Biomedical Engineering Reference
In-Depth Information
1.3.2 Determination of Total Phosphate
Important!
Use phosphate-free test tubes (cleaned with hot diluted
HCl) or plastic disposables.
ThisprocedureissimplerandmorereliablethanthatbyFiske and
Subbarow.
A6N HCl
Solutions/Reagents
B 2.5% ammonium molybdate (w/v) in ddH
2
O
C 10% ascorbic acid (w/v) in ddH
2
O
D 2% urea (w/v) in ddH
2
O
E
Reagent after ashing
: Soln. B, C, and ddH
2
Oaremixedin
a ratio of 1:1:8
E
Reagent without previous ashing
: Soln. A, B, C, and ddH
2
O
are mixed in a ratio of 1:1:1:7
EandE
arestableonlyfor1day.
Standard
10 mM KH
2
PO
4
conc. sulfuric acid
conc. nitric acid
Ashing
Ashing must be done if phosphate is at least partially covalently
bound as, for example, in nucleic acids, nucleotides, or phospho-
proteins.
Give 0.2 ml of conc. sulfuric acid to 1 - 2 ml of aqueous sample.
Concentrate the liquid carefully in a hood at about 130
◦
C and then
heat to 280
◦
C until white fog appears. After cooling, add one to
two drops of conc. nitric acid and heat again until nitrous gases
are visible. After cooling, add 2 ml of Soln. D, boil the solution for
a short period, and fill up to 5.0 ml with ddH
2
O.
Determination
Mix 2.0 ml of the sample solution after digestion or sample in
Soln. A with Soln. E and E
. Close the test tubes and incubate in
the dark at 37
◦
C for 1.5 - 2 h. After that, read samples, blank, and
standards at 750 nm.
The standard curve is made in the range of 50 - 350 nmoles
phosphate.
100 nmoles phosphate
=
µ
=
µ
9.497
g PO
4
3.097
g P
References
Chen PS, Toribara TY (1956) Anal Chem 28:1756
1.3.3 Phospholipid Determination
A
chloroform/methanol 1:2 (v/v)
Solutions/Reagents
chloroform
