Chapter 4 Size exclusion chromatography

Question 1

Objectives

Answer 1

Understand the principles of size-exclusion chromatography (SEC).

Describe the definition of parameters in gel filtration.

Describe how the SEC method can be used to determine molecular weight of a molecule/protein.

Question 2

Gel filtration

Answer 2

To separate protein mixture into peptides using chromatography

Question 3

Size exclusion chromatography (SEC) introduction

Answer 3

Also known as gel filtration or molecular sieving.

Proteins are sorted by molecular mass or size.

A larger protein has a shorter retention time in the column

Question 4

Retention time definition

Answer 4

Length of time protein is retained in column

Question 5

Illustration of size exclusion chromatography

Answer 5

Protein sample is driven through a molecular exclusion gel

Proteins are collected separately in different tubes

carbohydrate polymer beads, small molecules enter the aqueous space within the beads

large molecules cannot enter the beads

Question 6

Principle of size exclusion chromatography

Answer 6

Proteins larger than pore size will flow in between the beads

Proteins smaller than pore size will enter the pores (diffuse in and out of the beads)

Question 7

Graph of absorbance vs time of gel filtration

Answer 7

Sample injection

high molecular weights

intermediate molecular weights

low molecular weights

Question 8

4 phases of gel filtration

Answer 8

1. equilibration
2. sample application
3. sample separation
4. regeneration

Question 9

Column matrix

Answer 9

Material: cross linked dextran (sephadex), agarose, silica or polyacrylamide

Question 10

Why use these material for column matrix?

Answer 10

Matrix materials should not non-specifically absorb proteins - to enable separation of target proteins

Question 11

How do I know when my protein of interest is eluted?

Answer 11

Proteins larger than pores travel through space between beads and elute at Vo as buffer flows through column.

Proteins smaller than pores diffuse in and out of pores and elute after Vo but within Vt as buffer flows through column

Vt is total column volume
Vo is void column
Vt-Vo is volume of beads

Question 12

Elution volume Ve

Answer 12

An elution volume (Ve) is the volume of eluent required to cause elution of a substance at the peak of its elution profile

Question 13

5 line summary of the principles of size exclusion chromatography

Answer 13

SEC uses a stationary phase containing spherical gel beads of a certain pore size.

Proteins are sorted by molecular size and weight (MW).

Proteins larger than pores travel through space between beads and elute at Vo as buffer flows through column.

Proteins smaller than pores diffuse in and out of pores and elute after Vo but within Vt as buffer flows through column.

High molecular weight proteins elute first followed by intermediate molecular weight proteins, and lastly low molecular weight proteins.

Question 14

Protein purification with SEC

Answer 14

Know the identities of the proteins in the protein mixture

Know the specific MW of each protein and their concentration

multimers - a protein composed of several subunits.

Question 15

2 Factors that affect resolution during size exclusion chromatography

Answer 15

1. Flow rate

2. Sample volume

Question 16

How flow rate affects resolution of SEC?

Answer 16

Slower the flow rate, greater the resolution

Question 17

How sample volume affects resolution of SEC

Answer 17

The smaller the sample volume, the greater the resolution

Question 18

What are the different things SEC can do?

Answer 18

Separate small MW substances
separate multimeric forms of a protein
separate glycopeptides from non-glycosylated peptides
Separate salts from proteins

Question 19

Desalting protein samples by SEC

Answer 19

Salts and protein samples have a large difference in molecular size.

Select a gel porosity to exclude the protein sample.

During SEC, salts are trapped in the pores of the gel and take a longer time to elute compared with the proteins which are excluded and elute rapidly.

The desalting may be carried out at high flow rates without impaired resolution.

Very large volumes of sample may be desalted in a single step.

Question 20

What is dialysis (dialysis bag)

Answer 20

Identical to principal of desalting by SEC but proteins are retained instead of being eluted.

Place the protein solution in the dialysis bag made from a membrane with porosity that retains the protein sample.

During dialysis, salts diffuse out of the dialysis bag while proteins are trapped within.

Question 21

Dialysis bag mechanism

Answer 21

The concentrated solution in the dialysis bag is placed together into a beaker with buffer

over time, the contents inside the dialysis bag and the buffer environment will be at equilibrium, salt diffuse out of the membrane while protein stay inside the bag

Question 22

SEC application

Answer 22

Desalt urine to obtain proteins in urine

Question 23

Determination of molecular weight

of unknown protein using SEC

Answer 23

1. Prepare a set of standard proteins
2. Determine Vo of column using Blue dextran (MW> 1 million daltons)
3. Determine Ve of standard proteins.
4. Plot log MW of standard proteins vs Ve / Vo
5. Determine Ve of unknown protein.
6. Using the Ve / Vo of unknown protein, determine its log MW from the standard curve to calculate its MW

Question 24

How do I know when my protein of interest is eluted?

Answer 24

Figure out Ve from the graphs

Ve is between sample injection to first intermediate molecular weight absorbance curve raise (elution)