Size calibration curve

An internal calibration method based on the universal calibration concept " was used to establish a hydrodynamic-size calibration curve which gave the relationship between the retention volume (V ) and the... 

Figure 5. Hydrodynamic Size Calibration Curve Constructed Based on the Chromatogram of PAAM.

When these calculatirns are performed for a range of particle sizes, calibration curves of phase difference versus particle size can be generated for any desired optical configuration and particle refractive index. It... 

Calibration curve for the determination of formula weight by size-exclusion chromatography. 

FIGURE 2.4 Calibration curve of dextran on Sephacryi S-300 SF. Calibration curves were calculated from one chromatogram of a broad MWD reference sample using data for the molecular mass distribution as obtained by a calibrated gel filtration column ( , upper curve) and on-line MALLS ( ). The calibration curve was found useful for estimating the size of globular proteins. [Reproduced from Hagel et al. (1993), with permission.]... 

The range of pore sizes in which TSK-GEL PW and TSK-GEL PWxi columns are available permits a wide spectrum of water-soluble substances to be analyzed. Calibration curves for polyethylene glycols chromatographed on... 

Polysaccharides TSK-GEL GMPWxl TSK-GEL GSOOOPWxl -I- TSK-GEL G3000PWxi Large pore size, linear calibration curve, small particles, high resolving power... 

Synthetic polymers TSK-GEL GMPWxi TSK-GEL GSOOOPWxl G3000PWxl Large pore size, low adsorption, linear calibration curve... 

H type resins are available in different pore sizes. Examples of calibration curves for polystyrene standards are shown in Figs. 4.38 and 4.39. Other series of H type columns have similar calibration curves. Exclusion limits are listed in Tables 4.12-4.16. 

Another important parameter for column selection is the proper choice of sorbent porosity. The pore size of the sorbent determines the fractionation range of the column. The best way of doing this is by looking at the calibration curves of the columns, which are normally documented by the column vendor (cf. Fig. 9.3 for PSS SDV column calibration curves and PSS SDV fractionation ranges) (7). 

Figure 9.4 illustrates a simple way of selecting the best column for SEC work based on the calibration curves of two sorbents with different pore sizes (4). 

The linear column (PSS SDV 5 /mm linear) has a wider molar mass fractionation range while keeping the analysis time roughly the same. Therefore the slope of the calibration curve is much steeper and the resolution will be poorer in this case. The second column with a single pore size (PSS SDV 5 /mm 1000 A) separates only below 50,000 Da, but does this very efficiently in the same time. 

All packing materials produced at PSS are tested for all relevant properties. This includes physical tests (e.g., pressure stability, temperature stability, permeability, particle size distribution, porosity) as well as chromatographic tests using packed columns (plate count, resolution, peak symmetry, calibration curves). PSS uses inverse SEC methodology (26,27) to determine chromatographic-active sorbent properties such as surface area, pore volume, average pore size, and pore size distribution. Table 9.10 shows details on inverse SEC tests on PSS SDV sorbent as an example. Pig. 9.10 shows the dependence... 

A range of individual pore size PLgel packing materials is produced and their pore size distribution is conveniently represented by a SEC calibration curve as illustrated in Fig. 12.1. It should be pointed out that the descriptors used for the different pore sizes, 50 A, 100 A, and so on, are not the actual pore sizes of the beads but relate to the size of a polystyrene molecule just excluded from the packing material. This nomenclature comes from the original work carried out by Moore (3) and should only be viewed in the context of differentiating... 

FIGURE 12.1 SEC calibration curves for PLgel individual pore size columns (300 X 7.5 mm), eluent THE at 1.0 ml/min, polystyrene calibrants. 

Individual pore size columns have variable pore volume, and because the column dimensions are fixed, the combination of different columns must result in variable slope of the overall calibration curve and hence variable degrees of resolution as a function of molecular weight. 

The user must have a good understanding of the individual calibration curves for each individual pore size column in order to avoid mismatching them, which can result in artifacts in polymer distributions. 

The main criterium for column selection is pore size distribution as it is desirable to have maximum pore volume for separation in the molecular weight range of interest. Having determined the upper molecular weight limit required, a column with a suitable exclusion limit should be selected. In the case of individual pore size columns, it is then a question of selecting other columns with complementary calibration curves to comprise a column set covering the re-... 

With these facts in mind, it seems reasonable to calculate the pore volume from the calibration curve that is accessible for a certain molar mass interval of the calibration polymer. A diagram of these differences in elution volume for constant M or AM intervals looks like a pore size distribution, but it is not [see the excellent review of Hagel et al. (5)]. Absolute measurements of pore volume (e.g., by mercury porosimetry) show that there is a difference on principle. Contrary to the absolute pore size distribution, the distribution calcu-... 

The reason for such difficulties is the GPC mechanism itself. We do not separate by molar mass but by the size of the solvated molecules. Different solvation of chemical unlike molecules results in breaking the M sequence of the calibration curve this becomes visible especially in the low molar mass range. Sometimes such difficulties can be circumvented if a specific detector is used, e.g., if the sample absorbs in the ultraviolet (UV) range and the disturbing peaks are UV transparent. 

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