Debruyne I
Anal Biochem. 1983 Apr 15;130(2):454-60. doi: 10.1016/0003-2697(83)90615-2.
A sensitive technique for inorganic phosphate determination was developed. It is based on the formation of an insoluble rhodamine B-phosphomolybdate complex. After it is washed with 1 N HCl the precipitate is dissolved in acetone and rhodamine B is measured spectrophotometrically at 555 nm. In 1 N HCl, the complex is composed of three molecules rhodamine B and one molecule phosphomolybdate. Due to the high molar absorbance of rhodamine B in acetone and to the threefold amplification of dye concentration compared to Pi concentration in the precipitated complex, a molar absorption coefficient of 330,000 +/- 5000 M-1 cm-1 (SD) is obtained. This allows the determination of quantities as low as 1.5 nmol Pi with good precision, while quantities as low as 0.5 nmol Pi are detectable. The effect of anions and buffers was studied. Some possible applications of the method are illustrated, as, e.g., enzyme activity measurement at very low substrate concentration and determination of small quantities of Pi and total phosphate in (biological) samples.
开发了一种用于测定无机磷酸盐的灵敏技术。它基于不溶性罗丹明B - 磷钼酸盐络合物的形成。用1N盐酸洗涤后，沉淀溶解于丙酮中，并在555nm处用分光光度法测定罗丹明B。在1N盐酸中，该络合物由三个罗丹明B分子和一个磷钼酸盐分子组成。由于罗丹明B在丙酮中的高摩尔吸光度以及与沉淀络合物中磷酸根离子浓度相比染料浓度的三倍放大，获得了330,000±5000 M-1 cm-1（标准差）的摩尔吸收系数。这使得能够以良好的精密度测定低至1.5 nmol磷酸根离子的量，同时可检测低至0.5 nmol磷酸根离子的量。研究了阴离子和缓冲液的影响。举例说明了该方法的一些可能应用，例如在极低底物浓度下测量酶活性以及测定（生物）样品中的少量磷酸根离子和总磷酸盐。
