Fluorescence detection acids

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). 

In this chapter, we present the theory and results of measurements on humic acid fractions using fluorescence techniques. The fluorescence techniques are attractive for this application because of the natural fluorescence of humic materials, the hi sensitivity of fluorescence detection, and the ability to directly observe the morphology of the molecule in aqueous solutions without the need for drying or applying harsh chemical conditions. Several interesting types of information are obtained from fluorescence measurements ... 

We inferred that these properties might be exploited in a series of unique derivatizing reagents designed specifically for trace analysis of organic compounds using HPLC separation and fluorescence detection. The use of these pyridones for the analytical purposes reported here is based on their acidic properties. Treatment of a lH-2-pyridone with a base converts the pyridone to its salt. 

Figure 4.4 Release of amino acids from cortical slices exposed to 50 mM K+. Measurements by HPEC and fluorescence detection after reaction of amino acids with o-phthalaldehyde 1, aspartate 2, glutamate 3, asparagine 4, serine 5, glutamine 6, histidine 7, homoserine (internal standard) 8, glycine 9, threonine 10, arginine 11, taurine 12, alanine 13, GABA 14, tyrosine. Glutamate concentration is almost 1 pmol/gl which represents a release rate of 30 pmol/min/mg tissue...

Carvalho, P.R.N. and Collins, C.H., HPLC determination of carminic acid in foodstuffs and beverages using diode array and fluorescence detection, Chromatographia, 45, 63, 1997. 

The analysis of amino acids involves chromatographic issues similar to those encountered in analysis of simple amines. Underivatized amino acids have, with a few exceptions, weak UV absorbance and a strong tendency to interact with stationary phases in undesirable ways. Underivatized amino acids are normally separated with ion exchange chromatography, then visualized post-column by reaction with ninhydrin, o-phthaladehyde (OPA), or other agents. Underivatized tryptophan and the metabolites kynurenine, 3-hydroxykynurenine, kynurenic acid, and 3-hydroxyanthranilic acid, were separated on a Partisphere 5-p ODS column with fluorescent detection.121... 

Wu, S. and Dovichi, N. J., Capillary zone electrophoresis separation and laser-induced fluorescence detection of zeptomole quantities of fluorescein thiohy-dantoin derivatives of amino acids, Talanta, 39, 173, 1992. 

Kuhr, W. G. and Yeung, E. S., Indirect fluorescence detection of native amino acids in capillary zone electrophoresis, Anal. Chem., 60, 1832, 1988. 

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... 

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... 

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... 

B. Schmidt and D. Riesner, A fluorescence detection system for the analytical ultracentrifuge and its application to proteins, nucleic acids, viroids and viruses (in Ref. [77]). 

Marnela et al. [57] used an amino acid analyzer using fluorescence detection to determine penicillamine in urine. Urine is analyzed on a Kontron Chromakon 500 amino acid analyzer containing a column (20 cm x 3.2 mm) of AS70 resin in the Li (I) form. Buffers containing LiOH, citric acid, methanol, HC1, and Brij 35 at pH 2.60, 3.20, and 3.60 are used as mobile phases (0.4 mL/h). The fluorescence reagent is prepared by the method of Benson and Hare. Detection is at 450 nm (excitation at 350 nm). The analyte response is linear from 0.025 to 10 mM, with a limit of detection of 25 pM. 

Valproic acid has been determined in human serum using capillary electrophoresis and indirect laser induced fluorescence detection [26], The extract is injected at 75 mbar for 0.05 min onto a capillary column (74.4 cm x 50 pm i.d., effective length 56.2 cm). The optimized buffer 2.5 mM borate/phosphate of pH 8.4 with 6 pL fluorescein to generate the background signal. Separation was carried out at 30 kV and indirect fluorescence detection was achieved at 488/529 nm. A linear calibration was found in the range 4.5 144 pg/mL (0 = 0.9947) and detection and quantitation limits were 0.9 and 3.0 pg/mL. Polonski et al. [27] described a capillary isotache-phoresis method for sodium valproate in blood. The sample was injected into a column of an EKI 02 instrument for separation. The instrument incorporated a conductimetric detector. The mobile phase was 0.01 M histidine containing 0.1% methylhydroxycellulose at pH 5.5. The detection limit was 2 pg/mL. 

DeMar Jr. J.C., Disher, R.M., and Wensel, T.G. (1992) HPLC analysis of protein-linked fatty acids using fluorescence detection of 4-(diazomethyl)-7-diethylaminocoumarin derivatives Abstract 465. Biophys. J. 61(A81). 

Oser, A., Roth, W.K., and Valet, G. (1988) Sensitive non-radioactive dot-blot hybridization using DNA probes labeled with chelate group substituted psoralen and quantitative detection by europium ion fluorescence. Nucleic Acids Res. 16, 1181-1196. 

Petty et al. [293] used flow injection sample processing with fluorescence detection for the determination of total primary amines in sea water. The effects of carrier stream flow rate and dispersion tube length on sensitivity and sampling rates were studied. Relative selective responses of several amino acids and other primary amines were determined using two dispersion tube lengths. Linear calibration curves were obtained over the ranges 0-10 6 M and... 

Fogli et al. developed and validated an HPLC method with fluorescence detection for simultaneous routine TDM of anthracyclines and their metabolites.27 They coupled a Waters LC Module I Plus system equipped with a WISP 416 autosampler with a Model 474 scanning fluorescence spectrophotometer. The stationary phase was a Supelcosil LC-CN column (250 x 4.6 mm, 5 /um particle size) with a /iBondapak-CN guard column. The mobile phase consisted of 50mM monobasic sodium phosphate buffer and acetonitrile (65 35 v/v), adjusted to pH 4.0 with phosphoric acid. The flow rate was 1 mL/min. The fluorescence detection was set at excitation wavelengths of 233, 254, and 480 nm and at an emission wavelength of 560 nm. 

FMOC offers sensitivity comparable to OPA when used in conjunction with fluorescence detection, whilst also reacting with both primary and secondary amines. Moreover, the derivatives are stable for more than 30h in an acidic environment. 

FMOC-amino acids can be chromatographed using a C8 column and acetonitrile in sodium acetate buffer as the mobile phase. Fluorescence detection with excitation at 260nm and emission at 31 Onm gives the best results. 

K. Arlt, S. Brandt, and J. Kehr, Amino acid analysis in five pooled single plant cell samples using capillary electrophoresis coupled to laser induced fluorescence detection. J. Chromatogr. A 926, 319 325 (2001). 

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