ABSTRACT
Weber et al. [111, 147, 149-156] have extensively studied the biuret reaction as a means of detecting peptides separated by HPLC. They identified a number of operating parameters that influence sensitivity, including post-column temperature, reaction time, buffer composition, Cu(II) concentration, peptide structure, and pH. Post-Column Temperature. In general, most of the peptides studied possess increased anodic sensitivities when the post-column temperature is elevated (50°C compared to 30°C) demonstrating a kinetic barrier to complex formation [154]. At a higher temperature, more of the Cu(II)-peptide complexes form in the reaction window. At the cathode, results for nonelectroactive (in the absence of Cu(II)) and electroactive peptides vary. The nonelectroactive peptides have increased sensitivities at elevated temperatures because there is more current at the upstream electrode, therefore more Cu(III)-peptide is formed. However, the electroactive peptides generally are characterized by a decrease in cathode sensi-tivity at 50°C resulting from degradation of the Cu(III)-peptide complexes (Figure 11). Reaction Time. Intuitively, a longer post-column reactor will increase the electrochemical signal. This is true for larger peptides, N-acylated peptides, and peptides with
pyroglutamate in the first position, but for small peptides (up to eight amino acids) without those attributes listed, the longer post-column reaction time does not increase peak height and only leads to peak broadening [154]. Tsai et al. [151] examined precolumn formation of the biuret complex as an alternative to post-column derivatization. This allows for the increased reaction time needed for longer peptides (>eight amino acids) and those containing proline. Buffer Composition. Investigation of a number of buffer systems (pH 9.8) including bicarbonate/carbonate (pH 9.8), boric acid/sodium hydroxide (pH 9.8 and 8.0), boric acid/glycerol/sodium hydroxide (pH 9.8), and phosphate (pH 8) revealed the bicarbonate buffer system to be optimal [154]. Phosphate buffers appear to inhibit, in comparison to borate, the formation or the electrochemistry of the complex and should be avoided. Cu(II) Concentration. In addition to the above-mentioned factors affecting sensitivity, Chen and coworkers [155] determined the biuret reaction to be zero order with respect to Cu(II), implying that an excess concentration of Cu(II) will not improve reaction rate or sensitivity. Amino Acid Composition and Peptide Structure. Similar to some of the previously mentioned derivatization agents, peptides containing electroactive amino acids exhibit increased anodic sensitivities over nonelectroactive peptides in the presence of Cu(II), resulting from the additive effect of the amino acid and Cu(II) oxidation. At the cathode, sensitivities of electroactive peptides are equal to those for nonelectroactive peptides except for shorter tripeptides [157] where, in some cases, cathode signals are repressed by the presence of tyrosine.
