Derivatization procedures

The recent development and comparative application of modern separation techniques with regard to determination of alkylphosphonic acids and lewisite derivatives have been demonstrated. This report highlights advantages and shortcomings of GC equipped with mass spectrometry detector and HPLC as well as CE with UV-Vis detector. The comparison was made from the sampling point of view and separation/detection ability. The derivatization procedure for GC of main degradation products of nerve agents to determine in water samples was applied. Direct determination of lewisite derivatives by HPLC-UV was shown. Also optimization of indirect determination of alkylphosphonic acids in CE-UV was developed. Finally, the new instrumental development and future trends will be discussed. 

The GC separations, derivatization procedures, mass spectral interpretation, structure correlations, and other information presented in this book were collected or experimentally produced over the length of a 30-year career (F.G.K.) in GC/MS. It has not been possible to reference all sources therefore, in the acknowledgments, we thank those persons whose work has significantly influenced this publication. 

C. Aromatic Carboxylic Acids (See the following derivatization procedures.)... 

Aminophenols (as acetates see Chapter 32, Section IC, for acetate derivatization procedure)... 

Accordingly, the resulting current reflects the rate at which electrons move across the electrode-solution interface. Potentiostatic techniques can thus measure any chemical species that is electroactive, in other words, that can be made to reduce or oxidize. Knowledge of the reactivity of functional group in a given compound can be used to predict its electroactivity. Nonelectroactive compounds may also be detected in connection with indirect or derivatization procedures. 

A sensitive determination of alkanesulfonates combines RP-HPLC with an on-line derivatization procedure using fluorescent ion pairs followed by an online sandwich-type phase separation with chloroform as the solvent. The ion pairs are detected by fluorescence. With l-cyano-[2-(2-trimethylammonio)-ethyl]benz(/)isoindole as a fluorescent cationic dye a quantification limit for anionic surfactants including alkanesulfonates of less than 1 pg/L per compound for a 2.5-L water sample is established [30,31]. 

The detector must respond to the samples of interest and if not, then the necessary derivatization procedures must be carried out. 

In any derivatization procedure certain criteria must be met. Firstly, the reaction must be rapid and quantitative, or at the very least reproducible. Secondly, the reagents must be easily separated from the final products and they must produce a single product from each component. Thirdly, the derivatives must have detection properties that differ from those of the original components and the capability for their subsequent chromatographic separation must be preserved. 

There is a wide range of reagents available for derivatization and the analyst is again referred to the books by Frei and Lawrence (17) and Karl Blau and John Halket (47) for further reading. The references given here have been chosen as those that are most likely to include the complete details of the derivatizing procedures. They were not chosen as the most contemporary examples of analyses employing derivatization techniques. 

Post-column reaction is a common feature of many special types of analyses, the most well-known being the amino acid analyzer that uses ninhydrin with a post-column reactor to detect the separated amino acids. In general, derivatization and post-column reactor systems are techniques of last resort. In some applications they are unavoidable, but if possible, every effort should made to find a suitable detector for the actual sample materials before resorting to derivatization procedures. 

Quantitative accuracy and precision (see Section 2.5 below) often depend upon the selectivity of the detector because of the presence of background and/or co-eluted materials. The most widely used detector for HPLC, the UV detector, does not have such selectivity as it normally gives rise to relatively broad signals, and if more than one component is present, these overlap and deconvolution is difficult. The related technique of fluorescence has more selectivity, since both absorption and emission wavelengths are utilized, but is only applicable to a limited number of analytes, even when derivatization procedures are used. 

For example, if the internal standard is mnch more volatile than the analyte it is likely that more will be lost dnring storage, if it is mnch more polar it may be extracted either mnch more or less efficiently than the analyte during sample work-np, or if the internal standard is different chemically then derivatization procedures may be more or less efficient (it may be argued that as long as this differential behaviour is reproducible, it can be allowed for in the calcnlation procednres bnt significant extra work wonld be required to confirm such reproducibility). 

