NBD chloride reagent

Note The reagent can be employed for qualitative and quantitative analysis on silica gel and RP layers. Ammonia, amine and acid-containing mobile phases should be completely removed beforehand. Amino phases cannot be employed. The NBD-chloride reagent is not as sensitive as the DOOB reagent (qv.) on RP phases. 

Although the reagent itself is not fluorescent an excess of NBD-chloride can interfere in quantitative analysis. In such cases it should be checked whether prechromatographic derivatization produces better results [3, 4]. The reaction products can then be separated on polyamide layers. 

Note If netilmicin is to be chromatographed alone it is recommended that the methanol content of the mobile phase be increased (e.g. to 23 -I- 7), in order to increase the value of the hRf. The detection limit for the substances in the application tested was more sensitive using DOOB reagent on RP layers than when NBD chloride, fluorescamine or o-phthalaldehyde were employed. The derivatives so formed were stable and still fluoresced after several weeks if they were stored in the dark. 

The specific detection of aromatic nitro compounds is a second example. These can be converted by reduction to primary amines, which are then diazotized and coupled to yield azo dyes (cf. reagent sequence Titanium(III) chloride — Bratton-Marshall reagent ). Sodium nitrite —naphthol reagent, diazotized sulfanilic acid and other reagents specific for amino groups (e.g. ninhydrin, fluorescamine, DOOB, NBD chloride [9]) can also be used in the second stage of the reaction (Fig. 21). 

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