Amino acids derivatized, fluorescence detection

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... 

An alternative to derivatization of nonfluorescent compounds is to perform indirect fluorescence detection. The procedure is performed on-column, by incorporating a fluorescent ion into the electrolyte. When ionic analytes interact with the fluorophore, the result is either displacement of the fluorophore or ion pairing with it.47 Kuhr and Yeung51 explored indirect laser-induced fluorescence detection using 1.0 mM salicylate as the fluorophore in the electrolyte they analyzed 10 amino acids and obtained detection limits on the order of 10 5 M. 

The chromatographic analysis of amino acids with spectrophotometric detection usually requires the formation of derivatives, because of httle absorption of UV light above 210 run. Precolumn deiivatization is usually preferable. o-Phthalaldehyde (OPA) is the deiivatization reagent that probably has the best characteristics. It reacts with primary amino groups in the presence of a thiol at pH 9.5 and room temperature to form l-alkylthio-2-alkyl substituted isoindoles (Fig. 10.5). The derivatives show maximum absorption at 335 nm and are highly fluorescent, with excitation wavelength at 340 nm and emission at 445 nm. Mercaptoethanol has been more extensively used than other thiols for the derivatization, but the OPA-mercaptoethanol isoindoles are unstable. The stability of isoindoles is improved when A-acetyl-L-cysteine (NAC) is used instead of mercaptoethanol [12]. 

Derivatization is useful for detection of compounds such as amino acids and amines that lack easily detectable groups. For similar reasons, saccharides, as a class of compound, ehcit much interest. Two derivatization schemes have been reported using benzamide (61) and FMOC—hydrazine (62) to produce fluorescent products. 

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).

Derivatization of a racemic compound with an achiral group may play an important role in the analysis of a chiral compound (Fig. 7-15). In the case of substances with low or no UV-activity, the compounds can be rendered detectable by introducing an UV-absorbing or fluorescent group. If the racemate itself shows selectivity on a chiral stationary phase (CSP), this method can be applied to reduce the limit of detection. Examples have been reported in the literature, especially for the derivatization of amino acids which are difficult to detect using UV detection. Different derivatization strategies can be applied (Fig. 7-16). 

Table III. Detection Limits of 0PA/2-ME and NDA/CN Derivatized Amino Acids Using Conventional and Laser-Induced Fluorescence (LIF) Detection...

Anions of weak acids can be problematic for detection in suppressed IEC because weak ionization results in low conductivity and poor sensitivity. Converting such acids back to the sodium salt form may overcome this limitation. Caliamanis et al. have described the use of a second micromembrane suppressor to do this, and have applied the approach to the boric acid/sodium borate system, using sodium salt solutions of EDTA.88 Varying the pH and EDTA concentration allowed optimal detection. Another approach for analysis of weak acids is indirect suppressed conductivity IEC, which chemically separates high- and low-conductance analytes. This technique has potential for detection of weak mono- and dianions as well as amino acids.89 As an alternative to conductivity detection, ultraviolet and fluorescence derivatization reagents have been explored 90 this approach offers a means of enhancing sensitivity (typically into the low femtomoles range) as well as selectivity. 

Wen-Chen Z, Ling-Jun L, Xian-En Z et al (2008) Application of 2-(l l//-benzo[ i]carbazol-11 -yl) ethyl carbonochloridate as a precolumn derivatization reagent of amino acid by high performance liquid chromatography with fluorescence detection. Chin J Anal Chem 36 1071-1076... 

Zhang H, Le Potier I, Smadja C et al (2006) Fluorescent detection of peptides and amino acids for capillary electrophoresis via on-line derivatization with 4-fluoro-7-nitro-2,l, 3-benzoxadiazole. Anal Bioanal Chem 386 1387-1394... 

Compared to absorbance detection, direct detection of proteins rich in aromatic amino acids by the intrinsic fluorescence of tryptophan and tyrosine residues provides enhanced sensitivity without the complexity of pre- or postcolumn derivatization. The optimal excitation wavelengths for these amino acids are in the 270- to 280-nm range. 

Piepponen TP, Skujins A. 2001. Rapid and sensitive step gradient assays of glutamate, glycine, taurine and y-amino-butyric acid by high-performance liquid chromatography-fluorescence detection with o-phthalaldehyde-mercap-toethanol derivatization with emphasis on microdialysis samples. J Chromatogr B 757 277-283. 

Several kit solutions for indirect detection of amino acids and carbohydrates and fluorescence detection of amino acids after derivatization. 

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