Derivatisation, HPLC

Chassaigne H, and Lobinski R (1998) Characterization of horse kidney metallothionein isoforms by electrospray MS and reversed-phase HPLC-electrospray MS. Analyst 123 2125- 2130. Chemosphere (1999) Special issue - Sources of error in methylmercury determination during sample preparation, derivatisation and detection. Chemosphere 39 1037-1224. 

As compared to HPLC, cSFC shows higher efficiency, universal and selective detection, minimal derivatisation for separation and the ability to separate thermally labile organic compounds. Often, cSFC analyses are also considerably faster. This arises because higher mobile phase diffusion coefficients translate directly into higher optimum velocities. However, sensitivity, detection dynamic range and sample capacity... 

In order to achieve detection limits below the ng mL-1 range only amperometric, chemiluminescence, radiometric, or conventional fluorescence (CF) can be applied (Table 4.41). Fluorescence detectors are generally about 100 times more sensitive and more selective than UV detectors. The selectivity of fluorescence detection is due to the fact that only aromatic and conjugated molecules can be analysed, and by applying specific excitation and emission wavelengths the selectivity can even be increased. Pre- or postcolumn derivatisation in HPLC is a technique that is most commonly performed prior to UV absorption or fluorescence detection... 

In LIF detection systems, excitation power may be increased up to six orders of magnitude compared to CF detection. Most LC-LIF detection concerns under-ivatised polynuclear aromatic hydrocarbons (PAHs) and fluorescing dyes (e.g. polymethines). Because only a limited number of analytes possess native fluorescence, derivatisation of the analyte before detection is normally required in trace analysis of organic solutes by means of LIF detection. LIF detection in HPLC was reviewed... 

Oligomeric additives with broad MWD tend to be a problem in conventional HPLC conditions. In cases where no interest exists in the oligomer distribution it is common practice to solve the problem by creating a uniform structural unit useful for analysis. For example, isocratic (or gradient) LC-UV was used for the determination of the polymeric light stabiliser Tinuvin 622 in polyolefins using dissolution (toluene)/derivatisation (TBAH)-precipitation (alcohol) the diol formed was quantitatively determined by NPLC [653]. 

HPLC has also been used for analysing fatty acid mixtures [708] and for the characterisation of fatty acids and their derivatives [709]. Fatty acids are commonly analysed on polymeric RPLC columns. Only multiple unsaturated fatty acids can be detected by UV in HPLC the others require derivatisation into UV-absorbing or fluorescing derivatives. Simultaneous determination of saturated and unsaturated fatty acids (C12-C24) by means of RPLC has been reported [710]. Derivatisation is necessary. 

The main characteristics of on-line SPME-HPLC(-MS) are shown in Table 7.18. Most of the SPME fibres are compatible with HPLC solvents. SPME combined with HPLC provides a means by which simple, rapid concentration of analytes can be achieved together with a means of introduction of the concentrated analytes to the HPLC system. This eliminates the need for larger injection volumes, and avoids derivatisation if the analytes were to be detected by GC. An advantage of the SPME method over LLE methods is the absence of a solvent peak in chromatograms obtained after extraction by SPME. SPME is not suitable for organic solutions. As SPME is a microextraction technique, coupling to ft, HPLC may be envisaged. 

After passing through the column, the separated solutes are sensed by an in-line detector. The output of the detector is an electrical signal, the variation of which is displayed on a potentiometric recorder, a computing integrator or a vdu screen. Most of the popular detectors in hplc are selective devices, which means that they may not respond to all of the solutes that are present in a mixture. At present there is no universal detector for hplc that can compare with the sensitivity and performance of the flame ionisation detector used in gas chromatography. Some solutes are not easy to detect in hplc, and have to be converted into a detectable form after they emerge from the column. This approach is called post-column derivatisation. 

GC of aqueous simulant or water extract of olive oil using cold on-column injector. 1,4 butane diol internal standard Solvent extraction with dichloromethane and concurrent derivatisation with 9-(methylaminomethyl)anthracene. Fluorescent derivatives analysed by HPLC with fluorescence detection... 

The following pages give tables of substances that are electrochemically detectable after pre-column derivatisation (PRE-CD) or post-column derivatisation (POST-CD), (enzymatic-) reaction ((ENZ.-)POST-CR) or irradiation (HPLC-hv-EC) with corresponding data on reagents, enzymes, etc. The detection potentials are versus AgCl unless otherwise stated. 

The derivatised resin product was suspended in DCM (6 mL) for 30 min, after which TEA (0.06 mL) was added and the resultant suspension was gently stirred for 15 min at ambient temperature. The suspension was filtered, the spent resin washed with DCM (5mL) and DCM MeOH (1 1, 5mL), and the combined filtrate was evaporated to dryness in vacuo to afford the title compound (25 mg, 73%) as a white crystalline solid. RP-HPLC analysis (Gl) showed the exclusive presence (>98%) of Fmoc-Val-NHOH (Rt = 5.1 min). 

AEOs have been analysed by HPLC and UV or fluorescence detection after suitable derivatisation. The derivatising agents proposed so far are phenyl isocyanate [80,81], 1-anthroylnitrile [82], 3,5-dinitro-benzoyl chloride [83], naphthyl isocyanate [84] and naphthoyl chloride [84], However, the lack of fluorescence activity and the need for synthesis through a multistep reaction for some derivatising agents limits their application in a real-world analysis. In fact, only a few of them were applied in the determination of AEOs in environmental samples. Zanette et al. [84] developed derivatisation and separation... 

As mentioned above, the most commonly used method for the analysis of cationic surfactants has been HPLC coupled with conductometric, UV, or fluorescence detectors, the latter typically utilizing post-column ion-pair formation for enhanced sensitivity. Analysis by GC is only possible for cationic compounds after a derivatisation step [33] because of the ionic character of this compound. However, structural information might be lost. 

Aranda and Burk [81] have established an SPME-HPLC-FL method and on-line derivatisation to determine AEs (Brij 56) in water. The surfactant was extracted with a PDMS-DVB fibre and pre-column derivatisation with 1-naphthoyl chloride in the presence of 4-(dimethyl-amino)pyridine as catalyst. The method has a limit of detection of 0.1 mg L-1. 

For the analysis of AEO sediment extracts on HPLC-fluorescence (FL), it was necessary to derivatise using naphthoil chloride and IV-methylimidazole in order to incorporate a chromophore into the analytes [37]. 

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