Chemical derivatization methods

Many of the chemical derivatization methods employed in these strategies involve the use of an activation step that produces a reactive intermediary. The activated species then can be used to couple a molecule containing a nucleophile, such as a primary amine or a thiol group. The following sections describe the chemical modification methods suitable for derivatizing individual nucleic acids as well as oligonucleotide polymers. 

Fluorescent probes containing sulfhydryl-reactive groups can be coupled to DNA molecules containing thiol modification sites. The chemical derivatization methods outlined in Section 2.2 (this chapter) may be used to thiolate the oligo for subsequent modification with a fluorophore. Appropriate fluorescent compounds and their reaction conditions may be found in Chapter 9. The protocol discussed in the previous section can be used as a general guide for labeling DNA molecules. 

Extending the utility of fluorescence to various nonfluorophores is achieved via chemical derivatization methods, also termed labeling or tagging methods (Reaction 11.2). Numerous commercial fluorescent tags are available with disparate reactive functional groups. For example, derivatives of fluorescein, fluorescein isothiocyanate (FITC) are reactive toward nucleophiles such as amines and sulfhydryl groups. 

When tackling a complex chemical problem in many areas of chemistry, it is advisable to adopt a variety of methodologies, both chemical and instrumental, in combination, rather than relying on a single approach in attempting to solve the problem. In this context, the utilization of infrared spectroscopy in conjunction with chemical derivatization methods has proven to be a fruitful marriage in the solution of many chemical problems in the past. This is particularly true in the case of humic substances—the utility of infrared spectroscopy has been expanded considerably when used in conjunction with chemical derivatization as will be discussed later. 

Chemical derivatization methods as an aid to MS analysis continue to be important. Derivatization can be divided into tagging of reducing ends and protection of most or all of the functional groups. Permethylation is still an important type of full derivatization. 

Chemical derivatization methods provide a useful additional tool for protein structural analysis, particularly when conpled with the multistage tandem mass spectrometric capabilities of modern ion trap mass spectrometers. The objective of this chapter was to provide a brief overview of the chemical derivatization strategies that are employed currently to address the challenges associated with protein identification, characterization, and quantitative analysis as well as for the characterization of protein-protein interactions. 

Besides physical methods of detection, chemical derivatization methods can be employed to yield or complement results. Derivatization to colored, fluorescent, or UV absorbing compounds can be carried out pre- or postchromatographically. Prechromatographic derivatizations, during sample preparation or on the starting zone of the layer, are employed to increase the selectivity of the separation and the sensitivity of detection, and to improve the linearity and to stabilize labile substances. Postchromatographic derivatizations, however, have the aim to visualize the substances and to improve the sensitivity of detection. 

N. Shahidzadeh-Ahmadi, M. M. Chehimi, F. Arefi-Khonsari, N. Foulon-Belkacemi, J. Amouroux and M. Delamar, Colloids Surf. A105, 277 (1995). A. Chilkoti and B. Rattner, Chemical derivatization methods for enhancing the analytical capabilities of X-ray photoelectron spectroscopy and static secondary ion mass spectrometry, in Surface Characterization of Advanced Polymers (L. Sabbatini and P. G. Zambonin. eds.). VCH. Wein-heim, 1993, Chap. 6. 

There are ill-defined limits on EI/CI usage, based mostly on these issues of volatility and thermal stability. Sometimes these limits can be extended by preparation of a suitable chemical derivative. For example, polar carboxylic acids generally give either no or only a poor yield of molecular ions, but their conversion into methyl esters affords less polar, more volatile materials that can be examined easily by EL In the absence of an alternative method of ionization, EI/CI can still be used with clever manipulation of chemical derivatization techniques. 

Other spectroscopic methods such as infrared (ir), and nuclear magnetic resonance (nmr), circular dichroism (cd), and mass spectrometry (ms) are invaluable tools for identification and stmcture elucidation. Nmr spectroscopy allows for geometric assignment of the carbon—carbon double bonds, as well as relative stereochemistry of ring substituents. These spectroscopic methods coupled with traditional chemical derivatization techniques provide the framework by which new carotenoids are identified and characterized (16,17). 

Over the years the literature is filled with examples where the initial characterization was incorrect. One example is illustrated below. In 1940, Sethna and Shah presumed that they synthesized coumarins 42 and 43 from a reaction between P-orcacetophenone (44) and its 4-0-methyl ether 45 under standard Kostanecki-Robinson conditions, respectively. Three decades later Bose and Shah synthesized coumarin 43 via another route and concluded that the initial assignment made by Sethna and Shah was incorrect. After the Bose and Shah findings were published, Ahluwalia and Kumar concluded that the Sethna and Shah products were actually chromones 46 and 47 based on proton NMR data and chemical derivatization. Despite these shortcomings, the Kostanecki-Robinson reaction remains an effective method for formation of both coumarins and chromones. 

