Procedure and HPLC

System I acetonitrile/methanol (85 15). System 2 hexane/methanol (25 75). System 1 is used at the beginning of the run and is changed to system 2 after 11 min. 

A typical separation of spinach carotenoids supplemented with zeax-anthin is given in Fig. 6.2 this is derived from Krinsky and Welanki-war (1984). The identity of the peaks may be established by collecting 

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The method of Cosyns et al [28] was modified with fluorescence detection to increase sensitivity [18]. The plasma extraction procedure and HPLC... 

Extraction of Active Compounds from Food. Vitamins are the group of compounds more usually extracted from foods using SFE (83). A method for the analysis of the natural contents of vitamins A and E in milk powder based on SFE, a miniaturized alkaline saponification procedure, and HPLC was proposed by Turner and Mathiasson (84). Modifications of the sample matrix, the combination of static and dynamic extraction modes, and the effect of changes in extraction parameters such as temperature, flow rate, time, collection solvent, and collection temperature were optimized, obtaining recoveries of 99% and 96% for vitamins A and E, respectively. Another method for the determination of vitamins A and E based on the coupling of SFE-enzymic hydrolysis-HPLC has also been proposed providing recoveries between 79% and 152% (85). 

Macula. HPLC analysis was used to study the distribution of carotenoids in the macula (206). The extraction procedure and HPLC analysis of macular carotenoids obtained from human donor eyes on a 5-pm Spherisorb ODS-1 C18... 

L Ye, WO Landen Jr, J Lee, RR Eitenmiller. Vitamin E content of margarine and reduced fat products using a simplified extraction procedure and HPLC determination. J Liq Chromatogr Rel Technol 21 1227-1238, 1998. 

Marcomini, A., M. Zanette, Derivatization procedures and HPLC separations for environmental analysis of AE, Riv. Ital. Sostanze Grasse, 1994, 7/, 203-208. 

Tar. Before the development of gas chromatography (gc) and high pressure Hquid chromatography (hplc), the quantitative analyses of tar distillate oils involved tedious high efficiency fractionation and refractionation, followed by identification or estimation of individual components by ir or uv spectroscopy. In the 1990s, the main components of the distillate fractions of coal tars are deterrnined by gc and hplc (54). The analytical procedures included in the specifications for tar bulk products are given in the relevant Standardi2ation of Tar Products Tests Committee (STPTC) (33), ISO (55), and ASTM (35) standards. 

The recent development and comparative application of modern separation techniques with regard to determination of alkylphosphonic acids and lewisite derivatives have been demonstrated. This report highlights advantages and shortcomings of GC equipped with mass spectrometry detector and HPLC as well as CE with UV-Vis detector. The comparison was made from the sampling point of view and separation/detection ability. The derivatization procedure for GC of main degradation products of nerve agents to determine in water samples was applied. Direct determination of lewisite derivatives by HPLC-UV was shown. Also optimization of indirect determination of alkylphosphonic acids in CE-UV was developed. Finally, the new instrumental development and future trends will be discussed. 

Whilst for the analysis of plant material for cannabinoids both GC and HPLC are commonly used, in analytical procedures the employment of GC-based methods prevails for human forensic samples. Nonetheless, the usage of HPLC becomes more and more of interest in this field especially in combination with MS [115-120]. Besides the usage of deuterated samples as internal standards Fisher et al. [121] describe the use of a dibrominated THC-COOH (see 7.5). The usage of Thermospray-MS and electrochemical detection provide good performance and can replace the still-used conventional UV detector. Another advantage in the employment of HPLC rather than GC could be the integration of SPE cartridges, which are needed for sample preparation in the HPLC-system. 

Extraction procedures combined with GC, GC-MS, HPLC, and HPLC-MS etc. are commonly applied for homogeneity determination of PAHs, PCBs and other organic compounds see Section 3.4. For these approaches sample sizes up to 10 g and even more are often needed for reliable results (Schantz et al. 1995). 

The determination of the residue levels by GC and HPLC and evaluation of the residue levels were carried out by the procedures described for the plant material in Sections 2.2.3 and 2.2.4, respectively. 

MAE in conjunction with high-performance liquid chromatography/mass spectrometry (HPLC/MS) shortened the cleanup procedure and provided satisfactory recoveries (97-102%) for the parent imazethapyr and its two metabolites (hydroxy and glucose conjugate metabolites). 

In this study, HPLC/fluorescence was compared with HPLC/ESI-MS and HPLC/ESI-MS/MS. A summary of the procedure is described as follows. The sample was prepared using the method of Luke et al.f and the sample was then cleaned up using a 1-g aminopropyl SPE cartridge. The sample was loaded on to the... 

The PRISMA model was developed by Nyiredy for solvent optimization in TLC and HPLC [142,168-171]. The PRISMA model consists of three parts the selection of the chromatographic system, optimization of the selected mobile phases, and the selection of the development method. Since silica is the most widely used stationary phase in TLC, the optimization procedure always starts with this phase, although the method is equally applicable to all chemically bonded phases in the normal or reversed-phase mode. For the selection of suitable solvents the first experiments are carried out on TLC plates in unsaturated... 

