Dear Dilip,

To simplify..

Normal Phase silica  has Si with OH which makes it highly polar.  When you use normal phase silica, the compounds  'adsorbs' to the stationary phase and the elution is based on ' the least polar compounds are less adsborbed'.  Increasing mobile phase strength (increasing the polarity by using more and more polar solvents) helps in eluting more polar constituents.

The polarity of 'reverse phase silica' is based on the side chain length.  For example, C18 phase (which is Si-O-C18 chain) is the least polar, where the interaction is more like 'partition between a non polar - C18 phase and the mobile (solvent) phase.  The retentivity of a compound in this environment is dependent on the 'partition co-efficient' of the compound (in which phase  will it be more retentive?).  Accordingly, the most polar will be least retained in the column and the non-polar compound retained longer. 

Reducing the chain lengths (C8, C6, C4 and C2) increases the polarity of the stationary phase facilitating more retentivity of polar constituents. 

An anology to reverse phase hplc would be paper chromatography.  In the paper, water molecules retained by the stationary 'cellulosic' phase makes the compound retentivity( in a solvent partition between 'water' and 'organic mobile phases), to be dependent on 'partition co-efficient' of a given compound.

The choice of the stationary phase for separation will depend on  the 'polarity' of the constituent to be separated and of course on the complexity of your sample matrix.

Hope this information is useful

I think this will help you...

http://www.separations.us.tosohbioscience.com/ServiceSupport/TechSupport/ResourceCenter/PrinciplesofChromatography/ReversedPhase/

http://www.waters.com/waters/en_IN/HPLC-Separation-Modes/nav.htm?cid=10049076&locale=en_IN

http://wolfson.huji.ac.il/purification/PDF/ReversePhase/AmershamRPCManual.pdf

The only difference is in relative polarity of mobile and stationary phases. While in normal phase LC the mobile phase is nonpolar  (must be 100% without water wich is the main limitation of normal phase LC) and stationary phase is polar (silica etc.) in reverse phase the mobile phase is polar (water) while stationary phase is more hydrophobic.

You can also compare it with HILIC

http://en.wikipedia.org/wiki/Hydrophilic_interaction_chromatography

BTW none of the systems is limited to proteins

I agree to Tomás; the difference is the polarity of both stationary and mobile phases. But, you can also use water with normal phase LC, e.g. when separating different polar phospholipids (DPPC, DPPE, etc.) one can use chloroform/methanol/ammonia/water mixtures ...

Kind regards.

I agree with Tomas. To make it a bit simpler, normal phase as the name implies consists of a polar stationary phase, while reverse phase consist of a non polar stationary phase. separation in principle is therefore based on relative polarities of the analyte, and the mobile phase (given for instance that you are using the reverse phase and your staionary phase is non polar). Therefore, a polar analyte will interact more with your polar colunm in a normal phase and hence have a longer retention time and elute late and vice versa.

In few words,

Normal Phase - Less polar mobile phase compare to stationary phase

Reverse Phase Reverse of phase) - More polar mobile phase compare to stationary phase)

Normal Phase (silica)  =  Adsorption chromatography - least polar constituents elutes first and the most polar retained for longer time

Reverse phase (RP silica) = 'Partition Chromatography'  - least polar constituents are retained longer with the most polar constituents eluting  first

Reversed-phase chromatography (also called RPC, reverse-phase chromatography, or hydrophobic chromatography) includes any chromatographic method that uses a hydrophobic stationary phase. RPC refers to liquid (rather than gas) chromatography.

The term "reversed-phase" has a historical background. In the 1970s, most liquid chromatography was performed using a solid support stationary phase (also called a "column") containing unmodified silica or alumina resins. This method is now called "normal phase chromatography". In normal phase chromatography, the stationary phase is hydrophilic and therefore has a strong affinity for hydrophilic molecules in the mobile phase. Thus, the hydrophilic molecules in the mobile phase tend to bind (or "adsorb") to the column, while the hydrophobic molecules pass through the column and are eluted first. In normal phase chromatography, hydrophilic molecules can be eluted from the column by increasing the polarity of the solution in the mobile phase.

The introduction of a technique using alkyl chains covalently bonded to the solid support created a hydrophobic stationary phase, which has a stronger affinity for hydrophobic compounds. The use of a hydrophobic stationary phase can be considered the opposite, or "reverse", of normal phase chromatography - hence the term "reversed-phase chromatography". Reversed-phase chromatography employs a polar (aqueous) mobile phase. As a result, hydrophobic molecules in the polar mobile phase tend to adsorb to the hydrophobic stationary phase, and hydrophilic molecules in the mobile phase will pass through the column and are eluted first. Hydrophobic molecules can be eluted from the column by decreasing the polarity of the mobile phase using an organic (non-polar) solvent, which reduces hydrophobic interactions. The more hydrophobic the molecule, the more strongly it will bind to the stationary phase, and the higher the concentration of organic solvent that will be required to elute the molecule.

