Ultraviolet-Visible Derivatization

Ultraviolet-visible (UV-Vis) detection in liquid chromatography is often hampered by the poor spectral properties of the analytes at the applied analytical wavelengths. In this respect, a variety of pre- and postchromatographic derivatization reactions have been proposed for improving UV-Vis detectability. 

6-Trinitrobenzenesulfonic acid has been also used for the prechromato-graphic derivatization of aminoglycosides in alkaline media (228, 229). A major advantage of this label is the fact that hydroxyl groups cannot be concurrently derivatized, thus increasing, the selectivity. Unlike prementioned labels, 4-fluoro- 

3-nitrotrifluoromethylbenzene does not react with compounds such as amino acids that contain additional polar groups (230). This reagent docs not react with secondary amines either. 

Amino groups can also be derivatized using acyl chlorides that form amides. A number of suitable acyl chlorides including p-chloro-, p-methoxy-, p-nitroben-zoyl-, p-tolyl-, and p-nitro-benzenesulfonyl chloride have been successfully used for sensitive UV-Vis derivatization of nonabsorbing amine compounds (231). Among all those amide derivatives, p-methoxybenzamides appear more attractive because they exhibit high molar absorptivity at the convenient analytical wavelength of 254 nm. After derivatization in tetrahydrofuran-sodium hydroxide solu- 

Tertiary amines can also be selectively derivatized with citric acid on acetic acid anhydride, a mixture that is mixed with the eluent after chromatography and then heated to 393 K to develop a violet-red color (237). Absorbance is measure at 550 nm but some compounds can show strongly tailing peaks. 

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Hostettmann, K. et al., On-line high-performance liquid chromatography ultraviolet-visible spectroscopy of phenolic compounds in plant extracts using post-column derivatization, J. Chromatogr., 283, 137, 1984. 

Starting with a description of the analytical challenge in Chapter 19, the third part, which is devoted to analytical attitudes, proceeds with a detailed description in Chapter 20 of modern sample preparation procedures including solid-phase extraction, matrix solid-phase dispersion, use of restricted-access media, supercritical fluid extraction, and immunoaffinity cleanup. Flexible derivatization techniques including fluorescence, ultraviolet-visible, enzymatic, and photochemical derivatization procedures are presented in Chapter 21. 

Today, HPLC is the dominant analytical technique used for the analysis of most classes of compounds. The analyses can be carried out at room temperature and the collection of fractions for reanalysis or further manipulation is straightforward. The main reason for the slow acceptance of the HPLC technique for Upid analysis has been the detection system. Traditionally, HPLC used ultraviolet/visible (UV/vis) detection, which requires the presence of a chromophore in the analyte. Most lipid molecules do not contain chromo-phores and therefore would not be detected by UV/vis. Modern HPLC detection techniques, such as the use of a mass spectrometer as the detector, derivatization techniques to introduce chromophores, and the availability of pure solvents to reduce interference, have allowed HPLC to compete with and/or complement GC and other traditional methods of lipid analysis. In addition to analytical HPLC, preparative HPLC has been used extensively to collect pure samples of the lipids for the derivatization or synthesis of new compounds. 

Z. Zhang, N. Yoshida, T. Imae, Q. Xue, M. Bai, J. Jiang, and Z. Lui (2001). A self-assembled monolayer of an alkanoic acid-derivatized porphyrin on gold surface A structural investigation by surface plasmon resonance, ultraviolet-visible, and infrared spectroscopies. J. Colloid Interf. Sci. 243, 382. 

The derivatization of analytes is very important in several branches of analytical chemistry. It expands the fields of application of various spectroscopic techniques (ultraviolet-visible (UV-vis), fluorimetry, nuclear magnetic resonance (NMR), and mass spectroscopies), and in several cases increases also the selectivity and sensitivity of these techniques. Derivatization is also an inevitable tool in all chromatographic and electrophoretic techniques. In gas chromatography (GC), the main importance of derivatization is the improvement of the volatility/thermal stability of the analytes, and in all of the discussed separation techniques it has the potential of increasing the selectivity of the separation (including enantiomeric separations) and the sensitivity of the detection. 

