Gel Filtration Chromatography Lecture

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Transcript of Gel Filtration Chromatography Lecture

  • Chromatography literally means color writing.

    Chromatography was invented by the Russianbotanist Mikhail Tsvet in 1900. He used it to separate chlorophyll-containing extracts of plants.

    Key idea is that molecules of interest interactdifferentially with the stationary phase and a mobilephase, and thus can be separated. Gel-Filtration ChromatographyFirst, a review of the general principles of chromatography:

  • The Basic PrincipleIllustratedAs we discussgel-filtration(aka, size-exlclusion)chromatography, compare and contrast it with ion-exchange chromatographyfrom Lehningers Biochemistry by Michael M. Cox and David L. Nelson, W.H. Freeman, 2005

  • Partition Coefficient and Relative MobilityPartition coefficient describes the affinity of a compound for the stationary phase.

    or (Kav)= molecules adsorbed on stationary phase molecules in stationary and mobile phase

    Can have values between 0 and 1. Example, a molecule with = 0.4 will be 40% adsorbed on the stationary phase.

    Relative mobility or retention factor (Rf) describes the affinity of a molecule for the mobile phase.Rf = 1 (Recall Rf from TLC in Organic Chem)

  • Gel-Filtration ChromatographyThe principle is somewhat counterintuitive: a resin is selected that has pores through which smaller molecules can pass, but larger molecules are excluded and thus elute first.The ability of gel filtration to separate molecules according to size resides with the highly porous structure of gel filtration media and is basically a question of accessible volumes.Scanning electron micrograph of an agarose gel. Magnification x 50,000. Ref. Anders S. Medin, PhD Thesis, Uppsala University 1995.

  • Gel-Filtration Chromatographycontinued.from Lehningers Biochemistry by Michael M. Cox and David L. Nelson, W.H. Freeman, 2005

  • Gel-Filtration Chromatography: another view

  • The Role of the Partition Coefficient In Gel-Filtration Chromatographywhere Vt is total volume; Ve is the volume at which the molecule of interest elutes (assuming that it is within the fractionation range of the matrix); Vo is the volume of the space between the beads; and Vi is the volume of the space within the beads.see for example, http://www.wiley.com/college/fob/quiz/quiz05/5-6.html

  • You should learn thoroughly the first equation on the previous slide (Ve = Vo + KavVi), then you can obtain the other equations by reminding yourself of the two extreme values of the partition coefficient Kav. If Kav = 0 (i.e., if the molecule has no interaction with the resin and therefore passes around the beadsinstead of through the pores of the beads), then Ve = Vo, and the molecule will come out with the voidvolume.

    If Kav = 1 (i.e., if the molecule is so small that it has full access to the pores of the beads), then Ve = Vo + Vi = Vt,and the molecule will come out with the total volume.

  • For very small molecules that have full access to thepores of the beads (small dots), Ve = Vt excludedpartiallyincludedfullyincluded

  • High resolution modeGel filtration is commonly used in two different modes: 1. High resolution mode to separate large molecules like proteins, peptides oligonucleotides, polysaccharides etc.

  • Group separation mode2. Group separation mode to separate large molecules (Mr >5000) as a group from small molecules like salts and buffer ions.Group separation mode accepts much larger sample volumes than high resolution mode and can be performed at considerably higher flow rates.

  • Sephadex is a registered trademark of Pharmacia PL. Ultrogel is a registered trademark of Pharmacia-LKB. Bio Gel is a registered trademark of Bio-Rad Laboratories, Inc.http://instruct1.cit.cornell.edu/courses/biobm330/protlab/Gel_filtration.htmlFractionation Ranges

    Matrix nameBead typeApproximate fractionation range for peptides and globular proteins (molecular weight)Sephadex G-50dextran1500 - 30000Sephadex G-100dextran4000 - 150000Sephacryl S-200 HRdextran5000 - 250000Ultrogel AcA 54polyacrylamide/agarose6000 - 70000Ultrogel AcA 44polyacrylamide/agarose12000 - 130000Ultrogel AcA 34polyacrylamide/agarose20000 - 400000Bio-Gel P-60polyacrylamide3000 - 60000Bio Gel P-150polyacrylamide15000 - 150000Bio-Gel P-300polyacrylamide60000 - 400000