Lab 2 - SEC Chromatography

What does size exclusion chromatography (SEC) separate molecules based on?

Molecular size

What is the stationary phase?

The porous resin/beads packed inside the column

What is a fraction?

A portion of liquid collected as it exits the chromatography column

Which molecules elute first in SEC?

Larger molecules

Which molecules elute later?

Smaller molecules

Why do large molecules move through SEC faster?

They cannot enter the bead pores, so they take a shorter path around them

Why do smaller molecules move through SEC more slowly?

They enter the resin/bead pores, giving them a longer path through the column

What 2 molecules did we separate using SEC?

Hemoglobin and Vitamin B12

Approximately how large is hemoglobin?

~65,000 Da

Approximately how large is vitamin B12?

~1,350 Da

What color was the hemoglobin?

Red-brown

What color was the vitamin B12?

Pink

What should elute first: hemoglobin or B12?

Hemoglobin

Why does hemoglobin elute before B12?

Hemoglobin is much larger and is excluded from bead pores, so it travels through the column faster.

You observe a red-brown early fraction. What is likely present?

Hemoglobin

You observe a pink late fraction. What is likely present?

B12

What was the approximate exclusion limit of the SEC column?

~60,000 Da

What was done with the storage buffer initially inside the SEC column?

It was drained into the waste tube

How much protein mixture was initially loaded?

One drop

Why was the protein mixture loaded carefully onto the top of the column bed?

To avoid disturbing the resin bed and compromising the separation.

What was added after the protein entered the column bed?

250uL of column buffer, followed by another 250uL after draining

What was added after those initial washed?

3 mL column buffer

How many drops were collected in tubes 2-9?

5 drops per tube

How many drops were collected in tube 10?

10 drops or remainder of liquid