Fluorescence precolumn reaction

Others (i.e., Patch clamp ) Fluorescence Absorbance Luminescence Precolumn reaction Postcolumn reaction ... 

Further applications for the determination of the newer antidepressants have employed precolumn derivatization, which included a reaction with dansyl chloride or 4-(N-chloroformylmethyl-N-methyl) amino-7-nitro-2,l,3,-benzoxadiazole (NBD-COCL) followed by separation on ODS Cl8 analytical columns maintained at either 35°C or 70°C using either isocratic or gradient elution with fluorescence end-point detection. The compounds were isolated from human plasma or serum by LLE or SPE techniques using... 

Fig. 2 Postcolumn derivatization scheme for aflatoxin analysis 1, mobile phase 2, HPLC pump 3, injection valve 4, precolumn 5. analytical column 6, derivatizing agent solution 7, auxiliary HPLC pump 8, T-valve 9, oil or water bath 10, reaction coil 11, fluorescence detector 12, waste 13, chromatographic data handling system.

Colistin (COL) is a multicomponent antibiotic (polymyxins E) that is produced by strains of inverse Bacillus polymyxa. It consists of a mixture of several closely related decapeptides with a general structure composed of a cyclic heptapeptide moiety and a side chain acetylated at the N-terminus by a fatty acid. Up to 13 different components have been identified. The two main components of colistin are polymyxins El and E2 they include the same amino acids but a different fatty acid (216). A selective and sensitive HPLC method was developed for the determination of COL residues in milk and four bovine tissues (muscle, liver, kidney, and fat). The sample pretreatment consists of protein precipitation with trichloracetic acid (TCA), solid-phase purification on Cl 8 SPE cartridges, and precolumn derivatization of colistin with o-phthalaldehyde and 2-mercaptoethanol in borate buffer (pH 10.5). The last step was performed automatically, and the resulting reaction mixture was injected into a switching HPLC system including a precolumn and the reversed-phase analytical column. Fluorescence detection was used. The structural study of El and E2 derivatives was carried out by HPLC coupled with an electrospray MS. Recoveries from the preseparation procedure were higher than 60%. 

Used for the precolumn preparation of fluorescent derivatives of thiols optimum derivatization conditions were found to be pH 8.2 with a minimum of a 2.7-fold molar reagent excess using a reaction time of 1 h at 60°C under these conditions only free sulfhydryl groups are derivatized Reference 16... 

A variety of pre- and postcolumn sample derivatization schemes has been developed in CE, mainly for the attachment of fluorescent labels for detection purposes. Precolumn sample modification is usually less demanding than postcolumn reactions for reasons discussed in Sect. 3.2.4. 

The analysis of cell culture media and supernatants, as well as non-standard protein hydrolysates such as collagens and glycoproteins, has created the demand for techniques that accurately quantify additional amino acids not normally found in hydrolyzed samples, Methods of amino acid analysis (AAA) based on precolumn derivatization with 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate (AQC) have previously been shown to quantify hydrolyzed samples with a high degree of accuracy (1,2). The AQC-based method has also been shown to derivatize effectively in the presence of salts and lipids (3). Taking into account the above strengths, the excellent stability of the derivatives, and the unique fluorescence properties that allow for direct injection of the reaction mixture without cleanup, the AQC methodology represents an ideal choice for the analysis of complex samples. 

Of the two detection techniques mentioned above, fluorescence is preferred in general because it suffers from fewer operational difficulties. Many compounds do not display a native fluorescence however, this can be overcome if the analyte can be converted by chemical reaction into a fluorescent compound. This process is known as derivatisation and can be accomplished by either derivatising the sample prior to injection (precolumn) or after chromatographic separation (post-column). 

AQC (6-aminoquinolyl-A-hydroxysucdnimidyl carbamate) Fluorescent (ex. 245 nm, em. 395 nm) UV 245 nm 160 fmol 35-50 min precolumn Quaternary gradient elution required for complex, nonhydrolysate samples Tolerates salts and detergents, rapid reaction, stable product, good reagent separation, high sensitivity and accuracy... 

OPA (ortho-phthalaldehyde) Fluorescent (ex. 340 nm, em. 455 nm) 50 fmol 90 min postcolumn, 17-35 precolumn Secondary amine pretreatment, slow reaction, unstable derivative, background interference Good reagent separation, high sensitivity and reproducibility with automated system... 

LC Many solutes do not exhibit UV absorption or fluorescence, they can be converted in UV absorbing or fluorescent derivatives by pre or post column derivatization. The procedure has a wider range than in GC since the reaction can be performed following separation. When precolumn derivatization is carried out the chromatographic system is obviously different to the one selected for the nonderivatized solutes. 

Zika (1977) compared two precolumn reagents — fluorescamine and d2insyl chloride — and found the latter to be more suitable for seawater determinations. The long reaction times and the solvent extraction step involved prior to injection, together with the bulky nature of the derivative (leading to a loss of resolution), and the fact that the reagent and the reaction by-product fluoresce themselves, limits the usefulness of the method. 

High performance liquid chromatography methods are widely used to quantify the PSP toxins present in a shellhsh sample, but they can also serve to identify the toxin profile of a PSP bloom. PSP toxins have only a weak natural chromophore and must be modified before detection [16]. When oxidized in alkaline solution, they form a purine, which becomes fluorescent in acidic solution [6]. This derivatization reaction can be carried out precolumn or postcolumn, and the purines are monitorized in a fluorescence detector. 

Derivatization can be employed to advantage with LCD detection as well. Both precolumn or post-column reactions may be employed to produce electroactive compounds analogous to the formation of fluorescent derivatives. The UV-induced reaction chamber for ECDs described in Section 15.4 and the dual electrode detector oxidation-reduction... 

While one of the limitations of LIF detection is that few target molecules exhibit native fluorescence, especially biologically relevant proteins and DNA of clinical interest, several different labeling approaches both on- and off-chip have been demonstrated. For the former, the microchip fabrication steps provide the ability to incorporate additional structures into the design, in many cases without adding more steps (or cost) to the fabrication process. Examples include the additional channels and reaction chambers used to perform both precolumn" and postcolumn" labeling of amino acids. 

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