Amino group analysis, ninhydrin method

Fluorimetric methods for the determination of amino acids are generally more sensitive than colorimetric methods. Fluorescamine (4-phenyl-spiro[furan-2(3H),l -phthalan]-3,3 -dione) and o-phthaldialdehyde (OPA) substances are used for protein analysis. Fluorescamine reacts with amino groups to form fluorophores that excite at 390 nm and emit at 475 nm (Weigele et al., 1972). Applications of fluorescamine include monitoring the hydrolysis of K-casein (Beeby, 1980 Pearce, 1979) and quantification of proteins, peptides, amino acids in extracts (Creamer et al., 1985). OPA produces fluorescence on reaction with 2-mercaptoethanol and primary amines, with strong absorption at 340 nm. Lemieux et al. (1990) claimed that this method was more accurate, convenient, and simple for estimating free amino acids than the TNBS, ninhydrin, or fluorescamine methods. 

Figure 1. Calibration curve for amino group (dl-leucine) and ammonium ion (ammonium chloride) analysis by ninhydrin method.

Kaiser test, ninhydrin test, a simple and most frequently used method of on-resin monitoring in SPPS. A positive color reaction, performed with a small aliquot of the resin material, indicates unconverted amino groups. Samples containing <0.5% of unreacted amino groups can usually detected within minutes. A modified version of the Kaiser test allows quantitative analysis [E. Kaiser et al.. Anal. Biochem. 1970, 34, 595 V. K. Sarin et al.. Anal. Biochem. 1981, 117,147]. 

Fluorescamine (Fluram). Fluorescamine was developed from the study of ninhydrin chemistry as a reagent for detecting amino groups (Udenfriend et al., 1972) and has been successfully used in the reversed phase chromatographic analysis of proteins and peptides using an on-line post-column detection system. This reagent requires two separate pumps for the delivery of the mobile phase buffer and the reagent and this method may therefore be considered to be less... 

Even today, the postcolumn derivatization with ninhydrin introduced by Spademan, Stein, and Moore [29] represents the most common detection method for quantitative amino acid analysis. As a strong oxidant, ninhydrin reacts with the a-amino groups of eluting amino acids at temperatures around 130 °C, according to Eq. (5.3), releasing carbon dioxide. 

Earlier methods of estimating the a-amino nitrogen, due to Van Slyke, consisted of gasometric analysis of the nitrogen evolved when amino compounds were treated with nitrous acid or, alternatively of the carbon dioxide evolved on treatment with ninhydrin. The amino acids are readily absorbed on ion-exchange resins from which they can be eluted quantitatively either as a group... 

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