Hi,

after it is separated in aqueous layer, separate it and adjust its pH until your compound leaves the aqueous layer. It should work fine and then you filter the precipitate of do an extraction.

In the absence of counterions, the zwitterion is likely to have the lowest aqueous solubility of the 3 charge forms. If you use a large organic counterion, such as tetrabutylammonium for the negatively charged form at basic pH, for example, the compound should also be more soluble in the organic phase.

Crystallization of a zwitterion at the isoelectric point is tricky and often inefficient. If you only need a small amount of product and high yield is not the goal, AND you have a good estimate of pI, this can be the quickest way to get what you need. If you are doing this preparatively and/or if the compound does not crystallize well from your original mixture, ion exchange resins will probably be the most effective tools. (If you are trying to recover your product from a complex mixture, you will need to do IEX chromatography; please provide more details.)

When amino acid zwitterions are being synthesized, cation exchange resins such as Dowex 50, Amberlite IRC120 and others offer a very simple and efficient method for recovering products. The following process is frequently used following acid treatment, such as amino acid amide hydrolysis in HCl(aq):

RCH(NH[R'C=O])CO2H + xs HCl(aq) --> RCH(CO2H)NH3+ . Cl- + R'CO2H

The hydrolysis mixture consists of RCH(CO2H)NH3+ . Cl-, R'CO2H, water and excess HCl (e.g. R'=CH3 for an amino acid acetamide, where acetic acid is a volatile byproduct).

1) Evaporate excess volatile acid (rotary evaporation will remove water and HCl gas.) Purging HCl gas in a stream of N2(g) beforehand (fume hood) will reduce bumping during evaporation. Vent the vacuum pump exhaust to fume hood.

2) Dissolve the acid evaporation residue in a small volume of water

3) Calculate the number of millimoles of the zwitterionic substance you intend to isolate and pack a short column with 2-5 times this number of milli-equivalents of appropriately-washed NH4-form ion exchange resin (preparation usually done by washing with strong acid solution followed by water to give neutral eluate, then aqueous ammonia treatment to convert RSO3H groups to RSO3- .NH4+, and finally thorough washing with deionized water to give neutral filtrate.)

4) Slowly apply the solution of evaporated hydrolysis mixture residue onto the column, then wash the column with pure water until the eluate is free of Cl- (AgNO3 test) and the pH is neutral (or not lower than the pH of your DI water). At this point, the acidified zwitterion should be completely bound to the resin [RCH(CO2H)NH3+ . resin-SO3-]. (Check the column application eluate to be sure nothing has leaked through. TLC with ninhydrin detection works well for amino acids). You may also check dried drops of eluate with ninhydrin to be sure that displaced NH4+ ion is no longer eluting from the column.

Elute the washed column with aqueous NH4OH, using approximately twice the number of millimoles as meq of resin (e.g a column containing 50 mL of 0.8 meq/mL resin would be eluted with 2 x 50 x 0.8 = 80 mmol of NH4OH(aq), say 40 mL of 2 M). If the product has not completely eluted from the column by this time, more water should be used before you add more ammonia. Very hydrophobic amino acids may elute better if some methanol or ethanol is added to the water.

5) Evaporate the ammonia eluate. As with the HCl solution in step 1, this solution may behave better during rotary evaporation if you first bubble a stream of N2 gas into the eluate to purge excess NH3 (fume hood), followed by vacuum evaporation, only heating once all free ammonia is gone. Nearly all ammonium carboxylate salts (RCO2- . NH4+) decompose upon evaporation with heating to carboxylic acids, so this step is basically:

[RCH(CO2-)NH2 .NH4+](aq) --> RCH(CO2-)NH3+(s) + NH3(g)

i.e. once ammonia gas leaves, this residue is theoretically pure zwitterion.

6) At this point the purification method of choice is usually recrystallization, commonly from water-alcohol mixtures.

Based on the pH of the water-borne product, adjust the pH (usually at 7) to get desired product into the neutral mode and extract with organic solvent. If certain salts are accompanied, use dialysis. You could also salt out your product (though a bit messy at times, and less yielding but you will get your product).

Dear Rajiv L,

It mostly depends upon adjustment of pH. So titrate yours reaction mixture with bases like aq.NaOH and increase the pH(until it reaches basic pH) so that you could extract it into organic solvents.

Have any of the people suggesting pH adjustment and extraction with organic solvent ever actually tried this? You should understand based on principles that you should have learned in beginning organic chemistry that IT WILL NOT WORK! Organic/aqueous extraction following pH change work for weak acids or weak bases, but not zwitterions.

RCO2-Na+ (charged, water-soluble) + H+ -> RCO2H (neutral, soluble in organic solvent)

R'-NH3+ (charged, water-soluble) + -OH -> R'NH2 (neutral, soluble in organic solvent)

R"CH(NH2)CO2- (charged, water-soluble) + H+ -> R'CH(NH3+)CO2- (net neutral, but still charged; INsoluble in organic solvent but may partially crystallize)

R"CH(NH3+)CO2- + H+ -> R"CH(NH3+)CO2H (charged, water-soluble, organic solvent INsoluble)