Liquid chromatography of polymers under limiting conditions of desorption II. Tandem injection and quantitative molar mass determination

Liquid chromatography under limiting conditions of desorption (LC LCD) is a method which allows molar mass independent elution of various synthetic polymers. A narrow, slowly moving zone of small molecules, which promotes full adsorption of one kind of polymer species within column (an adsorli) acts as an impermeable barrier for the fast moving macromolecules. The latter accumulate on the barrier edge and elute nearly in total volume of liquid within column. At the same time, transport of less adsorptive macromolecules is not hampered so that these are eluted in the size exclusion (SEC) mode. As result, polymers differing in their polarity and adsorptivity can be easily separated without molar mass interference. Three methods of barrier creation are discussed and compared. It is shown that a fraction of sample may elute unretained if the adsorli sample solvent is used as a barrier in connection with a narrow-pore column packing. One part of excluded macromolecules likely breaks-out from the adsorli zone and this results in partial loss of sample and distortion of the LC LCD peaks. This problem can be avoided if the adsorli zone is injected immediately before sample solution. Applicability of the LC LCD method for polymer separation has been demonstrated with a model mixture of poly(methyl methacrylate) (adsorbing polymer) and polystyrene (non adsorbing polymer) using bare silica gel as a column packing with a combination of tetrahydrofuran (a desorption promoting liquid -a desorli) and toluene (adsorli). It has been shown that the LC LCD procedure with tandem injection allows simple and fast discrimination of polymer blend components with good repeatability and high sample recovery. For quantitative determination of molar masses of both LC LCD and SEC eluted polymers, an additional size exclusion chromatographic column can be applied either in a conventional way or in combination with a multi-angle light scattering detector. A single eluent is used in the latter column, which separates the mixed mobile phase, system peaks and the desorli zone from the polymer peaks so that measurements are free from disturbances caused by the changing eluent composition. The resulting LC LCD x SEC procedure has been successfully applied to poly(methyl methacrylate) samples.     

Characterization of branched ultrahigh molar mass polymers by asymmetrical flow field-flow fractionation and size exclusion chromatography

The molar mass distribution (MMD) of synthetic polymers is frequently analyzed by size exclusion chromatography (SEC) coupled to multi angle light scattering (MALS) detection. For ultrahigh molar mass (UHM) or branched polymers this method is not sufficient, because shear degradation and abnormal elution effects falsify the calculated molar mass distribution and information on branching. High temperatures above 130 °C have to be applied for dissolution and separation of semi-crystalline materials like polyolefins which requires special hardware setups. Asymmetrical flow field-flow fractionation (AF4) offers the possibility to overcome some of the main problems of SEC due to the absence of an obstructing porous stationary phase. The SEC-separation mainly depends on the pore size distribution of the used column set. The analyte molecules can enter the pores of the stationary phase in dependence on their hydrodynamic volume. The archived separation is a result of the retention time of the analyte species inside SEC-column which depends on the accessibility of the pores, the residence time inside the pores and the diffusion ability of the analyte molecules. The elution order in SEC is typically from low to high hydrodynamic volume. On the contrary AF4 separates according to the diffusion coefficient of the analyte molecules as long as the chosen conditions support the normal FFF-separation mechanism. The separation takes place in an empty channel and is caused by a cross-flow field perpendicular to the solvent flow. The analyte molecules will arrange in different channel heights depending on the diffusion coefficients. The parabolic-shaped flow profile inside the channel leads to different elution velocities. The species with low hydrodynamic volume will elute first while the species with high hydrodynamic volume elute later. The AF4 can be performed at ambient or high temperature (AT-/HT-AF4). We have analyzed one low molar mass polyethylene sample and a number of narrow distributed polystyrene standards as reference materials with known structure by AT/HT-SEC and AT/HT-AF4. Low density polyethylenes as well as polypropylene and polybutadiene, containing high degrees of branching and high molar masses, have been analyzed with both methods. As in SEC the relationship between the radius of gyration (R(g)) or the molar mass and the elution volume is curved up towards high elution volumes, a correct calculation of the MMD and the molar mass average or branching ratio is not possible using the data from the SEC measurements. In contrast to SEC, AF4 allows the precise determination of the MMD, the molar mass averages as well as the degree of branching because the molar mass vs. elution volume curve and the conformation plot is not falsified in this technique. In addition, higher molar masses can be detected using HT-AF4 due to the absence of significant shear degradation in the channel. As a result the average molar masses obtained from AF4 are higher compared to SEC. The analysis time in AF4 is comparable to that of SEC but the adjustable cross-flow program allows the user to influence the separation efficiency which is not possible in SEC without a costly change of the whole column combination.     

