The refractive index detector

The refractive index detector was one of the first on-line detectors to be developed and was described by Tiselius and Claesson (1) in 1942. It was also the first detector to be made commercially and was at one time the only on-line detector available for general use. It is universal, in the sense that it detects all solutes that have a refractive index different from that of the mobile phase and is probably the next most popular to the UV detector. 

This detector is almost universal, though not very sensitive, and its lack of sensitivity in most cases restricts its use to situations where other detectors fail to detect the compounds of interest. 

The refractive index detector is very sensitive to variations in temperature, but as long as this is realized and the detector kept properly thermostated, this is usually not a cause for concern. 

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Hplc techniques are used to routinely separate and quantify less volatile compounds. The hplc columns used to affect this separation are selected based on the constituents of interest. They are typically reverse phase or anion exchange in nature. The constituents routinely assayed in this type of analysis are those high in molecular weight or low in volatility. Specific compounds of interest include wood sugars, vanillin, and tannin complexes. The most common types of hplc detectors employed in the analysis of distilled spirits are the refractive index detector and the ultraviolet detector. Additionally, the recent introduction of the photodiode array detector is making a significant impact in the analysis of distilled spirits. 

Refractive index detectors. These bulk property detectors are based on the change of refractive index of the eluant from the column with respect to pure mobile phase. Although they are widely used, the refractive index detectors suffer from several disadvantages — lack of high sensitivity, lack of suitability for gradient elution, and the need for strict temperature control ( + 0.001 °C) to operate at their highest sensitivity. A pulseless pump, or a reciprocating pump equipped with a pulse dampener, must also be employed. The effect of these limitations may to some extent be overcome by the use of differential systems in which the column eluant is compared with a reference flow of pure mobile phase. The two chief types of RI detector are as follows. 

Thin-layer chromatography (TLC) is used both for characterization of alcohol sulfates and alcohol ether sulfates and for their analysis in mixtures. This technique, combined with the use of scanning densitometers, is a quantitative analytical method. TLC is preferred to HPLC in this case as anionic surfactants do not contain strong chromophores and the refractive index detector is of low sensitivity and not suitable for gradient elution. A recent development in HPLC detector technology, the evaporative light-scattering detector, will probably overcome these sensitivity problems. 

Sodium dodecyl sulfate present in hydrophilic ointments has been determined by TLC on silica gel with flame ionization detection, which was considered better than the colorimetric method. TLC is preferred to HPLC in this case due to the low sensitivity of the refractive index detector that makes difficult the analysis of small amounts of sodium dodecyl sulfate [284]. 

If the mixture to be separated contains fairly polar materials, the silica may need to be deactivated by a more polar solvent such as ethyl acetate, propanol or even methanol. As already discussed, polar solutes are avidly adsorbed by silica gel and thus the optimum concentration is likely to be low, e.g. l-4%v/v and consequently, a little difficult to control in a reproducible manner. Ethyl acetate is the most useful moderator as it is significantly less polar than propanol or methanol and thus, more controllable, but unfortunately adsorbs in the UV range and can only be used in the mobile phase at concentrations up to about 5%v/v. Above this concentration the mobile phase may be opaque to the detector and thus, the solutes will not be discernible against the background adsorption of the mobile phase. If a detector such as the refractive index detector is employed then there is no restriction on the concentration of the moderator. Propanol and methanol are transparent in the UV so their presence does not effect the performance of a UV detector. However, their polarity is much greater than that of ethyl acetate and thus, the adjustment of the optimum moderator concentration is more difficult and not easy to reproduce accurately. For more polar mixtures it is better to explore the possibility of a reverse phase (which will be discussed shortly) than attempt to utilize silica gel out of the range of solutes for which it is appropriate. 

The flow sensitivity of a detector will also be one of the factors that determines the long term noise and thus will influence the sensitivity or minimum detectable concentration of the detector.lt is usually measured as the change in detector output for unit change in flow rate through the sensor cell. Again, the refractive index detector is the most sensitive to flow rate changes. 

The refractive index detector, in general, is a choice of last resort and is used for those applications where, for one reason or another, all other detectors are inappropriate or impractical. However, the detector has one particular area of application for which it is unique and that is in the separation and analysis of polymers. In general, for those polymers that contain more than six monomer units, the refractive index is directly proportional to the concentration of the polymer and is practically independent of the molecular weight. Thus, a quantitative analysis of a polymer mixture can be obtained by the simple normalization of the peak areas in the chromatogram, there being no need for the use of individual response factors. Some typical specifications for the refractive index detector are as follows ... 

