Nonaqueous Capillary Zone Electrophoresis of Synthetic Organic Polypeptides

Abstract

Poly(Nepsilon-trifluoroacetyl-L-lysine) was used as a model solute to investigate the potential of nonaqueous capillary electrophoresis (NACE) for the characterization of synthetic organic polymers. The information obtained by NACE was compared to that derived from size exclusion chromatography (SEC) experiments, and the two techniques were found to be complimentary for polymer characterization. On one hand, NACE permitted (i) the separation of oligomers according to their molar mass and (ii) the separation of the polymers according to the nature of the end groups. On the other hand, SEC experiments were used for the characterization of the molar mass distribution for higher molar masses. Due to the tendency of the solutes (polypeptides) to adsorb onto the fused-silica capillary wall, careful attention was paid to the rinsing procedure of the capillary between runs in order to keep the capillary surface clean. For that purpose, the use of electrophoretic desorption under denaturating conditions was very effective. Optimization of the separation was performed by studying (i) the influence of the proportion of methanol in a methanoVacetonitrile mixture and (ii) the influence of acetic acid concentration in the background electrolyte. Highly resolved separation of the oligomers (up to a degree of polymerization n of approximately 50) was obtained by adding trifluoroacetic acid to the electrolyte. Important information concerning the polymer conformations could be obtained from the mobility data. Two different plots relating the effective mobility data to the degree of polymerization were proposed for monitoring the changes in polymer conformations as a function of the number of monomers.

Separation of living and dead polymers in synthetic polypeptide mixtures by nonaqueous capillary electrophoresis using differences in ionization states

“…The polymerization was initiated by n-hexylamine as described in detail in the previous papers [26,27]. After 48 h, total conversion was reached.…”

“…To ensure a good repeatability on uncoated capillaries, any adsorbed polymers were removed by an electrophoretic desorption in the presence of denaturing electrolyte [26]. This procedure was performed between each run for uncoated capillaries.…”

“…From these results, we were able to identify [27] and to quantify [28] the proportion of living polymers (polymers ending with an amine group) and dead polymers (polymers ending either with a neutral N-formyl group or a carboxylic group). However, the NACE conditions did not allow a complete separation of the two dead polymer families [26].…”

“…Moreover, NACE can give valuable information that is complementary to other separation techniques such as size-exclusion chromatography (SEC). Recently, we demonstrated [26] that PTLLs could be separated according to (i) their molar mass for DP , 50 and (ii) their functionality. From these results, we were able to identify [27] and to quantify [28] the proportion of living polymers (polymers ending with an amine group) and dead polymers (polymers ending either with a neutral N-formyl group or a carboxylic group).…”

Nonaqueous capillary electrophoresis in coated capillaries: An interesting alternative for proteomic applications

“…In addition, NACE can also be applied to separate more hydrophobic compounds such as higher fatty acids, steroid hormones, membrane proteins in general, which are insoluble in aqueous media and present a wide application in the proteomics [4]. On the other hand, only few studies are related to separation of peptides using NACE electrolytes, e.g., synthetic peptides [30].…”