Refractive index, monitoring

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. 

These detectors are less sensitive than solute property detectors with a maximum sensitivity of 1 in 106. Examples of this type of detector include those which monitor refractive index (RI), dielectric constant, or eluant density. The latter two are relatively insensitive and not generally used. 

Despite the differences in generation of the evanescent field, the basic binding experiment is basically the same for all the optical biosensors (see Fig. 5.3). One of the interacting partners, the receptor, is attached to the sensor surface while the analyte binds to the receptor from free solution. As the sensor monitors refractive index changes occturing in real time, the amount of receptor, analyte and the rate of binding can be determined. Indeed, the estimation of the interaction kinetics is one of the key advantage of this technique. 

Probe-beam deflection is a technique in which a monochromatic source, typically a He-Ne laser beam, passing parallel to the electrode surface, is used to monitor refractive index changes in the diffusion layer. It is a simple and cost-effective way of profiling the diffusion layer, and is used to monitor the diffusion layer ingress and egress of ions, particularly protons. It is often used in conjunction with electrochemical quartz crystal microbalance (EQCM) measurements that monitor mass changes within or on the electrode layer itself. 

In a similar manner the density of the contents of the detector cell will change with pressure. It follows that the detector would respond to fluctuations in pump pressure or indirectly to fluctuations in mobile phase flow rate if there was a significant flow resistance subsequent to the detector as in the case of multidimensional column systems. It follows that a bulk property detector, functioning on the measurement of density could only be employed with pumps having very constant flow rates and pressure control. The above argument applies equally to other bulk property detectors which monitor refractive index or dielectric constant and it can therefore be concluded that all bulk property detectors will have a limited sensitivity and have to be employed with very well controlled mobile phase supply systems. 

In LC, the most common means for monitoring the eluant is to pass it through a cell connected into an ultraviolet spectrometer. As substances elute from the column, their ultraviolet absorption is measured and recorded. Alternatively, the refractive index of the eluant is monitored since it varies from the value for a pure solvent when it contains organics from the column. 

Attenuated total reflection, on which atr—ftir is based, occurs when the rarer medium is absorbing and is characterized by a complex refractive index (40). The absorbing characteristics of this medium allow coupling to the evanescent field such that this field is attenuated to an extent dependent on k. The critical angle in the case of attenuated total reflection loses its meaning, but internal reflection still occurs. Thus, if the internally reflected beam is monitored, its intensity will reflect the loss associated with the internal reflection process at the interface with an absorbing medium. 

Source sampling of particulates requites isokinetic removal of a composite sample from the stack or vent effluent to determine representative emission rates. Samples are coUected either extractively or using an in-stack filter EPA Method 5 is representative of extractive sampling, EPA Method 17 of in-stack filtration. Other means of source sampling have been used, but they have been largely supplanted by EPA methods. Continuous in-stack monitors of opacity utilize attenuation of radiation across the effluent. Opacity measurements are affected by the particle size, shape, size distribution, refractive index, and the wavelength of the radiation (25,26). 

Optical properties also provide useful stmcture information about the fiber. The orientation of the molecular chains of a fiber can be estimated from differences in the refractive indexes measured with the optical microscope, using light polarized in the parallel and perpendicular directions relative to the fiber axis (46,47). The difference of the principal refractive indexes is called the birefringence, which is illustrated with typical fiber examples as foUows. Birefringence is used to monitor the orientation of nylon filament in melt spinning (48). 

Another classification of detector is the bulk-property detector, one that measures a change in some overall property of the system of mobile phase plus sample. The most commonly used bulk-property detector is the refractive-index (RI) detector. The RI detector, the closest thing to a universal detector in lc, monitors the difference between the refractive index of the effluent from the column and pure solvent. These detectors are not very good for detection of materials at low concentrations. Moreover, they are sensitive to fluctuations in temperature. 

Figure 14.17 Schematic diagram of the on-line coupled LC-GC system VI, valve foi switcliing the LC column outlet to the GC injector V2, valve for switching the LC column to back-flush mode V3, LC injection valve RI, refractive index monitor detector UV, ulti avio-let monitor detector FID, flame-ionization detector.

An infrared beam is directed through a crystal of refractive index (ni) onto a sample of smaller refractive index (n2). The intensity of the reflected beam is monitored as a function of the wavelength of the incident beam. These absorptions are used to identify the chemical structure. ATR has a sampling depth of about 0.3-3.0 microns. 

The dead point is obtained by including in the sample a trace of an unretained solute or, more often, one of the components of the mobile phase. For example, when using a methanol water mixture as the mobile phase, the dead point is obtained from the elution of a pure sample of methanol. The pure methanol can often be monitored, even by a UV detector, as the transient change in refractive index resulting from the methanol is sufficient to cause a disturbance that is detectable. 

The differential refractometer monitors the deflection of a light beam caused by the difference in refractive index between the contents of the sample cell and those of the reference cell. A beam of light from an... 

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. 

The molecular weight distribution of cell wall polysaccharides was estimated by gel filtration with a TOSOH TSK gel G4000 PWXL (7.8 x 300 mm) column equilibrated and eluted with 0.05 M sodium acetate, 0.01 M EDTA, 0.05 M NaCl (pH 5.0) in polyuronide and 0.05 M sodium citrate, 0.1 M NaCl (pH 5.5) in the hemicellulose fraction. Samples (1 mg/ml) of 100 ml were injected. The eluate was monitored by a refractive index detector (Shimadzu R1D-6A, Kyoto, Japan) and collected at the fraction size of 0.4 ml. 

In this study, four Styragel columns were utilized one column had a nominal porosity rating of 10, two colvtmns of 10, and the fourth column of 10 A. The refractometer was maintained at 37°C. A 5 ml syphon was used to monitor a solvent flow rate of 1 ml/min. The instrviment was run at the highest sensitivity setting because the refractive index difference between our solvent and polymer was only moderate and because a number of samples analyzed had a broad molecular weight distribution (MWD). 

Another variation of the preceding method is to apply HPLC to fractionate the cleaned-up aliphatic-aromatic fraction from flash colurim separation of soluble organic matter as it is performed in the Chevron laboratory, for example, as described in Reference 2. A Waters HPLC system equipped with a preparative Whatman Partisil 10 silica column (9.4 X 500 mm), a HPLC pump, and two detectors for separation monitoring (a UV and refractive index detector) are used, giving three fractions of aliphatic hydrocarbons, mono-, di-, and triaromatics and polar compounds. The hrst two fractions are eluted with hexane, whereas polar compounds are eluted with... 

---