Many chiral derivatization reagents have been developed for the enantioseparation of amino acids wherein ultraviolet-visible or fluorescence tags are introduced. The fluorescence derivatization is more effective for the determination of amino acid enantiomers in complex matrices in terms of sensitivity and/or selectivity. Table 1 shows the chiral derivatization reagents, whose structures are shown in Figure 2, used for the enantioseparation of amino acids. 

Then L, Ding L, Yu A, Yang R, Wang X, Li J, Jin H, Zhang H (2007) Continuous determination of total flavonoids in Platycladus orientalis (L.) Franco by dynamic microwave-assisted extraction coupled with on-line derivatization and ultraviolet-visible detection. Anal Chim Acta 596 164—170... 

The absorption characteristics have been used to estimate the number of nitroazidophenyl groups that are attached to protein derivatized with 4-azido-2-nitrofluorobenzene. The ultraviolet-visible absorption or the characteristic infrared band at approximately 2100 cm-, often broad or a doublet, can be used to follow photolyses. 

Flow injection methods can be coupled to any detection system available. The most common detection systems include a chemiluminescence detector, fluorometer, ultraviolet-visible (UV-Vis) spectrophotometers, and a sophisticated mass spectrometer. Flow injection methods coupled to these detectors have been applied in the analysis of parabens in consumer products such as food, cosmetics, and pharmaceutical formulations. However, parabens have been determined mostly by UV-Vis spectrophotometers and chemiluminescence detectors as reported in the literature. Other detectors such as electrochemical and fluorescence detectors have disadvantages of poor reproducibility and requirements for additional derivatization [21]. 

The derivatization process (5) is accomplished in aqueous media at basic pH (pH 7-10) in a matter of approximately 15 min to yield a 2-cyanobenz[f]isoindole (CBI), which is stable for 10 to 12 hr in solution. As shown in Figure 1, the absorption characteristics of the CBI adducts are also readily accessible for assay by standard fluorescence or ultraviolet detection. In addition to the absorption between 200 and 300 nm, there are two maxima in the visible spectrum at approximately 420 and 440 nm accessible for fluorescence or ultraviolet detection. A probable mechanism (5,11) for the CBI formation is illustrated in Scheme 1. 

Since BAs occurring in food do not exhibit satisfactory absorbance or fluorescence in the visible or ultraviolet range, chemical derivatization, either pre- (35-37) or postcolumn (38), is usually used for their detection in HPLC. The most frequently employed reagents for precolumn derivatization are fluorescamine, aminoquinolyl-lV-hydroxysuccinimidyl carbamate (AQC) (39, 40), 9-fluorenylmethyl chloroformate (FMOC) (41-43), 4-dimethylaminoazobenzene-4 -sul-fonyl chloride (dabsylchloride, DBS) (44), N-acetylcysteine (NAC) (45,46), and 5-dimethyl-amino-1-naphthalene-1-sulfonyl chloride (dansylchloride, DNS) (47,48), phthalaldehyde (PA), and orf/to-phthaldialdehyde (OPA) (49-51), together with thiols such as 3-mercaptopropionic acid (MPA) (37) and 2-mercaptoethanol (ME) (35,49). 

For the purification of compounds, methods including molecular filtration, solid phase extraction (SPE, SPME), solvent extraction, and a variety of basic chromatographic techniques (thin layer, low pressure, ion exchange, size exclusion, etc.), HPLC, and GC (with derivatization of nonvolatile compounds) can be used. Additionally, instrumentation to identify compounds is available, such as the different spectrometric applications, including infrared (IR), mass (MS), ultraviolet and visible (UV-Vis), and NMR spectroscopy. In recent years, the so-called hyphenated techniques (combined chromatographic and spectral methods such as... 

Analytical quantification of BAs may be difficult due to the complexity of some food matrices and the low concentrations of BAs generally encountered in the majority of foodstuffs. In addition, the low volatility of these compounds and the lack of chromophores for most of the BAs, does not allow the rapid direct detection by ultraviolet and visible (UV and vis) spectrometric or fluorimetric (FL) methods. In general, in order to obtain an optimal analysis, extraction, clean-up, concentration, and derivat-ization procedures are required. Extraction methods usually based on liquid-liquid or solid-phase extraction with C18 or ion-exchange cartridges can be applied to improve selectivity and sensitivity (Giannotti et al., 2008 Pena-Gallego, Hemdndez-Orte, Cacho, Ferreira, 2009). Alternative approaches, such as solid-phase microextraction... 

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