Chromatography Forum

Dear All,
We are developing method for analysis of 3-chloropropylaine HCl content in one of our intermediates. As the compound is non-UV active, the method is developed on HPLC-RI.
MP: 0.1% HFBA in water.
Column: C18, 250 mm x 4.6 mm, 5 micron
Diluent: Mobile phase

Whenever I inject 3-chloropropylamine HCl standard, I always observe two peaks, one eluting very early (about 2 min) and one eluting later (about 13 minutes). Initially, we thought that the early eluting peak belongs to HCl salt. Accordingly, we injected HCl (since the impurity contains about 28% HCl by weight, for 100 ppm of impurity, we checked the retention time of HCL at 28 ppm level) but found an almost negligible peak eluting at the retention time of early eluting peak, much smaller than that observed in 3-chloropropylamine HCl. We also tried to break the salt, isolating the impurity and injecting it. However, we still observed the peak but smaller than that observed in salt.
In salt the peak ratio was 28:72
In base the peak ratio was 7:92.
Shall we consider this peak as belonging to HCl or any other reaction is happening within the solution (we tried different diluents but the pattern remains same). Please suggest.

Best regards,

Andy

I speculate that the early-eluting peak is chloropropylamine with chloride as the counterion and the later-eluting peak is molecules of chloropropylamine that have exchanged chloride for heptafluorobutyrate as the counterion. In that case there should be a continuum connecting the two peaks, consisting of molecules that started with chloride but acquired heptafluorobutyrate partway down the column. Please advise if you do see such a continuum.

You can test this by adding some heptafluorobutyric acid to your sample prior to injecting it. That would permit the exchange of counterions to occur prior to the chromatography.

Negative peak could be H+, positive — Cl-. Please check any chloride salt instead of HCl or another acid.

Dear Andy and Dap,
thanks a lot for the suggestions. Based on your suggestions, we have put following two preparations in the running set,

1. triethylamine HCl salt solution to check the retention time of HCl.
2. HFBA spiked in 3-aminopropylamine HCl solution.

I'll share the results of the both tomorrow. Thanks a lot to both of you. It really helped a lot in finding the root cause.

Best regards,

Aniket