Page 35 - The Biochemistry of Inorganic Polyphosphates
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                                                                Chromatographic methods       19
                                                      Trimetaphosphate


                                               Tetrametaphosphate


                                           Pentametaphosphate                  Orthophosphate
                             First direction  Alkaline solvent  Hexametaphosphate  Pyrophosphate





                                                        Tripolyphosphate
                                                     Tetrapolyphosphate

                                                Pentapolyphosphate
                                              Hexapolyphosphate
                                         Heptapolyphosphate
                                                             Second direction
                                                             Acidic solvent

                        Figure 2.1 Separation of PolyPs and cyclophosphates by two-dimensional paper chromatography.
                        The basic solvent is isopropanol–isobutanol–water–25 % ammonia (40:20:39:1), while the acidic
                        solvent is isopropanol–water–25 % TCA–25 % ammonia (70:30:20:0.3) (Dirheimer, 1964).



                        cyclotriphosphate and orthophosphate, which are readily identified chromatographically,
                        especially when ‘Ebel’s basic solvent’ is used (Ebel, 1952a).
                          Ebel and co-workers have developed both paper chromatography (Ebel, 1949, 1951,
                        1952bc, 1953ab, 1954, 1958; Ebel and Dirheimer, 1957) and column chromatography
                        (Ebel and Bush, 1956; Ebel et al., 1962) techniques for the analysis of PolyPs. Paper
                        chromatographic separation of condensed phosphates only permits the separation and de-
                        termination of comparatively low-molecular-weight PolyPs and cyclophosphates (n = 2–9).
                        Using Ebel’s method of two-dimensional paper chromatography (Ebel, 1952b, 1953ab), it
                        is possible to separate the oligomeric PolyPs and cyclophosphates. An example of such a
                        separation, carried out by Dirheimer (1964), is shown in Figure 2.1.
                          In addition to paper chromatography, chromatography using ion-exchange resins has
                        been successly employed. Using this method, it is possible to separate polyphosphates with
                        values of n of 2 to 12 (Figure 2.2). Paper electrophoresis has also been employed to separate
                        oligomeric PolyPs and cyclophosphates, together with thin-layer chromatography (Wade
                        and Morgan 1955; Kulaev and Rozhanets, 1973; Kulaev et al., 1974a,c).
                          However, these methods are only capable of separating polyphosphates of fairly
                        low molecular weight. Two methods are currently available for the separation of
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