8 GEL FILTRATION AND GEL PERMEATION [Proc. Soc. Analyt. Chew. Gel Filtration and Gel Permeation The following are summaries of the papers presented at a Joint Meeting of the Midlands Section and the Special Techniques Group held on October 16th 1968 and reported in the November 1968 issue of Proceedings (p. 216). Sephadex Gel Filtration BY MRS. CELIA MALE (Pharmacia (Great Britain) Ltd. Paramount House 75 Uxbridge Road Ealing London W.5) SINCE its introduction in 1959 Sephadex gel filtration has become a widely used chrornato- graphic procedure both on an analytical and preparative scale. The product known as Sephadex is a white inert powder consisting of bead-shaped particles that swell in aqueous solvents to give a gel suspension. There are eight different Sephadex G-types that swell in aqueous solvents vix.G-10 G-15 G-25 G-50 G-75 G-100 G-150 and G-200. These differ only in their degree of cross-linkage and hence in their porosity and fractionation range. Dextran of bacterial origin is cross-linked with epichlorhydrin. January 19691 GEL FILTRATION AND GEL PERMEATION 9 Sephadex G-10 is the most highly cross-linked and the least porous (exclusion limit about molecular weight 700) while Sephadex G-200 is the least cross-linked and most porous gel (exclusion limit about molecular weight 800,000). Sephadex LH-20 is a lipophilic derivative produced by the introduction of hydroxy- propyl groups into Sephadex G-25. Sephadex LH-20 swells in polar organic solvents and has an exclusion limit of about molecular weight 5000. Slurries of swollen Sephadex particles can be packed into chromatographic columns or the superfine grade can be used for thin-layer chromatography.The resulting gel bed or layer can be used to separate molecules according to size. If no solute-gel interaction effects are in operation the elution position of molecules is determined by their molecular size. Large excluded molecules appear in the effluent first followed by molecules of de- creasing size. On each gel type the elution behaviour of a molecule can be characterised by a distribution coefficient K which is zero for excluded molecules and tends towards unity for very small freely diffusible molecules. Distribution coefficients greater than unity indicate that adsorption is taking place. The three main classes of separations that take place on Sephadex columns are “de-salting,” fractionation and those based on solute - gel matrix interaction effects.The separation of two or more species of molecule differing greatly in size with distri- bution coefficients approaching zero and unity has often been loosely referred to as de- salting because in the historical context the de-salting of high molecular weight materials such as proteins was one of the first practical applications of Sephadex gel filtration. De-salting is normally carried out on Sephadex G-25 and can be scaled-up to commercial proportions. The new Sephamatic gel filters are designed for industrial scale procedures. On the laboratory scale however this type of separation has numerous applications such as removal of low molecular weight impurities from macromolecules binding studies and labelling techniques.Sephadex gel filtration has become a widely used laboratory technique for the fraction- ation of complex mixtures or the separation of two or more components of a mixture in which the components do not differ greatly in molecular dimensions. The Sephadex gels cover a wide fractionation range. Sephadex G-10 can be used for the fractionation of molecules of molecular weight less than 700 while Sephadex G-200 the most porous gel is used for the fractionation of macromolecules. It is generally assumed that interaction effects occurring between the gel matrix and the molecular species under investigation are the basis of separations that do not fall into the above categories. Aromatic substances adsorb on to Sephadex gels and the separation of molecules containing aromatic groups but not necessarily differing in size is often possible.The presence of residual carboxyl groups on the Sephadex matrix causes adsorption of basic and exclusion of acidic compounds. These charge effects are predominant in distilled water and can be largely eliminated by the use of an eluant with an ionic strength of at least 0.02. Solute - gel interaction effects are greatest in the highly cross-linked gels particularly on Sephadex G-10. Gel Permeation Chromatography in Non-aqueous Solvents BY F. W. PEAKER (Chevnistvy Depavtment The Univevsity P.O. Box 363 Bivvnzxghavn 15) THE essential features of equipment required to carry out a gel permeation chromatographic analysis in organic solvents include a solvent de-gassing unit; a pump capable of operat- ing up to 300 p.s.i.; pressure-regulating valves; an injection loop; analysis and reference columns; a detector which is frequently a differential refractometer; and a syphon system to indicate each increment of elution volume passed through the system. Hot-wire detectors and infrared spectrometers have also been used to monitor the eluent but a differential refractometer with a sensitivity of lo-' units is probably the most versatile detector in general use. 10 GEL FILTRATION AND GEL PERMEATION [Proc. SOC. Analyt. Chem. High molecular weight material is excluded from the gel to a greater extent than material of lower molecular weight and a continuous recording of the refractive index of the eluent against elution volume is produced. This record may be converted into a weight distribution curve for the sample once the characteristics of the columns have been established by a study of the elution volumes of well characterised polymers of known molecular weight and narrow molecular weight distribution.After calibration the number weight viscosity and Z- average molecular weights of the samples can be calculated from the distribution curve. This method is most reliable in the measurement of the weight and viscosity average values as they are dependent upon the higher moments of the distribution curve. When calibration samples are not readily available and polystyrenes are the only materials available commercially a universal calibration curve can be drawn up by plotting the product of the limiting viscosity number and the molecular weight i.e. [q] x M against elution volume.This depends upon the assumption that gel permeation is determined by the equivalent hydrodynamic volume of the solute molecules in solution. This approach can be used to carry over the calibration to other polymers and also to branched-chain molecules. As an example of the use of this technique the degradation of natural and synthetic rubber by mechanical forces was discussed. The milling of a natural rubber brings about a narrowing of the molecular weight distribution to a state in which it is unaltered by further milling. In contrast polyisobutylene when degraded in solution produces a significant proportion of very low molecular weight material in the range 50 to 1300 molecular weight units. Cross-linked polystyrene is the most widely used column-packing material and can be made to be effective in the separation of materials of molecular weight of lo7 extending through a range of permeabilities to the least permeable gels capable of handling material of only 10 to 50 units. In this lowest range the separations must be regarded as purely preparative. Attempts at correlating molecular size with elution volume indicate the existence of inter- actions between the solute to be analysed and the packing material. Within an homologous series the elution volume increases with molecular weight but materials with identical mole- cular volumes frequently have different elution volumes depending upon the solvent used and the chemical nature of the solute. The presence of highly polar groups has a very pro- nounced effect.