Chromatography

• Chromatography


  Technique used to separate and analyze mixtures
• Intermolecular forces affecting chromatography


  • London Dispersion (induced dipole)
  • Dipole-dipole interactions
  • Hydrogen bonding
  • Ion-dipole interactions
• Frontal analysis


  Only the least-retained product is isolated from the others. The mixture to be separated is continuously fed into the column under conditions that favour the binding of all components but ONE.
• Frontal analysis


  • Charcoal filtration as a preparative mixture
• Displacement analysis


  Materials move down a column by being displaced by a more highly retained solute. Cannot achieve complete separation.
• Displacement analysis


  • Ion-exchange chromatography
• Competitive attraction in chromatographic elution

  Solutes partition between two phases (equilibrium). Separation based on relative retention. Making the column longer increases degree of separation. Many types of competitive reactions used; ionic interactions, polarity, physical (size).
• Theoretical plates


  Levels in the stationary phase. More levels/theoretical plates (smaller particles), the better the separation.
• Partition coefficient


  Solute in stationary phase/solute in mobile phase
• Retention time


  Measured using either the time or volume. VR = tR x flow rate
• You cannot keep increasing the length of the column to get good separation. You increase the time it takes for solution to go through column and the process becomes too slow.
• Short column


  • Bigger chance of products flowing together
  • Peaks are not as sharp (band broading)
• Chromatographic resolution


  How well a column retains each compound. How completely two neighbouring peaks are separated from each other (Rs), has to be baseline separation. Depends if being used for quantitative or qualitative results.

  
• Trailing-tail broading


  Due to diffusion in mobile phase. The reverse phase diffusion is more significant than the forward mobile phase movement (flow rate dependent).
• Principle divisions of chromatography based on mobile phase


  • Gas Chromatography
  • Liquid Chromatography

  
• Adsorption Chromatography


  Solid Stationary Phase. Normal Phase - silica gel (polar). Reverse Phase - silica gel with ester instead of -OH functional group (makes it non-polar). Separation due to a series of adsorption/desorption steps between the solute and fixed active sites of the stationary phase. Less tightly bound compounds eluted out by mobile phase at an earlier time.

  
• Silica gel - be careful when interacting with other very polar compounds as the bond may be too strong
• Solvent selection


  Normally use blend of two or more solvents. Solvent Strength - a measure of relative solvent polarity (ability to displace a solute). Polarity Index - a related index used for reverse phase methods. Normal Phase solvents (lower polarity) - hexane, chloroform, methanol, ethyl acetate. Reverse Phase solvents - acetonitrile, water, methanol.
• Separation is possible if the solutes have differing degrees of attraction to the phase.
• Partition Chromatography


  Support material coated with stationary phase that is insoluble in mobile phase. Normal phase - polar stationary phase with non-polar solvent. Reverse phase - non-polar stationary phase with polar solvent. Separation is based on solute partitioning between two liquid phases (relative solubility). More highly retained species have a greater affinity (solubility) for the stationary phase than the mobile phase (solvent) - based on differences in relative solubility.

  
• Droplet Countercurrent Chromatography (DCCC)


  Series of vertical tubes connected by capillaries. Liquid stationary phase stays in the tubes, the mobile phase is pumped through the system slowly. Mobile phase is denser than the stationary phase, then it is pumped from the top (MP less dense than SP, then pumped from the bottom). Theoretical plates = number of tubes. Large number of tubes to increase the efficiency. Low flow rate required - slow process.
• Centrifugal DCCC


  Use of centrifuge to enhance gravity, may achieve the efficiency of several hundred theoretical plates.
• Ion exchange Chromatography


  Stationary phase has ionically charged surface opposite to solutes. Polar group cation exchangers (Acidic) - SO3H, CO2H, OH, PO4H3. Polar group anion exchangers (tertiary/quaternary ammonium groups) - CH2-NR2, CH2-N+R3. Charged solutes are bound to stationary phase in exchange for associated counter-ions. Solutes are eluted by increasing the ionic strength of the solution/buffer.
• Proteins in ion exchange chromatography

  All proteins have ionisable groups on the surface. Groups are charged depending on pH, which changes the net charge of the protein. Each protein has a pH where the net charge is 0 (Isoelectric point, pI). Proteins positively charged at pH below isoelectric point - cation exchanger. Proteins negatively charged at pH higher than isoelectric point - anion exchanger.

