Analyte derivatization techniques

An important difference between Protein-Pak columns and other size exclusion columns is the silica backbone of the Protein-Pak columns. Because the silica structure is unaffected by the solvent, these columns do not swell or shrink as a function of the solvent. This is a general advantage compared to other size exclusion columns. However, silica-based columns can only be used up to pH 8, which limits their applicability. Also, surface silanols are accessible for interaction with the analytes, but this phenomenon has been minimized by proper derivatization techniques. Generally, a small amount of salt in the mobile phase eliminates interaction with silanols. 

Anions of weak acids can be problematic for detection in suppressed IEC because weak ionization results in low conductivity and poor sensitivity. Converting such acids back to the sodium salt form may overcome this limitation. Caliamanis et al. have described the use of a second micromembrane suppressor to do this, and have applied the approach to the boric acid/sodium borate system, using sodium salt solutions of EDTA.88 Varying the pH and EDTA concentration allowed optimal detection. Another approach for analysis of weak acids is indirect suppressed conductivity IEC, which chemically separates high- and low-conductance analytes. This technique has potential for detection of weak mono- and dianions as well as amino acids.89 As an alternative to conductivity detection, ultraviolet and fluorescence derivatization reagents have been explored 90 this approach offers a means of enhancing sensitivity (typically into the low femtomoles range) as well as selectivity. 

As can be seen in Table 3, a wide range of analytes derivatized with different labels have been detected using the POCL reaction. Most of these applications have employed flow injection or liquid chromatographic techniques. An area of growing interest is the combination of capillary electrophoresis with chemiluminescence. Several strategies have been used to detect analytes with fluorescent... 

Although the majority of analytes do not possess natural fluorescence, the fluorescence detector has gained popularity due to its high sensitivity. The development of derivatization procedures used to label the separated analytes with a fluorescent compound has facilitated the broad application of fluorescence detection. These labeling reactions can be performed either pre- or post-separation, and a variety of these derivatization techniques have been recently reviewed by Fukushima et al. [18]. The usefulness of fluorescence detectors has recently been further demonstrated by the Wainer group, who developed a simple HPLC technique for the determination of all-trani-retinol and tocopherols in human plasma using variable wavelength fluorescence detection [19]. 

Analytical chemistry applied to foods widely uses separation techniques in order to characterize foods on the basis of their major and minor constituent compounds. The minor compounds often highlight important differences rather than the major compounds. Chromatographic techniques have been widely applied however, when only gas chromatography (GC) was available, the application of this technique was severely limited by the thermal stability of the analytes. Derivatization reactions are able to somewhat protect organic molecules from degradation, but generally these procednres are rather time consuming and, sometimes, artifacts could be formed. 

False-negative results arising from disturbance of normal peak ratios of the analyte ions may be overcome by a variety of means including reinjection of the same derivative on another column, application of an alternative derivatization technique, and/or performing a second analysis with a different method. Thus, the identity, for example, of an illegal hormone residue in a suspect sample has been given by a series of different events including two values for the ratio to the front in two-dimensional HPTLC, a characteristic fluorescence after sulfuric... 

Starting with a description of the analytical challenge in Chapter 19, the third part, which is devoted to analytical attitudes, proceeds with a detailed description in Chapter 20 of modern sample preparation procedures including solid-phase extraction, matrix solid-phase dispersion, use of restricted-access media, supercritical fluid extraction, and immunoaffinity cleanup. Flexible derivatization techniques including fluorescence, ultraviolet-visible, enzymatic, and photochemical derivatization procedures are presented in Chapter 21. 

Air analysis for carbamate pesticides may be performed by sampling air over 1 pm PTFE membrane. The analytes collected over the membrane are extracted with methylene chloride, exchanged into methanol, and analyzed by HPLC using postcolumn derivatization technique as described above. Certain pesticides may be analyzed too by the colorimetric method (see Part 3 under individual compounds). 

CWC-related chemicals in aqueous liquid samples (water samples) are usually recovered by extraction with an organic solvent. Modem methods such as SPE and solid phase micro extraction (SPME) have also been presented 04 24). Organic extractions and these modem methods mainly recover nonpolar CWC-related chemicals, but leave behind the water-soluble and nonvolatile chemicals. These must also be recovered, however, because the agents tend to decompose (hydrolyze) rapidly under conditions in the environment. In the past few years, techniques such as CE and LC, relying on element specific or mass spectrometric detection, have been intensively developed to provide easy and effective ways of recovering these chemicals from water samples with only minor sample preparation (2S 44,1. For GC/MS analysis, the water must be displaced and the analytes derivatized. 

