Quantification of each component in a mixture
Different instruments used are as follows-
Solvent Reservoir– A glass reservoir is useful to contain mobile phase contents.
Pump- A pump can generate pressures up to 42000 kPa.
Sample Injector– A sample injector provides injection within the range of 0.1-100 mL of volume in a liquid sample along with high reproducibility and high pressure.
Columns consist of polished stainless steel, 50 to 300 mm long and 2 to 5 mm internal diameter. They consist of a particle size of 3–10 µm in its stationary phase.
Detector– Fluorescence, UV-spectroscopy, electrochemical sensors and mass-spectrometric are in use as detectors.
Data Collection Devices– The computer detects and integrates each component’s chromatography response, thereby quickly interpreting and reading the data.
How is it used?
In high-performance liquid chromatography, the pressurised liquid solvent, which contains the sample mixture, is passed through a column filled with a solid adsorbent material with a pump’s help. The different components in the sample mixture interact differently with the adsorbent material. It causes different flow rates for various parts present in the mix, thereby separating the elements.
Mobile phases include any miscible combination of water along with various organic solvents.
The most common organic solvent are-
- acetonitrile
- methanol
Some techniques use water-free mobile phases. Therefore, in these types, the mobile phase in aqueous components contains acids such as as-.
- Formic acid
- Phosphoric acid
- Trifluoroacetic acid
Sometimes salts are also used to assist the separation of the sample components in these types.
Different types of HPLC
Normal Phase
It separates analytical compounds based on their polarity. The stationary phase is polar, whereas the mobile phase is the nonpolar mobile phase.
They use silica in its stationary phase and compounds such as hexane, chloroform, diethyl ether, methylene chloride in its mobile phase.
Reverse Phase
It operates on the principle of hydrophobic interaction. In this type, the stationary phase is hydrophobic and nonpolar, whereas it uses polar liquids, such as acetonitrile or mixtures of methanol and water, as the mobile phase.
Size-exclusion
It separates particles based on their molecular size. Therefore larger molecules are washed through the column, whereas the smaller molecules penetrate in the porous of the packing particles.
Ion-Exchange
It is useful for ionisable or ionic samples. In these types, the ionically charged surface is the stationary phase, and an aqueous buffer is a mobile phase. The mobile phase has ionic strength as well as accurate pH, which thereby controls the elution time.
Uses of HPLC:
Manufacturing–
It is useful for the manufacturing of biological as well as pharmaceutical products.
Legal procedures
It is useful for legal purposes, such as during the detection of drugs in urine.
Scientific research
In research for separating complex biological sample and the synthetic chemicals from each other)
Medical procedures
It is also useful for medical purposes, such as detecting vitamin D and other substance levels in the blood serum.
Food and Flavour
They are used to measure the quality of soft drinks, and they also analyse the sugar and preservative content in fruit juices. They also explore the polycyclic compounds of the vegetables.
Clinical tests
They are used to detect the endogenous Neuropeptides in the extracellular fluid of the brain.
Environmental Applications
They are used to detect the phenolic compounds in the drinking water and bio-monitor the environment’s pollutants.
Conclusion
HPLC has a significant role in different industries, contributing to chemistry in various sectors with its uses and impact. Therefore, it is very accurate that it is a tremendous boon for chemistry.