Wolfgang Radke

Polymer separations by liquid interaction chromatography: Principles – prospects – limitations

Most heterogeneities of polymers with respect to different structural features cannot be resolved by only size exclusion chromatography (SEC), the most frequently applied mode of polymer chromatography. Instead, methods of interaction chromatography became increasingly important. However, despite the increasing applications the principles and potential of polymer interaction chromatography are still often unknown to a large number of polymer scientists. The present review will explain the principles of the different modes of polymer chromatography. Based on selected examples it will be shown which separation techniques can be successfully applied for separations with respect to the different structural features of polymers.

Determination of the DS distribution of non-degraded sodium carboxymethyl cellulose by gradient chromatography

Two series of sodium carboxymethyl celluloses (NaCMCs) derived from microcrystalline cellulose (Avicel samples, DP~160) and cotton linters (BWL samples, DP~1400) with average degrees of substitution in the range DS=0.45-1.55 were analyzed by gradient liquid adsorption chromatography (gradient LAC) in order to determine their chemical composition distributions (DS distributions or 1st order heterogeneities). Clear separations of samples having different average DS values were achieved for both sample series, allowing determination of the DS distributions of the samples. A slight molar mass influence on the eluent composition at elution was observed. From the DS distributions the DS standard deviations were calculated and taken as a measure for the extent of chemical heterogeneity of the single samples. While no noticeable dependence of the chemical heterogeneity on average DS was observed for Avicels, the heterogeneity decreases with increasing average DS for BWLs.

Molar mass characterization of sodium carboxymethyl cellulose by SEC-MALLS

Two series of sodium carboxymethyl celluloses (NaCMCs) derived from microcrystalline cellulose (Avicel samples) and cotton linters (BWL samples) with average degrees of substitution (DS) ranging from DS=0.45 to DS=1.55 were characterized by size exclusion chromatography with multi-angle laser light scattering detection (SEC-MALLS) in 100 mmol/L aqueous ammonium acetate (NH4OAc) as vaporizable eluent system. The application of vaporizable NH4OAc allows future use of the eluent system in two-dimensional separations employing evaporative light scattering detection (ELSD). The losses of samples during filtration and during the chromatographic experiment were determined. The scaling exponent as of the relation [Formula: see text] was approx. 0.61, showing that NaCMCs exhibit an expanded coil conformation in solution. No systematic dependencies of as on DS were observed. The dependences of molar mass on SEC-elution volume for samples of different DS can be well described by a common calibration curve, which is of advantage, as it allows the determination of molar masses of unknown samples by using the same calibration curve, irrespective of the DS of the NaCMC sample. Since no commercial NaCMC standards are available, correction factors were determined allowing converting a pullulan based calibration curve into a NaCMC calibration using the broad calibration approach. The weight average molar masses derived using the so established calibration curve closely agree with the ones determined by light scattering, proving the accuracy of the correction factors determined.

Application of two-dimensional chromatography to the characterization of macromolecules and biomacromolecules.

AbstractModern polymeric materials are heterogeneous with respect to different structural features, for instance molar mass, composition, and architecture. One-dimensional separation methods such as size-exclusion chromatography (SEC) are insufficient to fully resolve the multidimensional distributions of such complex materials. Therefore, two-dimensional separation methods are increasingly employed to characterize macromolecules. The present article describes in detail the advantages and experimental aspects of two-dimensional macromolecular separations. Selected examples will be discussed to explain the strategies used to separate macromolecules with respect to specific structural features. Graphical AbstractSchematic representation of a two-dimensional chromatogram of a four component mixture

Predicting the chromatographic retention of polymers: Application of the polymer model to poly(styrene/ethylacrylate)copolymers

The retention behavior of a range of statistical poly(styrene/ethylacrylate) copolymers is investigated, in order to determine the possibility to predict retention volumes of these copolymers based on a suitable chromatographic retention model. It was found that the composition of elution in gradient chromatography of the copolymers is closely related to the eluent composition at which, in isocratic chromatography, the transition from elution in adsorption to exclusion mode occurs. For homopolymers this transition takes place at a critical eluent composition at which the molar mass dependence of elution volume vanishes. Thus, similar critical eluent compositions can be defined for statistical copolymers. The existence of a critical eluent composition is further supported by the narrower peak width, indicating that the broad molar mass distribution of the samples does not contribute to the retention volume. It is shown that the existing retention model for homopolymers allows for correct quantitative predictions of retention volumes based on only three appropriate initial experiments. The selection of these initial experiments involves a gradient run and two isocratic experiments, one at the composition of elution calculated from first gradient run and second at a slightly higher eluent strength.

