Lipids refractive index detector

Another point of interest was the time required to equilibrate the system after changes were made in solvent composition. While the ChromSpher Lipids column had a column volume of ca. 3 ml, an increase in ACN concentration was not noted until the introduction of 7-8 ml of solvent (determined with refractive index detector). The problem of ACN-silver ion interaction and subsequent ACN retention is not new and may be noted in all forms of chromatography employing silver ions in the stationary phase. In the isocratic system, the column was equilibrated with the appropriate solvent mix for at least 0.5 h before sample injection. Since ACN dissolves very slowly into hexane, the ACN-hexane solvent mix was thoroughly stirred for 5 min before use. To obtain reproducible retention times, thorough mixing of the ACN and hexane is essential. 

Hopia AI, Ollilainen VM. Comparison of the evaporative light scattering detector (ELSD) and refractive index detector (RID) in lipid analysis. J Liq Chromatogr 1993 16 2469-2482. 

Refractive index detectors are mostly used for sugar and lipid analyses. The presence of dissolved solutes in the mobile phase will cause a change in the refractive index. However, it is important to realise that the intrinsic characteristic makes up for the fact that gradient elution is usually not employed with a refractive index detector, as a change in the mobile phase implies a change in the refractive index which can barely be differentiated from that resulting from the presence of dissolved solute(s). 

The evaporative light-scattering detector (ELSD) is a near universal detector suitable for the determination of (mainly) neutral compounds that are less volatile than the mobile phase used for the separation [151,152]. Primary uses include the detection of compounds with a weak response to the UV detector, especially carbohydrates, lipids, surfactants, polymers and petroleum products. Its greater sensitivity and ease of use in gradient elution separations makes it preferable to the refractive index detector for these applications. The ELSD is compatible with most volatile solvents used for normal and... 

Refractive index detectors also have several applications in lipid analysis. They are "universal" detectors, but lack sensitivity, require isocratic elution conditions and are sensitive to minor fluctuations in temperature. Their main value is probably in small-scale preparative applications, say with 1-2 mg of a lipid extract. For example, a refractive index detector was utilized with a column (4.6 x 250 mm) of Ultrasil Si (5 micron silica gel) and isocratic elution with isooctane-tetrahydrofuran-formic acid (90 10 0.5 by volume) to separate most of the common simple lipid classes encountered in animal tissue extracts, such as those of liver [304]. Cholesterol esters, triacylglycerols and cholesterol were each resolved and gave symmetrical peaks. 

Although refractive index detectors are common on analytical systems due to then-universal nature, they have a number of disadvantages for process use. Their advantage is their ability to detect compounds such as carbohydrates, lipids, and simple peptides, which possess no measurable UV absorption. Offset against this is their incompatibility with gradient operation and often their need for a flow splitter to cope with process scale flow rates. This latter is undesirable for any detector, due to the fact that the relative flow rate through the detector cell can vary depending on solvent viscosity and flow rate. 

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 equipment widely used for the detection of carbohydrates in the HPLC method is the differential refractive index (RI) detector. The principle involved in this detection depends on the continuous measurement of the variation of the RIs of the mobile phase containing the samples with little or no chromophores such as carbohydrates, lipids, and other polymer compounds that do not absorb UV light. RI detection method presents high degree of reproducibility and is very convenient for the analysis of polysaccharides. However, other detectors such as evaporative light scattering detector and pulsed amperometric detector have been used for the detection of polysaccharides [100]. 

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