Refractive index detector selection

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. 

Select the detector. To acquire molecular weight distribution data, use a general detector such as a refractive index detector. To acquire structural or compositional information, employ a more selective detector such as an ultraviolet (UV) or infrared (IR) detector. Viscometric and light-scattering detectors facilitate more accurate molecular weight measurement when appropriate standards are not available. 

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 ... 

Manufacturers publish their product s performance characteristics as specifications, which are often used by the customer for comparison during the selection process. Table 1 shows the specifications of an Agilent 1100 Series Quaternary Pump, which is quite representative of other high-end analytical pumps. Note pulsation is particularly detrimental to the performance of flow-sensitive detectors (e.g., mass spectrometer, refractive index detector). Differences in dwell volumes and composition accuracy between HPLC systems might cause problems during method transfers. 

Very often baseline problems are related to detector problems. Many detectors are available for HPLC systems. The most common are fixed and variable wavelength ultraviolet spectrophotometers, refractive index, and conductivity detectors. Electrochemical and fluorescence detectors are less frequently used, as they are more selective. Detector problems fall into two categories electrical and mechanical/optical. The instrument manufacturer should correct electrical problems. Mechanical or optical problems can usually be traced to the flow cell however, improvements in detector cell technology have made them more durable and easier to use. Detector-related problems include leaks, air bubbles, and cell contamination. These usually produce spikes or baseline noise on the chromatograms or decreased sensitivity. Some cells, especially those used in refractive index detectors, are sensitive to flow and pressure variations. Flow rates or backpressures that exceed the manufacturer s recommendation will break the cell window. Old or defective source lamps, as well as incorrect detector rise time, gain, or attenuation settings will reduce sensitivity and peak height. Faulty or reversed cable connections can also be the source of problems. 

Another detector, which has found considerable application, is based on the changes in the refractive index of the solvent that is caused by analyte molecules. In contrast to most of the other detectors listed in Table 32-1, the refractive index detector is general rather than selective and responds to the presence of all solutes. The disadvantage of this detector is its somewhat limited sensitivity. Several electrochemical detectors that are based on potentiometric, conductometric, and voltammetric measurements have also been introduced. An example of an amperometric detector is shown in Figure 32-9. 

Detection and Analysis of Sugars. Because the refractive index detectors used in HPLC are not very selective and are characterized by low sensitivities for different sugars and their various isomers, reactors containing immobilized enzymes are often coupled to the corresponding chromatographic columns to obtain significant improvements in selectivity and sensitivity. Examples include ... 

Non-selective detectors react to the bulk property of the solution passing through. A refractive index detector monitors the refractive index of the eluate. The pure mobile phase has a specific refractive index which changes when any compound is eluted. The detector senses this difference and records all peaks — hence the term non-selective or bulk-property detector. This is why the refractive index as well as the conductivity detector is not suited for gradient elution. 

The most commonly used analytical technique for sugars is HPLC with a refractive index detector (RID). Although the HPLC-RID method is simple, the RID lacks sensitivity and selectivity. Therefore, UV and fluorescence detection is frequently used, coupled with pre- or postcolumn derivatization, for analysis with higher sensitivity. Liquid chromatography-mass spectrometry (LC-MS) using electrospray ionization also requires pre- or postcolumn derivatization. LC-MS using atmospheric pressure chemical ionization does... 

Autoxidized trilinolein was separated into mono-, bis- and tris-hydroperoxides by preparative reversed phase HPLC using a 5 m C-18 column with UV at 235 nm and refractive index detectors (Chapter 2, F Figure 6.9). The monohydroperoxides of trilinolein were further resolved into the positional isomers components by normal phase HPLC on a 5 m silica column with UV detection (Figure 6.10). Complete and partial separation of various cis,trans- and trans,trans-2- and l(3)-mono-13- and 9-hydroperoxides were identified by lipolysis and capillary GC. The ratio of 1(3)- to 2-monohydroperoxides estimated by normal phase HPLC averaging 2.0 indicated that oxidation of trilinolein was not selective toward either the 1(3)- or 2-position. 

The methods evaluated for KHI analyses are methyl orange/colorimetric, iodine complexation, and size exclusion chromatography (SEC). The parameters investigated in evaluating the colorimetric method are the matrix effects and interferences from sulfide and organics present. The preliminary results for SEC determination of KHI are presented, and the limitations of Corona Aerosol Detector (CAD) and refractive index detector (RID) are discussed in terms of linearity and selectivity. 

When entering the field of liquid chromatography, the scientist is always faced with the problem of detector selection. The subject of detector choice will be dealt with later in this book but at this point, it should again be emphasized, that there is no ideal LC detector. Consequently, the practicing liquid chromatographer needs to have at least two, if not more, different types of detector available, or the full versatility of the technique will not be realized. It is therefore recommended that one of the detectors available should be a bulk property detector, which should probably be the refractive index detector. This would be a particularly appropriate detector if preparative or semi-preparative chromatography is likely to be required. If the separation and quantitative analysis of ionic materials are contemplated, then the refractive index detector might be replaced... 

The ideal HPLC detector should have the same characteristics as those required for GC detectors, i.e. rapid and reproducible response to solutes, a wide range of linear response, high sensitivity and stability of operation. No truly universal HPLC detector has yet been developed but the two most widely applicable types are those based on the absorption of UV or visible radiation by the solute species and those which monitor refractive index differences between solutes dissolved in the mobile phase and the pure mobile phase. Other detectors which are more selective in their response rely on such solute properties as fluorescence, electrical conductivity, diffusion currents (amperometric) and radioactivity. The characteristics of the various types of detector are summarized in Table 4.14. 

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