Molecular-mass calibration chromatography

The molecular masses of polygalacturonase and exopolygalacturonase were approximately determined by gel chromatography on Superose 12 using FPLC device (Pharmacia, Sweden) and the Calibration proteins II kit (Boehringer-Mannheim, Germany). 

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. 

A polymer, even in its pure state, corresponds to a distribution of macromolecules with different masses. Exclusion chromatography can determine the distribution in molecular weights and the most probable mass and the mean mass. For this type of application, a calibration curve is made using macromolecules of known masses by plotting the retention times (or volumes) on the abscissa and the logarithms of the molecular masses on the ordinate. As can be seen in Fig. 7.5, a linear relationship is obtained. Using this graph, an approximate mass of the unknown can be determined by use of the retention time (volume). This assumes that the mass and the molecular volumes are directly related. 

A series of reference proteins of known molecular masses are used to calibrate the column and Mr for an unknown protein is estimated from its position on the graph.195,196 Another modification of the method depends upon chromatography in a high concentration of the denaturing salt guanidinium chloride. The assumption is made that proteins are denatured into random coil conformations in this solvent.196... 

Size exclusion chromatography is the premier polymer characterization method for determining molar mass distributions. In SEC, the separation mechanism is based on molecular hydrodynamic volume. For homopolymers, condensation polymers and strictly alternating copolymers, there is a correspondence between elution volume and molar mass. Thus, chemically similar polymer standards of known molar mass can be used for calibration. However, for SEC of random and block copolymers and branched polymers, no simple correspondence exists between elution volume and molar mass because of the possible compositional heterogeneity of these materials. As a result, molar mass calibration with polymer standards can introduce a considerable amount of error. To address this problem, selective detection techniques have to be combined with SEC separation. 

Gel permeation chromatography (GPC) is the established method for the determination of molar mass averages and the molar mass distributions of polymers. GPC retention is based on the separation of macromolecules in solution by molecular sizes and, therefore, requires a molar mass calibration to transform elution time or elution volume into molar mass information. This kind of calibration is typically performed with narrow molecular mass distribution polymer standards, universal, or broad calibration methods or molar-mass-sensitive detectors like light-scattering or viscosity detectors. 

MALDI (matrix assisted laser desorption and ionization) combined with size-exclusion chromatography is increasingly being used to study the microstructure of polymers [542-544], MALDI allows the desorption and ionization of macromolecules with molecular mass up to hundreds of kilodaltons with little or no fragmentation. The MALDI mass spectra provides structural information for identification of the polymer repeat units or copolymer sequence, end group mass and mechanism of polymerization for polymers with a narrow polydispersity (< 1.25). For samples of high polydispersity MALDI fails to provide reliable information. The separation of polydisperse samples by size-exclusion chromatography provides fractions suitable for characterization by MALDI. MALDI can also be used for mass calibration of size-exclusion columns. 

Thus calibration of the apparatus with, for example, polystyrene standards with known values of K and a in the particular solvent at a particular temperature permits the absolute molecular mass to be calculated via the universal calibration method [9]. A further advantage of the detector is its ability to be used at temperatures up to 150°C, thus permitting the analysis of polyolefins. The study of branching in polymers is also possible with this system. This new detector probably represents the most significant advance in detectors for size exclusion chromatography in recent years. 

The pumps now available for liquid chromatography applications are very consistent in their flow delivery, but a very small error in the flow rate can lead to a much more significant error in the calculated average molecular masses. Consequently, the use of an internal marker for a flow rate correction plays an essential role in calibrating an SEC system and in the subsequent calculation of results. 

Figure 23-11 Calibration curve used to estimate molecular mass of an unknown protein by molecular exclusion chromatography.

At its simplest level, MS measures molecular masses. With calibration it can also determine quantities on a relative or absolute scale for pure compounds and, with varying degrees of success, for components in a mixture. The analysis of complex mixtures such as a protein digest may require coupling with a separative method such as chromatography (GC-MS or LC-MS) or electrophoresis, either off-line (SDS-PAGE) or on-line (CE-MS). Eurther experiments can provide detailed structural information, e.g. peptides... 

Gel permeation chromatography (GPC) analysis of PHA polymers. Polymers were isolated from cells as described previously (4) and molecular mass data of polyesters were obtained by GPC analysis using a Shimadzu lOA system fitted with a RID-IOA refractive-index detector with serial columns of ShodexK802 and K806M as described previously (6). Polystyrene standards with a low polydispersity were used to make a calibration curve. 

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