Amino acids separation fluorescence 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. 7-9. Separation of amino acids after derivatization 5 with OPA and mercaptoethanol. Column Superspher 100 RP-18 (4 pm) LiChroCART 250-4, mobile phase 50 mM sodium acetate buffer pH 7.0/methanol, flowrate 1.0 ml min temperature 40 °C detection fluorescence, excitation 340 nm/emission 445 nm. Sample amino acid standard sample (Merck KGaA Application note W219180).

Hu S, Li PCH (2000) Micellar electrokinetic capillary chromatographic separation and fluorescent detection of amino acids derivatized with 4-fluoro-7-nitro-2,l,3-benzoxadiazole. J Chromatogr A 876 183-191... 

The reaction of 2,4-dinitrofluorobenzene (DNFB) (Sanger s reagent [10]) with amino acids is another useful technique which is often employed for the analysis of N-terminal amino acids by TLC and column chromatography after derivatization. The reaction involved in product formation is shown in Fig.4.6. The separated derivatives are determined by measuring the quenching of fluorescence on TLC plates or by UV analysis after column chromatography. The generalized absorption curves of dinitrophenyl (DNP)-amino acids in acidic and alkaline solutions are shown in Fig. 4.7. 

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. 

Benturquia et al. [51] described simultaneous determination of vigabatrin and amino acid neurotransmitters in brain microdialysates by capillary electrophoresis and laser-induced fluorescence after precolumn derivatization with naphthalene-2,3-dicaroxaldehyde (NDA). Optimal separation and detection were obtained with a sodium borate buffer (pH 9.2) containing 60 mM sodium dodecyl sulfate and 5 mM hydroxy-propyl-/ -cyclodextrin with fluorescence detection at 589 nm and excitation at 543 nm. The assay was rectilinear over the concentration range of 1.5-200 /(M and the lower limit of detection was 0.13 yM. Both the within-day and day-to-day reproducibilities and accuracies were less than 14.3% and 4.9%, respectively. 

As with MID on MS, the more wavelengths that are simultaneously monitored, the greater is the likelihood of valid identification. Another analytical technique is the formation of derivatives which are fluorescent or absorb UV radiation at unique wavelengths. The compound of interest may be derivatized and injected onto the HPLC system the column separates the reactants and then passes them through the detector. The compound may also be derivatized "post column" as done by amino acid analyzers. The de-rivatizing reactant is metered to mix with the column effluent and is then sent to the detector. Ideally, only the derivatized products should be detectable. 

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