Calibration, size exclusion

Anionic pol5Tnerizations make the molecular weight standards that are used to calibrate size-exclusion chromatographs. Equation (13.38) predicts PD = 1.001 at In = 1000. Actual measurements give about 1.05. The difference is attributed to impurities in the feed that cause terminations and thus short chains. Also, the chromatograph has internal dispersion so that a truly monodisperse sample would show some spread. Even so, a PD of E05 is extremely narrow by pol5Tner standards. This does not mean it is narrow in an... 

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

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. 

Quality assurance for size exclusion supports is based primarily on the reproducibility of molecular weight calibrations. Although the reproducibility of the exclusion and inclusion limits is important, the distribution coefficients (Ko) of included standards are a better indication of duplication. Table 10.3 (page 314) shows such data for the SynChropak GPC and CATSEC supports. 

Most size exclusion chromatography (SEC) practitioners select their columns primarily to cover the molar mass area of interest and to ensure compatibility with the mobile phase(s) applied. A further parameter to judge is the column efficiency expressed, e.g., by the theoretical plate count or related values, which are measured by appropriate low molar mass probes. It follows the apparent linearity of the calibration dependence and the attainable selectivity of separation the latter parameter is in turn connected with the width of the molar mass range covered by the column and depends on both the pore size distribution and the pore volume of the packing material. Other important column parameters are the column production repeatability, availability, and price. Unfortunately, the interactive properties of SEC columns are often overlooked. 

B. Broad Standard Size Exclusion Chromatography (SEC) Calibration... 

Column manufacturers normally provide basic information about their columns, such as plate count, particle size, exclusion limit, and calibration curve. This information is necessary and fundamental, however, it is not sufficient to allow users to make an intelligent decision about a column for a specific application. For example, separation efficiency, the dependence of separation efficiency on the mobile phase, the ability to separate the system peaks from the polymer peak, the symmetry of the polymer peak, and the possible interaction with polymers are seldom provided. 

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. 

In principle all methods except viscosity measurement can be used to obtain absolute values of molar mass. Viscosity methods, by contrast, do not give absolute values, but rely on prior calibration using standards of known molar mass. The relationship between polymer solution viscosity and molar mass is merely empirical but the techniques are widely used because of their simplicity. All of the absolute methods are time-consuming and laborious and are not used on a routine basis. As well as the techniques already mentioned, there is the size-exclusion method of chromatography known as Gel-Permeation Chromatography (GPC). All of these methods are discussed in detail in the sections that follow. 

Molecular weight distributions were examined by high performance size-exclusion chromatography (HPSEC) as described [22] on two serial Shodex OHpak KB-803 and KB-805 columns (0.8 x 30 cm Showa Denko, Japan) with a OHpak KB-800P guard column (0.6 x 5 cm), equilibrated at 1 mL/min in 0.1 M UNO3. The system was calibrated with a pullulan calibration kit (Showa Denko). 

The validity of a practical method of direct viscosity calculation from size exclusion chromatographic analysis is demonstrated. The method is convenient to use and is not limited by the availability of narrow MWD standards. It is possible to accurately measure polymer Mark-Houwink constants using the suggested broad standard SEC-[n] and SEC-MW calibration procedure. 

A new direct method for using size exclusion chromatography (SEC) to evaluate polymer intrinsic viscosity [n] is discussed. Sample viscosity information is obtained by combining SEC elution curve data and calibration data using direct SEC-[n] calibration procedures without involving polymer molecular weight calculations. The practical utility, convenience and the expected precision of the proposed method are illustrated. 

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). 

Molecular Weight and Peak Broadening Calibration in Size Exclusion Chromatography... 

Russell, D. J., Calibration of high performance size exclusion chromatography for small epoxy molecules, /. Liquid Chromatogr., 11, 383, 1988. 

Mourey, T. H., Miller, S. M., and Balke, S. T., Size exclusion chromatography calibration assessment utilizing coupled molecular weight detectors, /. Liq. Chromatogr., 13, 435, 1990. 

Nave, R., Weber, K., and Potschka, M., Universal calibration of size-exclusion chromatography for proteins in guanidinium hydrochloride including the high-molecular-mass proteins titin and nebulin,. Chromatogr. A, 654, 229, 1993. 

Lederer, K., Imrich-Schwarz, G., and Dunky, M., Simultaneous calibration of separation and axial dispersion in size exclusion chromatography coupled with light-scattering, J. Appl. Polym. Sci., 32, 4751, 1986. 

Wu, C., Simultaneous calibration of size exclusion chromatography and dynamic light-scattering for the characterization of gelatin, Macromolecules, 26, 5423, 1993. 

Dubin, P. L. and Principi, J. M., Failure of universal calibration for size exclusion chromatography of rodlike macromolecules vs. random coils and globular proteins, Macromolecules, 22, 1891, 1989. 

Frederiksson, H., Andersson, R., Koch, K., and Amon, P, Calibration of a size-exclusion chromatography system using fractions with defined amylopectin unit chains, /. Chromatogr. A, 768, 325, 1997. 

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