Instead of reducing the phosphomolybdate complex with ascorbic acid (molybdenum blue) try oxidizing it with Ammonium Metavanadate and measure at 420 nm.

We have done both total P and Pi in our studies using ammonium molybdate protocol. Kindly refer to the following publication for detailed methods

http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0108459  

It works well for us. Kindly write to me for any further helps

All the best!

Best regards

Ceasar

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Have you think that percloric acid may creates an oxidizing environment that do no allows the ascorbic acid to reduce the phosphomolibdic complex. in this case I may suggest the use of a high concentration of ascorbic acid

You could try doing a simple sodium carbonate/bicarbonate extraction of lyophilized plant material, and measure the results using an ion chromatography machine (if you have access to one). This method will also allow you to quantify other free anions, such as nitrate, chloride and sulphate. I hope this helps.

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I agree with Luke you could try with a simple extraction. Mainly if you want to quantify the free phosphate which is more soluble. The colorometric method can be use

, although the IC is faster and you can determine other ions

We've experienced myriad interferences when using oxidizing acids (e.g., 3:1 nitric : sulfuric) to digest leaves for Ptot using the molybdate method--as far as we can tell, any trace of residual nitric acid following digestion will consume reducing agent more rapidly than the phospho-molybdate complex. Your symptoms sound a lot like ours. 

I'll second Armando Carrasquero's (above) suggestion--use extra reducing agent, and even consider adding some extra to the sample before adding the molybdate reagent to prevent oxidation of the molybdenum/antimony.  

Very sorry for the late reply and thank you all for your answers. I really appreciate the help.

+Bruce Neagle:  I don't really think ascorbic acid is the problem of the precipitation because the plant cells also contain vitamin C, don't they? I used the ascorbic acid method because I have read somewhere (don't remember exactly where) that ascorbic acid method has lower detection limit than vanadate method.

+ Luke Bell: Does this method measure only free phosphate (PO43-) or any phosphate containing compound? I am focusing on free phosphate only. I would prefer spectrophotometry method as it is simpler for me regarding equipment, but I will try this method if the previous one fails to work.

+ Armando Carrasquero + Timothy Veverica: After extraction of the sample, I neutralized the excess acid with KOH/triethanolamine to pH ~ 5-6. Also, the ascorbic acid concentration I used was 10.8 mg/ml reagent (according to Strickland and Parsons, 1972), which I think is quite high. The reagent gives good color with Na3PO4 solution at concentration up to 1 mmol/ml. When added to the samples, the reagent did give a faint blue color. The problem is there was a layer of blue-greenish precipitation at the bottom of the well, which interfered with the reading of the plate reader, leading to the results fluctuating and inaccurate. I suspect that some compounds in the samples, maybe phenolic, reacted with the reagent and caused this precipitation. The plant I am studying is tomato, whose leaves indeed contain many phenolic compounds. I wonder if switching to another extracting solvent like acetone or ethanol, or adding activated charcoal might solve this. Nevertheless, I will try your suggestion of adding more ascorbic acid and report back to you.

+ S. Antony Ceasar: Thank you very much for sharing with me your protocol. May I ask the purpose of adding β-mercaptoethanol and phenylmethylsulfonyl fluoride? Are they essential? These are cytotoxic compounds so I would like to avoid them.

The method that I mentioned measures free phosphate within your sample. Best of luck.

Duy,

Metavanadate method for phosphate is about 40 times less sensitive than the molybdenum blue method.  However, you may not need this increased sensitivity since TOTAL phosphate from leaves is about 1000 ppm on a dry basis.  I have no idea what is available!

Hi Mr. Duy Minh Pham,

Obviously, you have lots of help already. I just want to raise up a question from the view of analytical chemist:

How did you define 'free phosphate'? Your definition should guide you choose the right sample preparation and analytical method.

In my opinion, more friendly extraction method with approperate cleanup procedure (SPE) should be considered; IC (ion chromatography) are phosphate ISE (Ion Specific Electrodes) are recommended in your case. 

There is also a method for 'available Phosphate (Bray?)' for soils.