A simple, rapid three-stage assay has been developed to determine free and N-acetylated hexosamincs, individually as well as in mixtures. Previously, such mixtures had to be analyzed in two operations, most commonly by the Elson-Morgan (1) and the Morgan-Elson (2) procedures. Substituents on hydroxyls affect the nature and distribution of chromogens, and in tJhc Morgan-Elson method R 4-O-derivative inhibitn all color formation ~3~41. These methods also require a free reducing group, as does deamination of free hexosamiues by nitrous acid (5). Although ninhydrin reacts with amines without a free reducing group, ninhydrin does not react with N-acetylattd derivatives (6).
Rydon and Smith (7) reported t,hat pept~itles of all kinds, as ~11 as proteins and arylated amino acids, could be tlct'ected on paper chromatograms by chlorination with gaseous chlorine to form an N-chloropcptide, which reacts with iodide to form iodine; in the presence of amylosc, iodine forms the blue amylose-iodincx complex. Similarly, Powning and Irzykiewicaz (8) used Cl? in Ccl, as a dip to clctect chit'in oligosaccharides on paper chromatograms. Pan and Dutcher (9) found that NaOCl served as a chlorinating agent in place of Cl, and that a mild reducing agent such as 95% ethanol, 5% glucose, or 1% formaldehyde could be used to destroy excess NaOCl. In our work this spray t#echnique has been valuable for detecting both free amI N-acetylated hexosamines in monomeric or polymcaric forms on paper or thin-layer chromatograms. Zitomcr and I,aml)cart (10) rc~l~orted that, ammonia ill