Fractionation methods liquid chromatography

Several methods have been proposed to produce polyunsaturated fatty acid (PUFA) concentrates particularly high in eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA). Most PUFA enrichment methods are based upon a combination of techniques such as saponification, solvent extraction, urea fractionation, molecular distillation, fractionation distillation, liquid chromatography, and super critical carbondioxide extraction. Current evidence suggests that the physiological effects of omega-3 fatty acids are such that the annual world supply of fish oils will be grossly inadequate as a source of these materials, and alternative sources will be needed (Belarbi et al, 2000). 

SERS has also been applied as a sensitive, molecule-specific detection method in chromatography, e.g. thin layer, liquid, and gas chromatography. SERS-active colloids were deposited on the thin layer plates or mixed continuously with the liquid mobile phases. After adsorption of the analytes, characteristic spectra of the fractions were obtained and enabled unambiguous identification of very small amounts of substance. 

Figure 12.22 SFC-GC analysis of aromatic fraction of a gasoline fuel, (a) SFC trace (b) GC ttace of the aromatic cut. SFC conditions four columns (4.6 mm i.d.) in series (silica, silver-loaded silica, cation-exchange silica, amino-silica) 50 °C 2850 psi CO2 mobile phase at 2.5 niL/min FID detection. GC conditions methyl silicone column (50 m X 0.2 mm i.d.) injector split ratio, 80 1 injector temperature, 250 °C earner gas helium temperature programmed, — 50 °C (8 min) to 320 °C at a rate of 5 °C/min FID detection. Reprinted from Journal of Liquid Chromatography, 5, P. A. Peaden and M. L. Lee, Supercritical fluid chromatography methods and principles , pp. 179-221, 1987, by courtesy of Marcel Dekker Inc.

One of the first examples of the application of reverse-phase liquid chromatography-gas chromatography for this type of analysis was applied to atrazine (98). This method used a loop-type interface. The mobile phase was the most important parameter because retention in the LC column must be sufficient (there must be a high percentage of water), although a low percentage of water is only possible when the loop-type interface is used to transfer the LC fraction. The authors solved this problem by using methanol/water (60 40) with 5% 1-propanol and a precolumn. The experimental conditions employed are shown in Table 13.2. 

Although fractional crystallization has always been the most common method for the separation of diastereomers. When it can be used, binary-phase diagrams for the diastereomeric salts have been used to calculate the efficiency of optical resolution. However, its tediousness and the fact that it is limited to solids prompted a search for other methods. Fractional distillation has given only limited separation, but gas chromatography and preparative liquid chromatography have proved more useful and, in many cases, have supplanted fraetional crystallization, especially where the quantities to be resolved are small. 

In a study of the metabolism of methyl parathion in intact and subcellular fractions of isolated rat hepatocytes, a high performance liquid chromatography (HPLC) method has been developed that separates and quantitates methyl parathion and six of its hepatic biotransformation products (Anderson et al. 1992). The six biotransformation products identified are methyl paraoxon, desmethyl parathion, desmethyl paraoxon, 4-nitrophenol, />nitrophenyl glucuronide, and /wiitrophenyl sulfate. This method is not an EPA or other standardized method, and thus it has not been included in Table 7-1. 

Molecular weight of the components of the enzymatic complex was determined using a Sephadex G —75 column after its calibration by dextrans with molecular weight equal to 10,000, 40,000 and 70,000 and rafinose with molecular weight of 504. Fractions were also analyzed by the disk —electrophoresis method in PAAG (7) using 7.5% polyacrilamide gel (pH 4.3). Activity of pectinesterase was determined by titrometric method [8]. The enzymatically released methanol analyzed by gas—liquid chromatography [9]. 

In particular, for copolymers this required an orthogonal coupling of one GPC to another to achieve the desired cro fractionation before application of dual detectors. This method is really a new polymer analysis member of a family of approaches developed in the literature which we are now terming "Orthogonal Chromatogr hy . It not only provides both a cro fractionation approach for copolymers and a new way of determining the GPC s "imperfect resolution" it also enables separation mechanisms previously reserved for the liquid chromatography of small molecules to be used for polymer analysis. 

Radke et al. [28] described an automated medium-pressure liquid chromatograph, now commonly called the Kohnen-Willsch instrument. At present, the method is widely used to isolate different fractions of soluble organic matter (for instance, as described in Reference 29 to Reference 31). A combination of normal phase and reversed-phase liquid chromatography has been used by Garrigues et al. [32] to discriminate between different aromatic ring systems and degrees of methylamine in order to characterize thermal maturity of organic matter. 

A further extension of the DFG S19 method was achieved when polar analytes and those unsuitable for GC were determined by LC/MS or more preferably by liquid chromatography/tandem mass spectrometry (LC/MS/MS). Triple-quadrupole MS/MS and ion trap MS" have become more affordable and acceptable in the recent past. These techniques provide multiple analyte methods by employing modes such as time segments, scan events or multiple injections. By improving the selectivity and sensitivity of detection after HPLC separation, the DFG S19 extraction and cleanup scheme can be applied to polar or high molecular weight analytes, and cleanup steps such as Si02 fractionation or even GPC become unnecessary. 

Multidimensional liquid chromatography encompasses a variety of techniques used for seunple separation, cleanup and trace enrichment [12,279-289]. A characteristic feature of these methods is the use of two or more columns for the separation with either manual or automatic switching by a valve interface of fractions between columns. These techniques require only minor modification to existing equipment, and of equal importance, enable the sample preparation and separation procedures to be completely automated. 

SEC in combination with multidimensional liquid chromatography (LC-LC) may be used to carry out polymer/additive analysis. In this approach, the sample is dissolved before injection into the SEC system for prefractionation of the polymer fractions. High-MW components are separated from the additives. The additive fraction is collected, concentrated by evaporation, and injected to a multidimensional RPLC system consisting of two columns of different selectivity. The first column is used for sample prefractionation and cleanup, after which the additive fraction is transferred to the analytical column for the final separation. The total method (SEC, LC-LC) has been used for the analysis of the main phenolic compounds in complex pyrolysis oils with minimal sample preparation [974]. The identification is reliable because three analytical steps (SEC, RPLC and RPLC) with different selectivities are employed. The complexity of pyrolysis oils makes their analysis a demanding task, and careful sample preparation is typically required. 

Using the newer methods, such as gas chromatography, liquid-liquid chromatography, fluorometry, and mass spectrometry, it is possible to measure many compounds at the parts-per-billion level, and a few selected compounds with special characteristics at the parts-per-trillion level. Even with these sensitivities, however, a considerable concentration must usually be undertaken to permit the chemical or physical fractionation necessary to render the final analyses interpretable. A major effort has therefore been expended on the study of methods of separation and concentration, and this is discussed further in Chap. 8. 

The methylene blue reaction can also be used in a fractionation procedure for surfactants. The complexes with methylene blue can be collected in an organic solvent, concentrated, dissolved in methanol, and separated by high-performance liquid chromatography [205]. A variation of this method, permitting the collection of surfactant from large volumes of sample, should be workable in seawater. 

A more traditional but still successful method for the detection of a protein phosphorylation is by radioactive labeling with 35P. The labeled protein is digested, the peptides are separated by high-performance liquid chromatography, and the phosphorylated peptides are detected in specific fractions via their radioactivity. The fraction with the phosphorylated peptides can be further analyzed by mass spectrometry (Figeys et al., 1999). 

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