Molecular weight and elution volume

A major part of the development of SEC in the study of the molecular mass distribution of polyethylene has been concerned with developing effective calibration procedures. This requires correlation of elution volume and molecular weight. There are, however, no well-characterized narrow-distribution linear polyethylene standards for determining the elution... 

All polycondensation reactions were followed by FT-IR-spectroscopy. Polyamide formation could be demonstrated by the disappearance of the ester C = 0 stretching mode and simultaneous formation of amide C = 0 stretching modes32>. Additional proof for polymer formation is given by gel permeation chromatography. From the elution volumes a molecular weight between 2,000 and 10,000 can be estimated. 

The calculation library includes programs to process all data encountered in normal GPC operation for standards and unknowns. It includes routines to decode data, correct for baseline drift, interpolate for flow variation, calculate mean elution volume, calculate molecular weight averages, and calculate corrected molecular weight averages and distribu-... 

By appropriate calibration, the chromatogram is converted to a tme (fingerprint) molecular weight distribution curve, from which all pertinent averages are easily calculated. The most reliable calibration is based on the relationship between the product [ft]M (intrinsic viscosity multiplied by molecular weight) and elution volume (or time), following Flor/s equation (3-7) which relates intrinsic viscosity to hydrodynamic volume and molecular weight. 

The calibration curve can be divided into three sections. In the first, there is no separation of the small molecules as they are all totally occluded within the pores of the gel and there is no separation. The second has an approximately linear variation of the elution volume with molecular weight over a wide range of elution volume. The columns separate the polymer molecule over this particular range of molecular weights very well. In the final section of the... 

The maximum concentrations which can be injected without peak broadening and change in elution volume are listed for various injection volumes and molecular weights in Table 4.1(d). These values vary with column characteristics and are not transferable, but they illustrate how the column loading must be substantially reduced as the molecular mass of the sample increases. It arises from the reduction in pores within the gel which separate higher-molecular-mass species, and also because of localized viscosity effects. 

The complexes were further purified by size-exclusion HPLC. In this way colorless material was removed, and from the elution volume a molecular weight of approximately 335 kDa could be estimated for both complexes. The height of the protein absorbance in the UV as compared to that of other pigment-protein complexes suggested a BChl g content of 10 - 15 % by weight, corresponding to about 40 BChls per complex. This would indicate that the complexes contain the antenna complement... 

The proteins are the p subuiut (O), the 35-kDa protein (A), the 9-kDa protein ( ), and the 6-kDa protein ( ).(V) is a plot of apparent molecular mass vs elution volume for molecular weight standards. 

Calibration of a packed column also presents difficulties when using SEC to characterize EOR polymers. No large molecular weight, monodispersed water-soluble polymer standards are available to relate elution volume to molecular weight. Consequently, the usual SEC calibration methods are not applicable and special calibration techniques must be applied. 

Typically, the elution of small molecules has been described as a linear function of log M and the elution volume, Vg. Molecular weight calibration curves obtained with members of a specific homologous series often give very good linearity with remaining members of the series, within the limit of the included volume of the column system used. An example of this application with oligosaccharides and several monosaccharides is shown in Figure 1. The predictive ability of such log M curves decreases as the solute studied... 

Today, data acquisition and processing are usually computer controlled. There are four transformations required of the raw chromatographic data to provide results as usually reported see Figure 3.20 (79) (a) conversion of elution time to elution volume, (b) conversion of elution volume to molecular weight, (c) conversion of detector response to polymer concentration, and (d) conversion of polymer concentration to weight fraction. Quantification of plate count and resolution, as well as calibration are discussed further in ASTM D5296-97 (89). 

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. 

Fig. 5-5. Size exclusion chromatography calibration curves, plots of elution volume versus molecular weight for a series of macroporous polystyrene materials designed for reversed phase chromatography and as base matrices for subsequent modification. Calibration performed using a 300 mm x 7.5 mm ID column and polystyrene standards with a tetra-hydrofuran eluent.

Traditionally, column efficiency or plate counts in column chromatography were used to quantify how well a column was performing. This does not tell the entire story for GPC, however, because the ability of a column set to separate peaks is dependent on the molecular weight of the molecules one is trying to separate. We, therefore, chose both column efficiency and a parameter that we simply refer to as D a, where Di is the slope of the relationship between the log of the molecular weight of the narrow molecular weight polystyrene standards and the elution volume, and tris simply the band-broadening parameter (4), i.e., the square root of the peak variance. 

The need for a viscosimeter capable of online measurement of the intrinsic viscosity of a polymer sample arose when it was shown that polymer molecules elute in order of decreasing hydrodynamic volume.81 The hydrodynamic volume can be related to the product of intrinsic viscosity and molecular weight. 

It was shown that the logarithm of the product of intrinsic viscosity and molecular weight of polymers of different chemical and stereochemical composition, configuration, or molecular weight is linearly related to the elution volume.81 A plot of log (r) M) vs. elution volume provides a single calibration curve, useful for samples of similar composition, and is termed a universal... 

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