Accurate HPLC determination of piperazine residues in the presence of other secondary and primary amines

A reversed and normal phase HPLC separation technique with diode array detection was used for the investigation of interactions of diethylenediamine (piperazinc) and its DNS derivative with different adsorbents, used as HPLC packings. The chromatography in reversed phase mode with s deactivated C-18 column (BakerBond C-18 BDC, SUPELCOSIL (TM) LC-C18-DB) did not give positive results. Piperazine was not retained on the column and eluted at death time. On the other hand, the product of coupling reaction (DNS-diethylenediamine) was strongly retained on the packing, in spite of the addition to the mobile phase of 1-heptanesulfonic acid, to diminish ionic forces between DNS product and column packing, and increase the interactions with octadecyl chains. These results confirmed the published observation, that for the analysis of this compound the use of normal phase system is obligatory. This leads to the elimination of ionic bonding between DNS-diethylenediamine and column packing. For that propose several columns were tested, e.g., BakerBond C18 BDC, SUPELCOSIL (TM) LC-C18-DB, SUPELCOSIL (TM) LC-CN, and home-made SG-MIX phase. On the basis of these results, the SG-MIX column was selected for further studies comprising the optimization of mobile phase, allowing the obtaining of peaks with high symmetry and good resolution, which is indispensable for the analysis of products containing trace amounts of this compound.