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THE OF SODIUM POLYPHOSPHATES 543

The Hydrolysis of Sodium Polyphosphates

LEOPOLDO J. ANGHILERI * and ESTHER S. MILLER

University of Colorado Medical Center, Veterans Administration Hospital, Denver, Colorado, USA

(Z. Naturforsdb. 26 b, 543—545 [1971]; received February 19, 1971)

The hydrolysis of 32P-sodium polyphosphates (linear and cross-linked) in aqueous solution has been studied. The radiometric determinations indicate that the ortho- formation is a slow reaction, and that the amount formed by the linear variety is higher than that produced by the cross-linked form. There is a significant formation of metaphosphates during the hydrolysis of the cross-linked polyphosphate which is missing or at least reduced to a much lesser extent in the case of the linear polyphosphate.

Polyphosphates are widely known because their use in water treatment. Recently the attention was driven to some interesting biological properties of those compounds which are relevant to pathological processes as aortic calcification1 and malignant growths2. In all the cases these properties appear to be related to their ability to chelate divalent cations. Since the interpretation of the reactions in- volved as well as the effectiveness of their cation sequestering action is related to the chemical struc- ture, the usefulness of these compounds is depen- dent on their ability to withstand hydrolysis. With these facts in mind, the hydrolysis of linear and cross-linked sodium polyphosphates was radiometri- cally studied. Fig. 1. Unidimentional chromatography of polyphosphate hy- drolytic products: 1 G r a h a m's salt and high molecular weight polymers, 2 Tetrametaphosphate, 3 Trimetaphosphate, Materials and methods 4 Tripolyphosphate, 5 and 6 Orthophosphate. A = Linear sodium polyphosphate. B = Cross-linked sodium The 32P-labeled linear and cross-linked sodium polyphosphate. polyphosphates were prepared as described elsewhere 3. 10 mg of each variety of polyphosphate in 0.5 ml of dium polyphosphates. For a quantitative evaluation, distilled water were hydrolyzed by heating in a boiling after being scanned the chromatograms were cut in water-bath (95 — 96 °C). Samples were withdrawn at sections corresponding to the activity peaks. Then the different times and analyzed by ascending paper chro- matography at 4 °C. Whatman 3MM paper and radioactivity was eluted from the paper with 1% H3P04 4 E b e l's * solvend were used. After an overnight and counted in a liquid scintillation counter . The cor- run a fairly good separation of five well defined frac- responding quenching corrections were made by ad- tions was obtained: a) high polymers, b) tetrameta- dition of a 32P internal standard. The radioactivity phosphate, c) tripoly-, trimetaphosphate, d) pyrophos- values corresponding to the different fractions plotted phate, and e) orthophosphate. Fig. 1 shows the 3 hours as percentage of the total 32P in function of the hydro- hydrolysis roducts of both linear and cross-linked so- lysis time are shown in Fig. 2 and 3.

Reprints request to Dr. LEOPOLDO J. ANGHILERI, Klinikum 3 L. J. ANGHILERI, J. Lab. Comp. VI, 166 [1970], ESSen, Tumorforschung der Ruhr-Universität, D-4300 * Composition of the chromatographic solvents according to Essen, Hufelandstr. 55. EBEL 5: Basic: 40 ml isopropyl alcohol + 20 nd of isobutyl * Present address: Klinikum Essen, Tumorforschung der alcohol -f 39 ml of distilled water + 1 ml of ammonium Ruhr-Universität, 43 Essen, Hufelandstrasse 55, West Ger- hydroxide (22° Be). Acid: 75 ml of isopropyl alcohol + many. 25 ml of distilled water + 5 g of trichloracetic acid + 0.3 1 D. SCHIBLER, R. G. G. RUSSELL, and H. FLEISCH, Clin. Sei. ml of ammonium hydroxide (22° Be). 35, 363 [1968]. 4 T. CLAUSEN, An. Biochem. 22, 770 [1968]. 2 W. REGELSON, W. M. TUNIS, and S. KUHAR, Acta Unio. Int. contraCancerum 16, 729 [I960].

Dieses Werk wurde im Jahr 2013 vom Verlag Zeitschrift für Naturforschung This work has been digitalized and published in 2013 by Verlag Zeitschrift in Zusammenarbeit mit der Max-Planck-Gesellschaft zur Förderung der für Naturforschung in cooperation with the Max Planck Society for the Wissenschaften e.V. digitalisiert und unter folgender Lizenz veröffentlicht: Advancement of Science under a Creative Commons Attribution Creative Commons Namensnennung 4.0 Lizenz. 4.0 International License. 544 L. J. ANGHILERI AND E. S. MILLER

An ascending bidimensional chromatography using first the basic and then the acid solvent of EBEL 5 was performed on a sample of 1 hour hydrolysis. The radio- activity was located by autoradiography (Figs. 4 and 5) and the percentage of total 32P corresponding to each spot counted as described above (Table I).

jj- Polyphosphates

2 i> Hydrolysis Time [hours] •

Fig. 2. Hydrolysis of polyphosphates: Orthophospphate for- mation as a function of the time hydrolysis.