  
• Size exclusion Chromatography


  Stationary phase is porous polymer matrix. Pores filled with solvent-mobile phase. Basis of separation is pore size - larger molecules come out first. Exclusion limit - size of the largest molecule that can enter the pore.
• If the gel-filtration column is calibrated with proteins of similar shape and known molecular weights, the unknown molecular weight can be determined.
• Affinity Chromatography


  Ligand - donates electron pair to central metal atom or ion to form coordination complex. Interaction of ligand with the solute species. Column needs adequate space. Slow flow used. Regenerative column. Stationary phase is a cation-exchange resin loaded with a metal ion that can form coordinative bonds with molecules from the mobile phase.
• Types of ligand


  • Specific - binds to only one species
  • General - binds to specific groups on target species
• Samples and their ligands


  • Enzymes - Substrate, inhibitor, cofactor
  • Antibody - Antigen, virus, cell
  • Hormones - Receptor, carrier protein
• Spacer


  Carbon chain interposed between ligand and matrix. Used when active site is located deep within the sample molecules. Too long - can interact with the sample species on its own. Too short - ligand can't reach active site.
• Support Matrix


  Material that ligand or spacers are bound to. Should be rigid, stable and have high surface area. Eg. Agarose, cellulose, dextran, polyacrylamide.
• Elution methods


  • Biospecific - inhibitor (free ligand added to mobile phase), free ligand competes for solute, when molecular weight inhibitor available
  • Nonspecific - reagent that denatures solute is added (pH, temperature etc.), Denatured solute releases ligand
• Supercritical Fluid Chromatography


  High resolution, low temperature, short analysis time. Mobile phase pumped as liquid (pressurised liquid preheated to reach critical point). Low pressure across the column. SCF pass through the column via injection port. Pressure restrictor at the end of the column or detector to ensure supercritical condition.
• Supercritical state


  At some point, the supercritical state is reached. CO2 most widely used. CO2 has similar density to H2O. Above the critical temperature, vapour and liquid have the same density. Vapour cannot be liquified by increasing pressure.
• Mobile Phases of SCF Chromatography


  Important properties in; temperature, pressure, density, viscosity and diffusion coefficients. Above the critical point; solvating power approach that of liquid (strength of ion-dipole interaction), viscosity similar with gas, diffusivity intermediate between gas and solid. Able to dissolve a variety of solutes. Changing the applied pressure changes the density and solubility of SCF and chromatographic retention.

  
• Applications of packed column SCF chromatography


  • Thermally labile compounds
  • Compounds beyond the volatility range of gas chromatography
  • Reduction in time analysis (vs HPLC)
  • Longer capillary column option
• Open-tubular columns


  Immobilised polymeric films as the stationary phase
• Considerations when preparing protein samples


  • Temperature (keep in freezer to preserve)
  • Freeze-thaw (not in and out the freezer)
  • Physical denaturation
  • Solution effect - close to neutral
  • Dilution effect - >1mg/ml
  • Oxidation - use antioxidants
  • Heavy metals - use metal chelators
  • Microbial growth
  • Proteases
• Protein characteristics


  Differential solubility; salt, solvent, pH, temperature. Low ionic strength - solubility increases with salt concentration. High ionic strength - solubility decreases with salt concentration.
• Supercritical fluid chromatography (SCF)


  • Changing the applied pressure changes the density and soluability of SCF and chromatographic retention