Hi Reena Sharma

1-      Sensitive colorimetric method  for phosphate detection that use ammonium molybdate combined with Malachite Green dye.

2-      They can measure phosphate concentrations down to about 1 micromolar and can be done in a 1-cm cuvette or a microplate.

3-      Can be used the literature.

http://www.enzolifesciences.com/BML-AK111/biomol-green/

Best regard

Dear Sharma,

We work a lots with P analysis in our lab. I can send you the protocol if you give me your e-mail address :)

khwan

Relative rapid and simple method is colorimetric determination from samples prepared by wet digestion:

Pollman R. M.: Atomic absorption spectrophotometric determination of calcium and magnesium and colorimetric determination of phosphorus in cheese: collaborative study. J. Assoc. Anal. Chem. 74 (1991) 27-31

Estimation of total P in plant tissue

Digestion mixture:

Digestion mixture will be prepared by adding 0.42 g of Se and 14 g of LiSO4.2H2O to 350 ml of H2O2, mixed well and 420 ml of conc.H2SO4 will be added slowly to it keeping it in an ice bath. The reagent so prepared will be stored at 2oC and used for the digestion of plant material.

Digestion Method:

Dried ground plant leaf material or root, (0.1 g) will be taken in a digestion flask; 2 ml of digestion mixture will be added to the flask and placed it on a hot plate. The temperature will be increased gradually from 50oC to 200oC. When the mixture turned black, 0.5 ml of HClO4 will be added to each sample, and heated again until the material will be colorless. The flasks will be removed from the hot plate and cooled down. The solution will be diluted up to 50 ml in a volumetric flask and filtered.

Phosphorous Determination (Jackson, 1958):

Procedure:

Standard solutions of P will be prepared using any phosphorous containing salt like NaH2PO4, KH2PO4.Standards solutions of P will be of 2,4,6,8, 10, 12, 14, 16, 18 and 20 mg L-1. 4.5 mL of each standard solution will be taken in test tube. Then 0.5 mL Barton’s reagent will be added to it. For complete reaction leave it for 10 minutes. Then the OD will be read at 470 nm using a spectrophotometer. Standard curve will be made. Then blank will be run in the same way. Then samples will be run in the same way and OD will be read. Actual reading of samples will be noted by using standard curve, which will be prepared for standard solutions.

P will be calculated by using formulae.

P (mgKg-1) = (PPM mg-1) x dilfactor reading

Sample (g)

Barton’s reagent

 Dissolve 25g Ammonium molybdate in warmed 400 ml distilled H2O. (Solution A).

 Dissolve 1.25g Ammonium meta-vendate in 300 ml distilled boiling water.

 Cool and add 250 ml conc. HNO3. (Solution B).

Mix A and B solutions and then dilute up to 1L.

Thanks Zafar..

can we do digestion directly using conc. H2SO4.. I think Se, LiSO4.2H2O in H2O2 are acting as catalyst only, using them will decrease the digestion time but may interfare in results... We also test Total Kjeldahl Nitrogen following Bremmer and Kenny 1965. There Selenium catalyst is used to speed up the digestion process.

Digest for the determination of plant P

Weigh accurately a sufficient amount of oven-dried and ground plant sample (around 0.1g) and transfer to a digestion tube. Add 2 mL of concentrated H2SO4, swirl gently to wet the sample and leave to stand overnight. Place the tubes in a block digestor in a fume hood with extractor on and heat at 120 deg C for 30 minutes. Gradually increase the temperature to 330 deg C and boil for 30 minutes. Leave the digestion block switch on. The digestion is complete if there is no sign of solid particle in the resulting dark else continue strong heating 330 deg C treatment. Rapidly cool down the digest to room temperature in an air-draft with the hot tubes inside the hood and then carefully add 0.5ml of 30% H2O2 solution. Return the tubes to the hot block and heat until the dense white fume of H2O2 has subsided. Remove the tubes and cool to room temperature. If digest is still unclear repeat the H2O2-heating treatment.

When cold add about 10-20 mL distilled water to the clear digest and make up to 50 mL with distilled water, mix thoroughly and allow to stand overnight for the suspended silica to settle.

P (N and K also) can be determined in the digest.

Determination of P in digest

Reagents

• P-Catalyst solution (0.2743%)

• Sulphomolybdic solution

Add 12 g ammonium molybdate to about 50 mL distilled water in a 250 mL conical falsk and then cautiously add with stirring on a magnetic stirrer 70 mL conc. H2SO4. and dilute to 500ml

• Reducing solution

Pipette 25 mL of sulphomolybdic solution into a 100-mL volumetric flask. Add 530 mg of ascorbic acid and then 5 mL of a P-catalyst solution. Dilute to the mark with distilled water and mix.

Procedure

Dilute digest with distilled water as required and add 2 mL of reducing solution, mix thoroughly and determine P colorimetrically on a spectrophotometer.

Thanks...Tesha.

Colorimetry methos of K. P. Moore is simple and good

Atomic Absorption Techniques of E. A. Hanlon is also available.

Interesting discussion! Coincidently, I am currently reading the protocols for the P determination in plant and soil samples. Hi Patompong, could you please send me your protocol to [email protected]? Thanks!

Pls see attached

Hi Patompong! Could you send me the protocol, please. I'm doing plant nutrient analysis for now, and looking for good procedure for determining phosphorus content. Thanks! :D

[email protected]

Hi everybody! 

I am also interested in determined P content (total phosphorus) in fresh or dried plant tissues. Can anyone share a protocol?

Thanks!

 Hello all,

I found a couple of protocols online for P quantification by colorimetric determination but I was wondering if anybody has  a tested  protocol both for the digestion with hot plate and the Molybdenum-Blue Method 

I am working on that area estimation of soil and plant p content. Hi Patompong, could you please send me your protocol to [email protected] ?

Thanks!

Hi Reena Sharma

1-      Sensitive colorimetric method  for phosphate detection that use ammonium molybdate combined with Malachite Green dye.

2-      They can measure phosphate concentrations down to about 1 micromolar and can be done in a 1-cm cuvette or a microplate.

3-      Can be used the literature.

http://www.enzolifesciences.com/BML-AK111/biomol-green/

Best regard

Hi Patompong!

Could also you send to me the phosphorus assay protocol, please at my address [email protected] Thanks a lot in advance

Best Regards

Use vanado molybdate method

I also Use Molybdate method, any faster one recommendation?