• cn • L ]"Tr'po'y" +Trimetaphosphate

° j-Tetrametaphosphate

© CL] ® L I pyR°PH0SPHATE

Fig. 4. Bidimentional chromatography of linear sodium poly- phosphate hydrolytic products (Notations in Table I).

Results Hydrolysis Time [hours]

Fig. 3. Hydrolysis of polyphosphates: Lower polymers for- Fig. 2 shows that the hydrolysis to orthophos- mation as a function of the time of hydrolysis. phate is a slow rate reaction, being lower for the cross-linked form than for the linear one. Pyro- 1 = Orthophosphate, 2 = Pyrophosphate, 3 = Tripolyphos- phosphate also is formed slowly, but contrarily to phate, 4 = Tetrapolyphosphat, 5 = Pentapolyphosphate, 6 = Hexapolyphosphate, 7 = Trimetaphosphate, 8 = Artifact, orthophosphate its formation is higher for the cross- 9 = Tetrametaphosphate, 10 = Pentametaphosphate and 11 linked variety (Fig. 3). After heating hours a peak = G r a h a m's salt and high molecular weight polyphos- in tetrameta-, tripoly-, and trimetaphosphate con- phates. centration is readied. Beyond this point their con- centration as well as that of high polymers decreases while the orthophosphate formation increases.

The E b e l's acid solvent was chosen because the Cross- 8.4 9.5 11.2 10.3 6.5 4.8 2.0 1.3 1.6 43.1 fairly good separation obtained with a low poly- linked Linear 6.2 14.4 2.0 0.4 - - - - - 77.0 phosphate degradation by the solvent action. Since in radiochromatographic determinations very low Table I. Chromatographic analysis of 1 hour hydrolysis pro- amounts of material are used (approximately in the ducts of sodium polyphosphates (Figs. 4 and 5). — As per- centage of total 32P in each fraction. range 50 — 100 jug), the possibility of a polyphos- phate-solvent interaction should be considered. This

5 J. P. EBEL, Bull. Soc. chim. biol. 20, 991 [1943]. is the reason why despite the fact that the bidimen- THE HYDROLYSIS OF SODIUM POLYPHOSPHATES 545

phate to orthophosphate 6:

(re-1) -chain + orthophosphate -> f (n-2) -chain+orthophosphate n phosphorus atoms / polyphosphate \ rings (e.g. trimetha-) — -> short chain (tripoly-)-> —shorter chain (pyro-) +ortho

In addition, despite the fact that the branching point in the cross-linked form is more rapidly hydro- lyzed than the linear chains, a higher rate of ring formation can be expected. Therefore, since ring degradation is reflected in a high pyrophosphate formation it is reasonable to expect a higher pyro- phosphate concentration during the cross-linked polyphosphate hydrolysis. As seen in Fig. 3 this as- sumption is corroborated by the experimental re- sults. On the other hand, the bidimensional chro- matographic findings of higher concentration of metaphosphates, or of polyphosphates which could be the result of ring degradation (Table I) still strengthens this hydrolysis interpretation. It is interesting to point out that the linear form shows no hydrolytic formation of metaphosphates (Table I) and its high pyrophosphate concentration can be related to a degradation via tripolyphosphate formation. Concerning this it should be mentioned that trimetaphosphate has been found present in polyphosphate solutions 7 and coincidentally tripoly- Fig. 5. Bidimentional chromatography of croll-linked sodium phosphate is the intermediate in its hydrolysis to polyphosphate hydrolytic products. orthophosphate. sional chromatography produces a better separation, Finally it can be stated that the linear sodium its use for a quantitative evaluation is not recom- polyphosphate is hydrolyzed by boiling water mendable. As discussed later, the high pH of the (95 — 96 °C) through a degradation path which basic solvent could modify substantially the meta- produces a higher proportion of orthophosphate phosphate concentration. than with the cross-linked form. On the other hand, this variety forms more pyrophosphate. This dif- ference seems to be related to a metaphosphate for- Discussion mation missing or at least occurring to a much lesser It has been suggested that the polyphosphate extent during the linear polyphosphate hydrolysis. hydrolysis in neutral or alkaline solution proceeds Coincidentally, the biological fate of these two forms through a simultaneous splitting off of end groups, of sodium polyphosphate seems to fit to a similar producing orthophosphate and a chain with one less pattern of higher metaphosphates formation in the phosphorus atom. Simultaneously a ring formation case of cross-linked polyphosphate. Furthermore, occurs, which is followed by its degradation to the this characteristic hydrolytic behavior appears ex- corresponding short chain polyphosphate, which plaining the peculiar in vivo distribution of the ultimately degrades via the tripoly- and pyrophos- cross-linked polyphosphate 8.

6 J. F. MCCULLOUGH, J. R. VAN WAZER, and E. J. GRIFFITH, 7 R. N. BELL, Ind. Engng. Chem. 39, 136 [1947]. J. Amer. chem. Soc. 78, 4428 [1956]. 8 L. J. ANGHILERI and E. S. MILLER, to be published.