Precolumn techniques, derivatization

Chromatographic enantioseparations can be achieved when the diastereoisomeric interaction is established via a precolumn covalent derivatization, or a non-covalent association formed within the chromatographic system through the use of chiral mobile phase additives (primarily an LC technique) or chiral stationary phases. 

The reaction of a homochiral derivatizing agent with a heterochiral sample to yield covalently linked diastereoisomeric products is a precolumn technique used for the analysis of the individual enantiomers. The diastereoisomeric products are separable under a variety of chromatographic conditions (GC and LC), including both normal and reversed-phase procedures. Fig. 23 shows the LC separation of two diastereoisomeric products formed by the reaction of a heterochiral amine and a homochiral derivatization reagent. The derivatization reaction involves the formation of an amide by treating an A-substituted 5-prolyl chloride with racemic amphetamine (Fig. 24). 

The precolumn technique that is most frequently employed today was developed during the early 1980s [32,33]. For this method, The classical Edman reagent phenylisothiocyanate (PITC) is used for amino acid derivatization after hydrolysis. Separation of the PTC amino acids i then accomplished by HPLG, with detection at 254 nm. Although standard Cig columns available Irom a variety of vendors are suitable for separation of the PTC-derivatized amino acids, there are specific columns that have bqen optimized for this purpose (e.g.. Waters). Approximately 0.5 /ug of peptMe should be hydrolyzed for analyses using precolumn derivatization. ... 

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. 

Further applications for the determination of the newer antidepressants have employed precolumn derivatization, which included a reaction with dansyl chloride or 4-(N-chloroformylmethyl-N-methyl) amino-7-nitro-2,l,3,-benzoxadiazole (NBD-COCL) followed by separation on ODS Cl8 analytical columns maintained at either 35°C or 70°C using either isocratic or gradient elution with fluorescence end-point detection. The compounds were isolated from human plasma or serum by LLE or SPE techniques using... 

The separation mechanism in the LC analysis of aminoglycosides is usually highly dependent on the applied derivatization technique, either precolumn or postcolumn. This is due to the fact that a prerequisite of aminoglycosides analysis is most often suitable derivatization to produce fluorescent derivatives the presence of primary amine groups in most of the aminoglycoside antibiotics enables a number of derivatives to be readily formed. 

Increased use of liquid chromatography/mass spectrometry (lc/ms) for structural identification and trace analysis has become apparent. Thermo-spray lc/ms has been used to identify by-products in phenyl isocyanate precolumn derivatization reactions Liquid chromatography/thermospray mass spectrometric characterization of chemical adducts of DNA formed during in vitro reaction lias been proposed as an analytical technique to detect and identify those contaminants in aqueous environmental samples which have a propensity to be genotoxic, t.e.. to covalently bond to DNA. 

Offline precolumn derivatization is the most common alternative in this respect it involves separating the esters obtained from the organic acids by reversed-phase chromatography, which amply surpasses solvophobic chromatography (i.e., the use of undissociated acids as such) and allows gradient elution techniques to be applied, thanks to the wider lipophilicity range covered by the derivatized compounds. 

Direct determination of urea pesticides by high-performance liquid chromatography has been widely reported in the literature (10,32-36,127-130). Ultraviolet detection has often been used (32,33,35,36,60,127) with usually acceptable sensitivity, although this detector is nonspecific and the sensibility is, in general, low. To overcome this problem, several techniques have been assayed, such as precolumn enrichment (60), postcolumn derivatization (34,10), and the use of other detection techniques such as the electrochemical (129), photoconductivity (128,130), and fluorescence detectors (9,10,34). Table 9 summarizes representative papers using these techniques in HPLC analysis. 

High-performance LC is widely used, offline or online, in the determination of pesticides, either as a final measurement step or as a separation technique. The increase in the use of HPLC is mainly the result, on the one hand, of its suitability for determining thermally labile and polar pesticides that require derivatization prior to GC, and, on the other, of its compatibility with online precolumn extraction and cleanup and with MS systems (19). 

