The colorimetric estimation of inorganic phosphate
The Colorimetric Estimation of Inorganic Phosphate Introduction A phosphate, an inorganic chemical, is a salt of phosphoric acid It consists of one central phosphorus atom surrounded by four oxygen atoms in a tetrahedral arrangement. The phosphate ion carries a negative three formal
phosphorus is found as a free phosphate ion in solution and is called inorganic phosphate, to distinguish it from phosphates bound in various phosphate esters. Inorganic phosphate is generally denoted Pi Phosphate are most commonly found in the form of adenosine phosphates, (AMP, ADP and ATP) and in DNA and RNA
Principle The measurement of inorganic phosphate must be one of the comments determinations carried out in a biochemistry lab. It is certainly surprising how frequently such a measurement is required, and the production of a standard curve is a useful exercise for checking the validity of Beers Law. Inorganic phosphate reacts with ammonium molybdate in an acid solution to form phosphomolybdic
acid. A reducing agent such as metol is added which reduces the molybdenum to give a blue colour but does not affect the uncombined molybdic acid. + ammonium molybdate Acid solu phosphomolybdic acid hosphomolybdic acid + metol(reducing agent)
etol not combine with excess of molybdic acid blue colour Method TCA St.sol(w. s) Copper Blan k
1ml _ 3ml Std1 Std2 Std 3 0.9ml 0.7ml 0.5m l 0.1ml 0.3ml 0.5m l 3ml 3ml 3ml
Std 4 0.3m l Std5 un _ _ 0.7m 1ml l _
3ml 3ml 3ml acetate buffer(pH4 ) Ammoniu 0.5ml 0.5ml 0.5ml 0.5m 0.5m 0.5ml 0.5ml m l
l molybdat e Mix, incubate at RT for 10 min. 0.5ml 0.5ml 0.5ml 0.5m 0.5m 0.5ml 0.5ml Metol And then read at 880nm. l l Unknown _ _
_ _ _ _ 1ml Calculation C1 X V1 = C2 X V2 Introduction
Inorganic phosphate react with ammonium molybdate in an acid solution to form phosphomolybdic acid. A reducing agent such as metol is added which reduces the molybdenum to give a blue colour but does not affect the uncombined.
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