Chapter 4 Size exclusion chromatography Flashcards
Objectives
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.
Gel filtration
To separate protein mixture into peptides using chromatography
Size exclusion chromatography (SEC) introduction
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
Retention time definition
Length of time protein is retained in column
Illustration of size exclusion chromatography
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
Principle of size exclusion chromatography
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)
Graph of absorbance vs time of gel filtration
Sample injection
high molecular weights
intermediate molecular weights
low molecular weights
4 phases of gel filtration
- equilibration
- sample application
- sample separation
- regeneration
Column matrix
Material: cross linked dextran (sephadex), agarose, silica or polyacrylamide
Why use these material for column matrix?
Matrix materials should not non-specifically absorb proteins - to enable separation of target proteins
How do I know when my protein of interest is eluted?
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
Elution volume Ve
An elution volume (Ve) is the volume of eluent required to cause elution of a substance at the peak of its elution profile
5 line summary of the principles of size exclusion chromatography
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.
Protein purification with SEC
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.
2 Factors that affect resolution during size exclusion chromatography
- Flow rate
2. Sample volume
How flow rate affects resolution of SEC?
Slower the flow rate, greater the resolution
How sample volume affects resolution of SEC
The smaller the sample volume, the greater the resolution
What are the different things SEC can do?
Separate small MW substances
separate multimeric forms of a protein
separate glycopeptides from non-glycosylated peptides
Separate salts from proteins
Desalting protein samples by SEC
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.
What is dialysis (dialysis bag)
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.
Dialysis bag mechanism
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
SEC application
Desalt urine to obtain proteins in urine
Determination of molecular weight
of unknown protein using SEC
- Prepare a set of standard proteins
- Determine Vo of column using Blue dextran (MW> 1 million daltons)
- Determine Ve of standard proteins.
- Plot log MW of standard proteins vs Ve / Vo
- Determine Ve of unknown protein.
- Using the Ve / Vo of unknown protein, determine its log MW from the standard curve to calculate its MW
How do I know when my protein of interest is eluted?
Figure out Ve from the graphs
Ve is between sample injection to first intermediate molecular weight absorbance curve raise (elution)
