Lab 9 - Size Exclusion Chromatography

chromatography

powerful analytical technique that separates mixtures into individual
components

Preparatory chromatography

isolation or purification of target molecules

analytical chromatography

identify and quantify components of mixtures

stationary phase

solid phase, liquid adsorbed on a solid surface

mobile phase

liquid or gas phase

effective separation depends on mixture characteristics such as...

absorption (l-s), partition (l-s), affinity towards one protein vs others, differences in mw

liquid chromatography

liquid = mobile phase, thermally unstable, non-volatile sample

gas chromatography

gas = mobile (He, N2), mixtures of volatile liquids & solid material, highly sensitive yet simple, small molecules

Thin-Layer Chromatography (TLC)

silica gel that is immobilized on a glass slide

paper chromatography

Stationary phase is a thick filter paper and water drops settled in its pores,
mobile phase is the appropriate fluid

column chromatography

Stationary phase is a column on which the sample to be separated is loaded
and then washed with mobile phase

Ion exchange chromatography

Uses electrostatic interactions between charged protein groups, and solid support
material (matrix), matrix has ion load opposite of protein to be separated,

(+) ion-exchange matrix = anion exchange

(-) matrix = cation exchange

Affinity chromatography

The specific protein which makes a complex with the ligand attached to a solid
support (matrix), and retained in the column, while free proteins leave the column.
Usually an antigen-antibody interaction!
• The bound protein leaves the column by means of changing its ionic strength
through alteration of pH or addition of a salt solution

Gel permeation/Gel filtration/size exclusion

Uses matrix to separate macromolecules based on their differences in molecular
sizes.
• Matrix is made from dextran, agarose, or polyacrylamide
• Stationary phase is a column and consists of inert molecules with small pores

column chromatography (mobile phase)

• acts as a solvent – sample mixture can be introduced in the column.

• acts as a developing agent – helps in the separation of components in the
  sample to form bands.

• acts as an eluting agent – the components that are separated during the
  experiment are removed from the column
  ex: ethanol, acetone, water.

column chromatography (stationary phase)

solid material with good absorption

Shape and size of particle: Particles should have uniform shape and size
• Stability and inertness of particles: high mechanical stability and chemically
inert.
• It should be colorless, inexpensive and readily available.
• Should allow free flow of mobile phase
• It should be suitable for the separation of mixtures of various compounds.

SEC separates molecules based on…

largest to smallest molecular weight (big to small)

gel - porous beads

diff size molecules are included/excluded from pores in matrix

large molecules

eluted FIRST in void, do not enter pores

small molecules

elute slower, s=s

factors affecting separation

flow rate, sample volume, column length, pore size, exclusion limit

exclusion limit

determined by pore diameter. proteins bigger than EL eluted tgt in single peak

large molecules are eluted in..

void volume (V0) (matrix)

small molecules are eluted in..

total volume (Vt) (all)

what is elution volume (Ve)

solutes within the separation range of matrix that are fractionally excluded with a characteristic (Vt-Vo)

Kav

partition coefficient. specifies retention of molecules

gel permeation chromatography (GPC)

SEC when organic solvents are used

gel filtration

SEC with aqueous solvents

SEC protein mixture separation steps

apply protein mix into column → add buffer (so it can travel thru column) → collect eluted sample into diff tubes → identify proteins

hemoglobin

red-brown

mw = 65k daltons

eluted first

large (excluded)

Vitamin B12

pink

mw = 1,350 D

small (fractionated)

penetrates bead pores

eluted last

expected outcome