Several precolumn derivatization techniques are available for those who wish to trade extra sample preparation time for the expense and maintenance of post column pumps and reactors. The more popular derivatives are dansyl-(32), OPA-(33), PTH-(34) and PITC-(35) amino acids. There are problems and limitations with some of these systems, however, analysis time is only 15-25 min. compared to 90-240 min. of the ion-exchange post column systems. 

The analysis of cell culture media and supernatants, as well as non-standard protein hydrolysates such as collagens and glycoproteins, has created the demand for techniques that accurately quantify additional amino acids not normally found in hydrolyzed samples, Methods of amino acid analysis (AAA) based on precolumn derivatization with 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate (AQC) have previously been shown to quantify hydrolyzed samples with a high degree of accuracy (1,2). The AQC-based method has also been shown to derivatize effectively in the presence of salts and lipids (3). Taking into account the above strengths, the excellent stability of the derivatives, and the unique fluorescence properties that allow for direct injection of the reaction mixture without cleanup, the AQC methodology represents an ideal choice for the analysis of complex samples. 

The formation of new chromophores for the optimization of ultraviolet (UV) detection of analytes in HPLC implies the synthesis of derivatives with conjugated systems in the molecule. Compared with GC, there are no restrictions on the volatilities of these derivatives for HPLC analysis. They may be synthesized before analysis (precolumn derivatization) or after chromatographic separation (postcolumn derivatization). The latter technique is rarely used and then only for a few classes of compounds, but it permits us to... 

Precolumn derivatization is the generally accepted approach for the determination of amino acids, because it offers significant advantages increased detection sensitivity, enhanced selectivity, enhanced resolution, and limited needs for sophisticated instrumentation (in contrast with postcolumn derivatization techniques). 

Most amino acids react with ninhydrin at ambient temperatures to form a blue color that becomes purple on heating. However, proline and hydroxyproline yield yellow compounds that are measured at a different wavelength. Other postcolumn derivatizations use fluorogenic reagents, such as o-phthaldialdehyde or fluorescamine. Precolumn derivatization techniques using o-phthaldialdehyde, dansyl, phenyl isothiocyanate, or 9-fluorenylmethyl chloroformate derivatives have been used with reversed-phase HPLC. Electrochemical detection has also been coupled with derivatization methods to enhance analytical sensitivity. 

Thiols, as well as sulfide and sulfite, were determined in porewater samples by reversed phase high performance liquid chromatography (HPLC). The technique is based on precolumn derlvatization with an o phthalaldehyde/amine reagent (Figure 1) followed by HPLC and fluorometric detection. Derivatized porewater samples were Injected directly into the HPLC system the detection limit is 0.1 nM (for 100 ul injection). Details of the method are given in Mopper and Delmas (12). 

Derivatization reactions can be performed either pre- or postcolumn. As outlined by Brinkman, there are important advantages to using the postcolumn techniques whenever possible (68). First, the analytes can be separated in their original form, which often permits the adoption of published separation procedures. Second, artifact formation is generally not a serious problem, in contrast to precolumn derivatization, where it increases the separation difficulty and causes problems with quantitation. Third, the reaction does not need to be complete and the reaction products need not be stable the only requirement is reproducibility. Several reaction principles have been extensively applied. These include true chemical derivatization such as with dansyl chloride or o-phthalaldehyde UV irradiation, which can convert the analyte of interest into a more easily detectable species solid-phase reactions, including catalytic reactions such as with the use of immobilized enzymes and chemiluminescence techniques. 

It is felt that the precolumn sampling techniques deserve much attention in future studies, as they can serve a double function in biochemical investigations (a) removal of solvents or derivatization agents and (b) protection of the analytical column from non-volatile impurities. Chemical nature of the precolumn packing can also be varied to suit a particular sample type. Further investigations aiming at the optimization and automation of the precolumn sampling techniques appear desirable. 

With the postcolumn denvatization technique, a split of the effluent before derivatization enables easy measurement of the radioactive undenvatized amino acid The precolumn derivatization technique with OPA may be equally simple, but some aspects need to be considered and examined. 

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