The GC-MS techniques give much more chemical information than does measurement of only 8-OH-dGua. However, the hydrolysis and derivatization procedures are lengthy and tedious, and may destroy some modified bases, e.g. hydroxymethyluracil (Djuric etal., 1991). It has also been speculated that they might create artefacts, e.g. if the amounts of modified bases increase during the preparation procedures. Data surest that formic acid hydrolysis does not create additional 8-OH-Gua in DNA (Halliwell and Dizdaroglu, 1992) but the question is currently open as to whether the derivatization procedures might do so. 

E.R. de la Rie and S. Watts, GCMS analysis of terpenoid resins in situ derivatization procedures using quaternary ammonium hydroxides, Stud. Conserv., 47, 257 272 (2002). 

Kallin, E., Lonn, H., Norberg, T., and Elofsson, M. (1989) Derivatization procedures for reducing oligosaccharides, Part 3 Preparation of oligosaccharide glycosylamines, and their conversion into oligosaccharide-acrylamide copolymers./. Carbohydr. Chem. 8, 597-611. 

This elegant organic transformation is, along with the recent development of a Pt(II)-based derivatization procedure for amino acids (221), an impressive example that platinum-mediated C-H activation offers great potential for not only the functionalization of simple small molecules but also the synthesis of complicated organic targets. 

Most of the continuously monitored water contaminants are determined via gas chromatography-mass spectrometry (GC-MS). However, an adequate separation of polar compounds via GC typically requires derivatization of the polar moieties (e.g., BSTFA derivatives). In addition to this, as the analyte groups show different properties concerning the number and kind of functional groups, it is quite difficult to develop a universal derivatization procedure suitable for all the target analytes. Furthermore, the presence in wastewater of many other organic compounds requires the use of labeled standards, which can make application of this method unfeasible [35]. 

A disadvantage of the derivatization procedure used in the case of S. typhimurium (60) was that the trimethylsilylated dimethyl monophosphoryl lipid A derivative was not suitable for the characterization of the (free) hydroxyl group in position 4. This identification could be achieved, however, by the approach of Imoto et al. (35), who investigated the dimethyl monophosphoryl lipid A of E. coli and its per-O-acetylated derivative to compare the chemical shifts before and after acetylation. From the differences in the chemical shift observed in H-n.m.r., the authors could clearly demonstrate that, in their preparation, position 4 of GlcN(I) was free. All... 

The advantage of trimethylsilyl (TMS) derivatives lies in the simplicity of the derivatization procedure, which is carried out by the addition of N,0-bis(trimethylsilyl)trifluoroacetamide (BSTFA) in acetonitrile and heating for approximately 2 h at 150 °C under anhydrous conditions in a sealed tube. However, there may be problems owing to the formation of multiple derivatives of each amino acid. Another technique involves the formation of n-butyl esters of the amino acids and their subsequent trimethylsilylation by a similar procedure. The n-butyl esters are formed by heating the amino acids for 15 min in n-butanol and HC1 and these are then converted to the A-TMS-n-butyl ester derivatives. A-acyl amino acid alkyl esters are commonly used. Acetylation of the butyl, methyl or propyl esters of amino acids,... 

Reaction with phenylisothiocyanate (PITC) in alkaline conditions produces stable phenylthiocarbamyl (PTC) adducts which can be detected either in the ultraviolet below 250 nm or electrochemically. However, this method involves a complex derivatization procedure and offers poorer sensitivity than the alternatives available for individual amino acids. It is useful, however, in conjunction with the automated analysis of peptides when single derivatized residues can be cleaved and analysed after conversion in acidic conditions to phenylthiohydantoins. 

Nitrophenolic compounds were analyzed by GC-MS-SIM, after trimethylsilylation by the flash heater derivatization procedure, which is suitable for nitrophenols not easily derivatized by the conventional methods. The method is suitable for identification of complex mixtures and for quantitative analysis in the nanogram range510. 

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