Many of the mathematical and experimental considerations used in other chromatographic methods also apply to RPC (for example, the separation resolution is dependent on the length of the column). It can be used for the separation of a wide variety of molecules. It is not typically used for separation of proteins, because the organic solvents used in RPC can denature many proteins. For this reason, normal phase chromatography is more commonly used for separation of proteins.

Today, RPC is a frequently used analytical technique. There are a variety of stationary phases available for use in RPC, allowing great flexibility in the development of separation methods.

LC or HPLC is a separation method that involves  partitioning of mixture between the moving liquid phase (the mobile phase) and the surface of the particles (the stationary p. The solid phase could be silica gel- commonly referred to as normal;  and bonded phase-reverse phase. Mobile phase consists of non-polar and polar solvents depending on the molecular weight, solubility and polarity (ionic or neutral...) and detected on a variety of detectors. The choice of detector depends on the chemical nature of the eluent to be detected. Most often it is the UV detector that is use along with refractive index, fluorescence. In recent years M/S detector is gaining ground which eliminates the use of routinely used detectors.

Most of these information are available in the manuals provided  by HPLC equipment suppliers e.g. Varian .   

The normal and reversed phase HPLC is distinguished mainly in the material of the stationary phase. In normal phase, the stationary phase is polar while in reversed phase, the stationary phase is non-polar. This polarity forms the basis of separation in the column as the more the interaction, the more the time the eluent will stay in the column. For example, if we have a stationary phase being polar, the polar constituents of our sample interacts with the stationary phase because of the relationship between the polar components and the stationary phase, this will tell on the retention time which helps in the deduction of the eluents. Summarily, the difference between normal and reversed phase is the polarity of the stationary phase which in turn tells on the retention time.

Thank you very much for your kind support. really it's quite important.

But my question is reference with Suresh Govindaraghavan sir, recently i observe the similar material on RP and NP chromatography. Both principle is quite different in mechanism, most of time we studied and suggested that principle of HPLC is ADSORPTION. That's why  i want to know how they work?

Is this silica, that provide this option?

Please Update me.

Thank and Regard

@Simon Drescher 

but if you use water in normal phase chromatography, isn't it the HILIC?

@ Tomás Hluska

yes ... I think both "methods" - normal phase LC using e.g. CHCl3/MeOH and small amounts of water (1-5%) and HILIC using acetonitrile and water - overlap.

Kind regards.

Dear Dilip,

To simplify..

Normal Phase silica  has Si with OH which makes it highly polar.  When you use normal phase silica, the compounds  'adsorbs' to the stationary phase and the elution is based on ' the least polar compounds are less adsborbed'.  Increasing mobile phase strength (increasing the polarity by using more and more polar solvents) helps in eluting more polar constituents.

The polarity of 'reverse phase silica' is based on the side chain length.  For example, C18 phase (which is Si-O-C18 chain) is the least polar, where the interaction is more like 'partition between a non polar - C18 phase and the mobile (solvent) phase.  The retentivity of a compound in this environment is dependent on the 'partition co-efficient' of the compound (in which phase  will it be more retentive?).  Accordingly, the most polar will be least retained in the column and the non-polar compound retained longer. 

Reducing the chain lengths (C8, C6, C4 and C2) increases the polarity of the stationary phase facilitating more retentivity of polar constituents. 

An anology to reverse phase hplc would be paper chromatography.  In the paper, water molecules retained by the stationary 'cellulosic' phase makes the compound retentivity( in a solvent partition between 'water' and 'organic mobile phases), to be dependent on 'partition co-efficient' of a given compound.

The choice of the stationary phase for separation will depend on  the 'polarity' of the constituent to be separated and of course on the complexity of your sample matrix.

Hope this information is useful

Thank you very much Suresh Govindaraghavan sir.

Thanks and Regard

Thanks for peviously posted answers.

I general princple any compound injected in to HPLC (whether NPor RP) the compounds should eute frm the column.

It depends on the strength of the solvent ( e.g: in Methanol coldition Rt is  10.0mins, and the same in Acetonitrile it could be 6.50 min and in THF could be 3.7 min).

This clearly indicating that polar / non-polar compounds are eluting fom the column.

One thing, if we are not observing the eluting analytes and spending much time in the column will leads to band spreading and suprerss the height and misleads as hump in the base-line.

Hence every analyst has to look in to the data and change to sharp gradients

Thaks and regards

That's to much interesting question too me. However, this discussion makes me clear

Reversed-phase Chromatography uses a polar aqueous as a mobile phase. Where the stationary phase contains non polar solvent as well. However is a good choice to use this method. The Normal-phase chromatography works in the opposite manner.

Thank you very much Suresh Govindaraghavan sir, for sharing important information about chromatography...

Thank you everyone, especially Suresh sir for differentiating between NP vs RP in the simplest way possible. Sir, I have one query regarding reverse phase LC, what is the significance of end-capping of stationary phase of any column (say C-18) ? I need to know more on it?

Thanks everybody for the helpful discussions