Chemical Derivatisation

The reader is recommended to consult the relevant textbooks56 (see also Bibliography, Section 9.10) for a more comprehensive account of chemical derivatisation in liquid chromatography. 

J F Lawrence and R W Frei, Chemical Derivatisation in Liquid Chromatography, Elsevier, Amsterdam, 1976... 

An approach that has been developed in response to the desire to extend the utility of SCFs to polar compound analysis is coupling of chemical derivatisation reactions to SFE. Table 3.20 lists situations when derivatisation may be useful [353]. 

Substances that cannot be directly measured in UV/VIS or F mode can generally be quantitated after chemical derivatisation [404]. Transfer of derivatising reagents onto the plate can be accomplished by spraying or by immersing the plate using an automatic immersion device. Such a device controls the immersion rate,... 

Chemical derivatisation is sometimes exploited in the case of lack of UV absorbing moieties. Stafford... 

A multiwavelength approach might have been considered as an alternative to chemical derivatisation. Ruddle and Wilson [62] reported UV characterisation of PE extracts of three antioxidants (Topanol OC, Ionox 330 and Binox M), all with identical UV spectra and 7max = 277 nm, after reaction with nickel peroxide in alkaline ethanolic solutions, to induce marked differentiation in different solvents and allow positive identification. Nonionic surfactants of the type R0(CH2CH20) H were determined by UV spectrophotometry after derivatisation with tetrabromophenolphthalein ethyl ester potassium salt [34]. Magill and Becker [63] have described a rapid and sensitive spectrophotometric method to quantitate the peroxides present in the surfactants sorbitan monooleate and monostearate. The method, which relies on the peroxide conversion of iodide to iodine, works also for Polysorbate 60 and other surfactants and is more accurate than a titrimetric assay. 

Principles and Characteristics In the early mass-spectrometric ionisation techniques, such as El and Cl, the sample needs to be present in the ionisation source in its gaseous phase. Volatilisation by applying heat renders more difficult the analysis of thermally labile and involatile compounds, including highly polar samples and those of very high molecular mass. Although chemical derivatisation may be used to improve volatility and thermal stability, many compounds have eluded mass-spectrometric analysis until the emergence of fast atom bombardment (FAB) [72]. 

The most valuable pharmacological properties of the peptide type ergot alcaloids induced a variety of attempts of chemical derivatisation of the parent compounds (1,2 + lit. quoted therein). In one of these the bromination of a-ergo-cryptine led to a product of highly interesting pharmacology, namely bromocriptine. 

This chapter therefore, provides information based on actual experimentation only all substances listed have been shown (either from the literature or from experiments) to be electrochemically detectable either by native electro-activity or after (photo)chemical derivatisation or (enzymatic or chemical) reaction. 

D.G. Watson. Chemical derivatisation in gas chromatography. In P. Baugh, ed. Gas Chromatography A Practical Approach. IRL Press, Oxford 1995) 133-170. 

J. Drozd (1981). Chemical derivatisation in gas chromatography in Journal of Chromatography Library, Vol. 19. Amsterdam Elsevier. 

In both these areas, chemical derivatisation has traditionally played a role and with the advent of gas chromatography an even more important role. The reasons for preparing a derivative suitable for GC analysis are many and varied and have been discussed thoroughly in a number of books and reviews (l- >). For convenience they are summarised in Table I. As can be seen, two different types of chemical derivatisation techniques are mentioned under Item 4 of Criteria, There is the chemical derivatisation of a pesticide as a pre-requisite of the method of analysis, e.g. esterification of the chlorophenoxy acids, as well as derivatisation as a method for confirmation of identity. The former must meet all the requirements associated with a practical, viable analytical procedure while for the latter the emphasis is on speed, ease of operation and reproducibility. 

Table I. Advantages Gained by Chemical Derivatisation and Some Reaction Criteria...

Since well-established chemical derivatisation techniques already exist for the majority of the organochlorine insecticides (5, 1) it is not surprising that recent activity in this area has centred round compounds such as Kepone, Mirex, HCB, the PCBs, etc. which co-interfere in both the identification and quantitation of pesticide residues. 

The techniques and apphcations of derivatisation for drug analysis have been reviewed J. F. Lawrence and R. W. Frei, Chemical Denvatization in Liquid Chromatography, Amsterdam, Elsevier, 1976 R. W. Frei, J. Chromat, 1979, 165, 75-86 S. Ahuja, J. chromatogr. Sci., 1979, 77, 168-172 R. W. Frei and J. F. Lawrence, Chemical Derivatisation in Analytical Chemistry, New York, Plenum Press, Vol. 1,1981 Vol. 2,1982. 

