Post derivatization

Lord and Pawliszyn" developed a related technique called in-tube SPME in which analytes partition into a polymer coated on the inside of a fused-silica capillary. In automated SPME/HPLC the sample is injected directly into the SPME tube and the analyte is selectively eluted with either the mobile phase or a desorption solution of choice. A mixture of six phenylurea pesticides and eight carbamate pesticides was analyzed using this technique. Lee etal. utilized a novel technique of diazomethane gas-phase methylation post-SPE for the determination of acidic herbicides in water, and Nilsson et al. used SPME post-derivatization to extract benzyl ester herbicides. The successful analysis of volatile analytes indicates a potential for the analysis of fumigant pesticides such as formaldehyde, methyl bromide and phosphine. 

HPLC, using a reversed phase system consisting of a Zorbax NH2 column eluted with acetonitrile/water (4 1), was employed for the preparative separation of the calystegines [10], The lack of a chromophore renders the use of UV or fluorescence detection impossible without pre- or post-derivatization of the sample and the relatively low sensitivity of refractive index (RI) detection severely limits this technique for routine analysis. However, the rapid evolution of LC-MS methods may lead to their application in the future. 

HPLC analysis of calystegines is somewhat difficult due to their solubility and high polarity, which limit the selection of column packings and solvent mixtures. Moreover, their lack of chromophore excludes spectroscopic methods of detection without pre- or post derivatization of the samples. Gas chromatography on fused silica capillary column coupled with mass spectrometry is the most suitable analytical technique used for the determination of calystegines. However, the high polarity and the nonvolatility of the compounds preclude their analysis without derivatization and numerous derivatization reagents have been tested [9]. 

Gaba, S., Agarwal, S., Omray, A., Eapan, D., and Sethi, P. (1997). Estimation of l-ornithine-L-aspartate by post-derivatization preparative TLC in combined dosage forms. East. Pharm. 40 137-138. 

Pentenamer Ionomers. Unsaturated polypentenamer elastomers have been derivatized by post-synthesis reactions (72—74). Phosphonate, thioglycolate, sulfonate, and carboxylate derivatives have been prepared and converted into ionomers. 

Post-column in-line photochemical derivatization permits fluorescence detection of the common aflatoxins Bl, B2, Gl, and G2 (60). Chromatographic evidence indicates that photolysis causes the hydration of the nonfluorescent Bl and Gl components to B2a and G2a components, respectively. Analysis of naturally contaminated com samples show no interfering peaks and permits the deterrnination of 1 and 0.25 ppb for Bl and B2, respectively. 

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. 

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. 

In post-column derivatization the chromatographic system is modified to allow the reagent to mix with the column eluent, give the reaction mixture sufficient time to complete and finally pass the reaction mixture to the detector. 

Post-column reaction is a common feature of many special types of analyses, the most well-known being the amino acid analyzer that uses ninhydrin with a post-column reactor to detect the separated amino acids. In general, derivatization and post-column reactor systems are techniques of last resort. In some applications they are unavoidable, but if possible, every effort should made to find a suitable detector for the actual sample materials before resorting to derivatization procedures. 

The Ugi reaction is the four-component condensation of an amine, aldehyde or ketone, carboxylic acid and isocyanide to give an o -acylamino amide [22-24], Although this process has the potential to introduce considerable diversity, the products themselves are not heterocycles but through appropriate choice of substrates, latent functionality in one of the precursors can intercept either an intermediate or further derivatize the acylamino amide Ugi product through post-modification. Thus variants of the Ugi reaction have been investigated under microwave-assisted conditions for the synthesis of diverse heterocyclic libraries [16,19-24],... 

Ritter, W. Post-Chromatographic Derivatizations in Quantitative (HP)TLC , Proc. 2nd Int Synq>. lustrum. High ifomu Thm-Lay Chmmalogr. (HPTLC) p. 100-113, Interlaken, 1982. 

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... 

The application of the fluorescence derivatization technique in an HPLC method involves utilization of a post column reaction system (PCRS) as shown in Figure 3 to carry out the wet chemistry involved. The reaction is a 2-step process with oxidation of the toxins by periodate at pH 7.8 followed by acidification with nitric acid. Among the factors that influence toxin detection in the PCRS are periodate concentration, oxidation pH, oxidation temperature, reaction time, and final pH. By far, the most important of these factors is oxidation pH and, unfortunately, there is not one set of reaction conditions that is optimum for all of the PSP toxins. The reaction conditions outlined in Table I, while not optimized for any particular toxin, were developed to allow for adequate detection of all of the toxins involved. Care must be exercised in setting up an HPLC for the PSP toxins to duplicate the conditions as closely as possible to those specified in order to achieve consistent adequate detection limits. 

Fraser CA, Gardner GJ, Maxwell PS, Kubwabo C, Guevremont R, Siu KWM, and Berman SS (1995) Preparation and certification of a biological reference material (CARP-i) for polychlorinated dibenzo-p-dioxin and dibenzofuran congeners. Fresenius J Anal Chem 352 143-147. Gahr A, Huber N, and Niessner R (1998) Fluorimetric determination of bromate by ion-exchange separation and post-column derivatization. Mikrochrm Acta 129 281-290. 

At this point, the anaiyte may not be amenabie to UV, FL, or EC detection. In this case, the best course of action may be to choose LC/MS (see Section 4.2). However, one other option is to use a pre- " or post-coiumn derivatization step to increase the detectabiiity of the anaiyte with respect to FL or UV. Fluorescent or UV labels are available for carboxylic acids," amines, phenols, and thiols. The decision to use pre- or post-column derivatization is predicated upon the functionality of the analyte available for derivatization and the rate and extent of the reaction between each derivatizing agent and the analyte. 

The increased use of IV-methyl carbamate insecticides in agriculture demands the development of selective and sensitive analytical procedures to determine trace level residues of these compounds in crops and other food products. HPLC is the technique most widely used to circumvent heat sensitivity of these pesticides. However, HPLC with UV detection lacks the selectivity and sensitivity needed for their analysis. In the late 1970s and early 1980s, HPLC using post-column hydrolysis and derivatization was developed and refined with fluorescence detection to overcome these problems. The technique relies on the post-column hydrolysis of the carbamate moiety to methylamine with subsequent derivatization to a fluorescent isoindole product. This technique is currently the most widely used HPLC method for the determination of carbamates in water" and in fruits and vegetables." " ... 

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. 

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