The GC/MS procedures for methamphetamine are described in Table 4. The papers published in Japanese - have corresponding reports in English. - - Methamphetamine was detected and determined by mass fragmentography in rat hair after administration of the substance. Nine methods also detected the metabolite amphetamine or amphetamine alone. Suzuki et al. determined methamphetamine also in nail, sweat and saliva. The workup (EX after acid or alkaline hydrolysis) and derivatization technique (methanol-trifluoroacetic acid [TEA]) is rather uniform in most procedures. Nakahara et al. ° used methoxyphenamine excretion into beard hair to discuss several washing procedures. Alkaline or methanolic extraction are used with one exception. Derivatization is mainly made by fluorinated anhydrides. A review ° gives details on analytical procedures, incorporation rates of amphetamines from blood to hair, and relationship between drug history and drug distribution in hair. 

The use of high-performance liquid chromatography (HPLC) as an analytical separation technique has had an explosive growth in the biochemical literature. The many modes of HPLC permit the rapid separation of widely varying classes of compounds. In addition, since compounds which are ionic, nonvolatile, or thermally labile can be analyzed by HPLC, derivatization prior to chromatographic separation is not usually needed. 

The first group of reactions (acylation) includes the greatest number of examples. Numerous recommended reagents are listed in the Table 1 they belong to two classes of reagents anhydrides (X = OCOR") and chloroanhydrides (X = C1). Most widely used of them are acetic and trifluoroacetic anhydrides. The byproducts of acylation, in all cases, are acids these reactions need basic media (additives of pyridine or tert-amines) to prevent the formation of nonvolatile salts from the analytes. The technique of derivatization is extremely simple Samples mixtures are allowed to stand with acylating reagents for some minutes prior to analysis. 

Today, HPLC is the dominant analytical technique used for the analysis of most classes of compounds. The analyses can be carried out at room temperature and the collection of fractions for reanalysis or further manipulation is straightforward. The main reason for the slow acceptance of the HPLC technique for Upid analysis has been the detection system. Traditionally, HPLC used ultraviolet/visible (UV/vis) detection, which requires the presence of a chromophore in the analyte. Most lipid molecules do not contain chromo-phores and therefore would not be detected by UV/vis. Modern HPLC detection techniques, such as the use of a mass spectrometer as the detector, derivatization techniques to introduce chromophores, and the availability of pure solvents to reduce interference, have allowed HPLC to compete with and/or complement GC and other traditional methods of lipid analysis. In addition to analytical HPLC, preparative HPLC has been used extensively to collect pure samples of the lipids for the derivatization or synthesis of new compounds. 

Many derivatization techniques, based on chemical reactions or even heat and chemical reactions, can be applied to TLC or HPTLC to specifically determine some compounds or groups of compounds this is specific to TLC techniques. For instance, exposure to fluorescamine in acetone wiU convert sulfonamide antibiotics into fluorescent derivatives which can be visualized under UV radiation [8]. Other techniques are reported and, as suggested earher, the reader should refer to the latest edition of the Official Methods of Analysis pubhshed by the Association of Official Analytical Chemists (Arlington VA, U.S.A.) for complete details about aU the available techniques for the routine analysis of drugs. 

Most amino acids react with ninhydrin at ambient temperatures to form a blue color that becomes purple on heating. However, proline and hydroxyproline yield yellow compounds that are measured at a different wavelength. Other postcolumn derivatizations use fluorogenic reagents, such as o-phthaldialdehyde or fluorescamine. Precolumn derivatization techniques using o-phthaldialdehyde, dansyl, phenyl isothiocyanate, or 9-fluorenylmethyl chloroformate derivatives have been used with reversed-phase HPLC. Electrochemical detection has also been coupled with derivatization methods to enhance analytical sensitivity. 

For libraries with an upper mass limit of approximately 500 amu, GC-MS can prove advantageous. Capillary-GC on fused silica capillaries is characterized by high separation power and the ability to analyze relatively polar substances. In many cases, the problems of tailing, thermal instability and volatility associated with excessive analyte polarity can be overcome by the use of derivatization techniques. 

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