Quantification of underivatized fatty acids from vegetable oils by HPLC with UV detection.

We propose a chromatographic method for the separation of saturated and unsaturated fatty acids by a high-performance liquid chromatography system, equipped with a photo diode array detector. Central to the method is the use of an appropriate mobile phase composed of acetonitrile, methanol, and n-hexane in ratio 90:8:2 acidified with 0.2% acetic acid, which allows the detection of fatty acids without a preliminary derivatization with chromophores or fluorescent dyes. Calibration on solutions of standards mixtures gives a quantification limit (at a wavelength of 208 nm) of 0.232, 0.093, 0.039, 0.056, 0.068, 0.004, 0.0005, 0.067 mg/mL for the myristic, palmitic, palmitoleic, stearic, oleic, linoleic, linolenic, and erucic acids, respectively. The method, applied to different vegetable oils (olive, sunflower, soybean, and palm) was able to distinguish the main fatty acids and quantify their amount. Data reliability was tested by comparing our results (on the relative percentages of some fatty acids in the olive oil) with those obtained by gas chromatographic analysis. Differences of the order of 0.3%, 0.6%, 2%, and 6% were observed for the oleic, linoleic, palmitic, and linolenic acids. Although less accurate, our method proved to be a simple alternative to standard gas chromatographic technique, as it can be applied even using a simple UV detector.

Heme symmetry, vibronic structure, and dynamics in heme proteins: ferrous nicotinate horse myoglobin and soybean leghemoglobin.

We report the visible and Soret absorption bands, down to cryogenic temperatures, of the ferrous nicotinate adducts of native and deuteroheme reconstituted horse heart myoglobin in comparison with soybean leghemoglobin-a. The band profile in the visible region is analyzed in terms of vibronic coupling of the heme normal modes to the electronic transition in the framework of the Herzberg-Teller approximation. This theoretical approach makes use of the crude Born-Oppenheimer states and therefore neglects the mixing between electronic and vibrational coordinates; however, it takes into account the vibronic nature of the visible absorption bands and allows an estimate of the vibronic side bands for both Condon and non-Condon vibrational modes. In this framework, an x-y splitting of the Q transition for native and deuteroheme reconstituted horse myoglobin is clearly assessed and attributed to electronic perturbations that, in turn, are caused by a reduction of the typical D(4h) symmetry of the system due to heme distortions of B(1g)-type symmetry and/or to an x-y asymmetric position of the nicotinate ring; in deuteroheme reconstituted horse myoglobin the asymmetric heme peripheral substituents add to the above effect(s). On the contrary, in leghemoglobin-a no spectral splitting upon nicotinate binding is observed, pointing to a planar heme configuration in which only distortions of A(1g)-type symmetry are effective and to which the nicotinate ring is bound in an x - y symmetric position. The local dynamic properties of the heme pocket of the three proteins are investigated through the temperature dependence of spectral line broadening. Leghemoglobin-a behaves as a softer matrix with respect to horse myoglobin, thus validating the hypothesis of a looser heme pocket conformation in the former protein, which allows a nondistorted heme configuration and a symmetric binding of the bulky nicotinate ligand.