Affinity and Size chromatography

Matrix of size exclusion chromatography

crosslinked dextran with epichlorohydrin

size limit for crosslinked dextran with epichlorohydrin

6,000 daltons, meaning that only mol with a molecular weight smaller than 6,000 daltons can go in to pores

How size chromatography works

large mol pass through the stationary phase while the small ones get stuck in the pores and take longer to wash out

What can the matrix of size exclusion chromatography be made of

agarose, dextran, cross-liked acrylamides

exclusion limit

max size of the mol can enter the pores of the bead 

Pore volume

space within particles

void volume

also called the excluded volume, space between particles which is occupied by the mobile phase

matrix volume

solid component of the particles that fills the column bed

Things to avoid with size chromatography

air bubbles: disrupt the flow

Which is larger hemoglobin or vitamin B

hemoglobin

absorbance of hemoglobin

630

absorbance of vitamin B

420

Principle of affinity chromatography

The target protein binds to the ligand in the stationary phase, while the nonspecific proteins pass through

What is affinity based off of

strength of the protein to the ligand

Matrix of the affinity chromatography

Trisacryl M : polyacrylamide support

Ligand of the affinity chromatography

chibacron blue

What did the ligand chibacron blue bind to

albumin

What is special about the elution buffer

it contains salt

why is it important for the elution buffer to contain salt

The salt uses electrostatic interactions to dislodge the proteins from the ligand