Amino acids separation derivatization with

The mixture of free amino acids is reacted with OPA (Fig. 7-8) and a thiol compound. When an achiral thiol compound is used, a racemic isoindole derivative results. These derivatives from different amino acids can be used to enhance the sensitivity of fluorescence detection. Figure 7-9 shows the separation of 15 amino acids after derivatization with OPA and mercaptothiol the racemic amino acids may be separated on a reversed-phase column. If the thiol compound is unichiral, the amino acid enantiomers may be separated as the resultant diastereomeric isoindole compound in the same system. Figure 7-10 shows the separation of the same set of amino acids after derivatization with the unichiral thiol compound Wisobutyryl-L-cysteine (IBLC). 

Fig.7-10. Separation of amino acids after derivatization with OPA and A -isobu-tyryl-L-cysteine. Column Superspher 100 RP-18 (4 pm) LiChroCART 125-4, mobile phase 50 mM sodium acetate buffer pH 7.0/sodium acetate buffer pH 5.3/methanol, flowrate 1.0 ml min temperature 25 °C detection fluorescence, excitation 340 nm/emission 445 nm. Sample amino acid standard mixture. (Merck KGaA Application note W219189 reproduced with permission from H. P. Fitznar, Alfred-Wegener-Institute for Polar and Marine Research.)...

A simple and rapid method of separating optical isomers of amino acids on a reversed-phase plate, without using impregnated plates or a chiral mobile phase, was described by Nagata et al. [27]. Amino acids were derivatized with /-fluoro-2,4-dinitrophenyl-5-L-alanine amide (FDAA or Marfey s reagent). Each FDAA amino acid can be separated from the others by two-dimensional elution. Separation of L- and D-serine was achieved with 30% of acetonitrile solvent. The enantiomers of threonine, proline, and alanine were separated with 35% of acetonitrile solvent and those of methionine, valine, phenylalanine, and leucine with 40% of acetonitrile solvent. The spots were scraped off the plate after the... 

Fig. 11 Separation of amino acid standards derivatized with PITC. Eluent A sodium acetate/triethy-larnine buffer (pH 6.4) eluent B 60/40 acetonitrile/water. Pico-Tag column. Peak identification 1 Asp, 2 Glu, 3 Ser, 4 Gly, 5 His, 6 Arg, 7 Thr, 8 Ala, 9 Pro, 10 ammonia, 11 Tyr, 12 Val, 13 Met, 14 Cys, 15 lie, 16 Leu, 17 Phe, 18 Lys. (From Ref. 184. Copyright 1984 Elsevier Science.)...

Figure 2. Chromatographic separation of amino acids after derivatization with phenylisothiocyanate (PITC) A. Separation of 200 picomole standard amino acid mix H containing 18 amino acids. B. Separation of an extended amino acid mix containing 28 amino acids. The standard one-letter abbreviations are used for the usual amino acids. Nonstandard amino acids are Sp, phosphoserine Hp, hydroxyproline Citr, citrulline Tau, taurine aAba, a-amino butyric acid HKl HK2, hyi oxylysines Om, ornithine , artifacts from reagents.

Figure 6. Chromatographic separation of amino acids after derivatization with phenylisothiocyanate (PITC)using physiological conditions A. Free amino acids found in rat brain tissue. B. Free amino acids in ant hemolymph. C. Bone collagen hydrolysate.

The separation power and sensitivity of CE is illustrated in Figure 21.23. A mixture of 18 amino acids is separated in 30 min, at quantities ranging from 2 to 7 attomoles. The amino acids were derivatized with fluorescein isothiocyanate (Fl i C) to form fluorescent derivatives, and a fluorometric detector system was employed. The detection limits ranged down to 10""2o mol in 1 nL, corresponding to 10 " M and 6000 molecules ... 

MM2 calculations, and the separation factors and capacity ratios, measured during isocratic elution, were related to the energy value changes between an analyte and a model phase. Diastereomers have very different stereostructures. The contact surface area with bonded allg l groups affects the elution order. Amino acids were derivatized with a chiral derivatization reagent, such as o-phthalaldehyde-AT-acetyl-L-cysteine (ref. 11) and N-tert-butylthiocarbonyl-L-cysteine, prior to the chromatographic analysis. 

The capacity ratios and the separation factors of amino acid enantiomers derivatized with (+)-l-(9-fluororenyl)ethyl chloroformate measured in... 

There are some cases in which MS has been employed for the determination of amino acids without a separation step. Amino acid samples derivatized with Sanger s reagent can be separated based... 

Ion-exchange chromatogram from Beckman-Spinco model 121MB amino acid analyzer introduced in 1969. After separation, amino acids were derivatized with ninhydrin to form colored products detected by visible absorption. [Courtesy Beckman-Coulter, Fullerton CA.1... 

Racemic and nonracemic amino acids were derivatized with chiral reagent l-fluoro-2,4-dinitrophenyl-5-L-phenylalanine amide (FDPA) according to Marfey s method [11]. Twelve proteinaceous a-amino acids were derivatized with FDPA, a chiral variant of MR. The resulting diastereomers were separated... 