Simulating chromatographic separation of topologically different polymers

Chromatography which is sensitive to the sizes of macromolecules and to their adsorption serves as an appropriate method to separate complex polymers. Unfortunately, the molar mass also influences the chromatographic retention, thus making quite difficult the problem of separation of polydisperse polymers by their topology.By using a theory of chromatographic behavior of macromolecules, we simulate chromatograms of polydisperse polymers that differ solely in their topology, and discuss possibilities to separate complex polymers (such as eight-, tadpole-, theta-, manacle-shaped polymers, etc.) from their linear, branched, or macrocyclic precursors or topo-isomeric products.As follows from the simulations, two approaches towards the separation of polydisperse polymers by topology are especially promising. The first one is the chromatography at optimized (critical or near-critical) interaction conditions, where molar-mass effects are minimized; The second one consists in combing different chromatographic modes, which allows obtaining a separation by both molar mass and topology in a 2D chromatogram.Some of the simulated chromatographic separations are qualitatively very similar to the real ones, the others are the theoretical prediction.     

Characterization of synthetic copolymers by interaction polymer chromatography: Separation by microstructure

Gradient elution of synthetic polymers has been studied both theoretically and experimentally using normal and reversed-phase HPLC systems. An accurate equation describing the gradient elution of polymer-homologous series in the context of continuous random-flight model of a flexible polymer chain interacting with attractive surface of the porous material has been derived and experimentally verified against a series of narrow polystyrene standards. Both the theory and the experiment predict the existence of molar mass-independent gradient elution at critical point of adsorption (CPA). The extension of the theory to synthetic copolymers predicts the existence of the CPA for statistical copolymers and describes its dependence on chemical composition and microstructure (blockiness) of the polymer chains. One of the important theoretical conclusions is that blockiness always increases the retention, so that blockier polymer chains elute later than their more random counterparts with the same chemical composition. This prediction has been confirmed experimentally using block and statistical styrene-methylmethacrylate copolymers. Block copolymers do not have CPA and always elute between critical points of the corresponding homopolymers. The retention depends on the polymer molar mass and increases with the length of the blocks from a stronger absorbing monomer. These findings provide theoretical and experimental bases for separation of statistical and block copolymers by chemical composition and microstructure of polymer chains.     

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.     

Analytical techniques for polymers with complex architectures

Complex polymers are distributed in more than one direction of molecular heterogeneity. In addition to the molar mass distribution, they are frequently distributed with respect to chemical composition, functionality, and molecular architecture. For the characterization of the different types of molecular heterogeneity it is necessary to use a wide range of analytical techniques. Preferably, these techniques should be selective towards a specific type of heterogeneity. The combination of two selective analytical techniques is assumed to yield a two-dimensional information on the molecular heterogeneity. For the analysis of complex polymers different liquid chromatographic techniques have been developed, including size exclusion chromatography (SEC) separating with respect to hydrodynamic volume, and liquid adsorption chromatography (LAC) which is used to separate according to chemical composition. Liquid chromatography at the critical point of adsorption (LC-CC) has been shown to be a versatile method for the determination of the functionality type distribution of macromonomers, the molecular architecture of homopolymers and the chemical heterogeneity of block and graft copolymers. The present paper presents the principle ideas of combining different analytical techniques in multidimensional analysis schemes for the analysis of polymers with complex architectures. Branched block and graft copolymers can efficiently be analyzed with respect to chemical composition and molar mass by LC-CC and two-dimensional chromatography. The chemical heterogeneity as a function of molar mass can be determined by combining interaction chromatography and FTIR spectroscopy. For the analysis of star-like polymers LC-CC is shown to be a powerful technique when the molar mass of different segments (blocks, grafts) must be determined.     

