edited by ROBERT REEVES Marlborough School
250
S.Rossmore Avenue
LOS
Angeies. CA 90004
'Gel Filtration-An Innovative Separation Technique Fred Blurnenfeld and James Gardner Millburn High School, 462 Millburn Avenue, Millburn, NJ 07041
Chemistry teachers are continuously searching for new, interestine. -. economical.. ranid. and safe lahoratorv techniques for use in the classroom.'we were fortunate todiscover such a techniuue a few vears aeo. A two-dav workshoo was spent at a local hiotechnology company where we were introduced to research techniuues used bv biochemists.'l'he techniques explored i n c l u d 2 gel elec&ophoresis and various types of chromatography. Reviewing and evaluating the many capabilities of these technique^,',^ we concluded that gel-filtration chromatography offered promise for the high school science program. Prior t o the 1960's, paper chromatography was seldom used in the high school experimental work. Today, few students escape exposure to this simple procedure. We felt that eel filtration could be adooted as widelv. Durine the past three years, we have developed and tested several laboratory activities and demonstrations involvinr- re1 - filtration. These include rapid separations of
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1) a starch-glucosemixture
2) leaf pigments 3) a mixture of vitamin B-12 and blue and yellow dextrans 4) a mixture of organic and inorganic compounds
Gel filtration has several distinct advantages for use in the hizh school lab over other conventional methods of sevara1) Economical. Gel columns can be used several times before being discarded. The cost of a typical column containing gel is about $1.50. Eluting agents such as salt and ethanol are
inexpensive and plentiful. Materials used are for the most part found in an average high school chemistry or biology lahoratorv. 2) ?'#me.The experiments, including pre-laband pust-labdiarussims, ran becomplpted in a maximumof one hour's time. 3) A l e r h o n t m a/ r e p a r a r i m The mechnnrsm of aeparatlon can be directly related to some molecular property of the molecules being separated. 4) Safety. The solvents employed are nontoxic and the technioue does not require anv comdicated or daneerous equi~. . . mek. 5 ) Preoorotion. Gels and other low-concentrationsolutions are wnple t o prepare wnhglsssware found m a typical laboratory There is no need lor complex apparatus. Theoretlcal Aspects Gel filtration is a form of liouid column chromatomaohv that separates molecules primarily on the basis of their size. In eel filtration the stationarv ~ h a s econsists of oorous eel par&es. The mobile phase i i f i u n d in the area between dextran gel (Fig. 2). The cross links are formed by reaction of the dextran with epichlorohydrin, giving different porosities for different amounts of cross linking. The gels are supplied in a dry form, and can be swelled with avariety of solvents such as water, salt solution, alcohols, DMSO, and formamide. The table lists some different gel types and their properties. Note that the separation ranges are usually given for proteins and polysaccharides. hut we have found se~arationof other tvves of molecules readily achievable. Another eel we found useful was Senhadex LH-2W 3. This gel has h o t i hydrophilic and lipophiiic properties, swelling in water as well as polar oreanic solvents. This enables chromatography to be done with water-insoluble solutes. In gel filtration chromatography, the behavior of a solute is best characterized by a distribution coefficient ( K d ) .
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"Gel Filtration. Theory and Practice," Pharmacia Fine Chemicals Inc., 800 Centennial Ave., Piscataway, NJ 08854. Andrews, P., Biochem. J., 91: 222 (1964). Pharmacia Fine Chemicals Inc.. 800 Centennial Ave.. Piscataway. New Jersey. 08854. "University Kit Instruction Manual." Volume 62 Number 6 August 1965
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Properties of Sephadex Dry Bead
Sephadex Type
GI0 G25
Grade
Diameter pm
40-120 100-300 Medium 50-150 Fine 20-80 Superfine 10-40 GI00 40-120 Superfine 10-40 0150 40-120 Superfine 10-40 Coarse
Fractionaction Range (Molecular Welght) Globular Proteins Dsxirans
