Primesep 200 separates the very hydrophilic polyamines, 1,3-diaminopropane, N-(3-aminopropyl)-1,3-diaminepropane, N,N-bis-(3-aminopropyl)-1,3-diaminepropane. The retention and resolution are affected by either a TFA concentration gradient, or a pH gradient from pH 4 to 3. This flexibility is obtained by simple mobile phases of water, acetonitrile (MeCN, ACN), ammonium formate, ammonium acetate or trifluoracetic acid (TFA) with evaporative light scattering detection (ELSD).
HPLC Separation of Polyamines on Primesep 500 Column
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Polyamines are both naturally occurring and synthetic. The naturally occurring polyamines have a variety of functions including ion channel modulation and biosynthesis of cells. The synthetic polyamines are used in motor oil and epoxy resins. Because of their strong ionic characteristics, they are not retained on reverse-phase columns and strongly retained on anion-exchange columns. In order to separate polyamines with varying numbers of ionic groups, we used a Primesep 500 HPLC mixed-mode column with embedded carboxylic groups of pKa around 5. By lowering the pH of the mobile phase, we can suppress the carboxylic groups on the column making it less retentive to polyamines. We separate the double and triple charged polyamines at one pH, then do a step gradient to lower pH to reduce the retention time and separate higher charged polyamines. The detection can be achieved with CAD or MS.