Intro to chromatography

What are the two main phases in chromatography, and hat is the general role of the stationery phase?

Mobile phase and stationary phase. The stationary phase interacts with molecules in the mobile phase to slow their movement based on affinity.

How does the strength of interaction between a molecule and the stationery phase affect its movement through a column?

Stronger interaction = slower movement; weaker interaction = faster movement.

In protein column chromatography, what are the typical stationary phase beads made from?

Spherical beads made from polymers like polystyrene or agarose-based polysaccharides (e.g., Sepharose).

What are fractions in the context of column chromatography?

Collected samples from the column outflow, each containing separated components.

Name 3 types of protein column chromatography?

Ion exchange chromatography (IEX), affinity chromatography (AC), and size exclusion chromatography (SEC).

What property of proteins does Ion exchange chromatography exploit for separation?

Net charge of the protein at a given pH.

What are the 2 ways to elute a bound protein from an ion exchange column?

Changing the pH or increasing the salt concentration (e.g., NaCl).

What is the functional group used in cation exchange chromatography for binding positively charged proteins? give one example.

Sulfopropyl (SP) or Carboxymethyl (CM).

What is the functional group used in anion exchange chromatography for binding negatively charged proteins? (Give one example.)

Quaternary ammonium (Q) or Diethylaminoethyl (DEAE).

What is the basis for separation in affinity chromatography?

Specific, reversible binding between a protein and a ligand attached to the column.

What is a His-tag, and how is it used in affinity chromatography?

A short sequence of histidine residues added to a protein, allowing it to bind to nickel ions on an affinity column.

What is the purpose of the wash buffer in affinity chromatography?

To remove non-binding proteins from the column after sample application.

What is the elution buffer, and how does it work in affinity chromatography?

A buffer containing a high concentration of a competing ligand that displaces the bound protein, causing it to elute.

After elution in affinity chromatography, why might the protein sample still need further purification?

Because the elution buffer contains a high concentration of ligand that may interfere with downstream analysis.

What is the principle behind size exclusion chromatography (SEC)?

Separation based on molecular size, where smaller molecules enter bead pores and travel slower.

How do the beads in SEC differ from those in IEX or affinity chromatography?

SEC beads are porous and lack functional groups; they separate by size, not chemical interaction.

In SEC, which molecules elute first—large or small? Why?

Large molecules elute first because they cannot enter bead pores and take a shorter path

What is a common use of SEC in protein purification workflows?

Removing small molecules (e.g., salts, ligands) from a partially purified protein sample

1. In the Week 1 experiment described, what protein is purified and from what source?

Lysozyme from chicken egg white.

What type of ion exchange column (cation or anion) is used in the Week 1 experiment, and what functional group does it contain?

Cation exchange column with Sulfopropyl (SP) functional groups.