The use of ion-pairing agents in reversed-phase chromatography is quite common, and the effect of mobile phase composition
on reversed-phase separations has been extensively studied in recent years. The addition of neutral salts can greatly affect
the adsorption behaviour of ionizable compounds on reversed-phase packings.1-4 However, most of this research has involved small molecular weight organic compounds, not larger molecules such as peptides
or proteins. Additionally, the majority of this work has been conducted using only silica reversed-phase packing materials.
Therefore, in order to better understand the effect of neutral salts on peptide separations, a polymeric reversed-phase packing
material, Amberchrom™ HPR10, was selected for the purification of recombinant human insulin. The purifications were conducted
with and without the addition of a neutral salt in the mobile phase.
Experimental
Figure 1: Insulin purification with sodium salts.
Recombinant human insulin expressed from yeast (Sigma, St. Louis, Missouri, USA) was purified using sodium citrate, sodium
sulphate, ammonium acetate or ammonium sulphate in a buffered Milli-Q® water/acetonitrile gradient on an Amberchrom™ Profile™
HPR10 column. The influent purity of the human insulin was ~92%. All separations were done using an Agilent 1100 HPLC system
and the conditions are listed below.
Results and Conclusions
Figure 2: Insulin purification with ammonium salts.
As can be seen in Figures 1 and 2, the addition of 50 mM salt has a profound effect on the elution profile. In general, the
elution profiles are much tighter with the addition of salt to mobile phase. Also the amount of mass per fraction was much
higher when compared to the insulin purification done without salt.
Figure 3 compares the yield/purity curves for the different purification conditions. The ammonium salts provided superior
yields and purities when compared with the purifications using no salt or purifications using sodium salts. The sodium salts
did increase the mass per fraction and gave a tighter elution profile, however, the overall yield using these salts was inferior
to the other purifications. This was especially true with the sodium citrate purification. Ammonium sulphate provided the
best overall purification, although the yield at 100% purity was slightly lower than that provided by ammonium acetate.
John Fisher, John Maikner, Amie Gehris, Marlin Kinzey and Mark Vander Hoff, Rohm and Haas Company, Advanced Biosciences,
Spring House, Pennsylvania, USA.
