Sample preparation derivatization

Sample preparation derivatization with 9-fluorenylmethylchloroformate (FMOC-CL)... 

Chemical derivatization reactions in analytical chemistry have been used to enhance detection, improve volatility for GC, or enhance selectivity such as for chiral separations. Sample preparation derivatization reactions occur at an active hydrogen such as an alcohol, phenol, amine, or sulfhydryl. The reaction types are largely alkylations, acylations, silylations, and condensations. A comprehensive review of derivatization reaction is available (53). 

Sample preparation derivatization with 9-fluorenylmethylchloroformate (FMCXT-CL) Analysis HPLC, ODS Hypersil UV detection at 263 nm... 

Some features of analytical procedures developed for MLC are explained below, including sample preparation, derivatization of the drugs and optimization of the chromatographic separation. 

Appropriate GLC procedures have also been used for the analysis of pure vitamins, their optical isomers, pharmaceuticals, and, rarely, even foods. The main disadvantage of this group of chromatographic methods seems to be the tedious and relatively time-consuming sample preparation (derivatization and/or cleanup procedures) prior to the GLC analysis. Nevertheless, the newly developed and introduced GC-MS techniques show that GLC is a sufficiently sensitive tool for the trace analysis of pantothenic acid, its higher homolog and other related compounds such as acyl-CoA, even in biological samples (serum, brain, foodstuffs). 

Preparation of the TMS derivative Add 0.5 ml of TRI-SIL Z reagent (trimethylsilylimidazole in pyridine) to 1-5 mg of the sample. (This derivatizing preparation does not react with amino groups and tolerates the presence of water.) Heat in a sealed vial at 60° until the sample is dissolved. An alternate method is to let the reaction mixture stand at room temperature for at least 30 minutes (or overnight). This procedure is not appropriate for amino sugars. 

The sample preparation in LC analysis is as important as the chromatographic separation itself. The procedure will often require considerable skill copied with a basic understanding of chromatographic methodology. The analyst will need to have some familiarity with micro techniques including general micro-manipulation, microfiltration, centrifugation and derivatization. 

The use of internal standards is somewhat controversial.115 There is agreement that an internal standard may be used as a correction for injection volume or to correct for pipetting errors. If an internal standard is included before sample hydrolysis or derivatization, it must be verified that the recovery of the internal standard peak is highly predictable. Ideally, the internal standard is unaffected by sample handling. Using an internal standard to correct for adsorptive or chemical losses is not generally approved, since the concentration of the standard may be altered by the conditions of sample preparation. An example of internal vs. external standards is given in Chapter 4. 

Hawk, G.L. and Little, J.N., Derivatization in Gas and Liquid Chromatography in Zymark Sample Preparation Program (SPF CT 1 103), Zymark Corporation, Hopkinton, MA, 1982. 

MS detection allows for sensitive detection without derivatizing samples, also reducing sample preparation. Achiral/chiral LC/LC, especially when coupled to MS detection, can be expected to continue to play an important role in the analysis of enantiomers that are present in biological and/or environmental samples. 

Several limitations on the synthetic techniques that can be employed are imposed by the need for rapidity and minimization of handling because of the radiation hazard, and the low concentration and small physical quantities of the compounds. Purification steps should be eliminated if possible by optimizing yields. Where purification is unavoidable, simple procedures are employed such as use of anion exchange columns to remove perrhenate (the most common contaminant in the final product). A variety of disposable sample preparation columns are well suited to this purpose and are available containing small quantities of anion or cation exchange materials (0.1 to 0.5 g typically) such as quaternary ammonium-, primary ammonium-, or sulfonate-derivatized silica. Reversed phase columns are also often used (C8 or C18-derivatized silica). The purification is often thus reduced to a simple filtration step which can be performed aseptically. 

As a consequence of the development of extraction methods for STA based on mixed-mode SPE columns, as well as of the recent introduction of instruments for the automated sample preparation allowing efficient evaporation and derivatization of the extracts, full automation of STA methods based on GC-MS analysis is also available. It needs GC-MS instalments equipped with an HP PrepStation System. The samples directly injected by the PrepStation are analyzed by full scan GC-MS. Using macrocommands, peak identification and reporting of the results are also automated. Each ion of interest is automatically selected, retention time is calculated, and the peak area is determined. All data are checked for interference, peak selection, and baseline determination. 

The next crucial portion of the process is the sample preparation, which allows the analyte to be detected at a level that is appropriate for detection by the instrument. The sample must be prepared in such a way that the matrix does not interfere with the detection and measurement of the analytes. Often, this requires complete separation of the sample matrix from the analyte(s) of interest. In other cases, the analytes can be measured in situ or first derivatized and then measured in situ. Both the nature of the analyte and the matrix dictate the choice of sample preparation (see Ref. [1]). 

PITC has been used extensively in the sequencing of peptides and proteins and reactions under alkaline conditions with both primary and secondary amino acids. The methods of sample preparation and derivatization follow a stringent procedure which involves many labour-intensive stages. However, the resulting phenylthio-carbamyl-amino acids (PTC-AA s) are very stable, and the timing of the derivatization step is not as critical as when using OPA. 

Automated injectors are often used when large numbers of samples are to be run. Most designs involve the use of the loop injector coupled to a robotic needle that draws the samples from vials arranged in a carousel-type auto-sampler. Some designs even allow sample preparation schemes such as extraction and derivatization (chemical reactions) to occur prior to injection. 

As mentioned earlier, the response of each protein will vary. This is especially apparent with colorimetric assays or derivatization methods requiring a chemical reaction. These protein-to-protein reactivity differences mean that a protein assay suitable for one protein may not be suitable for another. Even for a given protein and a specific protein determination method, results may still vary based on limitations of the assay. Methods requiring extensive sample preparation including protein concentration, buffer exchange, and time-sensitive reactions are liable to be less reproducible than direct measurement techniques, which have fewer variable parameters. The application will determine the suitability of the method. 

Abstract Gas chromatography (GC) is commonly used for the analysis of a myriad of compounds in neurochemistry. In this chapter various aspects of GG, including inlets, columns and detectors are discussed. Appropriate sample preparation, including extraction and derivatization techniques are also covered. In the latter portion of the chapter, examples of the analysis of specific types of endogenous and exogenous compounds by GG are dealt with. 

A review of sample preparation techniques written recently by Smith (2003) purports that derivatization is not very useful and that with advances in separation techniques or by using a different analytical technique, such as HPLG, derivatization can be avoided. While a laboratory will examine almost any alternative to avoid derivatization according to Smith (2003), switching equipment can be prohibitively expensive, and so derivatization certainly still has an important role in most laboratories. 

Rosenfeld, J. M., Recent Developments in the Chemistry and Application of Analytical Derivatizations, In Sampling and Sample Preparation for Field and Laboratory Fundamentals and New Directions in Sample Preparation, Pawliszyn, J., Ed., Vol. XXXVll, Elsevier Science, Amsterdam, Netherlands, pp. 609-668, 2002. 

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