Presentation on theme: "1. 2 A form of “partition chromatography”. Stationary phase is a porous gelatinous matrix (in the form of beads). Sample components enter pores."— Presentation transcript:
A form of “partition chromatography”. Stationary phase is a porous gelatinous matrix (in the form of beads). Sample components enter pores and are temporarily (reversibly) trapped/retarded as mobile phase moves. Relative mobility through column is dependent on size of pores vs. size of solute molecules. Larger molecules exit column faster than smaller molecules – i.e. separation is based on molecular weights. 3
4 Gel Filtration (aka size exclusion) Ion Exchange Affinity
5 Gel filtration chromatography can be used to purify proteins.
Introduction to the theory and practice of gel filtration chromatography. Calibration & use of a sephadex G-75 column for the separation & recovery of two known proteins. Create a simple (2-point) K av vs. log 10 MW graph to estimate the MW of a hypothetical protein. 6
7 H 2 C CH CH 2 Cl O Epichlorohydrin + Dextran ( -1,6-glucose w/ -1,3 branches) Sephadex®
8 VtVtVtVt ViViViVi VoVoVoVo V t & V o are experimentally determined; V i is calculated: V i = V t – V o
10 Elution Volume / Fraction No. OD 400 (——) OD 620 (– – –) (Blue Dextran) (DNP-amino acid) 05101520302535 VoVoVoVo VtVtVtVt V o = 10 mL, V t = 29.5 mL V i = V t – V o V i = 29.5 – 10 = 19.5 mL
The MW of a given protein molecule is related to its elution volume (V e ); (high MW proteins elute with less volume than low MW proteins). Elution volumes can be used to predict the MW of unknown proteins. 11
12 K av = V e – V o V t – V o K av “partition coefficient” It represents the fraction of the stationary phase that is available for diffusion of a given solute (i.e. protein).
13 If log 10 MW of unknown protein = 5; Then MW of unknown protein = 10 5 = 100,000.
There are many different kinds/brands of gel media (see appendix XV): Variations in chemical composition. Variations in MW ranges & exclusion limits Sephadex is “biodegradable”; must be kept sterile. Gravity flow is inexpensive; pressurized flow systems are expensive. 14
15 Simple gravity flow. Fraction collector set at 25 drops 1 mL. Before getting started: Turn on spectrophotometer; program for dual absorbance (400 & 620 nm; see appendix I.D). Make 1 mL of a 200-fold(+) dilution of blue dextran/DNP-AA calibration mix. (10 & 5 mg/mL 50 & 25 µg/mL) Measure ABS @ 620 & 400 nm. ABS/mL will facilitate recovery estimates.