Elution volumes calibration

Elution volume calibrations were performed using radioactive tracers of the rare earth elements and 133Ba, with atomic-absorption or flame-emission analysis of iron, sodium, potassium, calcium, and magnesium. As shown in Fig. 5.14, any barium added to the second columns is eluted at the start of the light rare earth element fraction . To ensure barium removal the sample can be put through the first column again. 

The function can then be interpolated for particle sizes between those corresponding to the known latices. The relationship between particle diameter and y, the mean elution volume of the particles, is the particle size versus elution volume calibration. As discussed earlier, an HDC is calibrated using polystyrene latices, but the calibration is valid for other latices (23), and even nonlatex materials. (Colloidal gold that had been sized at 40 nm by electron microscopy was detected at 39 nm using the FlowSizer.)... 

Calibration Samples. Monodisperse polystyrene latices are available with known, narrow particle size distributions. Coefficients of variance about the mean diameter are typically less than 6% of diameter measured using electron microscopy (25). HDC typically cannot resolve differences in diameter of only 6%. Therefore, these polystyrenes are sufficiently narrow to be used as HDC calibration reference samples. However, doing so may result in incorporation of a systematic error in the particle size versus elution volume calibration, arising from known electron microscopy errors of as much as 5% for particles below 1 um (26). Therefore, accuracy can only be stated as relative to electron microscopy results for the calibration samples. FlowSizer performance specifications have been reported elsewhere (27) with diameter and mass percent results within 5% of those determined by electron microscopy for a series of these monodisperse polystyrene latices. 

This equation describes the elution volume calibration curve for Mx- The elution volume (Vg) that corresponds to a GPC peak in the unknown polymer is used to obtain a value of log Mg from the polystyrene standard curve (Fig. 4.27) that has been obtained in the same column and solvent, and Mx is then calculated from Eq. (4.149). An alternative procedure is simply to choose a number of values of Vg and construct a new calibration curve for the polymer under study from the standard curve such as Fig. 4.27 and Eq. (4.143). 

Construct an elution volume calibration curve for poly(vinyl bromide) in tetrahydrofuran at 25°C, given that the MHS constants for this system are K = 1.59 x 10 cm /g and a = 0.64 [A. Ciferri, M. Kryezewski, and G. Weil, J. Polym. ScL, 27,167 (1958)], and calculate the weight average molecular weight of the polymer. 

Another approach to determining the interdetector volume of a viscometer is first to establish an [t ] versus elution volume calibration curve using a series of narrow polymer standards of known intrinsic viscosities. A broad molecular weight standard is then injected and the interdetector volume is adjusted to obtain superimposition of the intrinsic viscosity calibration curve (37). 

With a light-scattering detector, a log M versus elution volume calibration curve is constructed from a series of narrow molecular weight distribution polymer standards. A broad molecular weight distribution standard is then injected, and an iterative procedure finds the interdetector volume that superimposes the broad MWD standard calibration curve onto the one established by the narrow standards (38). 

Figure 4.1 Instrumental broadening of the chromatograms polystyrene standard reference materials. (A) Effect of solution concentration on observed chromatogram Mp = 1.43 x 10. Concentrations (a) 0.50 (b) 0.20 (c) 0.05 (d) 0.02 wt vol" %. 10 fim particles mixed gel (PS/DVB) column, 60 cm long (Polymer Lab. Ltd.) flow rate 1 cm min" 140°C ODCB mobile phase RI detector. (B) Separation of the individual oligomers by degree of polymerization, from 3 to 9. Mp = 580. Concentration 0.2 wt vol" %, flow rate 1 cm min" 140°C ODCB mobile phase RI detector. 2 x 10 /zm columns (PS/DVB gel) 50 and lOOA. (C) Polystyrene molecular weight/elution volume calibration curve. Experimental conditions as in (A).

B) Cumulative mass distribution against elution volume. (Q Molecular mass/elution volume calibration curve, determined by compounding (B) with the fractionation mass distribution [10]. 

Similarly, Ogawa et al. [40] obtained a molecular mass-elution volume calibration curve for polypropylene from a plot of log(My) against elution volume curve for fractionated polystyrene. The relations [rj i M against elution volume and were compared. When both curves were converted into... 

The primary information obtained from SEC is not the molar mass, but the apparent concentration at an elution volume. Only with the matching SEC calibration and the concentration profile from the concentration detector can the molar mass average and the MMD be obtained. Size exclusion chromatography is therefore a relative method if no absolute detection is employed [1]. The SEC calibration is based on assigning a molar mass to an elution volume (calibration of x-axis). This is in contrast to HPLC, where the detector response (signal intensity and peak area) is calibrated and assigned to a concentration (calibration of y-axis). 

