GB1437377A - Method and apparatus for isoelectric focusing - Google Patents

Abstract

1437377 Isoelectric focusing LKB-PRODUKTER AB 16 May 1973 [16 May 1972] 23426/73 Heading C7B Components (e.g. proteins) in a sample solution having a density gradient are separated by isoelectric focusing in at least one focusing chamber. The sample solution is subject to a direct current electric field parallel to the density gradient, the focusing being carried out in at least two focusing steps with the mean cross-section of the focusing chamber perpendicular to the electric field in each step being smaller than in the nearest preceding step. Isoelectric focusing is an electro-phoretic method of separation, based on differences in isoelectric points of the sample components and is performed in an electric current path having a varying pH. After equilibrium has been achieved, each separated substance is at the point in the pH gradient which corresponds to its isoelectric point. The pH gradient can be obtained either by mixing solutions of varying pH in gradually varying proportions, or can be obtained by the influence of the electric field on a number of ampholyte substances having different isoelectric points. Fig. 1 shows two sections of a focusing column 14 having a cooling jacket 1, 2. The first and second focusing steps are performed with the column respectively horizontal and vertical. In the first step there is, as shown, a Hg electrode 7 at the bottom and a removable Pd-Ag electrode 12 at the top. For the second step, the electrode 12 is removed, the column carefully turned upright (the mercury electrode thus moving to the bottom of the column), a Pt wire electrode is inserted at 6 into the top of the column and the second focusing step performed. The separated fractions can be emptied from the column or, if the column is of quartz, can be subjected to U.V.-absorption analysis. Fig. 2 shows alternative apparatus comprising a prefocusing column 21 and final focusing column 25 provided with a cooling jacket. Electrodes 27 are separated from the column 21 by glass filter discs 24 and the anolyte and catholyte 22, 23 are circulated through cooled tubes by pumps 29. For the prefocusing, the device is turned 90 degrees around an axis perpendicular to the plane of the paper so that the column 25 has a horizontal axis but for final focusing is turned to the position shown. Sample solution, after prefocusing, is transferred from column 23 to 25 by suction, with the removed solution being replaced by a solution of a greater density supplied through the opening 30. Materials are specified for the electrodes. In a further alternative, the different focusing steps are carried out in a focusing chamber defined by an elastic material and in which, after each focusing step, the chamber is elongated in the direction of the electrical field with a simultaneous reduction in the mean cross-section perpendicular to the field.