Separation of Amino Acids and Amines

Ninhydrin (2,2-dihydroxyindane-13-dione) reacts with primary and secondary amino compounds to form characteristic colored compoimds. Since its discovery by Ruhemann in 1910 [1], this colorimetric reaction has been widely used for the detection of amino acids, peptides, proteins, and amines. By the reaction with primary amino acids, a typical 

FIGURE 6 1 Reaction of postcolumn derivatization reagents with amino acids. 

LC—MS/MS methods are powerful tools for the determination of various compounds in complex biological matrices. For the determination of amino acids by MS/MS detection, a volatile mobile phase is required, and therefore, an ion-pairing reversed-phase LC system with a volatile 

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TLC studies on amino acids can be performed on a variety of sorbents. Blackburn (1989) has provided 56 tables showing TLC data and separations of amino acids and amines using various combinations of layers, mobile phases, and detection reagents. Information on both free amino acids and amino acid derivatives was included. 

Crown-ether CSPs have the ability to include some chiral molecules stereoselectively. These CSPs are well suited for the separation of amino acids and compounds containing a primary amine at or near the stere-ogenic centre. The most used commercially available crown-ether CSP is Crownpak CR (-I-), developed by Daicel (Osaka, Japan). 

Molmh-Perl, 1., Quantitation of amino acids and amines in the same matrix hy high-performance hquid chromatography, either simultaneously or separately, J. Chromatogr., 987, 291-309, 2003. 

S Einarsson, B Josefsson, P Moller, D Sanchez. Separation of amino acid enantiomers and chiral amines using precolumn derivatization with (+)-l-(9-fluorenyl)ethyl chlo-roformate and reversed phase liquid chromatography. Anal Chem 59, 1191, 1987. 

O-Phthaldialdehyde (OPA) is an amine detection reagent that reacts in the presence of 2-mercaptoethanol to generate a fluorescent product (for preparation, see Section 4.1, 2-mercaptoethanol) (Fig. 91). The resultant fluorophore has an excitation wavelength of 360 nm and an emission point at 455 nm. OPA can be used as a sensitive detection reagent for the HPLC separation of amino acids, peptides, and proteins (Fried et al., 1985). It is also possible to measure the amine content in proteins and other molecules using a test tube or microplate format assay with OPA. Detection limits are typically in the microgram per milliliter range for proteins. 

Method. A standard amino-acid analyzer (Technicon or an equivalent) may be used. The reagents for development and the buffers are prepared as for analysis of amino acids. The analytical column (24 cm X 0.57 cm) consists of Zeocarb 226-4.5% DVB (average particle diameter, 24 jum). The two buffers are prepared by dissolving 8.74 g of potassium citrate, 60.36 g of potassium chloride, 10 ml of Brij and 100 ml of n-propanol (for the first buffer, 140 ml of n-propanol for the second buffer) in enough water to make a total volume of 11. The pH of each buffer is 7.4. For analysis the sample is adjusted to pH 7.4 and an aliquot portion is applied to the column. The column temperature is maintained at 43 °C for 103 min and is automatically switched to 75 °C for the remainder of the run. The flow-rate of the buffer is 42 ml/h. The first buffer is automatically replaced by the second after 120 min. The second buffer is necessary for the separation of tryptamine and cadaverine. The use of the increased temperature results in a shorter elution time. The retention times of some basic amino acids and amines are listed in Table 4.3. Absorption is monitored at 570 nm with a 1,5-cm flow cell. 

DNS derivatives of amino acids and peptides are used for the protection of amino groups, separation by TLC and fluorimetric analysis. DNS derivatives can be also used for MS identification and have been employed for the confirmation of a large number of biogenic amines [209]. The molecular ions of these derivatives are usually intense and are often accompanied by an ion at m/e 170 or 171 as the key fragment. BNS derivatives are also useful for MS identification of amines of biological interest [82,83]. 

In addition to the mobile phase composition, the effect of other parameters such as temperature, flow rate, pH, and structure of the analytes were also studied, but only a few reports were available in the literature. In 1995, Lin and Maddox [66] studied the effect of temperature on the chiral resolution of amino acids and esters. The temperature was varied from 5°C to 25°C and it was reported that the resolution improved at low temperature. Hyun et al. [48-50,67] carried out the effect of temperature on the chiral resolution of amino alcohols, amines, fluoroquinolones, and other drugs. Again, lowering of temperature resulted in better resolution. The effect of temperature on the chiral resolution of phenylalanine, phenylglycine, and 2-hydroxy-2-(4-hydroxy-phenyl)-ethyl amine is shown in Table 5 [50], which indicates an increase in retention factors at lower temperature, but the best separation occurred at 20°C. These experiments indicated the exothermic nature of chiral resolution on CCE-based CSPs. Lin and Maddox [66] also studied the effect of flow rate on the chiral resolution of... 

Since its initial application to the separation of amino acids, paper chromatography has been widely applied to the separation of many classes of compounds, including peptides, organic acids, inorganic ions, antibiotics, steroids, and amines.7 A number of monographs on paper chromatography are available.8... 

There are several types of chiral derivatizing reagents commonly used depending on the functional group involved. For amines, the formation of an amide from reaction with an acyl halide [147,148], chloroformate reaction to form a carbamate [149], and reaction with isocyanate to form the corresponding urea are common reactions [150]. Carboxyl groups can be effectively esterified with chiral alcohols [151-153]. Isocynates have been used as reagents for enantiomer separation of amino acids, iV-methylamino acids, and 3-hydroxy acids [154]. In addition to the above-mentioned reactions, many others have been used in the formation of derivatives for use on a variety of packed and capillary columns. For a more comprehensive list, refer to References 155-159. 

Ion-exchange chromatography is a separation method based on the exchanging of ions in a solution with ions of the same charge present in a porous insoluble solid. The method is used for the deionization of water [1,2], It is often employed for the separation and identihcation of the rare earth and transuranium elements [2], Additionally, ion-exchange chromatography is also used in clinical laboratories for the automated separation and analysis of amino acids and other physiologically important amines used for pharmaceutical purposes [3],... 

The ready availability of amino acids and their different functionalizations in the side chains allowed for a number of applications in the field of supported catalysis. While the relatively low cost of many amino acids apparently does not seem to justify the preparation of supported catalysts derived from amino acids, other reasons (as mentioned above) may drive towards the immobilization of chiral catalysts, for example to experiment with different solubilities, the easy separation of the product from the catalyst, and the catalyst s recyclability. The immobilization of these compounds on a support can also be seen as an attempt to develop a minimalist version of an enzyme, with the amino acid playing the role of the enzyme s active site and the polymer that of an oversimplified peptide backbone not directly involved in the catalytic activity [34]. It should be mentioned at this point that, in principle, amine-based catalysts offer also the possibility to be recovered by exploiting their solubility profiles in acids. 

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