Reverse phase chromatography

Chromatography

1. Reverse Phase Chromatography Separation Techniques Prepared by: Rizwana Sarwar Submitted to: Dr. Nawazish Hussain

2. Introduction of Chromatography & Reverse Phase Chromatography Reverse Phase Chromatography is also known as Adsorption chromatography. It depends on the chemical interactions between solute molecules and specifically designed ligands chemically grafted to a chromatography matrix. Reversed Phase Chromatography is a technique in which the binding of mobile phase solute to an immobilized n-alkyl hydrocarbon or aromatic ligand occurs via hydrophobic interaction.

3. Application of Reverse Phase Chromatography Reversed phase Chromatography has found both analytical and preparative applications in the area of biochemical separation and purification. Molecules that possess some degree of hydrophobic character, such as proteins, peptides and nucleic acids, can be separated by reversed phase chromatography with excellent recovery and resolution.

4. Mode of Separation Reverse Phase Chromatography A reversed phase chromatography medium consists of hydrophobic ligands chemically grafted to a porous, insoluble beaded matrix. The base matrix for the commercially available reversed phase media is generally composed of silica or a synthetic organic polymer such as polystyrene. The selectivity of the reversed phase medium is predominantly a function of the type of ligand grafted to the surface of the medium. Generally speaking, linear hydrocarbon chains (n-alkyl groups) are the most popular ligands used in reversed phase applications. Si Si O － Si － CH 2 (CH 2 ) 16 CH 3 CH 3 CH 3 O － Si － CH 2 (CH 2 ) 16 CH 3 CH 3 CH 3 O － Si － CH 2 (CH 2 ) 16 CH 3 CH 3 CH 3 CH 3 CH 3 O － Si － CH 3

5. Reverse Phase Media The reverse phase media has two parts. Matrix Ligand Synthetic organic polymers, e.g. beaded polystyrene, are also available as reversed phase media.

6. Normal and Reverse Phase Chromatography Normal Phase: In the 1970s most liquid chromatography was done on non-modified silica or alumina with a hydrophilic surface chemistry and a stronger affinity for polar compounds - hence it was considered "Normal". Reverse Phase: The introduction of alkyl chains bonded covalently to the support surface reversed the elution order. Now polar compounds are eluted first while non-polar compounds are retained (hydrophobic) - hence "Reversed Phase".

7. Theory of Reverse Phase Chromatography Step 1: Equilibrate the column packed with the RP medium under suitable initial mobile phase conditions of pH, ionic strength and polarity (mobile phase hydrophobicity ). The polarity of the mobile phase is controlled by adding organic modifiers acetonitrile . Step 2: The sample containing the solutes is applied the chromatographic bed is washed further with mobile phase A to remove unwanted and unbounded molecules. Step 3: Bound solutes are next desorbed from the reversed phase medium by adjusting the polarity of the mobile phase and elute from the column. Step 4: Removal substances not previously desorbed from column by changing mobile phase B. Step 5: Re-equilibration of the chromatographic medium from 100% mobile phase B back to the initial mobile phase conditions.

8. RP Mechanism (Simple) Non-polar compounds in the mixture will tend to form attractions with the hydrocarbon groups because of van der Waals dispersion forces. Non Polar compounds are less soluble in the solvent because of the need to break hydrogen bonds as they squeeze in between the water or methanol molecules. Therefore spend less time in solution in the solvent and spent more time way through the column.

9. Resolution & Retention Time Resolution is a combination of the degree of separation between the peaks eluted from the column ( selectivity), the ability of the column to produce narrow, symmetrical peaks ( efficiency), the amount (mass) of sample applied and the retention time of the samples on the column. Resolution (Rs) is defined as the distance between peak maxima compared with the average base width of the two peaks. Rs can be determined from a chromatogram, as shown in Figure. The resolution of two peaks is determined by three variables: the retention factor (k), number of theoretical plates (N), and the separation factor ( α ). The separation factor (selectivity) α = k 2 /k 1 (where k 2 >k 1 ). Retention Time: The retention time is defined as the time between the sample introduction and the elution from the column. The retention time can be increased by adding more polar solvent or water and decrease by adding organic solvent. The resolution can be described by the equation for two closely eluting compounds . The resolution of two peaks can be improved by increasing a, N, or k.

10. Some Terms to Understand Retention Factor (K) At any given time during the migration through the system, there is a distribution of molecules of each component between the two phases . K= n s /n m is called the retention factor . n s = Number of molecules in the stationary. n m = Number of molecules mobile phases at a given time. Efficiency (N) The efficiency of a packed column is expressed by the number of theoretical Plates. N The greater the number of theoretical plates a column has, the greater its efficiency and resolution. The number of theoretical plates, N, is given by N = 5.54 (V 1 /W 1/2 ) 2 where V 1 is the retention volume of the peak and W 1/2 is the peak width (volume) at half peak height. Selectivity Good selectivity is the degree of separation between peaks. It is an important factor in determining resolution and depends largely on the nature of the RPC medium, the nature and composition of the eluent and the gradient used for elution. The selectivity, a, for two peaks is given by α = k 2 ´ /k 1 ´ = V 2 - V /V 1 – V α = V 2 /V 1

11. Critical Parameters in RP-chromatography Column Length: The resolution of high molecular weight biomolecules in reversed phase separations is less sensitive to column length than is the resolution of small organic molecules. Proteins, large peptides and nucleic acids may be purified effectively on short columns and increasing column length does not improve resolution significantly . Gradient elution: Gradients in are usually performed either with increasing pressure ( increasing density ) or with increasing temperature, or with increased concentration of polar modifiers (such as methanol ). It enhance the peak. Elution mode is gradient elution by changing composition of the mobile phase. pH: Optimum pH is one of the most important parameters to establish . Selectivity can be achieved changing the pH, for the separation. Ion-pairing Agents: A common way to increase the hydrophobicity of charged components, enhance binding to the medium , and so alter retention time, is to add ion-pairing agents to the eluent . Since most proteins and peptides are slightly basic, ion-pairing agents are often acids such as trifluoroacetic acid (TFA) whereas a base such as triethylamine is used for negatively charged molecules .