Size exclusion chromatography-gradients, an alternative approach to polymer gradient chromatography: 2. Separation of poly(meth)acrylates using a size exclusion chromatography-solvent/non-solvent gradient.

A gradient ranging from methanol to tetrahydrofuran (THF) was applied to a series of poly(methyl methacrylate) (PMMA) standards, using the recently developed concept of SEC-gradients. Contrasting to conventional gradients the samples eluted before the solvent, i.e. within the elution range typical for separations by SEC, however, the high molar mass PMMAs were retarded as compared to experiments on the same column using pure THF as the eluent. The molar mass dependence on retention volume showed a complex behaviour with a nearly molar mass independent elution for high molar masses. This molar mass dependence was explained in terms of solubility and size exclusion effects. The solubility based SEC-gradient was proven to be useful to separate PMMA and poly(n-butyl crylate) (PnBuA) from a poly(t-butyl crylate) (PtBuA) sample. These samples could be separated neither by SEC in THF, due to their very similar hydrodynamic volumes, nor by an SEC-gradient at adsorbing conditions, due to a too low selectivity. The example shows that SEC-gradients can be applied not only in adsorption/desorption mode, but also in precipitation/dissolution mode without risking blocking capillaries or breakthrough peaks. Thus, the new approach is a valuable alternative to conventional gradient chromatography.

Characterization of sodium carboxymethyl cellulose by comprehensive two-dimensional liquid chromatography

Two series of sodium carboxymethyl celluloses (NaCMC) with average degrees of substitution (DS) ranging from 0.45 to 1.55 were synthesized from low molecular mass Avicel cellulose (Avicel samples) and from high molecular mass cotton linters (BWL samples). The samples were characterized by online two-dimensional liquid chromatography using gradient liquid adsorption chromatography in the first and size exclusion chromatography (SEC) in the second dimension. This method allows the simultaneous determination of the chemical composition (DS) and the molar mass distribution within the individual samples. Moreover information was obtained on the dependence of the elution volume in gradient chromatography on molar mass. As expected, evidence for a stronger influence of molar mass on gradient elution volume was found for the low molecular mass NaCMC as compared to the high molecular mass BWL samples. Finally the applicability of the method for the simultaneous separation of blends heterogeneous with respect to chemical composition (DS) and molar mass was demonstrated. Such blends cannot be efficiently separated by either SEC or gradient chromatography alone, nor by simply combining the results of both methods. Only the complete two-dimensional chromatogram can reveal the complexity of such blends, since it reveals the correlations between molar mass and chemical composition.

Critical chromatography in ternary solvents

The behaviour of PMMA standards in mixtures composed of two binary critical eluents was investigated. It was found that combinations of two critical eluents do not result in a molar mass independent elution (critical conditions). When mixing two binary critical eluents, elution might occur in SEC, LAC or close to critical conditions in a non-predictable fashion. It can be assumed that the deviation from critical conditions is caused by preferential adsorption of some eluent components to the stationary phase or the polymer. In order to have a fast estimate, in which mode of chromatography elution will occur when mixing two critical eluents, ternary critical conditions were estimated from gradient experiments. A good agreement was observed between the so obtained critical lines and the elution behaviour resulting in mixtures of two critical eluents.

Characterization of cellulose acetates according to DS and molar mass using two-dimensional chromatography

A two-dimensional liquid chromatographic method (2D LC) was developed to analyze the heterogeneities of cellulose acetates (CA) in the DS-range DS=1.5-2.9 with respect to both, molar mass and degree of substitution (DS). The method uses gradient liquid chromatography (HPLC) as the first dimension in order to separate by DS followed by separation of the different fractions by size (SEC) in the second dimension. The 2D experiments revealed different correlations between gradient and SEC elution volume. These correlations might arise from differences in the synthetic conditions. The newly developed 2D LC separation therefore provides new insights into the heterogeneity of CAs.

Determination of molar masses of macromolecules by size exclusion chromatography-light scattering not requiring knowledge of refractive index increments

A new approach for the calibration of SEC-light scattering (SEC-LS) setups is proposed, which requires solely the molar mass of a reference polymer. Neither the specific refractive index increment of the calibrant nor of the analyte is required. Comparison of the molar masses derived in different solvents for a large number of chemically different polymers shows that the new approach yields the same molar masses as if molar masses were derived using dn/dc to calibrate the light scattering setup. The approach therefore allows easier determination of molar masses by SEC-LS.