Ninhydrin amino group analysis

Detection powders and fingerprint development kits commonly contain cream- or yellow-colored ninhydrin crystals or a solution of dissolved ninhydrin. Ninhydrin (also known as 1,2,3-indantrione, monohydrate 2,2-dihydroxy-1,3-indandione triketohydrindene, monohydrate and triketohydrinden hydrate) has the structure presented in Fig. 13.3.1. Ninhydrin will react with a free a-amino group, -NH2. This group is contained in all amino acids, and analysis with ninhydrin is often performed to verify the presence of amino acids. When a-amino acids (i.e., amino acids with the structure NH2-CHR-COOH) react with ninhydrin, a characteris-... 

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]. 

Analytical procedures involving reduction and determination of mercaptan are not accurate determinations of cystine in permanent-waved hair or in hair treated with mercaptan because mixed disulfide is reduced to mercaptan during analysis adsorbed mercaptan can also interfere in the determination. Procedures that do not involve reduction of hair such as ninhydrin detection (alpha-amino group) or dinitrofluorobenzene (DNFB) reaction followed by chromatographic separation [1, 58] discriminate between mercaptans and, therefore, should be better analytical procedures for detecting the different types of mercaptans and disulfides actually present in permanent-waved hair. 

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... 

Gibson, M. Amino acid analysis by HPLC/ninhydrin and tandem mass spectrometry detection. In ABRF Amino Acid Analysis Research Group, http //www.abrf.org/ ResearchGroups/AminoAcidAnalysis/EPosters/Gibson MS.pdf (accessed September 2004). 

This reaction is commonly used in both qualitative and quantitative analysis of amino acids. Nineteen of the 20 protein-derived a-amino acids have primary amino groups and give the same purple-colored ninhydrin-derived anion. Proline, a secondary amine, gives a different, orange-colored compound. 

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. 

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