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nucleic acid quantitative measurement - fixed phosphorus method
"principle"
phosphorus measurement methods are many, Fiske-Subbarow phosphorus method is a classic but still commonly used method, it has sensitive and simple characteristics. Various phosphorus organicare hydrolyzed by sulphuric acid or perchloric acid, making organophosphate digestion into inorganic phosphorus. Inorbic phosphorus reacts with palladium phosphate (commonly used ammonium phosphate or sodium tantalum) to produce a phosphate compound under acidic conditions.
is treated with a reducing agent, and the phosphate ensent is reduced to produce tantalum, with the largest light absorption peak at 660nm. In a certain concentration range, the depth of color is directly related to the phosphorus content. Therefore, phosphorus can be quantitatively determined by phosphorus.
chemical reaction type:
(NH4) 2MoO4 plus H2SO4→ H2MoO4 plus (NH4)2SO4
H3PO4 plus 12H2MoO4→ H3P (Mo3O10) 4 plus 12H2O
reducing agent
H3P (Mo3O10) 4→ Mo2O3MoO3
nucleic acid is a class of phosphorus-containing compounds whose molecules contain a certain proportion of phosphorus, generally pure RNA and its. < a href> "> nucleotides phosphorus-containing mass score of 9.0%; "" and its nucleotide-containing phosphorus mass The score is 9.2%, that is, every 100g nucleic acid contains 9.0 to 9.2g phosphorus, that is, the nucleic acid content is about 11 times the phosphorus content, so the amount of phosphorus measured, you can find the nucleic acid amount. This is the theoretical basis of phosphorus method, phosphorus quantitative determination is one of the common means to determine nucleic acid content.
bioorganic phosphorus material sometimes contains inorganic phosphorus impurities, so when using the fixed phosphorus method to determine the amount of the organophosphorus substance, the total phosphorus content of the sample must be determined separately, that is, the sample after digestion of the measured amount of phosphorus, and the sample's inorganic phosphorus content, that is, the sample has not been digested directly measured phosphorus content. Subtracting the total amount of phosphorus from inorganic phosphorus is the phosphorus content of the organophosphorus substance.
Methods and steps
< p style- "text-align: left;" >1, phosphorus standard curve drawingpipe number name | 0 | 1 | . 2 | . 3 | 4 |
standard phosphorus solution/mL
0
0.5
1.0
1.5
2.0
2.5
6 mol/L sulphuric acid/mL
0.5
0.5
0.5
0.5
0.5
0.5
0.5
0.5
0.5
0.5
<: left;">0.5
0.5
reducer/mL
0.1
0.1
0.1
0.1
0.1
.1
0.1
.Distilled water/mL
3.9
3.4
2.9
2.4
1.9
1.4
0" .9
after each reaction fluid is added, it is 20min warmed at 30 degrees C, with a wavelength of 660nm color in the ionometer, and a metered absorption value. With the phosphorus content as the horizontal coordinate and the light absorption value as the ordinate, the standard curve of phosphorus is determined.
2, total phosphorus measurement
take 30 ml of Keshi bottle 2, I. , II. number, I. bottle for blank control, with a scale straw accurately absorb 1.0mL, placed in II. Kai's bottle, I. Bottles add distilled water 1.0mL, without sample liquid, two bottles are added to 6mol/ L sulphuric acid 1.0mL digestive rack with a small fire" >digestion, to the solution brown, slightly cooled, add 2 mol / L nitric acid 2 drops, and then continue to heat, until the escape of white smoke, the solution is colorless and transparent, indicating that the digestion is complete.
After cooling the Kei's besott, add 1.0mL distilled water, boiling water bath steamed 5min, so that the pyrophosphate decomposition. Take the Keshi bottle out of the boiling water bath and wait for it to cool down, turning the I. and II. bottles.