A new reagent for detection of peptides, nucleotides, and other N-H-containing compounds on paper chromatograms.

The detection of small quantities of nitrogen-containing compounds on paper chromatograms is a problem of long standing interest. The ninhydrin technique, first introduced by Consden, Gordon, and Martin (l), has partially solved this problem and has proven to be extremely valuable. However, the narrow range of compounds detected, the decreased sensitivity with increased molecular weight of peptide, and the failure to detect acylated amino acids and cyclic peptides have restricted the use of this procedure. In an attempt to obviate some of these limitations, Rydon and Smith (2) described the use of the reaction between chlorine gas and compounds of nitrogen with at least one N-H bond to form the N-chloro compound. This latter compound was detected by means of a starch-iodide reagent. These authors demonstrated that the technique is superior to the ninhydrin technique for the detection of cyclic peptides, acylated peptides and amino acids, proteins, and high molecular weight peptides. In addition, they predicted that the method could be applied to any compound containing nitrogen with at least one N-H bond, but they did not document this prediction. Furthermore, in some cases they claimed increased sensitivity for their technique as compared to the ninhydrin technique. Since the original publication by Rydon and Smith, two modifications by Reindel and Hoppe (3,4) have appeared. Although in principle the technique of Rydon and Smith has much to recommend it, in our hands it has proven extremely variable. Part of the difficulty stems from the variability of the chlorination procedure. Furthermore, when sufficient concentrations of chlorine gas and sufficient times of chlorination were employed, a background color was produced which, in many cases, made detection of the desired compound extremely difficult. When attempts to remove this interfering background color by extensive aeration were made, it was found that the N-chloro compound was partially or completely lost as a result of hydrolysis, resulting in decreased sensitivity or a negative test. During the course of formation of some N-chloro derivatives with tert-butyl hypochlorite (5), it occurred to one of us (R. H. M.) that this reagent might substitute for chlorine gas in the Rydon and Smith technique. Systematic studies to develop this idea were undertaken and resulted in the new methods described below.