Phospholipids refractive index detection

Adsorption chromatography using small particle silica or alumina has also been employed in the separation of biologically meaningful substances. Phospholipids, for example, have been separated on silica (38). One of the big problems for such substances is detection, since many of the compounds are not U.V. active. Generally, the refractive index detector is employed for isocratic operation, and the moving wire detector for gradient operation. Formation of U.V.-active derivatives is also possible (39). 

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. 

The mobile phase consists of one or more solvents that are pumped through the chromatographic system, resulting in the separation of analytes. Mobile phases may also contain modifiers. Examples of frequently used solvents include hexane, methanol, 2-propanol, acetonitrile (ACN), and water. Examples of modifiers include tri-fluoroacetic acid, acetic acid, or formic acid. In general, the composition of the mobile phase should be kept simple. Factors that influence the choice of mobile phase include the solubility of the sample in the mobile phase, the polarity of the mobile phase, ultraviolet absorption wavelength, refractive index, and viscosity of the solvents. The purity of the solvents in the mobile phase is also important because the region of UV that is used for the detection of lipids (200-215 nm) must be free of interferences. For phospholipids, the most popular solvent systems are transparent to UV in the range of 200-215 nm they include... 

As detection methods, ultraviolet (UV) detection, refractive index (RI), eLSD," MS, " and tandem MS " have been used. To quantify the phospholipid and glycohpid classes, ELSD has been very successful. But, ELSD does not always result in a linear response. It should be noted that, because of the absence of a specific absorption peak for hpids, and every peak in normal HPLC is, in fact, a mixed response, UV detection does not allow their quantitative estimation. ... 

---