Studies on an abnormally sharpened elution peak observed in counter-current chromatography

Counter-current chromatography (CCC) of the bromoacetylation product of 3,3',5-triiodo-L-thyronine (T3) produced an unusually sharp peak for the desired product, N-bromoacetyl T3 (BrAcT3). A series of experiments revealed that bromoacetic acid, probably present as a side reaction product in the sample solution, was responsible. This compound repressed the ionization of the carboxyl group of BrAcT3 forcing it into the less polar stationary phase until the bromoacetic acid had eluted completely from the apparatus. At this point, the sudden increase of pH and consequent ionization of the BrAcT3 allowed the ammonium salt of the latter to enter the more polar moving phase where it eluted rapidly from the column as a sharp peak. The same phenomenon was observed in the CCC fractionation of a series of indole auxins where addition of trifluoroacetic acid to the sample caused peak sharpening by the same process. The phenomenon recalls pH gradient elution and isoelectric focussing except that the substance responsible for the pH range here is added along with the sample in one bolus forming a sharp pH gradient at its trailing edge. As with gradient elution, the technique is of practical interest since it permits collection of the eluting compounds with increased detectability in fewer fractions. The technique can also enhance separation of compounds whose partition coefficients differ with a change in pH.