The most important aspect of dmg discovery in industry today is speed. Natural product extracts containing low yields of complex unidentified metabolites are inimical to this process. Aspects of the structure elucidation of the complex dolastatins we encountered in our research on cyanobacterial metabolites were presented to illustrate this point. All these molecules presented required extensive 2-D NMR analyses, chemical derivatisations or were based on structures which had been proven by prior synthetic endeavors. Many of the problems encountered in the initial structure elucidation of the dolastatins were related to the low amounts of material available for structural analyses, a clear consequence of the initial misidentification of the true source of these metabolites. 

Our next goal was to establish conditions that would permit acylation of the phosphopantetheinyl thiol with acetate, malonate and fiuther more advanced polyketide intermediates. The reasons for this are twofold. First, we wanted to look for evidence of specific binding of these acyl substituents consistent with the proposed role of the ACP in binding and stabilisation. Secondly, we wished to use these acyl ACPs in in vitro mechanistic studies of the assembly process. As is evident from the stracture of the act ACP, there is an exposed cysteine residue at position 17, and preliminary studies showed, as expected, that selective chemical derivatisation of the phosphopantetheine thiol in the presence of cysteine 17 was not possible. This was overcome by mutagenesis to replace cysteine 17 with a serine, and the C17S mutant has been used in many of our subsequent in vitro studies. 

The material which is most commonly used for HPLC column packing is silica. It may be used either urunodified or after chemical derivatisation... 

One of the main reasons for the widespread use of silica as an HPLC packing material is that it may be used either unmodified or after chemical derivatisation of the silanol groups, allowing the surface chemistry to be altered to suit different modes of chromatography. 

RI detectors are very useful instruments because they can be used to quantitate analytes which are otherwise difficult to detect. An example is the analysis of sugars which have poor LTV absorbances and thus cannot be detected by UV absorbance or fluorescence measurements without chemical derivatisation. The exception to this universality is when the solute and solvent have identical RIs, whereupon no signal would be observed. The RI of a number of common HPLC solvents is given in Table 6.2. 

Chemical derivatisation Solid phase extraction Soxhlet extraction Steam distillation Supercritical fluid extraction Homogenisation Precipitation Dialysis... 

Bolts J. M., Bocarsly A. B., Palazzotto M. C., Walton E. G., Lewis N. S. and Wrighton M. S. (1979), Chemically derivatised n-type silicon photoelectrodes. Stabilization to surface corrosion in aqueous electrolyte solutions and mediation of oxidation reactions by surface-attached electroactive ferrocene reagents , J. Am. Chem. Soc. 101, 1378-1385. 

Derivatisation the detectabdity of certain compounds can be improved by chemical derivatisation prior to the HPLC analysis, or after the separation but prior to the detection (post-column derivatisation) the latter can be easily automated and is shared commercially by several manufacturers. 

Through chemical derivatisation, more polar food compounds can be analysed, including sugars and carboxylic acids. Tedetti et al. reported the analysis of low molecular weight carboxylic acids by GC with prior derivatisation to dibutyl esters . Low ppb concentrations were determined. A typical chromatogram of the separation of a number of these acids and related polar compounds from a sea water sample is shown in Figure 3.9. 

These gas ionisation sources have been described in Section 2.1.5 on mass spectrometry. They are particularly suitable for the analysis of mixtures of volatile and low molecular weight compounds (<800) such as hydrocarbons, essential oils and relatively nonpolar drugs. Chemical derivatisation, e.g. trimethylsilylation, can often be employed to increase the volatility of compounds containing polar functional groups (-OH, -COOH, -NH2 etc) so that GC-MS can be used. 

Non-chemical derivatisation has been employed for the detection of photoionisable species. The effluent is passed through a quartz tube where it is irradiated with ultraviolet light and the photoionised products passed to the conductivity detector [64,65]. Krull has reported the detection of the active components, typically organonitro derivatives, of a large number of explosives and drugs by in situ LC decomposition [66]. 

The study of photochemical kinetics is a very important approach for elucidating reaction mechanisms and determining the quantitative parameters characteristic for the progress of the photochemical reaction. Since the excitation of electronically excited states can be achieved more selectively than by thermal pathways, irradiation allows reactions to be guided into specific reaction channels. Solvent interaction, matrices, and variation in chemical derivatisation will influence this reaction coordinate. 

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