Peptides ionic strength optimization

Capillary Zone Electrophoresis (CZE). CZE is a widely used CE technique and separates peptides and proteins based on differences in their charge-to-mass ratios. Separations occur in a capillary filled with a buffer of constant composition. For CZE, the run buffer choice is extremely important because it determines the charge on the analyte molecule and its migration rate. Thus, the type of buffer, its ionic strength, and its pH are optimized for particular separation problems. Buffers based on sodium phosphate, citrate, acetate, or combinations thereof with concentrations ranging from 10 to 200 mM are frequently used [14]. 

The ability to inhibit wall interaction is demonstrated in Fig. 5. The complete tryptic digest of cytochrome c is analyzed at pH 4.0, with optimization accomplished by modifying the ionic strength of the buffer and separation field strength. A dramatic improvement in the separation is achieved by increasing the ionic strength. From these data, the best separation conditions can easily be identified, and the complex mixture spiked with the isolated peptides to identify them within the mixture. 

Peptide mixtures are very susceptible to interaction with SEC supports and their general heterogeneity makes it difficult to optimize the mobile phase [35,39]. Each class of peptide (acidic, basic, hydrophobic, and neutral) was observed to react differently to changes in pH, ionic strength, or addition of organic solvent when they were run on a Superdex Peptide column [35], It was determined that acidic peptides were best run at high pH and required addition of 30% acetonitrile at low pH, whereas basic peptides had the best peak shapes at low pH. 

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