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Reverse Phase HPLC

Mixtures are formed by mixing of more than one component in different proportions. All individual components can not react with each other and show their properties. There are several physical and chemical methods to separate the components of a mixture such as filtration, fractional distillation, vacuum distillation, separating funnel, crystallization and chromatography. All these separation methods are based certain physical properties such as solubility, boiling point etc.  Out of all these methods, chromatography is one of the best methods of separation of components of a mixture. Chromatography is based on solubility of components in the given sample mixture. In a chromatographic technique there is a stationary phase and a mobile phase. Stationary phase is usually a solid or thick liquid which can stay fixed in one place. 

On the other hand, mobile phase or eluent can be a liquid or gas which can move through it. The unknown sample is placed on the stationary phase and gradually moves along in the same direction. If the analyte has no interaction with the stationary phase, it will come out of the system. In chromatography process we have to choose such stationary and mobile phases which have some interaction with the analytes. There are various types of chromatography such as ion exchange chromatography, column chromatography, paper chromatography, liquid chromatography, high pressure liquid chromatography (HPLC) etc.

 

Difference Between Normal and Reverse Phase HPLC

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HPLC is also known as high performance liquid chromatography. It is type of column chromatography or better we can say that it is an improved column chromatography. In normal HPLC, the solvent is moved in column under gravity. If same proceeds at high pressure at around 400 atm, it is called as high pressure liquid chromatography. It increases the speed of process. The columns of HPLC have very small particles as packing material that increases the surface area for interactions between the stationary phase and mobile phase that enhance the quality of separation of the components of the mixture. Highly automated detectors are used in HPLC that provide very sensitive and accurate results. 

On the basis of relative polarity of the solvent and the stationary phase, HPLC can be classified as normal phase and reverse phase HPLC. In normal phase HPLC is like other column chromatography with sensitive detectors and is not use commonly. Here the column is packed with tiny silica particles. Hexane a non-polar hydrocarbon is most common solvent for this. Columns of normal HPLC has an internal diameter of 4.6 mm and a length of 150 to 250 mm. The absorption of polar compound occurs quickly on polar silica compare to other non-polar component. Due to slow absorption of non-polar compounds, it passes more quickly through the column. In Reversed phase HPLC, the size of column is same as of normal HPLC but non-polar silica is used to pack the column and polar solvent like water with methanol can be used in reverse HPLC. Non-polar silica is prepared by the addition of long hydrocarbon chains to the surface of silica. So we can say that there will be strong attraction between the polar solvent and polar molecules in the column. But stationary phase that is hydrocarbon chains attached to the silica and the polar molecules in the mixture will not attract with each other. That allows the polar molecules to spend most of time with the solvent molecules. At the same time the non-polar components are attracted towards hydrocarbon groups due to Van Der Waals dispersion forces. Since these components of mixture are less soluble in the solvent so they squeeze in between the polar solvent molecules. So they quickly move down through the column. 

Reverse Phase HPLC Principle

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HPLC or high pressure liquid chromatography is an advance technique for the separation of components in the mixture. It can be classified as normal and reverse phase HPLC.  Out of these, reversed phase HPLC is the most commonly used form of HPLC. In High performance liquid chromatography the stationary phase can be porous solid surface active material or a liquid that is coated on an inert solid support like silica. The mobile phase moves through the packed stationary phase in the column at high pressure. 

Reverse Phase HPLC
 
So we can say that in reversed phase HPLC the mobile phase is more polar compare to the stationary phase. Whereas in normal or straight HPLC, the stationary phase is polar and mobile phase is less polar or non-polar. In reverse phase HPLC the stationary phases is bonded to the surface of a silica particles and hydrophobic in nature.

Reverse Phase HPLC Column

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In reverse phase HPLC the column is packed with very small silica particles which are non-polar due to long hydrocarbon chains. The polar solvent is used as mobile phase for this and these polar solvent molecules attract the polar components of mixture whereas non-polar molecules of mixture attracts towards long hydrocarbon chains of silica bed. 

Reverse Phase HPLC Column

Overall the hydrophobic molecules adsorbs on a hydrophobic solid column in a polar mobile phase.  As we decrease the mobile phase polarity with the addition of more organic solvent, hydrophobic interaction between the solute and the solid support reduces, causes de-sorption. 

Reverse Phase HPLC Method Development

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Reverse Phase HPLC Development

In reverse phase HPLC, the sample is injected at high pressure. The entire process is automated and due to high pressures, it is different from gas chromatography. The mobile phase is passed through the column to the detector. The time taken by a certain component to travel from column to detector is called as retention time. Retention time can be measured from the time at which the sample is injected and display shows a maximum peak height for given compound. Each compound exhibits different peaks due to different retention times. It depends on the used pressure used, nature of the stationary phase, composition of the solvent and temperature. There are special detectors used in HPLC to detect components like UV- visible absorption, fluorescence detector etc. 

Ion Pair Reagent for Reverse Phase HPLC

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Ion-exchange chromatography is used in HPLC analysis for ionic samples. For reversed phase partition chromatography, many ion-pair reagents have been developed. Ionic compounds form an ion-pair with ion-pair reagents which are present in the mobile phase results the neutralization of charges. As the hydrophobic character of the ion-pair increases, the affinity for the reverse stationary phase also increases.
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