Phosphorus assay in column chromatography.

Malerials-30 per cent hydrogen peroxide (Baker and Adamson, phosphorus-free) and the ammonium molybdate, (NHJ6lMo7024.4Hz0, wereused. The Fiske and SubbaRow (I) reagent was prepared by adding 0.5 gm. of purified (1) 1-amino-2naphthol-4-sulfonic acid (Eastman Organic Chemicals) with mechanical stirring to 200 ml. of freshly prepared 15 per cent sodium bisulfite (anhydrous), followed by 1.0 gm. of anhydrous sodium sulfite. The solution was filtered, stored in a dark bottle, and iresmy prepared weekly. Procedure for Total Phosphorus Analysis-Up to 2.0 ml. of the sample to be analyzed and 0.5 ml. of 10 N HsS04 were placed in a 12-ml. conical centrifuge tube and heated in a 150-160”-oven for at least 3 hours. 2 drops of 30 per cent HsOz were added, and the solution was returned to the oven for at least 1.5 hours more to complete the combustion and to decompose all the peroxide. 4.6 ml. of 0.22 per cent ammonium molybdate (or 4.4 ml. of Hz0 plus 0.2 ml. of 5 per cent ammonium molybdate) and 0.2 ml. of the Fiske-SubbaRow reagent were added, mixed thoroughly, and heated for 7 minutes in a boiling water bath, with marbles covering the tubes. The optical density at 830 rnp was recorded with the use of a Beckman model B photometer with the red-sensitive phototube. This procedure for color development at 100” is hereafter designated the heating method. A longer period of digestion at 150-160” did not influence the results. I f an aliquot greater than 2.0 ml. was to be analyzed, it was first evaporated to dryness in a mechanical convection oven at about 110”. 1.0 ml. of H20 and 0.5 ml. of the 10 N HzS04 were then added with rinsing of the walls of the tubes, and the assay was continued as above. When more than 1 mmole of ammonium formate was present, the sample was evaporated to dryness at 110” in order to remove the formate; then Hz0 and H2S04 were added and the analysis continued as above. If the highest accuracy was not required, the following manipulations simplified and speeded multiple total phosphorus determinations on the eluates from column chromatographic separations. Aliquots from successive fractions were transferred to the 12-ml. centrifuge tubes with an automatic syringe,’ and the various reagents were delivered from elevated storage bottles