I would like to determine if my compound of interest, farnesyl pyrophosphate, is degrading in our storage conditions. My plan is to run PEI-TLC using solvent isopropanol:ammonia:water, 6:3:1, by volume (after both Waechter 1999 PNAS and Popjak and Williams 1969 J Biol Chem).
However I'm not sure what would be the best stain to visualize both FPP as well as the free ortho- and/or pyrophosphate that may arise from degradation. I'm looking at stains recommended at http://www.cchem.berkeley.edu/rsgrp/TLCStainGeneralReference.pdf and wonder:
Would the cobalt(ii) chloride method visualize orthophosphate? Would the ammonium molybdate tin(ii) chloride method visualize phosphate esters? Can I do multiple stains if they stain different things? Should I just buy something like http://www.sigmaaldrich.com/catalog/product/sial/69888?lang=en®ion=US ?
It's most important for me to detect if there are single phosphate groups in the FPP solution.
Cobalt(II) chloride for organic phosphate esters.
- Spray solution: 1% anhydrous cobalt(II) chloride solution in acetone.
- After-treatment: Heat at 40-50°C. Blue spots. The reaction is not sensitive.
- R. Donner, K. Lohs, J. Chromatog. 17, 349 (1965).
Ammonium molybdate - tin(II) chloride for phosphoric acids.
- Spray solution I: 1% aqueous ammonium molybdate solution.
- Spray solution II: 1% solution of tin(II) chloride in 10% hydrochloric acid.
- Procedure: Spray with I, dry the chromatogram and spray with II. Heat, if necessary, at 105°C for 3 - 5 minutes.
- H. Seiler, Helv. Chim. Acta 44, 1753 (1961).
There's also a third option for phosphoric acid:
- Ammonium molybdate - crystal violet for phosphoric acid. PC.
- Spray solution: Mixture of 5 ml 1% aqueous ammonium molybdate solution, 5 ml 25% hydrochloric acid and 90 ml acetone.
- Solution a: Dissolve 2 g crystal violet (or brilliant green or iodine green) in 350 ml water.
- Solution b: Dissolve with heating 4 g ammonium molybdate in water, add 50 ml 10 N hydrochloric acid and fill up to 100 ml with water.
- Dip solution: Mix a and b, wait at least 3 hours and filter the solution.
- Procedure: Spray the chromatogram with the spray solution, heat 3-6 min at 85°C, dip into the dip solution and place immediately on a prepared clean filter paper.
I would appreciate advice on which of these visualization methods I should use.
Edit: forgot to say, FPP is mildly UV-reactive. Not sure if ortho or pyro-phosphate would be. Maybe would be best to take a UV picture first, then use the Ammonium molybdate - tin(II) chloride method?
Thank you!
I'd use Iodine in silica then Ceric Ammonium Molybdate to visualize those substrates. Members of my lab find that sequence helpful when analyzing fermentations that produce polyolefin pyrophosphates.
I've used the following one with much success for alkylphosphonic acids and their alkylesters:
dip your plate in a 1% ethanolic solution of 4-(4-nitrobenzyl)pyridine
heat the plate very well
dip the dried, HOT plate in concentrated ammonia solution
phosphonates will appear as deep blue spots on a colourless background
You may want to test first if this works for phosphoric acid species, I'm pretty sure it'll work for the (alkyl)(pyro)phosphates
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