Fatty acids HPLC analysis

Fatty Acid Ester Analysis by Ag-HPLC. Ag-HPLC analysis of CLA isomers as FAME continues to be the single largest application for this technology. An excellent example is illustrated in Figure 3.3, the only figure in this chapter duplicated... 

DeMar Jr. J.C., Disher, R.M., and Wensel, T.G. (1992) HPLC analysis of protein-linked fatty acids using fluorescence detection of 4-(diazomethyl)-7-diethylaminocoumarin derivatives Abstract 465. Biophys. J. 61(A81). 

Phosphorus, fatty acids, carbohydrates, glycerol, and amino acids were analyzed by the method described in our previous paper [8] and references cited therein. SDS-PAGE [8], TLC [9], HPLC [9], determination of phos-phomonoester [8], reducing sugar analysis [13], methylation analysis [14], and hexose analysis [15] were performed as described in the respective literature. Two dimensional TLC was performed on silica-gel plate (Merck Silicagel 60 F254 No. 5715) using the solvent systems, chloroform-methanol-acetic acid (65/10/1, v/v/v) for the first development and chloroform-methanol-25% ammonia solution (65/10/1) for the second. 

The marriage of HPLC to mass spectrometry (MS), now developed into a mature instrumentation, continues to greatly impact many of the separation sciences, especially in pharmaceutical analysis where it has been used in new drug discovery [23,24] and in drug metabolite identification [25-27]. HPLC-MS has also made an impact on lipid research, providing a convenient approach to the analysis of phospholipids and fatty acids [28,29]. It has also greatly benefited the field of proteomics [30-34], especially analysis of protein structure and function. 

Not only fatty acids but every unsaturated lipid molecule can undergo oxidation. So sterol oxidation products have also been studied in this case, HPLC is used both as a preparative step and for analytical purposes [33,34], HPLC as a preparative step was proposed by several researchers to improve the speed of analysis in the case of sterols, stigmastadienes, and waxes [35,36], Stigmastadiene, which is used as a marker in the refining process applied to vegetable oils, is determined by capillary GC however, the International Organization for Standardization (ISO) method (ISO 15788-2 2003) [37] uses HPLC as a rapid screening technique. 

HPLC has more or less supplanted GC as a method for quantifying drugs in pharmaceutical preparations. Many of the literature references to quantitative GC assays are thus old and the precision which is reported in these papers is difficult to evaluate based on the measurement of peak heights or manual integration. It is more difficult to achieve good precision in GC analysis than in HPLC analysis and the main sources of imprecision are the mode of sample introduction, which is best controlled by an autosampler, and the small volume of sample injected. However, it is possible to achieve levels of precision similar to those achieved using HPLC methods. For certain compounds that lack chromophores, which are required for detection in commonly used HPLC methods, quantitative GC may be the method of choice, for analysis of many amino acids, fatty acids, and sugars. There are a number... 

Low-wavelength UV detection (200-210 nm) is more sensitive and permits the use of gradients but precludes the use of certain common lipid solvents, such as chloroform and acetone, which are opaque in the UV region of interest. With low-wavelength UV detection, the response will also be somewhat dependent on fatty acid composition. For these reasons the mobile phases used in lipid analysis by HPLC may seem rather strange to workers familiar with the Thin Layer Chromatography (TLC) or open column separations. 

Free fatty acids are separable by GC by the inclusion of phosphoric acid in the packing so, for HPLC analysis, the phosphoric acid or other equivalent strong acid is included in the mobile phase. On a SUPELCOSIL LC 18 column, a model mixture of free fatty acids was separated with a mobile phase containing tetrahydrofuran, acetonitrile, water, and phosphoric acid (6 64 30 0.1) at pH 2 (Fig. 1) (15). Oleic and elaidic acids, palmitoleic and palmitelaidic acids, and linoleic and linoelaidic acids were well separated, but margarine fatty acids presented a difficult problem. Ultraviolet detection of 220 nm was used to prepare this chromatogram. 

Cooper and Anders (20) reported the HPLC analysis of unsaturated C l8 and C20 fatty acids. Since the methylene-interrupted polyunsaturated acids show no specific UV absorption, the 2-naphtacylesters were prepared for UV detection at 254 nm [the column was a 3-ft X 0.07-in.-lD stainless steel tube packed withCORASIL-Ci8, methanol/water(85 15) served as the eluent, and a flow rate of 12 ml/h was obtained at a pressure of 300 psig]. The lower detection limit was 4-90 ng of ester. 

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