Calibrate the system. Use narrowly dispersed molecular weight standards of the polymer of interest to construct a calibration curve of log molecular weight versus elution volume (Eig. 3.2). If a more sophisticated software system is available, a broad molecular weight standard may be used to calibrate the system. 

FIGURE 4.43 Calibration curves for globular proteins on toyopearl resins. Column 22 mm X 30 cm. Sample Protein standards. Elution 0.06 A1 phosphate buffer, pH 7, in 0.06 A1 KCI. Legend elution volume V column volume. 

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-... 

In SEC, universal calibration is often utilized to characterize a molecular weight distribution. For a universal calibration curve, one must determine the product of log(intrinsic viscosity molecular weight), or log([7j] M). The universal calibration method originally described by Benoit et al. (9) employs the hydro-dynamic radius or volume, the product of [tj] M as the separation parameter. The calibration curves for a variety of polymers will converge toward a single curve when plotted as log([7j] M) versus elution volume (VJ, rather than plotted the conventional way as log(M) versus V, (5). Universal calibration behavior is highly dependent on the absence of any secondary separation effects. Most failures of universal calibration are normally due to the absence of a pure size exclusion mechanism. 

The most widely used molecular weight characterization method has been GPC, which separates compounds based on hydrodynamic volume. State-of-the-art GPC instruments are equipped with a concentration detector (e.g., differential refractometer, UV, and/or IR) in combination with viscosity or light scattering. A viscosity detector provides in-line solution viscosity data at each elution volume, which in combination with a concentration measurement can be converted to specific viscosity. Since the polymer concentration at each elution volume is quite dilute, the specific viscosity is considered a reasonable approximation for the dilute solution s intrinsic viscosity. The plot of log[r]]M versus elution volume (where [) ] is the intrinsic viscosity) provides a universal calibration curve from which absolute molecular weights of a variety of polymers can be obtained. Unfortunately, many reported analyses for phenolic oligomers and resins are simply based on polystyrene standards and only provide relative molecular weights instead of absolute numbers. 

From this it follows that plotting a graph of log M[7]] against elution volume should give a straight line for all polymer-solvent combinations. This was shown by Benoit et al to be true for a large number of systems and is referred to as the universal calibration curve. It is illustrated in Figure 6.6. 

Up to this point, all chromatographic information is in terms of elution volume. If a calibration file is available to the program, the chromatogram may be converted to log hydrodynamic volume. The chromatogram in terms of log hydrodynamic volume may be written to a file (with extension HYD) for later use. 

Size exclusion chromatography (SEC) separates molecules of a polymer sample on the basis of hydrodynamic volume. When the chromatograph is equipped only with a concentration-sensitive detector, i.e. conventional SEC, a molecular weight distribution (MWD) can be obtained from the chromatogram only through use of a calibration function relating molecular weight and elution volume V (2). 

This behavior shows that the dimensions of these polymers are independent of pH, ionic strength (in the ranges studied) and presence or absence of Tergitol or polyethyleneoxide. This result is of considerable help in interpretation of GPC behavior since in the absence of polymer-glass substrate interactions, the molecular weight calibration curves (log MW vs. elution volume) should be independent of pH, ionic strength or the two nonionic surfactants investigated. 

For investigation of GPC behavior, a series of M.W. calibration curves (log MW vs. elution volume) was obtained for a number... 

In the presence of polyethylene oxide MW 300,000 at a concentration of 0.025 g liter , variations in pH and ionic strength have no effect on elution volumes and a single calibration curve is obtained as shown in Figure 4 and Table II. This behavior presumably also results from modification of the glass surface by the polyethylene oxide surfactant, but in this case charge effects appear to be completely suppressed and the effective pore diameter and volume reduced. Such an interpretation is also in accord with the fact that the elution voliomes are lower with polyethylene oxide than with Tergitol, since Tergitol is a much smaller molecule than the polyethylene oxide. 

Experimental variables such as temperature, flow rate, sample concentration and mobile phase composition can cause changes in the elution volume of a polymer [439,457,460-464]. Chromatographic measurements made with modem equipment are limited more by the errors in the absolute methods used to characterize the molecular weight of the calibration standards than any errors Inherent in the measurements themselves, since the determination of molecular weights by SEC is not an absolute method and is dependent on calibration [462]. The Influence of temperature on retention in SEC is not very great, since no strong sorptive interactions are involved in the retention mechanism. Temperature differences between the column and solvent delivery... 

---