Sample preparation and derivatization techniques

The most common analytical technique for the analysis of FFAs and their breakdown products has been chromatography. HPLC has been used for the analysis of FFAs (Christie, 1997 Lues et ah, 1998 Zeppa et ah, 2001). Analysis of short-chain fatty acids (C2-C4) may be relatively simple (Zeppa et ah, 2001). However, the analysis of long-chain fatty acids (>C6) may require derivatization. They are extracted using solvents, converted to bromophenacyl esters, and analyzed by reverse-phase HPLC. GC (with sample preparation and derivatization) has been the method of choice for analysis of fatty acids. An ideal but difficult procedure is to extract FFAs from the aqueous phase and organic phase and combine them (IDF, 1991). The challenge is to overcome the effects of partitioning and extraction of compounds that interfere with the analysis. ISO and IDF have detailed some of the extraction methods for lipids and liposoluble compounds in milk products (ISO, 2001b). Several other methods, which are mainly different in the extraction and derivatization steps, were reviewed by Collins et ah (2004). 

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. 

In some manufacturing process analysis applications the analyte requires sample preparation (dilution, derivatization, etc.) to afford a suitable analytical method. Derivatization, emission enhancement, and other extrinsic fluorescent approaches described previously are examples of such methods. On-line methods, in particular those requiring chemical reaction, are often reserved for unique cases where other PAT techniques (e.g., UV-vis, NIR, etc.) are insufficient (e.g., very low concentrations) and real-time process control is imperative. That is, there are several complexities to address with these types of on-line solutions to realize a robust process analysis method such as post reaction cleanup, filtering of reaction byproducts, etc. Nevertheless, real-time sample preparation is achieved via an on-line sample conditioning system. These systems can also address harsh process stream conditions (flow, pressure, temperature, etc.) that are either not appropriate for the desired measurement accuracy or precision or the mechanical limitations of the inline insertion probe or flow cell. This section summarizes some of the common LIF monitoring applications across various sectors. 

Barr et al. and Bravo et al. used a different technique for sample preparation and determination of alkyl phosphates in urine the urine was spiked with internal standard and then concentrated to dryness via azeotrope codistillation with acetonitrile. The residue was dissolved in acetonitrile, derivatized and then analyzed using GC-MS. Both studies reported satisfactory validation data with coefficients of variation of <20%. 

One of the most powerful techniques used in Upid analysis today is HPLC coupled with mass spectrometry (HPLC/MS). Several mass spectrometric ionization techniques, such as fast atom bombardment (FAB) [23], electrospray ionization (ESI) [29,30], ionspray ionization (ISI) [31], and atmospheric pressure chemical ionization (APCI) [22,30,32] have been used. By using HPLC/MS, one can get information on the molecular structure of the intact lipids, which helps differentiate molecular species within different lipid classes. By using tandem mass spectrometry (MS/MS), identification of molecular species of different sphingolipids can be achieved in an easier and more sensitive way. There are many other advantages of using MS, such as small sample size, minimal sample preparation, and lack of need for derivatization, speeds, and sensitivity. In the literature, sphingolipids of both animal and plant origin were analyzed by MS. 

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. 

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 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. 

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. 

In this work, we adapted a method for the analysis of beer aldehydes using solid-phase microextraction (SPMF) with on-fiber derivatization. This extraction technique does not require solvents, consists of a one-step sample preparation procedure, and provides high sensitivity and reproducibility. It enabled a detailed study of aldehyde level changes during packaged beer storage. 

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