Ion Exchange and Gel Filtration Chromatography

Ion Exchange Chromatography

• Ion exchange chromatography separates molecules based on their charge using a stationary phase with opposite charges.
• Ion exchangers are charged molecules bound to a matrix, like a framework.
• The stationary phase is usually supplied as a chloride instead of hydroxide because the chloride form is more stable.
• The process can be regulated with increasing pH or ionic strength of the solution to enhance efficiency and speed.
• Ion exchange chromatography is used for various applications:
  • Water softening by removing calcium, magnesium, and other multivalent ions.
  • Water demineralization by removing cations and anions.
  • Neutralization using cationic exchangers for alkali hydroxides and anionic exchangers for acids.
  • Separation of electrolytes from non-electrolytes.
  • Separation of carbohydrates and their derivatives.

Gel Filtration Chromatography

• Gel filtration chromatography separates molecules based on their size and molecular weight using a porous gel matrix.
• The stationary phase is a gel matrix, like a sieve, that allows smaller molecules to enter and elute later, while larger molecules are excluded and elute earlier.
• Different types of gels are used, each with a specific pore size and range of separation:
  • Dextran: Used for separating small peptides and globular proteins with small to average molecular mass.
  • Polyacrylamide: Similar to dextran, available in a wide range of pore sizes.
  • Agarose: Used for separating large globular proteins or long linear molecules like DNA.
• Gel filtration chromatography has several applications:
  • Purification of compounds, especially enzymes and proteins.
  • Isolation and quantification of components in a mixture.
  • Estimation of molecular weights, primarily for globular proteins.

Differential Scanning Calorimetry (DSC)

• DSC is used to determine the transition temperature (Tm) of phospholipids, which is the temperature at which acyl chains in the phospholipid become disordered.
• Tm is influenced by several factors:
  • Length of fatty acid chains: Longer chains increase interactions, requiring higher energy to melt.
  • Unsaturation (double bonds): Unsaturation reduces interactions, lowering the melting point.
  • Proportion of phosphotidyl ethanol amine to phosphotidyl chlorine: Phosphotidyl ethanol amine forms hydrogen bonds, increasing interactions and Tm.
  • Cholesterol: Cholesterol can increase or decrease membrane permeability depending on its distribution.

Slab and Disc Gel Electrophoresis

• Slab (thin layer gel) electrophoresis
  • Uses gels in strips with different thicknesses (0.1-0.25 mm).
  • Gels can be run vertically or horizontally.
  • Allows for analysis of multiple samples simultaneously.
• Disc (capillary) gel electrophoresis
  • Uses gels in tubes with a diameter of 7 mm and a length of 10-20 cm.
  • Allows for easy sample addition and recovery.
  • Limits analysis to one sample per tube and makes comparisons difficult.

Isoelectric Focusing (IEF)

• IEF separates proteins based on their isoelectric point (pI), the pH at which they have no net charge.
• A pH gradient is established using ampholytes, which migrate to their corresponding pI position, creating a pH gradient.
• Proteins migrate in the electric field until they reach their pI, where they stop moving.
• This technique can be used to separate proteins with similar molecular weights but different charges.

Explore the principles and applications of ion exchange and gel filtration chromatography in this quiz. Learn how these techniques separate molecules based on charge and size, respectively. This quiz covers essential concepts and methods used in biochemical applications.