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Expt. 4: Column Chromatographic Methods Column Chromatography
Chromatography is the process use to separate molecules based on SOME physical property of the molecule:
- Mass (i.e. size)
- Charge
- Affinity for ligands or substrates
- Hydrophobic interactions
Two phases in EVERY chromatography experiment:
- Stationary phase: a surface or resin that is inert
- Mobile phase: Comprised of the solvent and the sample (eluant). Introduction of the mobile phase can either be done by a gravity feed (siphoning) system or by a pumping device (usually peristaltic). In most of the figures for this chapter (see below), gravity feed systems are used.
Seperation occurs due to VARYING degrees of interaction of sample with the stationary phase.
Expt. 4: Column Chromatographic Methods Interaction of sample with stationary phase can be modulated by changing the solvent conditions (i.e. pH, ionic strength, competitive ligands, etc.).
For column chromatography: stationary phase is referred to as resin or gel or matrix.
Three primary types of RESINS:
- Gel Filtration = Size Exclusion (SEC) = Molecular Sieve
- Ion Exchange (IEC)
- Affinity (ligands, substrates, or “tags”)
Expt. 4: Column Chromatographic Methods Size Exclusion Chromatography Uses:
- Separation and purification of proteins.
- Determination of Molecular Weight.
- Desalting (i.e. removing small molecule salts) protein samples.
- Change the pH and ionic strength of the buffer that the protein is in.
Molecules separated according to their Stokes Radii.
Assume that the Mass of protein is proportional to its Volume.
For spherical (globular) proteins: radius of protein + hydration sphere is proportional to molecular mass or weight.
Expt. 4: Column Chromatographic Methods Suppose you have a spherical protein, with a Stokes radius of 5 nm = 50 Å. Then V = (4/3) π r^3 = 524 nm^3
Expt. 4: Column Chromatographic Methods SEC resins are hollow beads prepared by cross linking a polymer such as Dextran or Acrylamide. The beads have a Wiffle Ball-like structure with more or less discrete hole or pore sizes.
The pore size is determined by the cross- linker/polymer ratio:
- High ratio : small pore size Æ low molecular weight cut-offs.
- Low ratio : large pore size Æ high molecular weight cut-offs.
Cross-linker figure
Expt. 4: Column Chromatographic Methods Cross-linking results in a limited range of pore sizes, rather than a single precise size. This allows for a molecular weight range over which the resin is effective (see Table 4-1 below).
A word of caution: the larger the pore size (less cross-linking) the more collapsible the resin. Cross-linking adds structural integrity, especially for the dextran based resins (i.e. Sephadex). Under high pressure, the higher MWT cut off resins can collapse because of their LESSER degree of cross-linking, creating an almost impermeable barrier at the bottom of a column. Polyacrylamide (i.e. the Bio-Rad “P” series) resins are significantly less prone to collapse and are often preferred for these applications.
Expt. 4: Column Chromatographic Methods The ”Volumes” of SEC The degree of interaction of the protein with the SEC resin, therefore its ability to separate proteins of different sizes or masses is best measured by the VOLUME in which the sample elutes from the column.
Volume Definitions:
- V 0 : Void Volume - the volume outside of the beads.
- Vi: Included Volume – the volume within the beads.
- Vg: Gel Volume- the volume of the beads themselves ( ≤ 1% of total volume).
- Vtotal: Total Volume of column. Vt=V 0 +Vi+Vg. Since Vg ~ 0, then: Vt = V 0 + Vi.
- Ve: Elution Volume for a sample.
How are these volumes measured? Referring to Fig. 4-4, the volumes are calculated from the fraction (of a known volume) at which the material elutes from the column.
Expt. 4: Column Chromatographic Methods Fig. 4-
Void Volume (V 0 ) is determined using a material (usually a colored dye such as BLUE DEXTRAN) that is too large to interact with the resin, therefore is not retarded by the resin as it flows through the column.
Total Volume (Vt) is determined by using a material that is very small and interacts maximally with the resin. As in the above case, often a colored inorganic salt (FERRICYANIDE OR POTASSIUM CHROMATE) can be used. We will use Ferri-Cyanide (Fe(CN) 6 3-).
Expt. 4: Column Chromatographic Methods Calibration of SEC Columns: The knowledge of the Kav for a given protein can be very useful in the determination of the molecular weight (or mass) of a given protein in the following manner:
- A precise volume of a mixture of proteins, Blue Dextran, and a small molecular weight indicator are eluted from the column and their Kav values determined.
- A plot of Kav vs. Log Mol. Weight (MW) is constructed.
- The unknown protein is added to the column using the same volume as used for standards, then eluted from the column. The Kav is determined for the unknown and MW is calculated from the plot of Kav vs. Log MW.
Expt. 4: Column Chromatographic Methods
Fig. 4-5 Kav vs. Log MW
Expt. 4: Column Chromatographic Methods Problems with SEC
- Initial equilibration is long and tedious. Involves hydration (swelling), then pulling a vacuum on the resin to remove air from within beads.
- CANNOT allow resin to go dry. If so, then repeat equilibration process.
- For sugar based resins, algae and bacteria can grow on sugar matrix. Store using a 0.2% NaAzide solution.
- Packing of column is critical. Best if done in continuous manner so all of resin settles at same time. Eliminates “banding” in column.
- Sephadex resins: Volume decreases with increase in ionic strength.
- Flow rate decreases with increase in MW range (i.e. G200 runs slower than G25). Can compensate by using peristaltic pump, BUT…
- High pressure can collapse Sephadex beads at bottom of column (use polyacrylamide resins).
- Bands are broadened on SEC columns: sample is diluted on SEC columns due to thermal diffusion and frictional effects. Improperly poured columns or columns with
Expt. 4: Column Chromatographic Methods plugged “frits”can result in very erratic band migration.
- Air bubbles can form in column when taken from cold room to room temp due to expansion of gas volume.
Room temp Æ cold: OK Cold room Æ room temp: Bad
Expt. 4: Column Chromatographic Methods
Ion Exchange Chromatography: DEAE and CMC Uses:
- Crude purifications of cell lysates (i.e. batch cuts).
- Decrease volume of protein sample (i.e. concentrate).
- Purify proteins according to charge properties of protein and buffer conditions (pH and ionic strength).
Separation based on net charge on protein and electrostatic interactions between protein and charged groups on resin.
Positively Charged AA Negatively Charged AA Arg Asp Lys Glu His RS- Znet = (#)(Zpos) + (#)(Zneg)
Znet is related to pH and pI: pH < pI Znet > 0 pH = pI Znet = 0 pH > pI Znet < 0
Expt. 4: Column Chromatographic Methods
Often proteins are referred to as Acidic, Basic, or Neutral: Acidic (lots of Asp, Glu)
pI < 6
Neutral 6 < pI < 8 Basic (lots of Arg, Lys)
pI > 8
Strong vs. Weak Ion Exchangers (see Table 4- for names and structures). Strong: Used to remove small anions or cations. Irreversible binding of ions to resin. Often used for “stripping” purposes. Weak: Reversibly binds anions or cations (i.e. proteins with negative or positive net charge).
