Post-column on-line derivatization

The following experimental parameters should be maintained, namely  

Merit The main merit of post-column-on-line derivatization is that ideally the separation and detection processes can be optimized individually. 

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What do you understand by pre-column off-line derivatizatiori and post-column on-line derivatization ... 

Post-Column, On-Line, Continuous Photolytlc Derivatizations in HPLC-Electrochemical Detection (EC) and FIA-EC ... 

A study of residual analysis of thirty pesticides and their transformation products was based on SPE on-line with HPLC-UVD or post-column derivatization with o-phthalaldehyde (73) and fluorescence detection (FLD), according to EPA method 531.1 and others. The method allowed determination of many pesticides in river and well waters at 0.01 to 0.5 -ig/L levels195. An automatized procedure was proposed for determination... 

A fraction collector and a post-column derivatization system were included (Figure 2.1) for a comprehensive and multi-purpose instrument. However, the fraction collector is needed only when collecting components from the effluent, and is generally not included in an analytical system. The post-column derivatization system is connected only when required for the selective and sensitive detection of specially targeted compounds. Usually, most compounds are directly detected by an on-line spectroscopic or other detector. 

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. 

Hostettmann, K. et al., On-line high-performance liquid chromatography ultraviolet-visible spectroscopy of phenolic compounds in plant extracts using post-column derivatization, J. Chromatogr., 283, 137, 1984. 

The CUSAL-HPLC couple has been combined additionally with pre- or post-column derivatization. Thus, pre-column derivatization was used for the determination of colistin A and B in feeds following USAL, the analytes were derivatized with o-phthaldialdehyde/2-mercaptoethanol and separated by HPLC for fluorimetric detection [48]. The experimental set-up used is depicted in Fig. 4.1 OA. Another application of CUSAL-HPLC is the determination of A/-methylcarbamates in soils and food [49] (see Fig. 4.10B), where the analytes were also derivatized with o-phthaldialdehyde after separation for fluorescence-based monitoring. A number of steps of the process including leaching, filtration, solid-phase extraction, liquid chromatographic separation, post-column derivatization and fluorescence detection were performed on-line, all in an automated manner. 

Figure 4.10. On-line coupling of USAL and detection with derivatization (A) in the pre-column mode for the determination of coiistin A and B in feeds (B) in the post-column mode for the determination of N-methyicarbamates in soil and foods, AC — analytical column, C — carrier, DC — derivatization coil, EL — elution loop, F — filter, FD — fiuorimetric detector, HC — hydrolysis coil, HPiV — high-pressure injection valve, HPP — high-pressure pump, IV — injection valve, L — leachant, LC — leaching chamber, MP — mobile phase, OPA — o-phthaldiaidehyde, PC — preconcentration column, PL — propagating liquid, PP — peristaltic pump, S / — switching valve, UP — ultrasonic probe, W — waste and WB — water bath. (Reproduced with permission of Elsevier, Refs. [48,49].)...

The derivatization for HPLC is performed either offline (pre-column) before injection into the column, or on-line (post-column) by mixing the reagent with the column effluent. 

Kiss and Sass-Kiss (2005) developed an on-line post-column derivatization method using OPA and 2-mercaptoethanol as reagents. HPLC... 

Development of selective analytical method for the determination of artemisinin poses challenging problems because it lacks ultraviolet (UV) absorption or fluorescent chromophores and does not possess functional groups with potential for derivatization. This study reports the determination of artemisinin by liquid chromatography with on-line post-column UV irradiation and peroxyoxalate chemiluminescence (PO-CL) detection. A similar method as previously reported by us for the determination of organic peroxide. In this method, after artemisinin is eluted from the HPLC column, it is UV irradiated to generate hydrogen peroxide, which is determined by PO-CL detection. 

Di Pietra, A.M. Gatti, R. Andrisano, V. Cavrini, V. Application of high-performance liquid chromatography with diode-array detection and on-line post-column photochemical derivatization to the determination of analgesics. J.ChromatognA, 1996, 729, 355-361... 

Use of selective derivatization reactions prior to the separation allows the introduction of functional groups which can be detected with high selectivity. In some cases derivatization can be carried out on-line before (pre-column) or after the separation column (post-column) [23]. Use of fluorescent reagents which react selectively with, eg, aliphatic amines is an example of pre-column derivatization. 

The experience of the user should fit in with the method and the equipment. An HPLC newcomer should not get started with a method involving on-line sample concentration of a biological sample with double column switching and subsequent post-column derivatization. That is hardly the way to make you happy right from the start. 

Another sensitive method for detection of cysteinyl-containing leukotrienes in HPLC assays is measurement of fluorescence, which has been used in combination with UV absorbance and radioactivity monitoring [119]. Post-column derivatization of compounds containing free amino groups was performed on-line with fluoresca-mine. 

The methods of derivatization can be divided into several categories pre-and post-column methods and off-line and on-line methods. For example, the formation of volatile derivatives for GC is usually prepared off-line in separate vials before injection into the chromatograph (precolumn). There are a few exceptions where the reagents are mixed and injected together the derivatization reaction occurs in the hot GC injection port (on-line). 

Derivatizations for HPLC purposes are accomplished either off-line or on-line. An on-line process may involve either pre-column or post-column reaction depending on the objective. 

Hsieh, J.Y.-K. Lin, L. Matuszewski, B.K. High-throughput liquid chromatographic determination of rofecoxib in human plasma using a fully automated on-line solid-phase extraction system, J.Liq.Chromatogr.Rel.TechnoL, 2001, 24, 799-812. [LOQ 0.5 ng/mL plasma post-column photochemical derivatization fluorescence detection SPE]... 

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