I am collecting fractions from HPLC with an eluent of acetonitrile (containing 0.1% formic acid) and water (containing 0.1% formic acid). I want to remove formic acid from rotary evaporation. But it does not work effectively. How do you normally do it?
Hello Juan,
We faced the same problem and wanted to solve it by SPE at first (see https://www.researchgate.net/post/What_is_the_capacity_of_a_RP18_SPE_catridge). At the end, we were successful by freeze drying after removing the acetonitrile in a rotorvapor.
A rotary evaporator is not the best solution for mixed solvents, because of the conventional condenser and collecting flask. I used a centrifugal evaporator with a cryogenic trap to protect the pump, in the days before the introduction of suitable acid-resistant membrane pumps. Slow, but you can increase the evaporation rate by bleeding in air or an inert gas.
Note that under vacuum, formic acid doesn't azeotrope with water and it may be necessary to check that it doesn't become concentrated, in which state it is quite reactive (I'm writing from distant memory).
As Christopher mentioned, we were worried about the fact that no azeotrope exists with water and we did not find information about other azeotrope compositions. So we freeze-dried a volume of some hundreds of milliliters containing 0.1% formic acid (several hundred microliters) and the sample became dry. I have not yet seen the NMR spectra.
Hello Juan,
Preparative thin-layer chromatography (TLC) of the collected fractions can be helpful. By this you have completely dried analyte as zones on the TLC plate. The zones can be scratched, dissolved in a suitable solvent, and new solution can be reconstructed through filtration of the TLC layer material (generally silica gel layer).
If any please let me know.
This exchange recalls old, smelly and possible out-of-fashion methods for isolating metabolites for identification. The techniques included ion exchange chromatography and paper electrophoresis. My metabolites were amino acids (Lee & Pollitt, Biochem. J. 1972, 126, 79-87). Sometimes you either need a buffer or just don't want your solutions to get too acidic - if that's a problem in the present case you could add some pyridine before evaporating.
Good volatile buffers are pyridine acetate and formate. Everyone had a distillation setup at the time, and I don't know if you can nowadays buy ready-purified products that meet requirements.
Reasonable-grade pyridine and acetic acid can be purified by distilling through a short packed or Vigreux column. Formic acid is best distilled and stored as the azeotrope (about 76%) with water (you could buy it at that strength); the concentrated acid is unstable. Store the purified products in a flash-proof fridge in containers with non-contaminating stoppers. You make up the buffers by volume, to avoid introducing KCl from a pH electrode. The lab water still was fed with deionised water (ultrafiltration didn't exist...).
The nmr spectra (60 and 100 MHz continuous wave instruments) of my metabolites were suprisingly clean.
Hello Juan,
The 1H NMR spectrum showed no sign of formic acid after freeze drying.
The reason for using formic acid at all was that we transferred a separation method from LCMS to prep. HPLC. We did not want to alter the eluent composition as we could not detect the analytes on the preparative system.
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