Ninhydrin-positive compound analyzer

A stepwise eluent gradient can be generated by simply using a series of reservoirs with different eluent solutions all connected to the pump feed line and each line being actuated by a solenoid valve. (See description of the ninhydrin-positive compound analyzer.) This technique works well if the step changes do not upset the monitoring device however, it necessitates additional equipment. 

In general, the components being separated and quantified by these analyzers are of relatively low molecular weight (less than a molecular weight of 1000). In fact, the high-molecular-weight components are usually removed by ultrafiltration or precipitation for the ninhydrin-positive compound analyzer and, in some cases, for the other analyzers. The detected compounds are thus the metabolic and catabolic products of the life processes. 

A systematic investigation of the free amino acids of the Leguminosae led to the isolation of a novel ninhydrin-positive compound from the leaves of Derris elliptica Benth. (Papilionidae) (93). This substance was analyzed as C6H,3N04 (microanalysis and high resolution mass spectrometry) and was shown to be an amino alcohol. The absence of a carbonyl in the 1R, the loss of 31 mass units in the mass spectrum, and a positive periodate cleavage reaction were best embodied into a dihydroxydihydroxymethylpyrrolidine structure. The relative simplicity of the NMR spectra (three peaks in the 13C spectrum four spin-system in the H spectrum) pointed out a symmetrical structure. Inasmuch as the material was optically active ([a]D 56.4, c = 7, H20), meso structures were ruled out, and the 2R, 3R, 4R, 5R relative configuration was retained (93). This structure (53) was further confirmed by an X-ray determination (94). 

Two of these systems, an analyzer for the UV-absorbing constituents (UV-analyzer) and one for carbohydrates, will be discussed in some detail. Two others, one for ninhydrin-positive compounds (amino acids and related compounds) and an analyzer for organic acids, will be introduced as systems that have great potential but which have not been fully developed as yet. These four analytical systems certainly do not represent all the concepts for the use of liquid chromatography in body fluids analysis however, they are systems that have been used at least to some degree in clinical and medical research laboratories. 

Finally, Droge and coworkers26 showed that the 2-aminoethyl phosphate-substituted KDO group is the lateral KDO unit of the branched trisaccharide (see Fig. 7), as follows. LPS from Salmonella minnesota mR3 was subjected to periodate oxidation. This sample, together with a control that had not been oxidized, was then mildly hydrolyzed with acid (pH = 3.4) during 1 h at 100°. Following removal of lipid A, both samples were analyzed by gel-filtration on Sephadex G-10, and paper electrophoresis. As expected, the ninhydrin-positive material obtained from the control sample was identical with KDO 7-(2-aminoethyl phosphate) (17) as previously identified. This spot was absent from the periodate-treated sample. Instead, an almost neutral, ninhydrin-positive spot was observed. This material (compound 26) was eluted, subjected to reduction with sodium [3H]borohydride, and hydrolyzed under strongly acidic conditions (see Scheme 11). Fol-... 

So far, the tentative chromatographic method has been used to make most of the identifications of the ninhydrin-positive and organic acid components, especially for urine constituents. This simply requires that the unknown peak has the same elution volume as a known reference compound. A significant effort has been made to provide more definite identifications for the components separated by the UV- and carbohydrate analyzers. To date, this has included over 70 UV-absorbing compounds and 18 carbohydrates, some of which are listed in Tables 1-3 (B2, M2). Tentative identification of many more compounds has been made in all four systems, and, hopefully, the efforts in confirmative identification will continue. 

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