Phosphate
- Phosphate
-
Phosphorus
P
Phosphate
An inorganic chemical (a salt of phosphoric acid)
Essential element for all living organisms
- Quantification of phosphate
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The principle of P quantification is how stain phosphate by ammonium molybdate.
Phosphate in lipids extracted from litter (Prototype)
Basically written by Otaki M
Preparation
Solvents and chemicals are of highly-quality grade
Glassware is washed by phosphate-free detergent, rinsed 10 times with hot tap water, and then 2 times with deionized water[Lipid extract for lipids]
Chloroform : Methanol : Deionized water = 1 : 2 : 0.8, v/v/v)
[Fiske-Subbarow reagent]
0.25% 1-amino-2-naphthol-4-sulfonic acid
13.7% sodium metabisulfite
3% sodium sulfite[Phosphate standard]
1.2 mM phenyl phosphate disodium salt
Adjust 0 to 98 nmol phosphorus before useExtraction of total lipids from litter
Total lipids are extracted on the basis of a method proposed by Bligh and Dyer (1959) with modification (White et al. 1979). A total amount of 1.0 g litter si bottled into a 40-ml screw-capped centrifuge tube, and then 19.0-ml extract is poured into the tube and mixed with litter. Thereafter, 5-ml chloroform and 5-ml deionized water are added into the tube. After the samples are vortexed and centrifuged at 2000 rpm for 5 min (Kubota KS-5000), two layers are developed in the tube. The lower layer (bottom phase) that contained lipids is transferred into a new test tube.
Quantification of phosphate in the lipids
Each of 1/10 amount of total lipid extract is digested by perchloric acid. After the lower layer sample is evaporated and condensed, the sample is mixed with 500 μl 60% perchloric acid. The mixed samples were then heated at 190 °C for 30 minutes. During the digestion, the lipid extract colors black and contains particulates. These two signals indicate that the organic compounds are digested and leak phosphate ions. To the sample, 2300 μl 1.78 mM ammonium molybdate solution and 100 μl of Fiske-Subbarow reagent are added (Fiske and Subbarow 1925). Then the samples in tubes are heated at 90°C for 10 minutes on dry bath. Absorbance at 815 nm that reflects the amount of blue-colored PMo12O407- reacted with phosphate was measured using a spectrophotometer (U-1800, Hitachi). The absorbance of standard solutions and the samples are measured at 815 nm using a spectrophotometer. The content of phosphorus ions on each sample is calibrated by the standard curve ranging from 0 to 98 nmol phosphorus ions.
Protocol
Litter sample = 1.0 g freeze-dried and grinded litter
↓ put into 40-mL screw-capped glass tube
[Total lipids extracted by Bligh-Dyer method]
↓ 19 mL single-phased extract mixed with 10-mL methanol, 5-mL chloroform and 4 mL deionized water
↓ 29-mL two-phased extract mixed with 5-mL chloroform and 5-mL deionized water
↓ Lipid-contained lower layer istransferred into another tube
↓ The lower layer sample evapolated by jet nitrogen flow(1/10 amount of total lipid extract in the sample was used for quantifying phosphate content)
[Fiske-Subbarow colorimetric determination of phosphorus]
↓ add 500 μl 60% perchloric acid to the samplePhospholipids containing the total lipids are separated into organic compounds and phosphate ions
↓ Heated at 190°C for 30 minutes.
↓ Mixed with 2300 μl 1.78 mM ammonium molybdate
↓ The anion was then reduced by 100-μl Fiske-Subbarow reagent(to form the blue colored β-keggin ion (PMo12O407-)
↓ Measuring absorbance at 815 nm
Quantification
References
- Bligh EG, Dyer WJ (1959) A rapid method for total lipid extraction and purification. Can. J. Biochem. Physiol. 37: 911-917
- Fiske CH, SubbaRow YJ (1925) The colorimetric determination of phosphorus. J. Biol. Chem. 66: 375-400
- White DC, Davis WM, Nickels JS, King JD, Bobbie RJ (1979) Determination of the sedimentary microbial biomass by extractable lipid phosphate. Oceologia 40: 51-62
- References