Chromatography PlanarColumn Paper TLC (Thin layer chromatography)

Chromatography is separation method based on different retention of analytes on the stationary phase. Different chemical of physical phenomena can be used to retain an analyte. Depending on the phenomenon responsible for separation several types of chromatography are considered. Types of chromatography  Adsorption chromatography  Ion exchange chromatography  Size exclusion chromatography  Ion exclusion chromatography  Affinity chromatography

Ion exchange chromatography (IEC) The method is used to separate any biomolecules carrying a charge, such as proteins, polynucleotides and fragments of both. Principle of ion exchange Ion exchange is an equilibrium between an ion attached to a solid support (ion exchange resin) and an ion in a solution Res - H + + Na + Res - Na + + H + [Res - H + ][Na + ] K = [Res - Na + ][H + ] Selectivity coefficient Describes the relative selectivity of the resins for H + and Na +

Example: ion exchange between a sulfocationite and an amino acid

SO 3 - Na + Ion exchange resin Matrix Counter-ion Structure of Ion exchange resins Fixed ion Cross-linked polymer (polymethacryl, polydivinylbenzene) Anion exchanger: positively charged groups on the solid support attract solute anion. Cation exchangers: covalently bound, negatively charged sites attract solute cations.

TypeExample of active group pH range of operation Application example Strongly acidic cation exchanger RSO Amino acids, inorganic separations Weakly acidic cation exchanger RCOO Transition elements, organic bases Strongly basic anion exchanger RN(CH 3 ) Alkaloids, fatty acids Weakly basic anion exchanger RC 2 H 4 N(C 2 H 5 ) Organic acids, aminoacids Ion Exchange Resins Examples: Polystyrene resins. Ion exchange groups

Requirements to a IE resin for biopolymer separations 1.Mechanical stability 2.Reduced unspecific adsorption 3.Higher saturation capacity 4.Fast mass transfer 5.Sustainability under sanitation conditions

Stationary phases for biopolymer separations Polysaccharides (cellulose, agarose, dextran) Synthetic organic polymers (polyacrylamide, polymethacrylate, polystyrene) inorganic materials (silica, hydroxyapatite) Particle size: 2 – 200  m analytical chromatography: 2-10 nm preparative chromatography: nm Pore size: 10 – 100 nm

SEPARATION OF PROTEINS BY IEC Pros: High resolution. A dilute protein or polynucleotide solution can be rapidly concentrated and simultaneously purified. Cheap mobile phases. Simple salt buffers are sufficient Cons: Ion-exchange chromatography is incompatible with mass spectrometry

SEPARATION OF PROTEINS BY IEC General cases where IEC is applied for protein and biomolecular purification and separation 1.De novo purification. 2.Purification of a protein with known pI, size, and primary structure 3.Preparative and industrial separation of proteins: 4.High resolution separation of protein variants and/or isoforms

1. Loading with salt 2. Equilibration 3. Loading of protein 4. Washout of unbound material 5. Elution 6. Regeneration 7. (Sanitization) Operation of Ion Exchange Column

Step 1. Loading with salt Charged ligands must be entirely saturated with counter-ions. 10 – 100 mM buffer + 1M NaCl or KCl At least 1 volume of column should be prepared

Step 2. Equilibration At least 10 column volumes must be eluted through a column to get an equilibrium conditions Stable and reproducible separation requires equilibrium to be established between the mobile phase and the stationary phase

Step 3. Loading of protein Loading of the protein solution should be performed at the same pH and conductivity as the equilibration buffer. Often this is not possible. Then the protein solution can be desalted by size exclusion chromatography, dialysis and the same ion composition can be obtained leading to most robust loading conditions.

Step 4. Washout of unbound material At least 1 column volume of equilibration buffer with eventually supplements first step of purification

Step 5. Elution Separation of the target compound from other bound concomitants 1.Elution by salt gradient (change salt concentration gradually displacing bound compounds in an order of increasing affinity ) 2. Elution by pH gradient (change the pH of the mobile phase creating conditions for target protein to desorb)

Step 6. Regeneration Preparation a column to new run of separation Regeneration solution must be strong enough to assure the desorption of all the sample components from the column

Step 6. Sanitation Working with biochemical media poses a danger to microorganism development in a column. To prevent the contamination of target analytes with pathogenic microorganism a regular sterilization of IEC equipment is necessary.