Achiral Columns Together with Chiral Mobile Phases. Ligand-exchange chromatography for chiral separation has been introduced (59), and has been appHed to the resolution of several a-amino acids. Prior derivatization is sometimes necessary. Preparative resolutions are possible, but the method is sensitive to small variations in the mobile phase and sometimes gives poor reproducibiUty. 

Fig. 7 Typical reversed-pbase separation of amino acids. Precolumn derivatization of a standard amino acid mixture was achieved employing FMOC. Resolution was achieved by gradient elution with acetonitrile, methanol, and acetate buffer (pH 4.2) on a C,8 column. Standard three-letter abbreviations for amino acids are used also, CySO H = cysteic acid. (From Ref. 164. Copyright 1983 Elsevier Science.)...

Amino acids are derivatized two ways to increase sensitivity. Free amino acids in solution are reacted with o-phthaldehyde (OPA) to form a fluorescent derivative that excites at UV,230nm, and emits at FL, 418 nm. These OPA derivatives are separated on Ci8 in a complex mixture of An/MeOU/ DMSO/water at pH 2.65. PTH amino acids are formed from the N-terminai end of peptides during Edman degradation for structure analysis of peptides and proteins. HPLC is used to identify which amino acids are released. PTH amino acids are separated at UV, 254 nm, on a Ci8 column with a gradient from 10% THF/water containing 5 mM acetic acid to 10% THF/AN.The separation with reequilibration takes 60min. Work with short 3-pm columns has reduced this separation to a 10-min gradient. 

Figure 10.3. Examples of chromatograms. (a) The separation of 20 essential amino acids (in derivatized form) by RPLC using 1 mm inside diameter by 150 mm long microbore column packed with 4 ft m silica support (with C18 CBP) particles and a water/ acetonitrile mobile phase gradient. (6) The fractionation of gasoline using SFC with C02 mobile phase (temperature and pressure programmed) in 0.25 mm x 50 cm packed column with 5 fim polymeric support particles. (Courtesy of Frank J. Yang.)...

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. 

Figure 5. Separation of amino acids derivatized with o-phthaldialdehyde (OTA) by reverse-phase chromatography. Extracts of nodules were derivatized according to the procedures described by Hill et al, (51, except the final volume was reduced from 5 mL to 0.5 mL or less and the concentrations of OTA and ethanethiol in the derivaUzing solution were increased to 60 mgjmL and 0.1 mglmLy respectively. A sample volume of 20 fiL was injected on column and the derivatized amino acids were eluted with an increasing gradient of acetonitrile from 12% to 100%.

The most widely used technique for the estimation of amino acid isomers is gas-liquid chromatography. Two basic strategies have been used to separate isomers by this technique. Both approaches appeared in the mid-1960 s, and both involve derivatiza-tion of the amino acids to suitably volatile acyl-esters (46,47). One method is similar in concept to the separation of disastereo-mers by ion exchange chromatography discussed above. In one step of the two step derivatization, the amino acids are esterified with an optically active agent. This procedure creates molecules with two centers of asymmetry which can be separated on a non-optically active liquid (stationary) phase (46). This method allows any laboratory equipped with a gas chromatograph to perform Isomer analyses. 

The second gas chromatographic method employs an optically active liquid phase (47). The amino acids are derivatized to acyl-esters to improve volatility however, in this case, both the acylation and esterification agents are optically inactive. Because the L-amino acid derivatives interact preferentially with the optically active stationary phase, the Isomers can be separated. However, use of this technique was limited because the optically active liquid phases were not stable to sufficiently high temperatures to separate all the derivatized amino acids. In 1977, Bayer and coworkers reported covalent coupling of an optically active moiety (L-valine) to a silicone liquid phase (48). This phase has high thermal stability and allows analysis in approximately one hour. 

Gil-Av et al. argued that the separation of the antipodes on a suitable asymmetric stationary phase would involve reversible association between the enantiomers and the asymmetric stationary phase molecules. The two antipodes would form diastereoisomeric interaction with somewhat different interatomic distances. Hence, there would be different polar, dispersive and/or steoric interactions between substituents situated round the asymmetric centers of the solute and stationary phase molecules, respectively. Such differences would effect the standard free energy of distribution and the magnitude of the distribution coefficients of the enantiomers. Gil-Av et al. used A-TFA-D-isoleucine lauryl ester and A-TFA-L-isoleucine lauryl ester as the stationary phase which were coated on the walls of a capillary column 100 m long, 250 pm I.D. and was shown to have an efficiency of about 98,000 theoretical plates. The samples of the derivatized amino acids were injected with a split ratio of 1 100. The separation was carried out isothermally at 90°C and the analysis time was just over 4 hours. The results obtained for the... 

Chirobiotic T exhibits a unique chiral selectivity for a number of classes of solutes. It is particularly useful for resolving the enantiomers of underivatized amino acids, N-derivatized amino acids and small peptides but is widely applicable to many classes of compounds. Chirobiotic T can also be used with a very wide range of solvents, and can be used with the polar organic system, in the reversed phase mode and in the normal phase mode. In addition, the chiral selectivity changes greatly from one development mode to another. The optimization protocol as seen in the chart Chirobiotic T is very similar to that used for Chirobiotic V and the same adjustments are made to optimize the separation. 

---