Chemiluminescence detection of arginine-containing peptides separated with monolithic high-performance liquid chromatography

The selective determination of arginine-containing peptides is hindered by the high pKa of the guanidine functional group, as most peptides are not stable at the pH often required to create an effective nucleophile [1]. Modifications of the Sakaguchi reaction-the spectrophotometric determination of arginine with hypohalites and phenols-have been extended to other guanidine compounds or arginine residues in proteins, but many inaccuracies have been uncovered [2] and [3]. For example, the 149-154 hexapeptide fragment (Ala-Asn-Thr-Pro-Asp-Arg) from chymotrypsin gave twice the response of the corresponding hydrolysate solution [2]. Alternatively, arginine residues will react with 9,10-phenanthrenequinone [2] and [4], p-nitrophenylglyoxal [3], or benzoin [1], [5] and [6] to form colored or fluorescent products; these reagents are generally more sensitive and accurate than the Sakaguchi reaction. Flow-analysis procedures for arginine or arginine-containing peptides that incorporate these chemistries require prior derivatization [6] or postcolumn reaction manifolds with numerous reagent lines [5] and [7] and in some cases a heated reaction coil [5]. We have recently developed an exceedingly simple and rapid method for the determination of arginine (in the presence of other amino acids) with a limit of detection of 0.1 µM, based on the chemiluminescent oxidation of arginine with hypobromite [8]. In this paper we present the determination of arginine-containing peptides separated with monolithic column chromatography. The highly porous monolithic column was selected as high flow rates can be applied at low pressure, without a significant decrease in efficiency. Therefore, in addition to a significant reduction in analysis time compared to conventional chromatography, the flow rates are more compatible with chemiluminescence detectors that have been developed for flow-injection analysis.