Refractive index, detector constant

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. 

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. 

As a consequence, some steps must be taken when using the refractive index detector (a) solvent delivery must be degassed and pulse free (b) the composition of the mobile phase should remain constant during a single run (isocratic elution) (c) the detector must be carefully thermostated and (d) the temperature of the column must also be kept constant, since a variation may affect the equilibrium of the solvent components between both phases and therefore the composition of the eluate from the column. 

Chromatographic System (See Chromatography, Appendix IIA.) Use a liquid chromatograph equipped with a refractive index detector that can be maintained at a constant temperature of 25°, a 25-cm x 4.6-mm (id) column packed with 10- im porous silica gel bonded with aminopropylsilane (Alltech 35643, or equivalent), and a guard column that contains the same packing. Maintain the column at a constant temperature of 25° 2°, and the flow rate at about 2.0 mL/min. Inject 20 pL of System Suitability Preparation into the chromatograph, and record the peak responses as directed under Procedure. The relative standard deviation for replicate injections is not more than 2.0%, and the alpha-Cyclodextrin and beta-Cyclodextrin peaks exhibit baseline separation, the relative retention times being about 0.8 and 1.0, respectively. 

From the Th-FFF retention data, it is possible to obtain a molar mass distribution after a suitable calibration for the determination of the Mark-Houwink constants (straight-line plot of log(D/DT) vs. log M [15]). Another possibility is to couple an absolute molar mass detector like MALLS (see Sect. 4.3.2) or a suitable detector combination such as an on-line viscometer coupled with a refractive index detector. This possibility does not require prior knowledge of DT... 

A uv or refractive index detector can also be incorporated into the stem, or the fractions can simply be analysed by tic, as for flash. "omatography. If the conditions are kept constant the results from mplc 133 very consistent, so if you wish to repeat separations of the same iixture, it is very easy to predict which fractions will contain each rmponent... 

Liquid chromatographic detectors are of two basic types. Bulk-properiy detectors respond to a mobile-phase bulk property, such as refractive index, dielectric constant, or density, that is modulated by the presence of solutes. In contrast, solute-property detectors respond to some property of solutes, such as UV absorbance, fluorescence, or diffusion current, that is not possessed by the mobile phase. 

Bulk property detectors such as the refractive index detector or the dielectric constant detector are often particularly pressure sensitive and for that reason wide diameter exit tubes are strongly recommended, however, such detectors are not useful for multidimensional analyses. It follows that the pressure sensitivity of the detector should be specified by the manufacturer. The pressure response Dp should be given as the output in mV for unit pressure change in ihe detector cell. The pressure response should be given in both mV/psi and mV/kg/m2, It is also recommended that the pressure noise is given in terms of that pressure change which would provide a signal equivalent to the noise, i.e. 

One of the first on-line liquid chromatography detectors to be developed in the early forties was, in fact, a bulk property detector, the refractive index detector (1). Bulk property detectors continuously monitor some physical property of the column eluent and by the use of a suitable transducer provide a voltage - time output that is either proportional to the physical property being measured, or made proportional to the concentrations of the solute eluted. The properties of the mobile pheuse that are most commonly monitored in commercially available bulk property detectors are refractive index, electrical conductivity, and dielectric constant, the dielectric constant detector being the least popular of the three. 

The SEC mechanism demands only an isocratic (constant composition) solvent system with normally a single solvent. The most frequently used organic solvents are THF, chloroform, toluene, esters, ketones, DMF, etc. The key solvent parameters of interest in SEC are (i) solubility parameter (ii) refractive index (iii) UV/IR absorbance (iv) viscosity and (v) boiling point. Sample solutions are typically prepared at concentrations in the region of 0.5-5 mg mL-1. In general an injection volume of 25-100p,L per 300 x 7.5 mm column should be employed. For SEC operation with polyolefins chlorinated solvents (for detector sensitivity and increased boiling point) and elevated temperatures (110 to 150 °C) are required to dissolve olefin polymer. HFIP is the preferred solvent for SEC analysis of polyesters and polyamides. 

Another important physical property of liquids is the refractive index. Since the refractive index is a constant for a particular liquid at a given temperature, it can be used to help identify substances, check for purity, and measure concentrations. One type of detector found in some liquid chromatograph instruments (Chapter 13) uses refractive index. 

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