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Francisco who developed the technique. In CACE, opposing chromatographic and electrophoretic flows are counterbalanced to obtain a steady state, and each solute is concentrated at its own equilibrium position. O'Farrell studied the behavior of the colored protein ferritin on a column packed with a restrictive gel permeation matrix (Biogel P-10) layered on top of a more porous gel (Biogel A-50m). Ferritin chromatographs rapidly through the restrictive upper layer but more slowly through the porous lower part of the column. When an appropriate voltage is applied, the solute's upward electrophoresis rate is greater than the downward chromatographic flow in the bottom matrix, but it is not high enough to counteract the more rapid chromatographic flow in the top matrix. The net movement of the ferritin is thus downward in the top part of the column and upward in the bottom part, and the ferritin is concentrated at an equilibrium position at the interface of the two gel beds. Preliminary results indicate that CACE has both the high solute capacity and the high resolution required for use in preparative-scale purifications. A small chromatographic column, 50 cm X 7 mm, has a capacity of more than 1 g ferritin in an expanded equilibrium zone occupying roughly 10 mL (40% of the column volume). This corresponds to an equilibrium concentration of about 100 mg/mL. "For this size of apparatus," writes O'Farrell, "the CACE method has a capacity about three orders of magnitude higher than electrophoresis, and is roughly equivalent to the highest capacity chromatographic methods." O'Farrell uses the behavior of four colored proteins (ferritin, hemoglobin, myoglobin, and cytochrome c) on a column of mixed gel beds with five different interfaces to demonstrate the resolution of his technique. But because these particular proteins differ considerably in molecular weight and should separate fairly easily on a conventional GPC column, it isn't clear from this example what additional separation power CACE offers over conventional gel permeation chromatography, according to Fred Régnier, professor of biochemistry at Purdue University and an expert on the chromatography of proteins. Hemoglobin and myoglobin are electrophoretically similar, however, so CACE should be very useful for resolving proteins that are relatively difficult to separate by electrophoresis. "Even if it does nothing more," says Régnier, "it is a very good, powerful
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way to do electrophoresis." One important aspect of CACE, detection of the solute band, has yet to be investigated. (So far, only colored proteins have been used in testing the method, making band detection a trivial task.) O'Farrell favors the use of colored markers, a mixture of diverse polyionic compounds (such as the ampholytes used in isoelectric focusing) that has been modified to make the compounds colored. This mixture should separate into many colored bands on the CACE column, and the proteins of interest should focus into an equilibrium zone that is bracketed by at least two of the colored markers. Other methods, such as spectrophotometric, conductivity, or thermocouple detectors could probably also be used, although detection of the solute within the chromatographic matrix might cause some problems with the spectrophotometry detector. Once the equilibrium zone is established and detected, the purified protein is isolated by turning off the applied voltage and collecting the solute band as it flows off the column with the solvent. O'Farrell suggests that the purified material also could be withdrawn from a port at the equilibrium position in a simple continuous-flow apparatus. Other possibilities, according to O'Farrell, include the use of additional varieties of chromatographic matrixes (which would make it possible to purify other classes of compounds, in addition to proteins, on the basis of hydrophobicity, affinity, or charge) and the simultaneous purification of several components of a mixture. Régnier, however, believes that multiple-component separation may not be as simple as it sounds and may not be worth the effort required to establish the necessary equilibrium conditions. It also may be difficult to remove several bands of purified material without losing resolution. Although the principles of CACE were patented in 1981, the method has not received much attention until recently. "The power of CACE is in its resolution and capacity, but determination of the precise equilibrium conditions for a particular component takes some work," says O'Farrell. "Its most likely use will be for the purification of materials that have already been characterized, but with refinement it could have major applications in industry. Last year it was presented at a conference, and all of a sudden everybody is interested in it." M.D.W. (1) O'Farrell, Patrick H. Science 1985,227, 1586-89.
