Derivatization techniques

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. 

For more specific analysis, chromatographic methods have been developed. Using reverse-phase columns and uv detection, hplc methods have been appHed to the analysis of nicotinic acid and nicotinamide in biological fluids such as blood and urine and in foods such as coffee and meat. Derivatization techniques have also been employed to improve sensitivity (55). For example, the reaction of nicotinic amide with DCCI (AT-dicyclohexyl-0-methoxycoumarin-4-yl)methyl isourea to yield the fluorescent coumarin ester has been reported (56). After separation on a reversed-phase column, detection limits of 10 pmol for nicotinic acid have been reported (57). 

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). 

An important difference between Protein-Pak columns and other size exclusion columns is the silica backbone of the Protein-Pak columns. Because the silica structure is unaffected by the solvent, these columns do not swell or shrink as a function of the solvent. This is a general advantage compared to other size exclusion columns. However, silica-based columns can only be used up to pH 8, which limits their applicability. Also, surface silanols are accessible for interaction with the analytes, but this phenomenon has been minimized by proper derivatization techniques. Generally, a small amount of salt in the mobile phase eliminates interaction with silanols. 

This chapter will focus on topic 3, which is normally regarded to be chiral derivatization chromatography, but will also cover other topics that might be considered when applying derivatization techniques. The goal for the separation of the race-mates may be their analysis or their preparation. Both topics will be covered in this chapter. 

Derivatization techniques are divided into pre-column and post-column techniques. Post-column derivatization is especially useful to enhance the detection of compounds, whilst pre-column derivatization is the method of choice for enan-tioseparations via derivatization. 

An excellent discussion on derivatization techniques has been given by Lawrence (17) including a detailed discussion on pre-column derivatization (18) and post-column derivatization (19). Probably, the more popular procedures are those that produce fluorescing derivatives to improve detector sensitivity. One of the more commonly used reagents is dansyl chloride (20), 5-dimethylamino-naphthalene-1-sulphonyl chloride (sometimes called DNS-chloride or DNS-C1). The reagent reacts with phenols and primary and secondary amines under slightly basic conditions forming sulphonate esters or sulphonamides. 

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. 

The first part of the book consists of a detailed treatment of the fundamentals of thin-layer chromatography, and of measurement techniques and apparatus for the qualitative and quantitative evaluation of thin-layer chromatograms. In situ prechromatographic derivatization techniques used to improve the selectivity of the separation, to increase the sensitivity of detection, and to enhance the precision of the subsequent quantitative analysis are summarized in numerous tables. 

The application of the fluorescence derivatization technique in an HPLC method involves utilization of a post column reaction system (PCRS) as shown in Figure 3 to carry out the wet chemistry involved. The reaction is a 2-step process with oxidation of the toxins by periodate at pH 7.8 followed by acidification with nitric acid. Among the factors that influence toxin detection in the PCRS are periodate concentration, oxidation pH, oxidation temperature, reaction time, and final pH. By far, the most important of these factors is oxidation pH and, unfortunately, there is not one set of reaction conditions that is optimum for all of the PSP toxins. The reaction conditions outlined in Table I, while not optimized for any particular toxin, were developed to allow for adequate detection of all of the toxins involved. Care must be exercised in setting up an HPLC for the PSP toxins to duplicate the conditions as closely as possible to those specified in order to achieve consistent adequate detection limits. 

Detection-Qriented Derivatization Techniques in Liquid Chromatography, edited by Henk Lingeman... 

A derivatization technique is commonly applied to an agrochemical with certain reactive functional groups (e.g., carboxylic acid, amine, phenol) to make the compound amenable to either gas chromatography (GC) or LC analysis. An in-depth discussion of derivatization reactions used in the analysis of agrochemicals is beyond the scope of this article. For more information on this topic, the reader is referred to Knapp. °... 

Possanzini, M. and Di Palo, V., Improved HPLC determination of aliphatic amines in air by diffusion and derivatization techniques, Chromatographia, 29,151, 1990. 

Cooper, J. D. H., Ogden, G., McIntosh, J., and Tumell, D. C., The stability of the o-phthaldehyde/2-mercaptoethanol derivatives of amino acids an investigation using high-pressure liquid chromatography with a precolumn derivatization technique, Anal. Biochem., 142, 98, 1984. 

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. 

An on-column acetic anhydride derivatization technique has been described for pseudoephedrine hydrochloride. Immediately after injection of a solution of pseudoephedrine onto a 20% SE-30, 1.8 m x 7 mm i.d. glass column at 125°C, an injection of acetic anhydride was made. The pseudoephedrine derivative formed on column has a retention time of 55.5 minutes as compared to a retention time of 8.7 minutes for underivatized pseudoephedrine.34... 

It should be noted that the majority of the derivatization techniques modify the peptide s N-terminus. The reason is that the N-terminal amine group is easier to modify than the C-terminal carboxyl group. Also, due to differences in pKa value in the e-amino group of lysine, there are possible reaction that modify the N-terminus only, while the lysine side chains remain intact. Modifications of carboxyl groups... 

Abstract Gas chromatography (GC) is commonly used for the analysis of a myriad of compounds in neurochemistry. In this chapter various aspects of GG, including inlets, columns and detectors are discussed. Appropriate sample preparation, including extraction and derivatization techniques are also covered. In the latter portion of the chapter, examples of the analysis of specific types of endogenous and exogenous compounds by GG are dealt with. 

The main derivatization techniques used are alkylation, acylation, silylation, or condensation (Drozd, 1975 Ahuja, 1976 Perry and Feit, 1978 Coutts and Baker, 1982). One of the few fundamental additions to derivatization reagents since the 1970s is the use of chloroformates (Wells, 1999). Chloroformates are useful in aqueous media, and a reaction with them proceeds rapidly at room temperature (Husek, 1998). Some examples of derivatization techniques are shown in O Figure 1-3. 

Perry JA, Feit CA. 1978. Derivatization techniques in gas-liquid chromatography. GLC and HPLC Determination of Therapeutic Agents, Part 1, Tsuji K. Marozowich W, editors. New York Marcel Dekker pp. 137—208. 

Wells RJ. 1999. Recent advances in non-silylation derivatization techniques for gas chromatography. J Chromatogr A 843 1. 

C. Dilution, Isolation, Enrichments, and Derivatization Techniques I. Pipetting and Liquid handling... 

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