Post-column derivatization method

Fluorescence detection can be up to four orders of magnitude more sensitive than UV absorbance, especially where laser induced excitation is used, mass detection limits being as low as 10-20—10 21 mole. Pre- and post-column derivatization methods are being developed to extend the applicability of fluorescence detection to non-fluorescent substances. Several types of electrochemical and mass spectrometric detector have also been designed. Detector characteristics are summarized in Table 4.21. 

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

A post-column derivatization method was recently proposed for analysis of BAs-NQS derivatives in wine. A C8 (150 x 4.6mm, 4 xm) column is used with a binary gradient composed of A) 15% acetonitrile and 85% aqueous solution (15 mM sodium heptanesulfonate and 10 mM phosphoric acid) (v/v), and B) 70% acetonitrile and 30% aqueous solution (8mM sodium heptanesulfonate and 10 mM phosphoric acid) (v/v). Eluent A is programmed at 100% for 8 min, then... 

These approaches can be designated as post-column derivatization methods and can be performed with the appropriate devices. Due to the relatively slow reaction rates of many biochemical reactions, off-line detection may be preferable. In this case, incubation and reaction times can be adjusted independently of the separation speed. In Fig. 5, the separation of eight microcystins (cyclic peptides) and similar compounds is illustrated. With a group-selective antibody against microcystins, the respective toxin peaks can be unambiguously detected. Moreover, the ELISA detection is about 100 times more sensitive and much more selective than UV detection [18]. 

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. 

With the development of HPLC, a new dimension was added to the tools available for the study of natural products. HPLC is ideally suited to the analysis of non-volatile, sensitive compounds frequently found in biological systems. Unlike other available separation techniques such as TLC and electrophoresis, HPLC methods provide both qualitative and quantitative data and can be easily automated. The basis for the HPLC method for the PSP toxins was established in the late 1970 s when Buckley et al. (2) reported the post-column derivatization of the PSP toxins based on an alkaline oxidation reaction described by Bates and Rapoport (3). Based on this foundation, a series of investigations were conducted to develop a rapid, efficient HPLC method to detect the multiple toxins involved in PSP. Originally, a variety of silica-based, bonded stationary phases were utilized with a low-pressure post-column reaction system (PCRS) (4,5), Later, with improvements in toxin separation mechanisms and the utilization of a high efficiency PCRS, a... 

A method to determine nabam by HPLC after acidic hydrolysis to ethylenediamine and post-column derivatization with o-phthalaldehyde-mercaptoethanol has also been reported. " ... 

Reversed-phase HPLC followed by post-column derivatization and subsequent fluorescence detection is the most common technique for quantitative determination of oxime carbamate insecticides in biological and environmental samples. However, for fast, sensitive, and specific analysis of biological and environmental samples, detection by MS and MS/MS is preferred over fluorescence detection. Thus, descriptions and recommendations for establishing and optimizing HPLC fluorescence, HPLC/ MS, and HPLC/MS/MS analyses are discussed first. This is followed by specific rationales for methods and descriptions of the recommended residue methods that are applicable to most oxime carbamates in plant, animal tissue, soil, and water matrices. 

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. 

In contrast to the post-column derivatization, the inherent sensitivity of this method is higher since the column effluent is not diluted. Adequate sensitivity can therefore be achieved by UV monitoring at 330nm. For higher sensitivity, fluorescence detection is often chosen, and emission is measured at wavelengths above 430nm. moreover, baseline quality is superior. 

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... 

As already mentioned the EP wants to replace old TEC tests with separation methods of higher efficiency for example, the purity of amino acids is currently evaluated by a TEC test for ninhydrin-positive substances that is only able to find and limit amino acids to 0.5%. However, this test is only valid in the case the amino acids are produced by the cleavage of peptides/proteins and purification. The ninhydrin method is also used in the amino acid analysis of peptides, utilizing a cation-exchange chromatography with a post-column derivatization and a subsequent UVA is detection. This method is often used in industries for purity evaluation of amino acids. 

Although a number of chromatographic methods have been reported for determinations of aminoglycoside antibiotics in blood serum and urine, the application of chromatographic methods to residue analysis has been very limited. Shaikh et al (93) recently described an HPLC method for neomycin in animal tissue, and Lachatre et al (94) described a method for nine aminoglycosides in plasma, urine, and renal cortex tissue. Both procedures use post column derivatization with a-pthalaldehyde and fluorescence detection. 

Dichloroacetamide present in chlorinated and chloraminated drinking waters was determined (at detection level of 23 pg L ) through an LC/fluorimetric method and a post-column derivatization reaction with o-phthaldialdehyde in the presence of sulfite at pH 11.5, that leads to the formation of a highly fluorescent isoindole fluorophore [225]. 

Reviews appeared on the following subjects Analysis of lipid hydroperoxides , the difficulties encountered for hydroperoxide analysis in a plasma matrix , post-column derivatization after GLC of lipid hydroperoxides and methods for detection and characterization of hydroperoxy groups in oxidized polyolefins . 

---