Autoxidation of unsaturated fatty acids is well known. Modifications of the extraction and separation procedures were made to investigate the possibility that these oxygenated fatty acids (OFAs) arise as artefacts. When exposure to light and air were minimized, no changes were noted in TLC and HPLC. 

Principles and Characteristics Plasma source techniques are more widely used in connection with liquid chromatography than atomic absorption spectrometry (see Section 7.3.3). ICP is a natural complement to liquid chromatography, and HPLC-ICP procedures... 

Food products are systematically checked for safety indicators, including pesticides contents and chemical compounds. For this procedure highly sensitive GC and HPLC methods are used. 

Matrix effect is a phrase normally used to describe the effect of some portion of a sample matrix that causes erroneous assay results if care is not taken to avoid the problem or correct for it by some mechanism. The most common matrix effects are those that result in ion suppression and subsequent false negative results. Ion enhancement may lead to false positive results.126 127 Several reports about matrix effects include suggestions on what can cause them and how to avoid them.126-147 While various ways to detect matrix effects have been reported, Matuszewski et al.140 described a clear way to measure the matrix effect (ME) for an analyte, recovery (RE) from the extraction procedure, and overall process efficiency (PE) of a procedure. Their method is to prepare three sets of samples and assay them using the planned HPLC/MS/MS method. The first set is the neat solution standards diluted into the mobile phase before injection to obtain the A results. The second set is the analyte spiked into the blank plasma extract (after extraction) to obtain the B results. The third set is the analyte spiked into the blank plasma before the extraction step (C results) these samples are extracted and assayed along with the two other sets. The three data sets allow for the following calculations ... 

In addition to the above-mentioned restrictions, eluent selection for LC and HPLC is especially important. While the gas used in GC will not interfere with analysis, it is possible for eluent components used in LC or HPLC to interfere with follow-on analysis. This will be true for both MS and IR analysis. Usually, however, all samples can be accommodated if sufficient thought is exercised in selecting both the method of separation and the method of introduction into the follow-on analytical procedure. 

This problem has been confirmed recently in a study of the mechanism of covalent reaction between nylon 6.6 and the sulpha toe thylsulphone dye Cl Reactive Blue 19 (7.37). Acid hydrolysis of the dyed fibre and HPLC analysis of the hydrolysate yielded the 6-aminohexylaminoethylsulphonyl derivative of Blue 19. Even when the dyeing procedure was optimised to achieve maximal exhaustion and fixation to the fibre [128], only about 30% of the N-terminal amino groups in the nylon 6.6 were accessible because of mutual blocking effects between these bulky anionic dye molecules. 

The term liquid chromatography (LC) is applied to any chromatographic procedure in which the moving phase is liquid, as opposed to gas chromatography (GC) where a gas is utilized as a mobile phase (see discussion in Chapter 14). Classical column chromatography (see Section 15.1), paper chromatography—a forerunner of thin-layer chromatography (see Chapter 13), and HPLC are all examples of LC. This should clarify why it is inappropriate to further abbreviate HPLC to LC unfortunately, it is still commonly done. 

For solution-phase libraries that are composed of mixtures of compounds, the difficulty of analysis escalates with increasing numbers of compounds. Typically, large mixtures of compounds are not analyzed before screening, whereas small ones may be analyzed for reaction completeness using mass spectrometry, HPLC, NMR, or combinations thereof. The identification and analysis of active compounds from these mixtures is painstakingly tedious, and often complete characterization is possible only after deconvolution procedures and resynthesis of the active compound. For solid-phase libraries, the methods currendy developed are discussed below. 

Numerous CE separations have been published for synthetic colours, sweeteners and preservatives (Frazier et al., 2000a Sadecka and Polonsky, 2000 Frazier et al., 2000b). A rapid CZE separation with diode array detection for six common synthetic food dyes in beverages, jellies and symps was described by Perez-Urquiza and Beltran (2000). Kuo et al. (1998) separated eight colours within 10 minutes using a pH 9.5 borax-NaOH buffer containing 5 mM /3-cyclodextrin. This latter method was suitable for separation of synthetic food colours in ice-cream bars and fmit soda drinks with very limited sample preparation. However the procedure was not validated for quantitative analysis. A review of natural colours and pigments analysis was made by Watanabe and Terabe (2000). Da Costa et al. (2000) reviewed the analysis of anthocyanin colours by CE and HPLC but concluded that the latter technique is more robust and applicable to complex sample types. Caramel type IV in soft drinks was identified and quantified by CE (Royle et al., 1998). 

Procedure The HPLC is carried out using (a) a Vydac C18 column, for proteins and peptides, maintained at 40 °C, (b) as the mobile phase at a flow rate of 1 ml per minute, a mixture of 48 volumes of mobile phase A and 52 volumes of mobile phase B prepared and maintained at a temperature of not less than 20 °C, and (c) a detection wavelength of 214 nm. 

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