Molecular size-elution volume relationships

Three main parameters have been considered for small molecules molecular mass, molecular chain length and molecular volume. Molecular mass is clearly of relatively limited value, but is the simplest parameter. 

Hendrickson and Moore [11] studied the behaviour of small molecules in THF, and concluded that five basic forces might be expected to modify the elution volume of compounds apart from molecular size  

These workers initially calculated the effective linear chain lengths (A) of molecules from the atomic radii and bond angles of the structural components of the molecule. This simple model was inadequate, and chain lengths were therefore also expressed in terms of carbon numbers. However, empirical correction factors were derived experimentally to bring certain types of compounds into agreement with the calibration plot derived from n-alkanes. 

A detailed, more recent study is given by Ogawa and Sakai [18] for the elution behaviour of small molecules in chloroform on two TSK G2000 Hg columns at a flow rate of ImLmin . Log o molar volume was plotted 

An extreme example of anomalous behaviour is that of sulphur, which is retained beyond the point of total permeation. This led to the proposal that sulphur should be used as an added internal standard in polymer characterization [18]. 

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Gel permeation chromatograms actually give information about molecular size. For any polymer, size is determined hy a number of factors. These include not only molar mass but also temperature and thermodynamic quality of the solvent. Hence the relationship between size and molar mass is unique for each particular polymer-solvent combination, and we caimot assume that because two peaks of different polymers, even in the same solvent at the same temperature, have the same elution volume their molecules have the same molar mass. 

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. 

Figure3.13 Typical relationship between molecular weight and elution volume of polymers on a size-exclusion column. Adapted from Miller (1988).

Fig. 4. Nucleotide-dependent self-association. Size exclusion chromatography serves to estimate the molecular size of the proteins. The logarithm of the molecular weight is plotted versus the elution volume of seven different marker proteins ( ) and a linear relationship is fitted. Then, in six seperate experiments the elution volume of hGBPI is determined in the presence of different nucleotides (O), from left to right, GDP.AlFx, GTP, GppNHp, GMP, GDP, and without any nucleotide. On the basis of the calibration line the elution volume allows to estimate the size of the assemblies. The three arrows each indicate the groups of tetramer, dimmer, and monomer hGBPI.

Low Molecular Weight Compounds. GPC may be used for low molecular weight compounds since efficient gels with small pore sizes are available. Since these gels are less cross-linked than the regular ones, they are more sensitive to swelling. This property r tricts the choice of solvents. In this case, elution volumes are less controlled by steric exclusion mechanism and the relationship between molecular si and molecular weight is not well-defined and depends on... 

It should be noted that the relationships between molar mass and retention volume for lignin sulfonates shown in Figures 3 and 4 are strictly only valid for the samples studied in these experiments because lignin sulfonates are polyelectrolytes and thus interact with each other and with the gel matrix of the column. The shape of the calibration curve is thus affected by, among other things, the size and concentration of the sample (2). Interactions between molecular species can be eliminated by eluting with a suitable electrolyte. 

In SEC, mass is not measured so much as the hydrodynamic volume of the polymer molecules, that is, how much space a particular polymer molecule takes up when it is in solution. However, the approximate molecular weight can be calculated from SEC data because the exact relationship between molecular weight and hydrodynamic volume for polystyrene can be found. For this, polystyrene is used as a standard. But the relationship between hydrodynamic volume and molecular weight is not the same for all polymers, so only an approximate measurement can be arrived at. Another drawback is the possibility of interaction between the stationary phase and the analyte. Any interaction leads to a later elution time and thus mimics a smaller analyte size. 

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