Amino acids ninhydrin postcolumn derivatization

Apart from ninhydrin, many other derivatization reagents have been used both precolumn and postcolumn derivatization modes have been extensively employed. Derivatization procedures offer significant advantages in both separation and detection aspects and, thus, will be discussed in further detail. The rest of the entry will be divided into two sections separation of underivatized amino acids, where the determination of free amino acids and postcolumn derivatization procedures are described and separation of derivatized amino acids, where precolumn derivatization approaches are discussed. 

Fig. 2 Column Micropak AA (sulfonated polystyrene) solvent A 02M sodium citrate, pH 3.25 solvent B IM sodium citrate, pH 7.40. Gradient 5 min 100% A 100-75% A in 20 min 75-70% A in 5 min 70-35% A in 5 min 10 min 35% 35-0% A in 1 min. T = 50°C for 25 min, then 90°C. Detection after ninhydrin postcolumn reaction. (Reprinted from Amino acid analysis with ninhydrin postcolumn derivatization, LC at Work, Varian Associates with permission.)...

Since the amino acid is destroyed during ninhydrin derivatization and forms the same reaction product regardless of the original amino acid (except for the secondary amino acids), ninhydrin derivatization wdl only work as a postcolumn method. 

Detection of amino acids is typically by UV absorption after postcolumn reaction with nin-hydrin. Precolumn derivatization with ninhydrin is not possible, because the amino acids do not actually form an adduct with the ninhydrin. Rather, the reaction of all primary amino acids results in the formation of a chromophoric compound named Ruhemann s purple. This chro-mophore has an absorption maximum at 570 nm. The secondary amino acid, proline, is not able to react in the same fashion and results in an intermediate reaction product with an absorption maximum at 440 nm. See Fig. 5. Detection limits afforded by postcolumn reaction with ninhydrin are typically in the range of over 100 picomoles injected. Lower detection limits can be realized with postcolumn reaction with fluorescamine (115) or o-phthalaldehyde (OPA) (116). Detection limits down to 5 picomoles are possible. However, the detection limits afforded by ninhydrin are sufficient for the overwhelming majority of applications in food analysis. 

Amino acid analyzers have improved the analysis of free amino acids to a great extent. They offer superior sensitivity, speed, and accuracy to conventional methods. Many such systems are based on IEC. Postcolumn detection is done by ninhydrin derivatization followed by photometric measurement at 570 and 440 nm for primary and secondary amino acids, respectively. Amino acid analyzers are now common and are being manufactured by many companies (e.g., Hitachi, Beckman, PerkinElmer, HP, Pharmacia, etc.). Numerous authors have used amino acid analyzers to monitor proteolysis in several kinds of cheeses (Ardo and Gripon, 1995 Edwards and Kosikowski, 1983 Fenelon et al., 2000 Gardiner et al., 1998 Kaiser et al., 1992 Yvon et al., 1997). A comparison of amino acid analyzers and several other methods for amino acid analysis is available from Biitikofer and Ardo (1999) and Lemieux et al. (1990). 

Postcolumn Reactors. Another growing field is the use of postcolumn reactors to produce a species that can be measured by one of the standard detectors, such as UV/visible, fluorescence, or electrochemical. Probably the earliest example of the use of postcolumn reactions was in the determination of amino acids by colorimetry using ninhydrin as the reactant. See the section on derivatization in Chapter 11, as well as the paper in Analytical Chemistry,57 or the book edited by Krull58 for further details. 

Amino acids react with many reagents to form stable derivatives and strong chromophores (see Table 1). Derivatization can precede (precolumn) or follow (inline postcolumn) the chromatographic separation. Both precolumn and postcolumn systems are currently employed ninhydrin and PITC analyzers are widely used, whereas AQC, OPA and OPA-FMOC systems provide the highest sensitivity. 

Sample Derivatization. For HPLC analyses, many analytes are chemically derivatized before or after chromatographic separation to increase their ability to be detected. For example, in automated amino acid analyzers, eluted amino acids are reacted with ninhydrin in a postcolumn reactor (see Chapter 20). The resulting chromogenic species are then detected with a photometer. Other examples include labeling amino acids or other primary amines with dansyl or fluorescamine tags either before or after the chromatographic step. 

Only a few amino acids are detected by the UV or visible spectrophotometers, fluorometers, or electrochemical detectors that are routinely used with HPLC analyzers. Consequently, amino acids typically are postcolumn derivatized for analysis by HPLC. The most widely used reagent for this purpose is ninhydrin. A number of colored products are formed, but the major one is presumed to result from deamination and condensation as follows ... 

Most amino acids react with ninhydrin at ambient temperatures to form a blue color that becomes purple on heating. However, proline and hydroxyproline yield yellow compounds that are measured at a different wavelength. Other postcolumn derivatizations use fluorogenic reagents, such as o-phthaldialdehyde or fluorescamine. Precolumn derivatization techniques using o-phthaldialdehyde, dansyl, phenyl isothiocyanate, or 9-fluorenylmethyl chloroformate derivatives have been used with reversed-phase HPLC. Electrochemical detection has also been coupled with derivatization methods to enhance analytical sensitivity. 

Dyes for postcolumn derivatization Ninhydrin has been the standard reagent for detecting amino acids... 

The amino acid produced in the fermentation broth can be quantified by employing different colorimetric assays (e.g., ninhydrin, 8-hydroxy quinoline), microbiological assays, or more specifically by HPLC methods using pre- or postcolumn derivatization with OPA, dansyl chloride, etc. 

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. 

Fluorescamine was developed by Weigele et al. in 1972 [8], based on the fact that strongly fluorescent pyrrolinones were formed by the reaction of ninhydrin, phenylacetaldehyde, and primary amines. The reagent, 4-phenylspiro[furan-2(3H),l -phthalan]-3,3 -dione (fluorescamine), is nonfluorescent, and it reacts with primary amines, amino acids, and peptides under aqueous conditions in a few minutes at room temperature to form intensely fluorescent substances (Figure 6.1). On the other hand, nonfluorescent derivatives are formed by the reaction of fluorescamine and secondary amino compoimds. Therefore, fluorescamine can be used for the selective determination of primary amino compounds, and the fluorophore produced by the reaction is the expected pyrrolinone. Because the reaction is sufficiently rapid and the hydrolysis products are nonfluorescent, the fluorescamine reaction is applicable for the postcolumn fluorescence derivatization of primary amino compounds [9]. The amino acids are separated by a cation-exchange column similar to the ninhydrin method, and the column effluent is mixed with an alkaline-buffered solution and fluorescamine reagent. The fluorescent derivatives are detected at 480 nm with excitation at 390 nm. 

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