Molecular-weight resolution

The use of hexafluoroisopropanol (HFIP) as an SEC eluent has become popular for the analysis of polyesters and polyamides. Conventional PS/DVB-based SEC columns have been widely used for HFIP applications, although the relatively high polarity of HFIP has led to some practical difficulties (1) the SEC calibration curve can exhibit excessive curvature, (2) polydisperse samples can exhibit dislocations or shoulders on the peaks, and (3) low molecular weight resolution can be lost, causing additive/system peaks to coelute with the low molecular weight tail of the polymer distribution... 

Minimum molecular weight resolution is slightly more difficult to calculate than resolution and it may be of no greater value in daily testing. Nonetheless, it should be standard practice to determine the void volume, pore volume, and calibration curve for any size exclusion column that is used in the laboratory. Thereafter, the slope (m) can be calculated and the minimum molecular weight resolution easily computed. 

Molecular Weight Precision per Time. Several techniques are available for measurement of molecular weight of polymers. SEC and CZE are based on the separation of analytes and are related to e technique reported in this study. In process analysis, the analysis time and molecular weight resolution are two important parameters for consideration. In contrast to the resolution defined in separation techniques such as SEC and CZE, but identical to that used in mass spectromet (40), we define molecular weight resolution, R, as given by equation 1... 

While SEC and CZE are capable of physically resolving components, and FIA is not, the point made in Table I is that at a given Mi the reliability of a molecular weight determination improves with increasing resolution per time, Rs/t (equations 1 and 2). In addition, SEC is limited by pore size for the molecular weight resolution of analytes, as well as polymer/solvent interactions that will affect the hydrodynamic radius of the polymer, while CZE experiences the artifact of non-ideal flow rate. However, FIA-based methods operate at zero back pressure, with a lower probability of clogging than most sample modulated analyzers such as S. Further, IA with G detection provides a means for the analysis of mixtures using multivariate statistical techniques (24). 

Amine-terminated EO/PO colpoly-mers, MW 900,2000,4000 elution in order of increasing molecular weight resolution of... 

All have molecular weights of 226 to the nearest integer (C = 12, H = 1, S = 32), but the exact molecular weights differ slightly. A resolution of 2500 is necessary to separate molecules 1, 2 and 3 but 75,000 is required to separate molecule 4 from molecule 3 which explains why high resolution mass spectrometers are sdiiglit. 

Analytical Approaches. Different analytical techniques have been appHed to each fraction to determine its molecular composition. As the molecular weight increases, complexity increasingly shifts the level of analytical detail from quantification of most individual species in the naphtha to average molecular descriptions in the vacuum residuum. For the naphtha, classical techniques allow the isolation and identification of individual compounds by physical properties. Gas chromatographic (gc) resolution allows almost every compound having less than eight carbon atoms to be measured separately. The combination of gc with mass spectrometry (gc/ms) can be used for quantitation purposes when compounds are not well-resolved by gc. 

Figure 18.3 Protein crystals contain large channels and holes filled with solvent molecules, as shown in this diagram of the molecular packing in crystals of the enzyme glycolate oxidase. The subunits (colored disks) form octamers of molecular weight around 300 kDa, with a hole in the middle of each of about 15 A diameter. Between the molecules there are channels (white) of around 70 A diameter through the crystal. (Courtesy of Ylva Lindqvist, who determined the structure of this enzyme to 2.0 A resolution in the laboratory of Carl Branden, Uppsala.)...

Molecular weight assignments for such resins will be dependent on the method used for measurement. Each manufacturer has his own relative scale. In practice, absolute molecular weight determination is not important. The method used must discriminate between resins with sufficient resolution to allow prediction of resin performance in the applications of interest. 

Traditionally, Sephadex has been used for molecular weight analyses of proteins (Determan and Brewer, 1975) and clinical dextran (Granath and Kvist, 1967). Today, Sepharose, Sephacryl, or Superose is used for the assay of broad MWDs. Superdex is the premiere choice for attaining the highest resolution of components of similar size (see Fig. 2.1). 

Errors in the molecular weight data from HPSEC are usually due to improperly prepared samples, column dispersity, or flow rate variations. The sample to be analyzed should be completely dissolved in the mobile phase and filtered prior to injection onto the column. A plugged column inlet frit will invalidate results. In addition, do not load the column with excess sample. Column overloading affects the accuracy of data by broadening peaks, reducing resolution, and increasing elution volume. For best results, the concentration of the injected sample should be as low as possible while still providing adequate... 

As with other size-exclusion techniques, the pore size of the selected Zorbax GF column should provide resolution over the molecular size range of the proteins that are to be separated. The Zorbax GF-250 column separates proteins in the range of 4000 to 400,000 Da. The Zorbax GF-450 provides separation over the range of 10,000 to 1,000,000 Da. When these two columns are coupled, they can be used to separate proteins with molecular weights of 4000 to 1,000,000. 

Small particle size resins provide higher resolution, as demonstrated in Fig. 4.41. Low molecular weight polystyrene standards are better separated on a GIOOOHxl column packed with 5 /u,m resin than a GlOOOHg column packed with 10 /Ltm resin when compared in the same analysis time. Therefore, smaller particle size resins generally attain a better required resolution in a shorter time. In this context, SuperH columns are best, and Hhr and Hxl columns are second best. Most analyses have been carried out on these three series of H type columns. However, the performance of columns packed with smaller particle size resins is susceptible to some experimental conditions such as the sample concentration of solution, injection volume, and detector cell volume. They must be kept as low as possible to obtain the maximum resolution. Chain scissions of polymer molecules are also easier to occur in columns packed with smaller particle size resins. The flow rate should be kept low in order to prevent this problem, particularly in the analyses of high molecular weight polymers. 

The sample concentration also should be kept as low as possible, particularly in analyses of polymers on columns packed with small particle size resins. The maximum sample concentration to achieve maximum resolution decreases as the sample molecular weight becomes higher and the resin particle size becomes smaller. It is usually in the range of 0.05-5 mg/ml, depending on the sample molecular weight and resin particle size. 

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