HPLC is a type of chromatography, which is a highly improved form of liquid column chromatography. Rather than to flow solvent under gravity through a column, it is forced through at high pressure which makes rapid and precise separation. It is capable of isolating and identifying the analytes present in the sample mixture.
Principle of high-performance liquid chromatography (HPLC):
The principle of HPLC is based on adsorption and partition chromatography. It depends on the nature of the stationary phase, where the stationary phase is a solid principle-based on adsorption and where the stationary phase is a liquid principle based on partition.
HPLC is a technique of analytical chemistry used to separate, identify, and quantify each solute present in a sample, depending on its interaction with the stationary phase and polarity. At high pressure, the pump flows a mobile phase with the sample mixture through a column and allows for better separation. Each component of the sample interacts with the adsorbent (column packing material). The solute that interacts strongly with the stationary phase will go slowly through the column than a solute with low interaction. This difference causes the separation rate of the different components.
Different types of HPLC chromatography:
There are four types of HPLC based on the phase system (stationary) in the process are as follows.
Normal-phase chromatography:
In this type of HPLC chromatography, a moderately polar mobile phase and polar stationary phase are used to separate the analytes which are freely soluble in solvents. In normal phase chromatography, less polar analytes elute first than the polar analytes. The use of more polar solvents in the mobile phase decreases the retention time (Rt) of analytes. It is better for the separation of analytes that differ in the number of functional groups as well as for proteins.
Reversed-phase chromatography:
This is a vital analytical technique of HPLC that is commonly used, in which the non-polar stationary phase and polar mobile phase used. In this method, the compounds are separated based on their polarity. The retention time is more for more non-polar compounds, while polar compounds elute more readily. The more hydrophobic the substances, the more strongly it will attach to the column, and the higher the concentration of organic solvent that will be required to elute the analytes. The RP-HPLC is most popular because it applies to the broad range of molecules. It cannot apply for the proteins because the organic solvent causes the denaturation of proteins.
Size-exclusion chromatography:
Size-exclusion HPLC or gel filtration chromatography technique applies to isolate the particles based on size. The large molecules flow rapidly throughout the column than the smaller molecules; it is a non-absorptive interaction with the samples. This is a vital analytical technique to determine the molecular weight of proteins as well as polysaccharides.
Ion-exchange chromatography:
Ion-exchange HPLC is the most useful for analysis of water, and purification of proteins. It separates the polar molecules and ions, based on similarity to the ion-exchanger. It is used for any type of charged molecules. Ion-exchange chromatography has two types, cation and anion chromatography. Cation exchange chromatography holds the positive charged and anion exchange chromatography hold anion with the positively charged functional group.
Instrumentation of HPLC:
The major components involved in the HPLC system are a reservoir, pump, injector, column, detector, and data acquisition.
Mobile phase reservoir: The reservoir is used to hold the solvent or mobile phase, often it has a glass bottle. It can be single, binary, or quaternary.
Degasser: It is used to remove the gases or dissolved gases from the mobile phase.
Pump: A pump is used to maintain a constant flow of the mobile phase through the HPLC system (e.g. Reciprocating, syringe, and pneumatic pump). The mobile phase composition in the method can be isocratic or gradient.
The injector (sample loop): It is positioned before the column and is used to introduce a variable and reproducible volume of the sample (E.g. Rheodyne, septum, stop-flow). It can be manual or auto-sampler.
Column: The column consists of particles (usually silica gel) that known as a stationary phase. It is a key component as it is responsible for the separation of the solutes. Some of the types of HPLC columns are C8, C18-octadecyl silane, cyano, amino, ion-exchange columns, etc.
Column oven: Nowadays it is a necessary component in HPLC which is used to control the column temperature, which is required for the sensitive molecules and viscous buffer.
Detector: Detector is used to detect the compound present in the eluent that comes from the column. It must be non-destructive, highly sensitive, and cover a broad molecular range. Some of the examples are UV-Vis, photodiode-Array detection (PDA), fluorescence, and refractive index, etc.
Data acquisition system: It used to control/adjusts the operating parameters, store data, and perform calculations such as peak area, height, theoretical plate number, tailing and fronting, etc.
The major advantages of HPLC:
- HPLC is used for the separation and analysis of very complex mixtures that are not possible with other conventional techniques.
- HPLC provides a simple, automated, and highly accurate method of recognizing certain chemical components.
- The minimum amount of solvent or mobile phase required for the process.
- Compared to other chromatographic methods, such as paper chromatography, thin-layer chromatography (TLC), and column chromatography, it is very fast and provides high resolution.
- It can be upgraded to liquid chromatography-mass spectrometry (LC-MS) to provide information about the molecular weight, and structure of specific compounds.
- Repetitive and reproducible analysis can be possible using a single HPLC column.
The major applications of HPLC:
HPLC is now one of the most powerful tools in analytical chemistry which is used in many applications that are for both quantification and identification. HPLC is applied in pharmaceuticals, environmental, drug science, forensic, clinical diagnosis, food industries, water purification, research purpose, agriculture, and it’s also used by several industries for the analysis as well as to maintain the quality of the products.
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