Chemistry of inorganic compounds – Phosphorus or compound thereof – Oxygen containing
Patent
1980-03-21
1982-06-15
Vertiz, O. R.
Chemistry of inorganic compounds
Phosphorus or compound thereof
Oxygen containing
423311, C01B 2532
Patent
active
043350860
DESCRIPTION:
BRIEF SUMMARY
This invention relates to the preparation of hydroxyapatite (HA). HA is of great utility in the chromatography of complex substances.
HA is a form of calcium phosphate and may be prepared from another form, brushite (CaHPO.sub.4.2H.sub.2 O). For use, it is desirable that HA should be in crystalline form with a substantial uniformity in the size of the crystals and I believe that it is important to retain the gross morphology of the brushite from which HA is derived.
The basic method for the preparation of commercial HA is described by Tiselius et al, Arch. Biochem. and Biophys., 65 (1956) 132 and involves boiling brushite in an excess of NaOH. I have confirmed the observation of Jenkins, Biochem. Preparations, 9 (1962) 83, that the NaOH must be added before the heating is started; X-ray diffraction showed that boiling without alkali leads to a rapid partial conversion of brushite to other forms of calcium phosphate, as described by W. E. Brown et al, Nature, 196 (1962) 150, with disruption of the brushite morphology that is needed for an adequate flow rate when using the product. It can be demonstrated that, while heating in the presence of alkali, the material comprises a mixture of brushite and HA. In the Tiselius method, before the materials boils, at a temperature of 70.degree.-80.degree. C., observation indicates that the conversion to HA is complete but that the HA crystallites are still very small. Beyond this stage, titration of samples showed that there was little further incorporation of alkali. In order to obtain satisfactory crystallites, it is necessary to subject the product of the first boiling stage to repeated boiling in dilute phosphate buffer. Although it is possible to prepare HA from brushite by alkali treatment in the cold, the crystallites which are formed initially do not grow larger.
These observations suggest that the main effect of the boiling stages, recommended by Tiselius et al without explanation and followed by all authors since, is to ripen the HA crystallites to a size giving optimum binding strength. Although it has been stated that the final preparation contains incompletely converted brushite crystals, X-ray results do not support this, and the low Ca/P ratio of the Tiselius material compared with the stoichiometric value of 1.67 is now believed to be due to deficiencies in the lattice.
A shorter process for the preparation of HA is described by R. K. Main et al, J. Am. Chem. Soc, 81 (1959) 6490, but this still involves a period of boiling and there are numerous washing stages. Again, an increase in elution molarity and crystallite size on boiling can be observed, but in this case the Ca/P ratio changes very little. It is noteworthy that, at the start of the boiling period, this material gave stronger binding than Tiselius HA at a similar stage. Furthermore, X-ray diffraction showed that the conversion to HA proceeds differently. The method uses ammonia which is added continuously so as to maintain a pH of about 8. At a very early stage of the conversion, when the temperature had risen only to 50.degree. C., I have observed, by X-ray diffraction, that the brushite is mixed not with HA but with octacalcium phosphate (OCP), Ca.sub.8 H.sub.2 (PO.sub.4).sub.6.5H.sub.2 O. Amorphous material was also present, giving a high background. At later stages, the patterns showed HA mixed with a trace of OCP which finally disappeared.
This sequence of transitions has been reported by Brown et al, supra, but only in the absence of added alkali. OCP may be formed in the Tiselius procedure also, but the higher pH used there may make the OCP stage too transient for it to be recorded. It seems clear that a more rapid production of suitably sized HA crystallites is associated with a detectable OCP intermediate. However, the procedure of Main et al shares the disadvantage of the Tiselius method that prolonged boiling and washing can lead to a break-up of the massed crystallites with consequent reduction in flow rate. Further, when the system is boiled, "bumping" (which varies in extent with
REFERENCES:
patent: 3737516 (1973-06-01), Jenner
patent: 4046858 (1977-09-01), Barsa et al.
patent: 4139599 (1979-02-01), Tomlinson et al.
Main et al., "A Modified Calcium Phosphate for Column Chromatography of Polynucleotides and Proteins", Journal of the American Chemical Society, vol. 81, pp. 6490-6495 (1959).
Mazin et al., Anal. Biochem., vol. 61, pp. 62-70 (1974).
Journal of Physical Chemistry, vol. 77, No. 19 (1973), The American Chemical Society (Easton PA., U.S.A.).
Boskey et al., "Conversion of Amorphous Calcium Phosphate to Microcrystalline Hydroxyapatite, A pH Dependent Solution-Mediated, Solid-Solid Conversion", pp. 2313-2317.
Langel Wayne A.
Vertiz O. R.
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