Ngày nay, những phương pháp này phần lớn được thay thế bởi HPLC. Có một ngoại lệ, tuy nhiên: sự phân ly liên quan đến phóng xạ nền trọng lượng phân tử thấp và các sản phẩm. Kể từ khi phương tiện truyền thông tách giấy và TLC là dùng một lần, và tách biệt có thể được thực hiện trong một khu vực hạn chế của phòng thí nghiệm, những phương pháp này vẫn còn thích hợp hơn cho công việc liên quan đến đồng vị phóng xạ. | SEPARATION METHODS IN ENZYME ASSAYS 225 thin-layer modes of chromatography were very commonly used for the separation of low molecular weight substrates and products of enzymatic reactions. Today these methods have largely been replaced by HPLC. There is one exception however separations involving radiolabeled low molecular weight substrates and products. Since paper and TLC separating media are disposable and the separation can be performed in a restricted area of the laboratory these methods are still preferable for work involving radioisotopes. The theory and practice of paper chromatography and TLC will be familiar to most readers from courses in general and organic chemistry. Basically separation is accomplished through the differential interactions of molecules in the sample with ion exchange or silica-based resins that are coated onto paper sheets or plastic or glass plates. A capillary tube is used to spot samples onto the medium at a marked location near one end of the sheet which is placed in a developing tank with some solvent system typically a mixture of aqueous and organic solvents in contact with the end of the sheet closest to the spotted samples Figure steps 1 and 2 . The tank is sealed and the solvent moves up the sheet through capillary action bringing different solutes in the sample along at different rates depending on their degree of interaction with the stationary phase media components. After a fixed time the sheet is removed from the tank and dried. The locations of solutes that have migrated during the chromatography are observed by autoradiography by illuminating the sheet with ultraviolet light or by spraying the sheet with a chemical . ninhydrin that will react with specific solutes to form a colored spot Figure step 3 . The spot locations are then marked on the sheet and the spots can be cut out or scraped off for counting in a scintillation counter. Alternatively the radioactivity of the entire sheet can be quantified by
