Inorganic phosphorous - (Jun/30/2012 )
I'm trying out for the quantification of inorganic phosphorus in my samples. I've tried it out with Fiske Subbarow and Munoz et al methods.....But i'm finding it difficult to get consistence reading in both the methods. I know there are number of factors that could have went wrong but I'm zeroing in on improper cleaning of my Test tubes..... can anyone tell me what the best method is to clean the test tubes off inorganic phosphates? Will acid -water do the trick? or any thing else?
we washed with phosphate-free detergent (not really phosphate-free when using sensitive phosphate assay) and rinsed with copious amounts of deionized water.
we measured inorganic phosphate using a method modified from chen, et al, anal. chem., 28: 1756-58 (1956). it is more sensitive than fiske-subbarow.
Determination of inorganic phosphorus in biological fluids
Starting by "Chen P.S. et al.(1956). Microdetermination of phoshorus. Analyt. Chemistry 28:1756-58", I modified it can be placed on board to several automatic chemical instruments.
Molybdate solution pH 1.0 : dissolve 0.5 g (0.4 mM) of ammonium molybdate <(NH4)6Mo7O24-4H2O> in 1 liter of 0.21 M H2SO4 (pipet 12.0 ml of reagent grade d. 1.8 g/ml, 95% purity H2SO4 into 500 ml H2O. cool to room temperature and dilute to 1 liter), stable indefinitely if stored at 4-25 °C.
Detergent : dilute 25 ml of Triton X-100 to 100 ml with H2O. Stable for 2 years if stored at 4 °C.
Working solution : 24 hrs before to use mix 100 ml of molybdate solution with 0.9 ml (0.225% final concentration) of detergent. Stable 6 months at 2-8 °C.
Label three tubes "reagent blank" (RB), "standard" (ST - KH2PO4), and "sample" (S), add 1.0 ml of working solution and the dispense 10 ul of standard/calibrator in ST e 10 ul of serum/plasma or 1:20 dilute urine(*) in S. Mix and incubate for 5 min. at 25-37 °C. Measure Abs at 340 nm (334-365) of ST and sample against RB within 60 min. and multiply for ST concentration.
(*) For urine collect for 24 hrs in collection bottle containing 10 ml of 10% (v/v) HCl to avoid phosphate precipitation. Mix well contained bottle and dilute urine 1:20 with dH2O and multiply the result by 20.
In the phosphorus determination advisable devoted glassworks use it. Carefully goes washed with ho diluted (6 mol/L^-1) hydrochloric acid, rinsed with deionized water and preserved, filled with water, up to the moment of the use. Wash polyethylene stoppers in 6 mol(L^-1 HCl, rinse with demineralized water, then wash in 8 mol/L^-1 nitric acid and again rinse with demineralized water. Pleonastic is cannot use detergents containing phosphate.