Acetic acid, rotary evaporation, and a pH sensitive compound

[RFRansom]

Hello you simply marvelous people;

Here's my problem: I'm using normal phase silica MPLC with chloroform and methanol:water:acetic acid as the mobile phases for my final purification step of a cyclic peptide. It works marvelously, and alternative methods without the acetic acid simply haven't so far for separating the compound of interest from a closely related compound that co-elutes on C18 RP.

My issue is that the compound does a rearrangement that breaks the ring upon prolonged exposure to pH below 3.0, especially with even modest heating, and during the course of rotary evaporation the chloroform and methanol volatilize long before the acetic acid, leaving a solution that's approximately half water, half acetic acid. What I'd like to do is neutralize the acetic acid with ammonia, yielding a neutral ammonium acetate that will disappear into thin air during the final lyophilization. However, titrating the pH of a mixture that's only about 10% 6:4 water:acetic acid, the rest solvents is, well, challenging. I know I should be able to figure out how much ammonia solution at a given concentration I need to add to neutralize my 4% solution, but I'm not sure I am calculating this right.

For example, VWR has ACS Ammonium Hydroxide 28-30% w/w. Assuming 30% ammonium hydroxide:

(300 g/L)(35 g/mol)-1 = 8.6 M

a 4% v/v solution of acetic acid:

(1.05 g/ml)(40 ml) = (42 g/L)(60 g/mol)-1 = 0.7 M

Since the Ka of acetic acid is 1.75 x 10-5, and ammonia is very similarly Kb = 1.80 x 10-5, making the solution equimolar in both:

0.7/8.6 = 0.081, i.e., add 81 ml to 1 L 4% acetic acid

should result in a final solution that's a bit, but not much, more basic than pH 7.0 (the compound is also labile at pH > 9.0). Correct?
[the amount of concentrated ammonia seems excessive]

I'd also like to get away from using halogenated hydrocarbons for obvious reasons, though I do reuse my chloroform for defatting partitioning after washing it a few times with water. I've tried some of the other TLC mobile phase systems developed over the years for these compounds that use solvents such as ethyl acetate, but with little success.

Suggestions welcome, my sincere thanks to all you experts who take time to answer my amateurish biochemist-turned-organic-separations-chemist questions. Y'all rock!

R

[H.Thomas]

Hi,

I think your ammonia calculation is wrong. 30% is for NH3, so MW is 17.

300 g/kg * 0.89 kg/L (density) = 267 g/L = 15.7 mol/L

[JMB]

Could you not just,

1) remove the chloroform/ methanol on the rotovap at RT
and
2) lyophilize the water/acetic acid at very low frozen temperatures?

You may possibly have to dilute the water/acetic mixture with additional water to maintain it in the frozen state.

Regards,
JMB

[RFRansom]

Hello Y'all;

I think I got scored on twice (to my delight).

Oooops on the ammonia vs. ammonium hydroxide. Nice.

Remove chloroform first. Yup. Using that suggestion I simply diluted the fraction with an equal volume of water, resulting in a phase separation, drained off the chloroform, and froze at -80 °C then lyophilized with the pump set to exhaust vapors rather than maintain a high vacuum. Frankly, the difference was only a few microns.

[bow] Thank you both for your timely and cogent suggestions, y'all rock!

R