HPLC Principle and Types of HPLC Chromatography
Introduction to HPLC:
HPLC is a technique used to separate, identify, quantitative and for qualitative analysis of each molecule in the sample, depends on its interaction with the stationary phase and polarity. Pump flows a mobile phase by high pressure with the sample mixture through a column and allows for better separation, so it is also called as high-pressure liquid chromatography. Each molecule of the sample interacts slightly with the adsorbent material, the molecule that interacts strongly with the stationary phase will go slowly through the column than a molecule with low interaction. this difference causes the separation rate of the different analytes.
The types of HPLC Chromatography are as follows:
Normal Phase Chromatography:
In this type of chromatography, the moderately polar mobile phase and the polar stationary phase are used to separate the analytes which are freely soluble in moderator solvents. The use of more polar solvents in the mobile phase decreases the retention time (RT) of analytes. In NP-chromatography less polar analytes elute first than the polar analytes. The NP-Chromatography is better for the separation of analytes that differ in the number of functional groups. It is used for protein separation.
This is a vital analytical technique that is commonly used, in this method analytes be separated on the base of polarity. The non-polar stationary phase and polar mobile phase uses in RP-Chromatography. Retention time is more for analytes which more non-polar, while polar analytes elute more readily. The more hydrophobic the analytes, 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-Chromatography most popular because it applies to the wide range of molecules. It cannot apply for the proteins because the organic solvent causes the denaturation of proteins.
SE-Chromatography or Gel filtration chromatography technique applies to separate the particles on the basis of size. The large molecules flow rapidly throughout the column than the smaller molecules, SE-Chromatography is 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:
This method most useful for the analysis of water, protein purification. 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. cations exchange chromatography holds the positive charged and anion exchange chromatography hold anion with the positively charged functional group.
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