This is a related question to an older question of mine: https://www.researchgate.net/post/How_to_measure_free_phosphate_Pi_content_in_leaf_tissue.
I am trying to measure free phosphate (Pi) concentration in tomato leaf using molypdenum blue method (ammonium molypdate + ascorbic acid). I followed several published papers to extract Pi from tomato leaf with HClO4 10% (Bozzo et al., 2006, Plant, Cell and Environment 29, 303-313) and TCA 10% (Wilcox et al., 2000, Crop Sci., 1601-1605). Strangely enough, both methods caused serious precipitations with the molypdenum blue reagent, which interfered with OD reading. Increasing ascorbic acid up to 8% in the reagent solution and reducing extraction:reagent ratio to 1:10 (v/v) did not help. You can view the demonstration in the attached files.
Recently I tested the extraction with acetic acid 1%. The result was surprisingly good. There was no precipitation with molypdenum reagent and the color was strong. However, I couldn't find any reference using acetic acid to extract Pi from plant, which seems strange. My question is whether acetic acid 1% is capable of fully extracting Pi from leaf cell, as well as killing cell enzymes which might change Pi concentration upon extraction, and whether it hydrolizes any organic phosphate compound and change the free phosphate content. Any suggestion about the problem with perchloric acid and TCA or a better extraction solvent for this assay is also welcomed.
I am surprised you got any phosphate color at all considering the molybdenum blue reaction needs a pH of 0.1 while acetic acid's pKa is ~2.4. You may have triggered another reaction especially since it is also used under alkaline (?) conditions to quantitate Antimony and Tannins.
You may want to oxidize any phosphomolybdic complex with 1% Ammonium Metavanadate at quantitate the yellow complex at 420 nm. It is a much more pH stable reaction. Check the AOAC (they have a section on plants) and the APHA (Standard Methods of Water and Wastewater Analysis).
Thank you for your answer. I am very sorry I didn't explain accurately. The molypdenum blue reagent I use also contains ~1.3 M H2SO4 and 0.2 mM potassium antimonyl-tartrate according to the formula by Strickland and Parsons (A Practical Handbook of Seawater Analysis, 1972). I haven't seen any reference indicating the required pH for this reagent so I haven't tested the reagent pH yet, but I believe that amount of H2SO4 is enough to bring the pH down quite low. in some papers like Bozzo et al. (2006), the authors didn't even use H2SO4 at all.
I use molypdenum blue method because according to Burns and Hutsby ( Critical comparison of the vanadomolybdate and the molybdenum blue methods for the analysis of phosphate in plant sap, 1986), molypdenum blue method is 10 times more sensitive than vanadomolypdate method, and the vanadomolypdate method also has the tendency to overestimate phosphate concentration when the sample background color is high, which is because of the interference of many other compounds at 420 nm absorption spectrum. In most of my references in plant biochemistry area they also used molypdenum blue method for spectrophotometric phosphate assay, due to plant cells containing a lot of interfering compounds.
As I recall the %P in plant tissue was about 0.05 to 0.10% (500 - 1000 ppm). But this was the AOAC procedure where the plant tissue dried, ground, and ashed. The resulting ash was dissolved in 1/3 HCl, with gentle heating, diluted to 100 mL in water. An aliquot was tested using the Ammonium Metavanadate procedure. Ashing ensures that ANY potential organic interferences are destroyed. You may have no choice in using this method.
Remember, P is also part of the DNA/RNA matrix so ashing (or even wet digestion) would be most appropriate.
In my study I want to measure free phosphate (orthophosphate) in leaf only, therefore I do not perform ashing step, and I also cannot use any extracting solvent that may hydrolize organic phosphate compounds. Free phosphate level in tomato leaf, according to Bozzo et al. (2006), is only around 10 ppm FW (probably around 100 ppm DW), so I think molypdenum blue method which has higher sensitivity would be better.
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