Postcolumn techniques, derivatization

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. 

As detection method in separation techniques, especially in thin-layer chromatography, high-performance liquid chromatography (HPLC) and electrophoresis. In HPLC applications for non-fluo-rescent compounds, either precolumn or postcolumn fluorescent derivatization is used. In capillary electrophoresis, the use of a laser to directly excite fluorescent compounds allows a extremely high sensitivity (femtomole to attomole levels of analytes can be detected in nanoliter volmnes). 

Reaction detectors are a convenient means of performing online postcolumn derivatization in HPLC. The derivative reaction is performed after the separation of the sample by the column and prior to detection in a continuous reactor. The mobile phase flow is not interrupted during the analysis and reaction, although it may be augmented by the addition of a secondary solvent to aid the reaction or to conform to the requirements of the detector. Reaction detectors are finding increasing application for the analysis of trace components in complex matrices where both high detection sensitivity and selectivity are needed. Many suitable reaction techniques have been published for this purpose [641-650]. 

Continuous monitoring methods based on amperometric (Nikolic et al. 1992) or spectrophotometric (Kuban 1992 Ma and Liu 1992) techniques for the quantification of free cyanide are also available. Ion chromatography with amperometric determination provides good sensitivity (2 ppb) and selectivity for free cyanide and the weak complexes of cadmium and zinc (Rocklin and Johnson 1983). Postcolumn derivatization and fluorescence detection provides low detection limits as well (0.1 ppb) (Gamoh and Imamichi 1991). 

Fluorescence is not widely used as a general detection technique for polypeptides because only tyrosine and tryptophan residues possess native fluorescence. However, fluorescence can be used to detect the presence of these residues in peptides and to obtain information on their location in proteins. Fluorescence detectors are occasionally used in combination with postcolumn reaction systems to increase detection sensitivity for polypeptides. Fluorescamine, o-phthalaldehyde, and napthalenedialdehyde all react with primary amine groups to produce highly fluorescent derivatives.33,34 These reagents can be delivered by a secondary HPLC pump and mixed with the column effluent using a low-volume tee. The derivatization reaction is carried out in a packed bed or open-tube reactor. 

For many years, automated amino acid analyzer using lEC and postcolumn derivatization with nin-hydrin (or less frequently with fluorescamine) has been the most popular technique for amino acid determination. Amino acids are separated in their free form by employing stepwise elution with sodium- or lithium-based buffers. 

Niacin can be detected by UV, ED, or FLD. UV is a widespread technique but it needs a longer preparation step and it does not reach high sensitivity. The FLD is more sensitive but it needs a pre or postcolumn derivatization to make niacin fluorescent. Krishnan et al. [599] describe a postcol-umn derivatization using cyanogens bromide and p-aminophenol, but this method involves toxic reagents. Mawatari et al. [600], instead, propose a fast, highly specific derivatization procedure, which involves UV irradiation at 300 nm in the presence of hydrogen peroxide and copper(II) ions. 

UV absorption occurs only below 220nm, thereby it is affected by the interference from mobile phase and from artifacts in complex foods. A multiwavelength UV detection has been experimented successfully for unambiguous evaluation of pantothenic acid [609]. However, UV detection presents a low sensitivity, compared to other techniques, like FLD or MS. FLD is applied by using a postcolumn derivatization. Pantothenic acid is converted to 3-alanine by hot alkaline hydrolysis and a reaction with OPA [610]. Also MS is successfully applied to increase the sensitivity of pantothenic acid analysis. 

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. 

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. 

Carbamates and substituted ureas are a numerous group of pesticides widely used to control weeds, pests, and diseases in fruit trees, vegetables, and cereals. Carbamate residues in foods are commonly extracted with water-miscible solvents and determined by using a liquid chromatograph equipped with a sensitive detector, frequently a UV detector. In addition, to obtain adequate detection selectivity, the postcolumn fluorimetric labeling technique is used for methyl carbamates. Substituted ureas are normally extracted from foods with organic solvents, and they can be determined directly by HPLC-UV or after postcolumn derivatization by fluorescence determination of their derivatives. 

Air analysis for carbamate pesticides may be performed by sampling air over 1 pm PTFE membrane. The analytes collected over the membrane are extracted with methylene chloride, exchanged into methanol, and analyzed by HPLC using postcolumn derivatization technique as described above. Certain pesticides may be analyzed too by the colorimetric method (see Part 3 under individual compounds). 

Derivatization is a technique that is most commonly performed prior to UV absorption or fluorescence detection. It is not restricted to these detection modes, however, and postcolumn electrochemical31 and postcolumn chemiluminescence32 detection have also been reported. Table 3.6 provides a list of some of the more common derivatizing agents and the compounds with which they react.33 Derivatization has also been used to aid in the detection of compounds such as amino acids,34 amines,35 saccharides,36 thiols,37 carboxylic acids,38 steroids,39 alcohols,40 fatty acids 41 and several inorganic species.42... 

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. 

Postcolumn reaction detection was reported used by 9% of the respondents to the detector survey (47). The most popular LC detectors are solute property detectors. From a cursory glance at the popular detection techniques already discussed, it is apparent there are many classes of important compounds for which there is no sensitive solute-property detector. For this reason, many types of postcolumn chemistries have been devised to derivatize separated solutes to form a detectable species. Postcolumn reaction detection has been thoroughly reviewed (68,69). 

The compound formed must be easily separated through the selected chromatographic technique (or analyzed if the derivatization is postcolumn). 

Carbamates, urea, and triazine type compounds can be analyzed on an HPLC. For carbamates postcolumn derivatization technique may be applied. Carbamates separated on a C—18 column are hydrolyzed with NaOH and the products amines are then derivatized with cr-phthalaldehyde and 2-mercaptoethanol to... 

---