Synthesis of polymeric particles and their application as pseudostationary phase in capillary electrochromatography for peptide separation

GRELA D; COLLAZO, D; ZANNONI, V; VIZIOLI, NM

Natal

Simposio; 21st International Symposium on Electro- and Liquid Phase- Separation Techniques and 20th Latin-American Symposium on Biotechnology, Biomedical, Biopharmaceutical, and Industrial Applications of Capillary Electrophoresis and Microchip Technology; 2014

At the present time, pseudostationary phases have turned an interesting alternative in capillary electrochromatography (CEC). On one hand, pseudostationary phases provide sites for analyte interaction and not require either frits or packing; on the other hand, this system does not need a column change, since a renewed column is used for each analysis.[1]The aim of this work was to evaluate the interaction between a mixture of five synthetic peptides and synthesized polymer particles acting as pseudostationary phase in CEC.Particles (Z-Average 440 nm - 640nm) were prepared from methacrylic acid and ethylenglycol dimethacrylate with benzoyl peroxide as radical initiator by utilizing a precipitation polymerization technique. The methacrylic acid (pKa 4.65) provides acid groups to the polymer. Different total monomer concentrations were tested, from 2 % to 6 %, in order to modify the size of the particles.After synthesis, the resulting particles were removed from its synthesis medium eliminating the solvent and unreacted monomers. Then, an aqueous suspension of particles was used as pseudostationary phase in CEC. Once the capillary was filled with the background electrolyte (BGE), particles in suspension were introduced hydrodinamically at 0.7 psi for 4.0 s. Immediately, sample was also introduced hydrodinamically at 0.3 psi for 3.0 s, and then voltage was applied. This procedure is known as "partial filling technique". BGE was composed of 25 mM of sodium phosphate buffer solution, and was adjusting at different pHs (from 5.0 to 8.0).The results showed that at pH 7.0 electroosmotic flow (EOF) was not affected by the presence of the particles and peaks corresponding to bradykinin and luteinizing hormone-releasing hormone (LHRH) were not present. Angiotensin I, oxytocin and methionine-enkephaline presented no change in their migration time. However, angiotensin I and oxytocin peak areas decreased.These findings suggest that particles have a strong interaction with basic peptides, bradykinin and LHRH, at pH 7.0 (particles with negative charge and peptides with positive charge). This behavior was observed with peptides migrating faster in the capillary, which were the ones with high positive net charge, and decrease as long as peptides presented less positive net charge.[1] Nilsson, C., Birnbaum, S., Nilsson, S. Electrophoresis 2009, 32, 1141-1147.