Direction of temperature gradient for normal-phase temperature gradient interaction chromatography in polystyrene fractionation

Temperature gradient interaction chromatography (TGIC) is a powerful technique for molecular weight fractionation of polymers, in which the interaction strength is controlled by varying the column temperature. In the present paper, the effects of the sign of the temperature dependence of the retention and the direction of the temperature gradient (raising or lowering) on TGIC in the normal-phase mode were studied for the molecular weight fractionation of polystyrene samples in organic mobile phases. It was found that a positive temperature gradient was effective in the system consisting of amino-modified silica (NH(2)) column and the eluent mixture of tetrahydrofuran and n-hexane where retention decreased with increasing temperature. A negative temperature gradient was effective for the systems consisting of a bare-silica column//chloroform/n-hexane and NH(2)-column//chloroform/n-hexane, where retention increased with increasing temperature. Increasing retention with increasing temperature has been found, so far, only for a water-soluble polymer (PEO) in an aqueous mobile phase in RP-TGIC.     

Coarse grained models for flexible liquid crystals: parameterization of the bond fluctuation model

We extend the bond fluctuation model, originally devised to investigate polymer systems, to contain anisotropic interactions suitable for the simulation of large flexible molecules such as liquid crystalline polymers and dendrimers. This extended model coarse grains the interaction between the flexible chains at a similar level of detail to the mesogenic units. Suitable interaction parameters are obtained by performing trial simulations on a low molar mass liquid crystalline system. The phase diagram of this system is determined as a function of the molecular stiffness. The nematic to isotropic transition temperature is found to increase with increasing stiffness.     

Crystallization Elution Fractionation and Thermal Gradient Interaction Chromatography. Techniques Comparison

Summary: The chemical composition distribution has been shown to be the most critical and discriminating parameter in understanding the performance of industrial polyolefins with non homogeneous comonomer incorporation. The chemical composition distribution is being analyzed by well known techniques such as temperature rising elution fractionation, TREF, crystallization analysis fractionation, CRYSTAF and crystallization elution fractionation, CEF. These techniques separate according to crystallizability and provide a powerful and predictable separation of components based on the presence of branches, irregularities or tacticity differences, independently of the molar mass. TREF, CRYSTAF and CEF can not be used, however, for the separation of more amorphous resins, and may not always provide the best solution for complex multi-component resins due to the existence of some co-crystallization. The application of high temperature interactive HPLC to polyolefins opened a new route to characterize these types of polymers. The use of solvent gradient HPLC for separation of polyethylene and polypropylene and the developments in HPLC on carbon based columns extended further the application of high temperature HPLC in polyolefins. A new approach has been developed recently using the carbon based column but replacing solvent gradient by a thermal gradient which facilitates the analysis of polyethylene copolymers and provides a powerful tool for the analysis of elastomers. Thermal gradient interaction chromatography (TGIC) is being compared with TREF and CEF with the analysis of model samples. The advantages/disadvantages of each technique are being investigated and discussed. The combination of TGIC and TREF/CEF provides an extended range of separation of polyolefins.     

Enthalpy assisted size exclusion chromatography. Part 2. Adsorption retention mechanism

A novel high performance liquid chromatographic method for separation of synthetic polymers has been tested. It involves combination of the enthalpic and entropic retention mechanisms, resulting in increased selectivity of separation within a specific molar mass range. In this present case, the enthalpic retention mechanism is adsorption of macromolecules on a bare silica gel column packing. Under critical conditions of enthalpic interactions, homopolymers are known to elute irrespective of their molar mass. However, in the vicinity of critical conditions, a situation can be identified when retention volumes (V(R)) rapidly decrease with increasing molar mass. Typically, this happens for polymer species close to or above their exclusion limit observed with the same column in the absence of enthalpic interactions between macromolecules and packing, that is near "ideal SEC" conditions. The dependence of polymer retention volume on molar mass closely resembles size exclusion conditions. However, the witnessed rate of change in V(R )with polymer molar mass is more pronounced, thus indicating increased selectivity of separation. This situation not only offers the benefit of more selective separation according to molar mass but efficient discrimination of macromolecules possessing different nature and interactivity with the column packing can be accomplished as well.     

