Elution behavior

Hm TABLE 4.7 Comparison of Amino Acid Elution Behavior... 

For precipitated protein, buffered solutions containing chaotropic reagents such as 0.1% SDS, 8 M urea, or 6 M guanidine or proteolytic enzymes such as pepsin may be used. However, an extended washing with buffer is required to remove SDS and guanidine. Unexpected elution behavior can occur if these reagents are not removed completely. 

Ligures 6.36 to 6.38 (pages 209 and 210) show the ehromatograms of polymethaerylates using the Asahipak GL-HQ series. As polymethaerylates also have hydrophobie strueture, aeetonitrile is added to the eluent by up to 10%. As a result, normal GPC elution behavior is obtained. 

As known, SEC separates molecules and particles according to their hydro-dynamic volume in solution. In an ideal case, the SEC separation is based solely on entropy changes and is not accompanied with any enthalpic processes. In real systems, however, enthalpic interactions among components of the chromatographic system often play a nonnegligible role and affect the corresponding retention volumes (Vr) of samples. This is clearly evident from the elution behavior of small molecules, which depends rather strongly on their chemical nature and on the properties of eluent used. This is the case even for... 

The elution behavior of various polymers near their critical adsorption point with silica gel packings and various eluents has been studied (12). It was of interest to apply hybrid column systems composed of active ( critical ) packings (silica gels) in combination with nonactive (nonadsorptive) PS/DVB and DVB-based gels. Some PS/DVB and DVB gels exhibited rather strong... 

FIGURE 22.7 Elution behavior of polystyrene standards in THE in PCHdC with different packing diameters , 1.40 /tm A, 1.91 m and , 0.87 /tm. Theoretical curves according to Eq. (I), where C = 3.7. (Reprinted from j. Chromatogr., 506,554, Copyright 1990, with permission from Elsevier Science.)... 

The model, therefore, predicts the elution behavior of solutes during a chromatographic process over a swollen gel as the stationary phase as a function of solute size and of the gel nanomorphology. On the reverse, from the elution behavior of solutes of known molecular size it is possible to extract the polymer chain concentration from chromatographic experiments, where an unknown swollen gel is the stationary phase. This is the basis of the ISEC, which is so often mentioned through this chapter [16,17,105,106]. 

In multi-residue analysis, an analyte is identified by its relative retention time, e.g., relative to aldrin when using ECD or relative to parathion or chlorpyrifos when using a flame photometric detection (FPD) and NPD. Such relative retention times are taken from corresponding lists for the columns used. Further evidence for the identity of an analyte is provided by the selectivity of the different detectors (Modules D1 to D3), by its elution behavior during column chromatography (Modules Cl and C2) and in some cases even by the peak form in a gas chromatogram. In a specific analysis for only some individual analytes, their retention times are compared directly with the corresponding retention times of the analytes from standard solutions. 

With high-fat materials and small Dump volumes, the CPC eluate may contain more than 0.5 g of lipids. This may seriously affect the elution behavior of the analytes when the CPC eluate is cleaned up further with Modules Cl and C2. 

Mori, S., Elution behavior of oligomers on a polyvinyl alccohol gel column with chloroform, methanol, and their mixtures, ]. Liq. Chromatogr., 11, 1205, 1988. 

A number of studies have reported the abilities of different eluting salts to produce both different resolution and different selectivities.17 23 As expected, polyvalent ions are stronger eluters than monovalent ions. Some studies have suggested a correlation between eluting ability and ranking in the Hofmeister series. Others fail to observe this correlation, but nevertheless note differences in elution behavior. Overall, it is impossible to predict which eluting ions, if any, may produce a... 

The relationship between molecular size and elution behavior can be used to estimate the molecular weight of an analyte. A calibration plot of log molecular weight (MW) vs. retention volume (or Ku) exhibits a linear segment between V, and Vi (Figure 7.1). If the plot is constructed with standard proteins with shapes similar to that of an analyte protein, the molecular weight of the analyte can be estimated by interpolation of its retention volume on the plot. The relationship between log MW and KD is linear for KD values between about 0.2 to 0.8. 

The chain lengths of different molecules were then compared with the chain lengths of hydrocarbons to calculate the carbon number which relates the elution volume of the compound to the elution volume of a real or imaginary n-hydrocarbon. The "effective" carbon number for benzene based on its elution volume was experimentally found to be 2.85 - i.e. it eluted near the elution volume for propane which has a carbon number of 3.0 while the calculations based on bond angles and radii of atoms indicated that benzene would have a carbon number of 3.55. Thus corrections to the calculations for carbon number were required. These were derived from the experimentally observed elution behavior of various molecules. 

The elution volumes for n-hydrocarbons show a straight line relationship vs the logarithms of their molar volumes. Molar volumes, calculated from the densities of compounds other than n-hydrocarbons, must be modified to have the elution volumes of these compounds conform to the same calibration line (elution volume vs log molar volume) as that for the n-hydrocarbons. W. W. Schulz (1 ) related the elution behavior of branched alkanes in the range of Cy-C] ] to the average numbers of gauche arrangements (Zg) which the molecule can assume. 

In summary, the approach outlined here is a straightforward method for determining representative values of viscosity ratios [ n ] MA /[ h ] LB I certainly g values significantly less than 1.0 are expected for such highly branched polymers (33). However, the anomalous dependence of g (v) on M[v1a suggests that 1) the core/shell hydrodynamic configuration and/or chromatographic artifacts invalidate universal calibration, and/or 2) the LB elution behavior does not conform to that of polystyrene in the assumed, constant manner. Further work is necessary to elucidate these points. 

It is essential that the eluent is inert and does not react with the sample components or the adsorbent. It must be borne in mind that aliphatic ketones like acetone or butanone easily undergo condensation reactions on active adsorbents and thus change the elution behavior ol he chromatographic system. r... 

Polyelectrolyte contraction can be followed by determining the Kj as a function of mobile phase ionic strength (104). In practice, however, the mobile phase ionic strength must be sufficiently high to ensure that the chain is in a contracted state. In this way, small changes in ionic strength, which may be inadvertently introduced during mobile phase preparation, will not affect the elution behavior of the sample. Also, if the ionic... 

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