Derivatization pre-column

FIGURE 6.31 Three-dimensional view of the inlet region of the silicon FFE device. To avoid the dielectric breakdown of Si, it has been deposited with a composite layer of Si02 and Si3N4 [89]. Reprinted with permission from the American Chemical Society. 

FIGURE 6.33 Schematic of the microchip with integrated pre-column reactor. The reaction chamber is 2 mm long and 96 im wide (at half-depth). The separation column is 15.4 mm long and 31 pm wide. The channels are 6.2 pm deep [106]. Reprinted with 

Pre-column OPA derivatization was also employed to analyze biogenic amines prior to MEKC separation on a PDMS chip [654]. Pre-column OPA derivatization and MEKC were also performed on a glass chip to analyze amino acids. Usually, OPA was used for fluorescent detection. However, in this report, amperometric detection was used as the OPA derivatives were also electroactive. Voltage (needed for separation) programming was used to decrease the migration time of late migrating species [655]. 

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EC, electrochemical detection Flu, fluorescence detection MS, mass specu-omeu-ic detection pre-Flu, fluorescence detection after pre-column derivatization post-Flu, fluorescence detection after post-column derivatization UV, UV absorbance detection. 

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. 

Pre-column derivatization offers some general advantages ... 

Chemical derivatization can be carried out before the separation (pre-column derivatization) or after the separation and before detection (post-column derivatization). If derivatization is carried out prior to separation, then a phase system must now be selected to separate the... 

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. 

The derivatives have an optimum fluorescence at an excitation wavelength of 340 nm and an emission wavelength of 455 nm. The adduct is relatively stable at a pH of 9-11 but it rapidly degrades to a non-fluorescent residue at low pH values. Consequently, when used as a pre-column derivatizing reagent the pH of the mobile phase should be kept fairly high, o-phthalaldehyde has been employed for derivatization in the analysis of dopamine (29), catecholamines (30) and histamines (31). 

Excess of the reagent hydrolyses to a non-fluorescent residue and the reagent itself does not fluoresce. The optimum wavelength of the excitation light is 390 nm and that of the emitted light 475 nm. This regent is, however, less sensitive than Fluoropa and the derivative is unstable consequently, it must be injected onto the column immediately after formation if used in pre-column derivatization. It has been used successfully in the separation and analysis of polyamines (32), catecholamines (33) and amino acids (34). 

Katayama, M., Masuda, Y., and Taniguchi, H., Determination of alcohols by high-performance liquid chromatography after pre-column derivatization with 2-(4-carboxyphenyl)-5,6-dimethylbenzimidazole,/. Chromatogr., 585,219, 1991. 

The most popular current techniques for amino acid analysis rely on liquid chromatography and there are two basic analytical methods. The first is based on ion-exchange chromatography with post-column derivatization. The second uses pre-column derivatization followed by reversed-phase HPLC. Derivatization is necessary because amino acids, with very few exceptions, do not absorb in the UV-visible region, nor do they possess natural fluorescence. 

Model RR/066 351 and 352 pumps models 750/16 variable-wavelength UV monitor detector 750/11 variable filter UV detector, MPD 880S multiwave plasma detector, 750/14 mass detector 750/350/06 electrochemical detector refractive index detector HPLC columns column heaters, autosamplers, pre-columns derivatization systems, solvent degassers, preparative HPLC systems... 

The process of chemically modifying the test molecules before the separation procedure is known as preparing derivatives or pre-column derivatization. In liquid chromatography this is done to permit the test molecules to be more easily detected after separation and to increase the sensitivity of the detection system and less frequently to alter the separation process. 

The use of reverse-phase columns with pre-column derivatization of the amino acids offers an acceptable alternative to the dedicated instrumentation of an amino acid analyser or separation by HPLC followed by post-column derivatization. 

Acetic anhydride is a useful GC pre-column derivatizing reagent for amines, phenols and alcohols. In the presence of aqueous base only amines and phenols are derivatized, but... 

A comparative study was made of the RP-HPLC analysis of free amino acids in physiological concentrations in biological fluids, with pre-column derivatization by one of the four major reagents o-phthalaldehyde (73) in the presence of 2-mercaptoethanol, 9-fluorenylmethyl chloroformate (90), dansyl chloride (92) and phenyl isothiocyanate (97, R = Ph) (these reagents are discussed separately below). Duration of the analysis was 13-40 min. Sensitivity with the latter reagent was inferior to the other three however, its use is convenient in clinical analysis, where sample availability is rarely a problem. The derivatives of 73 were unstable and required automatized derivatization lines. Only 92 allowed reliable quantation of cystine. All four HPLC methods compared favorably with the conventional ion-exchange amino acid analysis188. 

Peptides containing a tryptophan residue at the N-end can be determined by pre-column derivatization with glyoxal and RP-HPLC-FLD, as shown in reaction 9, giving single fluorescent peaks LOD 0.55-3.82 nM (SNR 3) for 100 pL injection volume221. 

Diazotization and formation of diazo dyes affords a general approach to pre-column derivatization, to be followed by direct phase or RP-HPLC with UVD or FLD. Thus,... 

Pre-column derivatization with either 134 or 135 followed by CZE and LIF detection was proposed for amino acids. The amino group of the analyte displaces the succinyloxy moiety of the reagent yielding a carboxamide325. See also Section IV.D.3.C for other acylating reagents derived from A-hydroxysuccinimide (95 and 96). 

Srinivas NR, Shyu WC, Barbhaiya RH. 1995. Gas chromatographic determination of enantiomers as diastereo-mers following pre-column derivatization and applications to pharmacokinetic studies a review. Biomed Chromatogr 9 1. 

Guo X, Eukushima T, Li F, Imai K. 2003. Determination of fluoxetine and norfluoxetine in rat plasma by HPLC with pre-column derivatization and fluorescence detection. Bio... 

Song Y, Quan Z, Evans JL, Byrd EA, Liu YM. 2004. Enhancing capillary liquid chromatography/tandem mass spectrometry of biogenic amines by pre-column derivatization with 7-fLuoro-4-nitrobenzoxadiazole. Rapid Commun Mass Spectrom 18 989. 

Automation in derivatization has led to the development of pre-colnmn and post-column HPLC techniqnes. In post-column derivatization, the separated components elnting from the colnmn are derivatized on-line in a heated mixing coil prior to entering the detector. Pre-column derivatization can be performed in many autosamplers or automated liquid handlers. 

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