Characterization of EO-PO Block Copolymers by Liquid Chromatography Under Critical Conditions

Summary: Block copolymers of ethylene oxide (EO) and propylene oxide (PO) are characterized by liquid chromatography under critical conditions (LCCC) for EO. At the critical adsorption point (CAP) for one structural unit, the non-critical block can elute in size exclusion (SEC) or adsorption (LAC) mode. Depending on the molar mass and architecture of the polymers, different strategies are applied. For samples with a higher molar mass, the SEC separation is the method of choice, while lower molar masses also allow a LAC separation. Examples for both situations are given, which show, that these approaches yield different information. In the SEC mode, homopolymers and diblocks can be separated from the triblocks. In LAC mode, a baseline resolution of individual oligomers can be achieved, in which homopolymers, diblocks and triblocks with the same number of repeat units of the non-critical block have the same elution volume.     

Crystallisation behaviour of high density polyethylene blends with bimodal molar mass distribution 1. Basic characteristics and isothermal crystallisation

Isothermal crystallisation of high density polyethylene (HDPE) blends and their parent polymers was investigated. The blends having broad bimodal molar mass distributions and various compositions were prepared by blending a high molar mass (M_w=330 kg/mol, M_w/M_n=4.8) and a low molar mass HDPE (M_w=34 kg/mol, M_w/M_n=10) in different ratios in xylene solution. The blends and their parent components were characterised by size-exclusion chromatography, dynamic rheological and density measurements. Crystallisation kinetics were studied using a polarised light microscope equipped with an in-house built hot stage and by differential scanning calorimetry. The Avrami theory was applied for crystallisation kinetics analysis. Such crystallisation kinetics parameters as nucleation rate, nucleation density, the Avrami index and cystallisation rate contant were determined for the blends and their parent polymers.According to the results obtained an increasing polydispersity of the sample had a slight increasing effect on the Avrami index, indicating gain in prevalence of the thermal nucleation over the athermal one. In all samples nucleation density increased continuously during crystallisation verifying that the presence of a certain thermal nucleation was typical for all the materials studied. Both the crystallisation rate constant and the nucleation rate decreased with increasing molar mass of the sample. The nucleation density increased proportionally to the increase in average molar mass and the values were larger at lower crystallisation temperatures.The formed supermolecular structure was found to be sensitive to the blend composition and crystallisation temperature. Irregular banded or non-banded spherulites were observed in the materials. Banding of spherulites was typical for the samples having higher average molar mass. The superstructures observed in this work were smaller and vaguer than the superstructures reported in the earlier studies of polyethylene materials having similar average molar mass but narrow molar mass distribution.     

Molar mass of main-chain bile acid-based oligo-esters measured by SEC, MALDI-TOF spectrometry and NMR spectroscopy: a comparative study

Bile acid-based polymers are promising new materials for biomedical applications. The determination of their molar mass, as for other novel polymers, has been difficult, due to the lack of suitable standards for size exclusion chromatography (SEC). In order to solve this problem, a family of main-chain bile acid-based oligo-esters has been synthesized by acyclic diene metathesis to be used as analogues in such analysis. These oligomers have been characterized by SEC, MALDI-TOF mass spectrometry and NMR spectroscopy. The results show that SEC with polystyrene standards tends to overestimate the molar mass of these materials and that a correction factor between 0.50 and 0.60 should be used for more accuracy.     

An SEC/MALS study of alternan degradation during size-exclusion chromatographic analysis

Ultrahigh-molar-mass (M) polymers such as DNA, cellulose, and polyolefins are routinely analyzed using size-exclusion chromatography (SEC) to obtain molar mass averages, distributions, and architectural information. It has long been contended that high-M polymers can degrade during SEC analysis; if true, the inaccurate molar mass information obtained can adversely affect decisions regarding processing and end-use properties of the macromolecules. However, most evidence to the effect of degradation has been circumstantial and open to alternative interpretation. For example, the shift in SEC elution volume as a function of increased chromatographic flow rate, observed using only a concentration-sensitive detector, may be the result of degradation or of elution via a nondegradatory slalom chromatography mechanism. Here, using both concentration-sensitive and multiangle static light-scattering detection, we provide unambiguous evidence that the polysaccharide alternan actually degrades during SEC analysis. The decrease in molar mass and size of alternan with increasing flow rate, measured using light scattering, allows ruling out an SC mode of elution and can only be interpreted as due to degradation. These findings demonstrate the extreme fragility of ultrahigh-M polymers and the care that must be taken for accurate characterization. Figure Scission of alternan chains in liquid chromatography.     

