Derivatization, chemical post column

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... 

Homish, R. E. and Wiest, J. R., Quantitation of spectinomycin residues in bovine tissues by ion-exchange high-performance liquid chromatography with post-column derivatization and confirmation by reversed-phase high performance chromatography-atmospheric pressure chemical ionization tandem mass spectrometry, /. Chromatogr. A, 812, 123, 1998. 

Hi. Lysine. Gamma radiolysis of aerated aqueous solution of lysine (94) has been shown, as inferred from iodometric measurements, to give rise to hydroperoxides in a similar yield to that observed for valine and leucine. However, attempts to isolate by HPLC the peroxidic derivatives using the post-column derivatization chemiluminescence detection approach were unsuccessful. This was assumed to be due to the instability of the lysine hydroperoxides under the conditions of HPLC analysis. Indirect evidence for the OH-mediated formation of hydroperoxides was provided by the isolation of four hydroxylated derivatives of lysine as 9-fluoromethyl chloroformate (FMOC) derivatives . Interestingly, NaBILj reduction of the irradiated lysine solutions before FMOC derivatization is accompanied by a notable increase in the yields of hydroxylysine isomers. Among the latter oxidized compounds, 3-hydroxy lysine was characterized by extensive H NMR and ESI-MS measurements whereas one diastereomer of 4-hydroxylysine and the two isomeric forms of 5-hydroxylysine were identified by comparison of their HPLC features as FMOC derivatives with those of authentic samples prepared by chemical synthesis. A reasonable mechanism for the formation of the four different hydroxylysines and, therefore, of related hydroperoxides 98-100, involves initial OH-mediated hydrogen abstraction followed by O2 addition to the carbon-centered radicals 95-97 thus formed and subsequent reduction of the resulting peroxyl radicals (equation 55). 

RG Luchtefeld. An HPLC detection system for phenylurea herbicides using post-column photolysis and chemical derivatization. J Chromatogr Sci 23 516-520, 1985. 

Although an excellent detector for PAEis, the fluorometer is not widely used in environmental analysis, as the number of environmental pollutants with fluorescent spectra is limited. The sensitivity and selectivity of the fluorometer are also used in the A-methyl carbamate pesticide analysis (EPA Method 8318). These compounds do not have the capacity to fluoresce however, when appropriately derivatized (chemically altered), they can be detected fluorome-trically. The process of derivatization takes place after analytes have been separated in the column and before they enter the detector. This technique, called post column derivatization, expands the range of applications for the otherwise limited use of the fluorometer. 

Microfluidic Lab-on-a-chip for Chemical/Biological Analysis and Discovery 6.2.8.2 Post-Column Derivatization... 

Derivatization Pre-column of post-column chemical derivatization To transform the analyte into a form to improve sensitivity or selectivity... 

Vassilakis, I. Tsipi, D. ScouUos, N. Determination of a variety of chemical classes of pesticides in surface and ground waters by off-line solid-phase extraction, GC with ECD and NP-detection and HPLC with post-column derivatization and fluorescence detection. J. Chromatogr. A, 1998, 823, 49-58. 

Recently, Talamond et al. [257] published a procedure for determining phytic acid in foods that involves anion exchange chromatography and suppressed conductivity detection and, thus, avoids post-column derivatization. Separation is carried out with an OmniPac PAX-100 using a NaOH gradient in the presence of 1 % (v/v) 2-propanol. The ASRS-1 suppressor is chemically regenerated with 25 mmol/L sulfuric acid. 

One of the weak points of fluorescence is that relatively few compounds fluoresce in a practical range of wavelengths. However, chemical derivatization allows many nonfluorescent molecules containing derivatiz-able functional groups to be detected, thus expanding the number of applications. Fluorescence derivatization can be accomplished either via precolumn or post-column methods. 

Furthermore, the post column reaction system must not contribute significantly to peak dispersion. Chemical derivatization is often an essential procedure in order to obtain adequate detector sensitivity, however, because it adds to the complexity of the analysis and can also reduce the resolving power of the column, it should only be resorted to when absolutely necessary. 

Future developments of electrochemical detector cells and applications to a wider range of compounds can be foreseen and could lead to greater exploitation of these detectors. j There is still scope for improvement in cell design, particularly in the improvement in flow patterns to reduce noise./ Novel electrode materials could be profitably exploited, for example, if electrocatalysis towards a given substrate could be demonstrated or low background currents achieved. Chemical derivatization has been frequently exploited in polarography to expand its range of applicability and similar techniques, particularly if implemented post-column and in-line, could likewise expand the areas of application of electrochemical detection in HPLC. 

Many chemicals are not detected using a fluorescence detection scheme unless they are chemically derivatized with a fluorescent label. A reaction may be done before a separation is performed, but there are advantages if it is done after the separation. In chromatography this is known as post-column reaction. This is a chemical reaction performed "on-the-fly" in the sample stream as it moves towards a detector, and it must create a fluorescent product only when sample is present. One of the most common reagent used for post-column derivatization is o-phthaldialdehyde (OPA), which reacts with primary amines to create a fluorescent product. In electrophoresis this process might be called an in-capillary reaction, since due to the electric field the separation... 

Fluorescence High sensitivity, but of limited applicability because so few analytes naturally fluoresce. Range of applications may be considerably increased if analytes are chemically modified to form fluorophores by either pre- or post-column derivatization. 

Many methods have been employed for the detection of these compounds such as high-performance liquid chromatography with pre or post column derivatization [22-24], calorimetry [25], fluorometry [20, 26], chemiluminescene [27], capillary electrophoresis [28, 29] and NMR spectrometry [30]. Electrochemical methods are other choices for their determination because they are inexpensive, simple, fast and sensitive. Experiments using mercury, mercury amalgam and a chemically modified electrode as the working electrode [31-33] have been performed for thiol-compound determination. However, mercury has restrictions due to its toxicity and the fast deterioration of the electrode response. Using a chemically modified electrode [34, 35] is not practical for use in flow injection analysis and liquid chromatography. Therefore, the BDD thin film electrode has been introduced it is known as a unique electrode material for a number of electrochemical applications [12, 36, 37]. 

Tertiary aliphatic amines are rather difficult to detect, but they can be detected at 254 run after quatemization with p-NBBr [66]. Pilocarpine and isopilocarpine were derivatized in a sealed ampoule for 24 h at 40 °C, then separated by ion-pair HPLC. Tertiary amines can also be determined by post-chromatographic derivatization. Kudoh et al. described a method [67] involving derivatization with 1% dtric acid in acetic anhydride at 120 C, separation on a chemically bonded silica gel Nucleosil 5N(CH3)2, column, and detection at 550 nm. 

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