Lipids ultraviolet detector

There are excellent HPLC systems available on the market today, yet there is one area of concern with this instrumentation, and this rests with the detection units. Certainly the most widely used detector system employs a low dead-volume micro-ultraviolet detector. This latter unit operates near 200 nm and detects mainly unsaturated linkages in phospholipids (or lipid) samples. Some contribution by carbonyl functions can be expected. This approach is an advantage when the sample under study contains olefinic groups, but will not detect those with saturated side (hydrocarbon) chains. An alternative detector is the refractive index monitor which is often called a universal detector, since it is based on the concept that the refractive index of the solvent changes when a solute is present. The drawback of the latter unit lies in its sensitivity, which is approximately 15- to 20-fold less than that of the ultraviolet monitor. 

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. 

Detection. Detection of lipids separated by open tubular SFC has been by flame ionization or mass spectrometry. For packed columns, ultraviolet (UV) or detectors or ELSDs have been employed. ELSDs have been used extensively for detection of lipids in connection with HPLC, and it is of interest to apply this detector also to packed column SFC. 

HPLC 1s carried out with a high speed chemical derivatization chromatograph (HLC 803, Toyo Soda Mfg. Co.) equipped with two detectors of ultraviolet and visible wave length, but an enzymatic reaction is performed using a Teflon tube (0.5 mm I.O. or 0.4 mn I.D. x 10,000 mm or 20,000 mm) in the thermostated water bath as shown in Fig. 1. In this equipped configuration with two detectors, elution patterns of protein and one of the lipid components can be obtained in one analysis as described in Section 2.6. 

HPLC has been used for measuring various compounds, for example, carbohydrates, vitamins, additives, mycotoxins, amino acids, proteins, triglycerides in fats and oils, lipids, chiral compounds, and pigments. Several sensitive and selective detectors such as ultraviolet-visible, FL, electrochemical, and diffractometric are available to utilize with HPLC depending on the compound to be analyzed. Various HPLC methods based on these detectors have been published for the measurement of vitamin E in biological and pharmaceutical samples and food products. Excellent literature reviews of HPLC based on various detectors in the analysis of vitamin E content in various matrices have been reported (Abidi, 2000 Aust et al., 2001 Ruperez et al., 2001 Lai and Franke, 2013). Table 19.5 reports several recent HPLC methods for the analysis of vitamin E and similar compounds in various matrices. 

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