Download 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 charge
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 Beer’s 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.
Pi + ammonium molybdate
Acid solu
phosphomolybdic acid
phosphomolybdic acid + metol(reducing agent)
Metol not combine with excess of molybdic acid
blue colour
Method
Blank
TCA
1ml
St.sol(w.s) _
Copper
3ml
Std1
0.9ml
0.1ml
3ml
Std2
0.7ml
0.3ml
3ml
Std3
0.5ml
0.5ml
3ml
Std4
Std5
0.3ml _
0.7ml 1ml
3ml 3ml
un
_
_
3ml
acetate
buffer(pH4)
Ammonium 0.5ml
molybdate
0.5ml 0.5ml
0.5ml 0.5ml 0.5ml
0.5ml
0.5ml
0.5ml 0.5ml
_
_
0.5ml 0.5ml 0.5ml
_
_
_
0.5ml
1ml
Metol
Unknown
_
Mix, incubate at RT for 10 min.
And then read at 880nm.
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.