A Stable Metaphosphate Preparation for Use as a Protein Precipitant. ∗

Abstract

That metaphosphoric acid causes essentially quantitative precipitation of proteins, in pH ranges acid to the isoelectric point of the protein, has long been known. Its use as a means of removal of the protein from blood serum preliminary to analysis for other components of the blood has been studied by Folin 1 and by Hiller and Van Slyke. 2 Due to its instability in aqueous solution and to the difficulty of obtaining a uniform commercial reagent, meta-phosphoric acid has lost favor as a standard protein precipitant, although its action closely simulates that of the more expensive trichloracetic acid commonly used (in 10% solution).

The object of this paper is to describe a method of preparation and manner of use of metaphosphoric acid which will largely eliminate the objections which have tended to prevent its use as a routine protein precipitant.

Metaphosphoric acid is commonly prepared by heating the ortho-acid to drive off one mole of water per mole of ortho phosphoric acid, or by the addition of one mole of water to one mole of P₂O₅. The acid so formed is a strong acid (pKa=ca. 1.5) and may be highly polymerized. When placed in solution in water, it is hydrolysed rapidly back to the ortho-acid. The commercial meta acid, obtained as sticks of glacial metaphosphoric acid, always contains sodium, usually containing approximately one atom of Na per 2 atoms of P. It is sometimes given the formula, NaPO₃. HPO_a. Titration curves made on solutions of such products, however, show that all of the acid which is present is in the ortho form. The remainder of the phosphorus is normally present as sodium meta phosphate. When a solution of the “glacial” acid is boiled, all the meta phosphate present is changed quickly to ortho, the same reaction being accomplished at room temperature in the course of 2-3 months.