Size exclusion chromatography calibration

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

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... 

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). 

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. 

Figure 1. Size exclusion chromatography of Ru3(C0)i2 catalyzed polymerization of MeNH-[H2SiNMe]x-H as a function of time. Polystyrene standards used for calibration.

Standard Test Method for Molecular Weight Averages and Molecular Weight Distribution of Certain Polymers by Liquid Size-Exclusion Chromatography (Gel Permeation Chromatograph -GPC) Using Universal Calibration" ASTM D 3593 80, Annual Book of ASTM Standards, ASTM, Philadelphia, PA, revised annually. 

A viscosity online detector in a size exclusion chromatography (SEC) instrument allows for a universal calibration for polymers with known K- and a-values. For polymers that are only soluble at high temperature, e.g., polyolefines, high-temperature detectors are available, which can be operated up to 200°C. In addition to molar mass measurements, viscosity detectors have also been employed successfully to obtain structural information of branched polymers [28]. 

Molecular weight measurements were determined by size exclusion chromatography on a high pressure liquid chromatograph equipped with a differential refracto-meter. A Waters Styragel HR 5 i column set (106 104 500 A) was employed and calibrated with PS standards. THF was used as solvent at a flow rate of 1 ml/min. 

L. K. Kostanski, D. M. Keller, and A. E. Hamielec. Size-Exclusion Chromatography A Review of Calibration Methodologies. J. Biochem. Biophys. Meth., 58(2004) 159-186. 

Novolac molecular weights were measured in THF at 35°C by high pressure size exclusion chromatography using a Waters Model 510 pump (flow rate=1.0 ml/min), 401 differential viscometer detector and a set of Dupont PSM 60 silanized columns. A universal calibration curve was obtained with a kit of 10 narrow molecular weight distribution, linear polystyrene standards from Toya Soda Company. Data acquisition and analysis were performed on an AT T 6312 computer using ASYST Unical 3.02 software supplied with the Viscotek instrument. 

Size-exclusion chromatography (SEC) has been used to characterize the unimer-micelle distribution. However, SEC is not an absolute method and thus requires calibration. Since it is practically impossible to calibrate a SEC apparatus for the unimers and micelles formed by a block copolymer, only indicative MW values can be obtained. Moreover, several authors have noted a strong perturbation of the unimer-micelle equilibrium during SEC experiments even when interaction of the material with the SEC column was minimized [4,61,62],... 

Comparison of Size Exclusion Chromatography Calibration Techniques... 

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