Specific elution volumes

Table V. Specific elution volumes (V and selectivity factors of...

Identification by comparison of elution data. The basic quantity characterizing the compounds qualitatively is the elution volume Fr, which represents the volume of gas necessary for the elution of the analyzed component. For the sake of comparison the specific elution volume Fg is also used it is calculated from the elution volume by correcting it for the amount of carrier gas necessary for passage through the column and the pressure drop in the column, and it is calculated for a temperature of 0 °C and for 1 g of the stationary phase. In practice, this is done as follows the value of the specific elution volume is calculated for one component of a series, and this value is taken as unity, the other values then being calculated with respect to it (the so-called elution rations r are obtained). 

The separation of mixtures involving N-methyl-JLtetrahydropyridines into their pure components by means of gas-liquid chromatography was discussed in a report by Holik et al. (87). They found that, using tris(/3-cyanoethoxymethyl)-y-picoline as the stationary phase, the primary factors involved in the specific retention volumes of these enamines is the electronic effect of a methyl substituent and the nitrogen atom on the carbon-carbon double bond. It was observed that 1,3-dimethyl-Zl -tetrahydropyridine (141) has a smaller specific retention volume and, hence, is eluted before... 

The most widely used molecular weight characterization method has been GPC, which separates compounds based on hydrodynamic volume. State-of-the-art GPC instruments are equipped with a concentration detector (e.g., differential refractometer, UV, and/or IR) in combination with viscosity or light scattering. A viscosity detector provides in-line solution viscosity data at each elution volume, which in combination with a concentration measurement can be converted to specific viscosity. Since the polymer concentration at each elution volume is quite dilute, the specific viscosity is considered a reasonable approximation for the dilute solution s intrinsic viscosity. The plot of log[r]]M versus elution volume (where [) ] is the intrinsic viscosity) provides a universal calibration curve from which absolute molecular weights of a variety of polymers can be obtained. Unfortunately, many reported analyses for phenolic oligomers and resins are simply based on polystyrene standards and only provide relative molecular weights instead of absolute numbers. 

The elution volume of a solute is determined mainly by its relative molecular mass and it has been shown that the elution volume is approximately a linear function of the logarithm of the relative molecular mass. It is possible to determine the relative molecular mass of a test molecule using a calibration curve prepared from the elution volumes of several reference substances of known relative molecular mass. This should be done using the same column and conditions (Figure 3.37) and in practice it may be possible to calibrate the column and separate the test substance at the same time by incorporating the reference compounds in the sample. Such a method is rapid and inexpensive and does not demand a highly purified sample, provided that there is a specific method for detecting the molecule in the eluate. 

However, the calculation requires different algorithms for each specific case. Hence, an explicit mathematical solution providing basing retention data (elution volumes or times, bandwidths, and resolution) is much more frequently used in analytical gradient chromatography, even at a cost of some simplifications [9-11]. 

Sample molecules that are too large to enter the pores of the support material, which is commercially available in various pore dimensions, are not retained and leave the column first. The required elution volume Ve is correspondingly small. Small molecules are retained most strongly because they can enter all the pores of the support material. Sample molecules of medium size can partly penetrate into the stationary phase and elute according to their depth of penetration into the pores (Fig. 7.3). No specific interactions should take place between the molecules of the dendrimer sample and the stationary phase in GPC since this can impair the efficiency of separation by the exclusion principal. After separation the eluate flows through a concentration-dependent detector (e.g. a UV/VIS detector) interfaced with a computer. One obtains a chromatogram which, to a first approximation, reflects the relative contents of molecules of molar mass M. If macromolecules of suitable molar mass and narrow molar mass distribution are available for calibration of the column, the relative GPC molar mass of the investigated dendrimer can be determined via the calibration function log(M) =f( Vc). 

Calibration Samples. Monodisperse polystyrene latices are available with known, narrow particle size distributions. Coefficients of variance about the mean diameter are typically less than 6% of diameter measured using electron microscopy (25). HDC typically cannot resolve differences in diameter of only 6%. Therefore, these polystyrenes are sufficiently narrow to be used as HDC calibration reference samples. However, doing so may result in incorporation of a systematic error in the particle size versus elution volume calibration, arising from known electron microscopy errors of as much as 5% for particles below 1 um (26). Therefore, accuracy can only be stated as relative to electron microscopy results for the calibration samples. FlowSizer performance specifications have been reported elsewhere (27) with diameter and mass percent results within 5% of those determined by electron microscopy for a series of these monodisperse polystyrene latices. 

The nature of most of the compounds separated from these samples is unknown and this emphasizes the advantage of a broad specificity antiserum in the detection of urinary THC metabolites. Indeed, none of the metabolites (11-hydroxy-THC, 8,11-dihydroxy-THC and THC-ll-oic acid) known to cross-react with the RIA antiserum, are seen to be present in the hydrolyzed urine sample (Figure 12). These compounds as shown in Figure 11 have elution volumes greater than 40 ml. On the basis of these results an RIA specific for these compounds would not have found any detectable material in the urine. To develop the combination of HPLC and RIA further, an antiserum exhibiting complete crossreactivity with all metabolites would be of greatest... 

The gas chromatographic method is based on the relation between the differential enthalpy of adsorption at zero coverage and the temperature dependence of the Henry s law constant, kn, as expressed in the form of Equation (4.3). In the low-pressure region, where Henry s law applies, the specific retention volume, Vj, is a linear function of kH (Purnell, 1962 Littlewood, 1970). This relationship makes it possible to make use of elution chromatography since... 

Run the sample through an FFF separation and collect fractions at designated elution volume intervals corresponding to specific size ranges... 

In such cases, as stated by Meira and co-workers [10], the quality of results depends on computational refinements. It is necessary to add specific constraints related to the chromatographic problem rejection of negative values or unrealistic fluctuations in the weight distribution. The normal way is to invert the large matrix defining the spreading function for any position on the elution volume scale. With modem computational facilities, that becomes easy, but it is still not trivial to obtain stable results, and proper filtering processes are useful. 

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