The normalization method is the easiest and most straightforward to use but, unfortunately, it is also the least likely to be appropriate for most LC analyses. To be applicable, the detector must have the same response to all the components of the sample. An exceptional example, where the normalization procedure is frequently used, is in the analysis of polymers by exclusion chromatography using the refractive index detector. The refractive index of a specific polymer is a constant for all polymers of that type having more than 6 monomer units. Under these conditions normalization is the obvious quantitative method to use. 

This classification is concerned with whether the detector monitors a property of the solute (analyte), e.g. the UV detector, or a change in some property of the solvent (mobile phase) caused by the presence of an analyte, e.g. the refractive index detector. 

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). 

Detection requirements in preparative-scale chromatography also differ from analytical erations where detectors are selected for their sensitivity. Sensitivity is not of overriding importance in preparative-scale chromatography the ability to accommodate large column flow rates and a wide linear response range are more useful. The sensitivity of the refractive index detector is usually quite adequate for prqtaratlve work but the ... 

C. The Rheodyne Model 7010 injection valve, equipped with a 20-pl loop, was switched to injection at the apex of the sample band, as observed on the refractive index detector. The complex kinetics of the production of mono-, di-, and tri-brominated glycols is shown in Figure 14. Optimization of parameters such as the flow rate of acid resulted in a 15% reduction in batch cycle time and eliminated the need for manual analysis and intervention to obtain a desired endpoint composition. 

Concentration-sensitive detectors, such as the refractive index detector or UV-VIS spectrophotometer... 

There are many HPLC detectors that can turn the presence of your compound into an electrical signal to be written on a chart recorder. Time was the refractive index detector was common. Clean eluent, used as a reference, went through one side of the detector, and the eluent with the samples went through the other side. A difference in the refractive index between the sample and reference caused an electrical signal to be generated and sent to a chart recorder. If you ve read the section on gas chromatography and looked ahead at infrared, you shouldn t be surprised to find both a sample and a reference. I did tell you the reference/sample pair is common in instrumentation. 

Slow elution of chemicals adsorbed on the column, 2) temperature effects, such as with the refractive index detector, and 3) a contaminated detector... 

The refractive index detector operates by comparing the refractive index of the mobile phase prior to the column with the refractive index of the column eluate. This detector responds to nearly all solutes but it is highly temperature-sensitive (Skoog et al., 1998). This type ofdetector can be used for sugars and fatty acids. 

Other analysis methods dependent on multiple detectors can be implemented using this automated system. Two methods under development are the use of a continuous viscometer detector with a refractive index detector to yield absolute molecular weight and branching, utilizing the universal calibration curve concept (4), and the use of a UV or IR detector with the refractive index detector to measure compositional distribution as a function of molecular weight. 

The refractive index detector, considered to be almost universal, is often used in series with a UV detector in the isocratic mode to provide a supplementary chromatogram. This detector, which is not highly sensitive, has to be temperature controlled, as does the column (0.1 °C). The baseline of the chromatogram has to be set to an intermediate position because it can lead to either positive or negative signals (Fig. 3.18). The detector can only be used in the isocratic mode because in gradient elution the composition of the mobile phase changes with time, as does the refractive index. Compensation, which is easily obtained in the case of a mobile phase of constant composition, is difficult to carry out when the composition at the end of the column differs from that at the inlet. 

In the beginning, the refractive index detector was the most used detection system, although it has two important drawbacks (1) solvent gradients cannot be used, and (2) it has low sensitivity and different responses to saturated and highly unsaturated TGs (112). Moreover, use of the ultraviolet (UV) detector is difficult, because the most adequate solvents also absorb in the same range and therefore cause an important baseline drift with gradient elution systems (106). 

Whether eluted from columns or from thin-layer plates, the quantitative determination of sugars was traditionally based on colorimetric reactions involving the use of chemical reagents, e.g., anthrone. These detection methods have been largely replaced in modem HPLC by the refractive index detector, although ultraviolet detectors are also employed. Recently we have also seen the introduction of other types of detector (e.g., the mass detector), as will be discussed later. 

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