Thin-Layer Gel Permeation Chromatography: A Technique to Separate Molecules According to Molecular Size Thin~layergel permeation chromatagraphy (TLGPC) is an excellent technique for separating molecules according to molecular size. I t is fast, selective and reasonably inexpensive.An important advantage of TLGPC over column gel permeation chromatography is that a number of samples can be run a t the same time. Solute separations occurring during TLGPC are due to partitioning of the molecules between a stationary phase immobilized by porous heads and the mobile phase. Entry into the stationary phase is determined by the relative sizes of the solute molecules and the pores within the gel. As a rule, smaller molecules will enter more pores than larger ones and will he retained within the stationary phase more of the time. This results in a slower rate of migration for the small molecules than for the larger molecules in the mixture. Since molecular size is essentially proportional to the molecular weight, this technique can be used to obtain molecular weights for molecules once the gel has been calibrated with suitable standards. In practice one plots the reciprocal of the migration rate versus the log of molecular weight Salukq of known molecular weight as well as one whose molecular weight is to he determined are included on the same plate. The preparation of TLGPC plates involves placing two, parallel 0.5-1.0 mm layers of masking tape on thesides of aglass plate. A slurry of pre-swollen gel, such as Sephadex G 150, is then spread on the plate with a glass stirring rod. The masking tape acts as a euidr to achieve alaver of uniform thickness. Next. filter Dmer bridees (Whatman II3MM) are soaked in the solvent. attached to e k s of thegel and cbinected to buff& reservairs.'~hepi& is then tilted to the desiredangle (10-20°), and pH %0 0.05 M phosphate buffer is added to the top reservoir. After a short equilibration period, sammples containing 100 fig of protein in 5 p1 of huffer are applied to the gel. The inclusion of avisible reference polymer such as hemoglobin is useful in determining when the solutes have travelled far enough for accurate measurement of migration distances. Other useful standards are cytochrome C, ribonuclease, chymotrypsinogen, and ovalbumin. At the experiment's end, solute proteins are located and identified by first placing a sheet of Whatman #1 filter paper on the gel surface for about 1 min, next drying the paper, and then dipping it into a suitable protein stain. Several copies can be made y repeating this process. Thin laver eel oermeation chromatoeraohv has several advantaees over similar column svstems in the teachine labaratorv.
--,.
Additional information regarding applications of or construction plans for TLGPC are available on request
Bibliography Kremmer, Tlbor. and Baross, LBszl6, "Gel Chromatography Theory, Methodology, Applications", John Wiley & Sons, New York, 1979. Neil J. Moir California Polytechnid State University San Luis Obispo. CA 93407
430
Journal of Chemical Education