More recently, liquid chromatography/mass spectrometry (LC/MS) and liquid chromatography/tandem mass spectrometry (LC/MS/MS) have been evaluated as possible alternative methods for carfentrazone-ethyl compounds in crop matrices. The LC/MS methods allow the chemical derivatization step for the acid metabolites to be avoided, reducing the analysis time. These new methods provide excellent sensitivity and method recovery for carfentrazone-ethyl. However, the final sample extracts, after being cleaned up extensively using three SPE cartridges, still exhibited ionization suppression due to the matrix background for the acid metabolites. Acceptable method recoveries (70-120%) of carfentrazone-ethyl metabolites have not yet been obtained. 

N. A. Parris, Instrumental Liquid Chromatography, a Practical Manual on High-Performance Liquid Chromatographic Methods (Journal of Chromatography Library, Vol. 27), Elsevier, Amsterdam, 2nd revised ed., 1984 J. Drozd, Chemical Derivatization in Gas Chromatography (Journal of Chromatography Library, Vol. 19), Elsevier, Amsterdam, 1981 J. F. Lawrence and... 

In the PO-CL system, the compounds showing native fluorescence or that fluoresce after chemical derivatization can be detected. As examples of the PO-CL detection of native fluorescence compounds, dipyridamole and benzydamine in rat plasma [57] and fluphenazine [58] have been reported in the former method, the detection limits of dipyridamole and benzydamine were 345 pM and 147 nM in plasma, respectively. Diamino- and aminopyrenes were sensitively determined using TCPO and their detection limits were in the sub-fmol range [59], Carcinogenic compounds such as 1- nitropyrene and its metabolites, can also be determined by the HPLC-PO-CL system. Nonfluorescent nitropyrenes were converted into the corresponding fluorescent aminopyrenes by online reduction on a Zn column followed by detection 2-50-fmol detection limits were achieved in the determination of ethanol extracts from airborne particulates (Fig. 13) [60],... 

Methods currently available for chemiluminescent detection of nucleic acids are not based on derivatization techniques that directly recognize one of the nucleic acid bases or nucleotides. For chemical derivatization-based chemiluminescent detection, the specific reactivity of alkyl glyoxals and arylglyoxals with adenine or guanine nucleotides has been investigated. 

We will discuss in some detail examples where various methods of separation, including chemical derivatization, were complemented by spectroscopic identification. However, even the use of the most advanced analytical methods frequently yields only partial... 

Some derivatization methods mentioned in other sections of this review include chemical ionization by nitric oxide (MS) or epoxidation (MS), formation of jr-complexes for NMR (shift agents) etc. Also, the Diels-Alder reaction, which was mentioned several times as a tool for derivatization of conjugated dienes and polyenes, was extensively described and reviewed in the literature. 

Various chemical derivatizations of natural carotenoids may serve to improve separation and lead to better characterization of structure. These methods are discussed in Section VI. 

There are two basic aims of chemical derivatization simplification of the fragmentation pattern by either promoting or demoting formation of a chosen ion series, and stable-isotopic labelling for simple assignment of ions to proper ion series (Fig. 6.21). Both methods may provide good results and their advantages and limitations will be discussed. 

Most HPLC instruments monitor sample elution via ultraviolet (UV) light absorption, so the technique is most useful for molecules that absorb UV. Pure amino acids generally do not absorb UV therefore, they normally must be chemically derivatized (structurally altered) before HPLC analysis is possible. The need to derivatize increases the complexity of the methods. Examples of derivatizing agents include o-phthaldehyde, dansyl chloride, and phenylisothiocyanate. Peptides, proteins, amino acids cleaved from polypeptide chains, nucleotides, and nucleic acid fragments all absorb UV, so derivatization is not required for these molecules. 

As mentioned earlier, the response of each protein will vary. This is especially apparent with colorimetric assays or derivatization methods requiring a chemical reaction. These protein-to-protein reactivity differences mean that a protein assay suitable for one protein may not be suitable for another. Even for a given protein and a specific protein determination method, results may still vary based on limitations of the assay. Methods requiring extensive sample preparation including protein concentration, buffer exchange, and time-sensitive reactions are liable to be less reproducible than direct measurement techniques, which have fewer variable parameters. The application will determine the suitability of the method. 

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