Adsorption Chromatography

INTRODUCTION

• Adsorption chromatography is probably one of the oldest types of chromatography around.
  
• It utilizes a mobile liquid or gaseous phase that is adsorbed onto the surface of a stationary solid phase.
  
• The equilibriation between the mobile and stationary phase accounts for the separation of different solutes.
  

PRINCIPLE

• Classic form of Chromatography by Tswett  
  

Principle

• Based on the principle that certain solid materials, collectively known as adsorbents have the ability to hold molecules at their surface.
  
• This adsorbing process which involves weak non-ionic attractive forces of van der Waal's and hydrogen bond occurs at specific adsorption site.
  
• These sites have the ability to discriminate between the molecules and the adsorption of the molecule depends on the strength of the interaction.
  
• As elutes continuously pass down the column, difference in their binding eventually lead to the separation for the analytes.
  
• Binding depends on the functional groups present in the molecules
  
  • Hydroxyl, aromatic groups - increase interaction with the binding surface
    
  • Aliphatic groups - decrease interaction with the binding surface
    

STATIONARY PHSAE / ADSORBENT

Silica

• with Silanol groups (Si-OH) groups on its surface
  
• Are slightly acidic in nature
  
• Can interact with polar functional group of the analyte or eluent.
  
• The topology (arrangement of Si-OH explains their differential separation
  
• Silica is acidic - good for the separation of basic substances
  
• Available for both LPLC & HPLC
  

Alumina

• Available for both LPLC & HPLC
  
• Alumina is basic - good for the separation of acidic substances
  

Carbon

• Available for both LPLC & HPLC
  

MOBILE PHASE ELUENT

• Depends on k' (partition ratio / capacity ratio) of analyte. (which is the time spent by analyte in the stationary phase relative to the time spend in the mobile phase)
  
• Eluent with polarity comparable to the most polar compound in the analyte mixture is selected as mobile phase.
  
  • Alcohol is selected for analytes with hydroxyl (-OH) groups
    
  • Acetone/esters is selected for analytes with carbonyl groups (=C=O) groups
    
  • Hexane / heptane /toluene (hydrocarbons) is selected for non polar analytes
    
  • Mixture of solvents also can be used in gradient elution.
    
• Presence of small amounts of water in the MP is beneficial when silica is the stationary phase – which selectively block more active Silanol groups leaving a more selective population of weaker binding sites.
  

MODES/ APPLICATION -USES

Modes

• Column
  
• Thin layer
  

Application/USES

• Most commonly used to separate non ionic water insoluble substances
  
  • Triglycerides
    
  • PTH
    
  • Aminoacids
    
  • Vitamins
    
  • Many drugs
    

TWO MAJOR TYPES

• Hydroxylapatite Chromatography
  
• Hydrophobic interaction  Chromatography
  

HYDROXY(L)APATITE CHROMATOGRAPHY

• Also known as hydroxyapatite chromatography
  
• Adsorbent used
  
  • Crystalline  hydroxyapatite (Ca10(PO4)6(OH)2)
    
• Mechanism
  
  • Involves both Ca & PO4 ions on the surface
    
  • Involve dipole-dipole interaction
    
  • Involve electrostatic interaction
    

APPLICATION/ USES OF HC

• Used to separate single stranded DNA from double stranded DNA
  
• Both single stranded and double stranded binds at low phosphate buffer concentration
  
• At increased buffer concentration single stranded DNA is selectively desorbed
  
• Further increase in buffer concentration desorbs double stranded DNA also
  
• Used of Cot Analysis
  
• So DNA can be separated from mixture of RNA and protein of cell Extracts.
  

COMMERCIAL

• Suitable for both LPLC & HPLC
  
• Crystalline or spheroidal hydroxy apatite is bonded to the agarose matrix
  
• Adsorption capacity is maximum around the neutral pH using 20 mM phosphate buffer
  
• Elution is done at 500 nM
  

HYDROPHOBIC INTERACTION CHROMATOGRAPHY

• Used to purify protein exploiting their surface hydrophobicity (non polar amino acids)
  
• In aqous solution these hydrophobic regions of proteins are covered with an ordered film of water molecules that effectivel mast these hydrophobic groups
  
• Exposed by the addition of salt ions
  
• Then these hydrophobic regions interact together and is the basis of salting out by the addition of ammonium sulphate
  
• If hydrophobic groups are attached to a suitable matrix the hydrophobic groups on the proteins will interact with them on the matrix to facilitate a protein-matrix attraction
  
• Most commonly used stationary phases are
  
  • Alkyl (hexyl , Octyl)
    
  • Phenyl groups
    
  • Attached to Agarose matrix
    

Commercial

LPLC(HIC)

• Phenyl Sepharose , Phenyl SPW
  

HPLC (HIC)

• Biogel TSK phenyl, Spherogel TSK Phenyl
  
• Ammonium sulphate is present in the sample since it fractionated before analysis with it which help to expose the hydrophobic centers.
  

Elution process.

• Gradually decrease the ionic strength
  
• Increase the pH
  
• Selective displacement by displaces that has s stronger affinity for the stationary phase than has the protein
  
  • Non ionic detergents like
    
    • Tween 20
      
    • Trinton –X100
      
  • Aliphatic Alcohols like
    
    • Butanol
      
    • Ethylene Glycol
      
  • Aliphatic Amines
    
    • Butylamine
      
• Some of these elution condition may cause protein denaturation
  
• Protein purified -->Aldolase, Transferrin, Cytochrom c, thyroglobulin etc.