Size Exclusion Chromatography

Size Exclusion Chromatography

Separation based on molecular size

How does SEC work?

Larger molecules can’t fit in the pores, so they elute faster

Smaller molecules get stuck in the pores, so they elute slower

Affinity Chromatography

Separation based on affinity of the target molecule to the stationary phase

How does affinity chromatography work?

The stationary phase is coated with molecules that bind to the protein of interest

The target protein binds to the stationary phase while everything else elutes out

A solution is added to break the bond and elute the target molecule

Ion Exchange Chromatography

Separation based on charge

How does Ion Exchange Chromatography work?

Cation exchange - negative beads attract positively charged protons

Anion exchange - positive beads attract negatively charged protons

Elution

Removing molecules from a chromatography column using the mobile phase, aka the buffer

What is an exclusion limit?

Maximum molecular size that can enter the pores of the beads in the column

Exclusion limit for SEC lab

60,000 Da

→ Hemoglobin is 65,000 Da, so it got eluted first

What were the results of the Size Exclusion Chromatography (SEC) experiment?

Hemoglobin eluted first because it was larger (65,000 Da)

Vitamin B12 eluted last because it was smaller (1,350 Da)

What was the sample used in the Size Exclusion Chromatography (SEC) experiment?

Mixture of Vitamin B12 and hemoglobin

What are the mobile and stationary phases in Size Exclusion Chromatography (SEC)?

Mobile phase - the buffer

Stationary phase - the beads

Reagents used in SEC lab

• Column buffer

• Protein Sample Mix

Aim of SEC lab

Observe molecules based on size using chromatography and understanding elution patterns through the use of hemoglobin and vitamin B12.

Principle of SEC Lab

Size Exclusion Chromatography separates molecules by size—large molecules elute first, small ones elute later.

Methods of SEC lab

We loaded a protein sample onto a column, added buffer, and collected fractions to observe size-based separation.

What color was hemoglobin in the SEC lab?

Brown

What color was Vitamin B12 in the SEC lab?

Pink