Intro to chromatography

Question 1

Properties influencing separation

Answer 1

• Solubility
• Ionic charge
• Molecular size
• Adsorption properties
• Binding specificity for other molecules

Question 2

Examples of separation methods

Answer 2

• Column chromatography
• High-performance liquid chromatography
• Dialysis
• Electrophoresis
• Paper electrophoresis
• Gel electrophoresis
• Capillary electrophoresis

Question 3

Column chromatography

Answer 3

• Column packed with a porous solid matrix
  called stationary phase ( e.g SiO2)
• The mixture of substances to be separated is dissolved in a small amount of solvent and loaded onto the column
• A mixture of solvents called the mobile phase is passed through the column
• Substances separate based on their
  interactions with the stationary phase
• Pure substances are collected in fractions

Question 4

High performance liquid chromatography

Answer 4

• Relies on pumps to pass the mobile phase through the column
• Faster and higher resolution separations
• Limitation to the amount of sample separated (analytical technique)

Question 5

Normal phase liquid chromatography

Answer 5

Stationary phase is more polar than the mobile phase

Question 6

Reversed-phase liquid chromatography

Answer 6

Stationary phase less polar than the mobile phase

Question 7

Principles of chromatography

Answer 7

• The stationary phase has certain physical and chemical characteristics that allow it to interact in various ways with different proteins
• Common types of chromatographic stationary phases: Gel filtration, Hydrophobic interaction, Ion exchange, Affinity, Reversed-phase

Question 8

Gel filtration

Answer 8

• Separation based on size
• The stationary phase is made from a gel that has pores. Smaller molecules can pass through the pores, larger molecules are excluded
• Small-sized molecules enter many pores in the gel, travel through slowly, and elute later
• Medium-sized molecules enter some pores in the gel and travel less slowly
• Large-sized molecules enter few pores in the gel, travel faster and elute earlier

Question 9

How to determine molecular mass by gel filtration

Answer 9

• A gel filtration column can be calibrated with known standards to allow rough measurement of molecular mass
• There is an inverse logarithmic relationship between the size of the molecule and the volume eluted from the column: standard curves to estimate molecular mass

Question 10

Dialysis

Answer 10

• Separates molecules according to size through the use of semipermeable membranes containing pores of less than macromolecular dimensions
• Pores in the membrane allow solvents, salts, and small molecules to diffuse across but block larger molecules

Question 11

Equilibrium dialysis in drug discovery

Answer 11

• If the ligand and protein do not bind the ligand is free to cross the membrane. At equilibrium, the concentration of the ligand in the assay chamber will be exactly half that initially placed in the sample chamber
• If the ligand and protein form a complex, the bound ligand will be unable to diffuse across the membrane and will remain in the sample chamber. The ligand concentration will still be equivalent on either side of the membrane upon equilibrium. In this case, the ligand concentration in the assay chamber is reduced by the total amount of ligand bound to the protein divided by two.

Question 12

Principles of hydrophobic interaction chromatography (HIC) and reversed-phase chromatography

Answer 12

• Separates molecules according to differences in their hydrophobicity
• More hydrophobic molecules interact longer with the stationary phase

Question 13

Hydrophobic interaction chromatography

Answer 13

• The interaction between hydrophobic proteins and an HIC medium is influenced significantly by the presence of certain salts in the running buffer
• High salt concentration enhances hydrophobic interactions while low salt concentration decreases interactions
• As the ionic strength of the buffer is reduced the protein with the lowest degree of hydrophobicity is reduced

Question 14

Reversed-phase chromatography

Answer 14

• RPC separates molecules according to differences in their hydrophobicity and offers high-resolution separation and analysis
• The stationary phase is more hydrophobic so non-polar organic solvents must be used to reverse the hydrophobic effect
• Separation depends on a reversible hydrophobic interaction between sample molecules in the mobile and stationary phase

Question 15

Adsorption (normal phase)

Answer 15

• Molecules adsorbed on the surface of insoluble substances through dipole-dipole and hydrogen bonding associations
• Separation based on the partition of various substances between polar stationary phase and less polar solvent