Sample size and retention values in gradient liquid chromatography of synthetic polymers

Summary Retention times in gradient liquid chromatography of synthetic polymers are often dependent on sample size. They increase with column load if the separation mechanism is governed by a solution process but decrease with increasing load if the mechanism is governed by adsorption. Since retention times independent of sample size are a prerequisite for peak identification as well as for the correct measurement of elution bands of samples with a broad distribution, measures to counteract sample-size effects deserve attention. Usually both solubility and adsorption are effective in gradient liquid chromatography of synthetic polymers. An appropriate balance of both effects is suitable for diminishing the influence of sample size on retention time of synthetic polymers. Ternary gradients allowing independent control of solubility and adsorption are promising.     

朝鲜淫羊藿的色谱指纹图谱及液相色谱/电喷雾串联质谱分析

建立了长白山区朝鲜淫羊藿药材的高效液相色谱指纹图谱的分析方法. 确定了18批朝鲜淫羊藿药材的13个共有峰, 该指纹图谱的精密度、稳定性和重现性的相对标准偏差均低于3.0%. 结合液相色谱/电喷雾串联质谱对特征峰进行了结构确认, 并根据电喷雾串联质谱数据推测了13个特征化合物的结构. 结果表明采用高效液相色谱与质谱联用技术对朝鲜淫羊藿色谱指纹图谱中的特征峰进行结构确认, 使其色谱指纹图谱的特征性更强, 更适合于药材质量的鉴别与评价.     

Separation of minor macromolecular constituents from multicomponent polymer systems by means of liquid chromatography under limiting conditions of enthalpic interactions

Minor (<1%) macromolecular constituents may significantly affect physical/utility properties of the multicomponent polymer systems. Separation and molecular characterization of the small amounts of macromolecular additives from the dominant polymer matrices represents an exacting analytical problem. Recently a series of unconventional liquid chromatographic methods was developed for separation of the constituents of polymer blends; their generic name is Liquid chromatography under limiting conditions of enthalpic interactions, LC LC. The LC LC procedures employ the difference in elution rate of the low molecular substances and the macromolecules within the column packed with porous particles. Small molecules permeate practically all pores of the packing and therefore they elute slowly. Polymer species are partially of fully pore excluded and in absence of enthalpic interactions they are rapidly transported along the column. The appropriately chosen low molecular substances promote interactions of macromolecules within the column. If eluted in front of sample, the interaction promoting low molecular substance may create a sort of slowly eluting barrier that is “impermeable” for the interacting macromolecules and efficiently decelerates their fast transport. The blocking action of a barrier differs for macromolecules of distinct nature, which elute from the column with a different rate to be mutually separated irrespectively of their molar mass. In present work, different approaches to the LC LC separations are compared from the point of view of their applicability to complex polymer systems, in which one constituent is present at very low concentration, and also in light of sample recovery. The practical examples are the two- and three-component polymer blends of polystyrenes, poly(methyl methacrylate)s and poly(vinyl acetate)s of different molar mass averages and distributions, as well as the diblock copolymers polystyrene-block-poly(methyl methacrylate) that contain their parent homopolymers.     

Temperature Gradient Interaction Chromatography: A Perspective

Chromatographia - The application of temperature gradient interaction chromatography (TGIC) as an advanced technique for the characterisation of polymers is discussed, in comparison to other liquid...     

Optimisation of ambient and high temperature asymmetric flow field-flow fractionation with dual/multi-angle light scattering and infrared/refractive index detection

Asymmetric flow field-flow fractionation (AF4) enables to analyse polymers with very high molar masses under mild conditions in comparison to size exclusion chromatography (SEC). Conventionally, membranes for AF4 are made from cellulose. Recently, a novel ceramic membrane has been developed which can withstand high temperatures above 130 °C and chlorinated organic solvents, thus making it possible to characterise semicrystalline polyolefins by HT-AF4. Two ceramic membranes and one cellulose membrane were compared with regard to their quality of molar mass separation and the loss of the polymer material through the pores. Separating polystyrene standards as model compounds at different cross-flow gradients the complex relationship between cross-flow velocity, separation efficiency, the molar mass and peak broadening could be elucidated in detail. Moreover, the dependence of signal quality and reproducibility on sample concentration and mass loading was investigated because the evaluation of the obtained fractograms substantially depends on the signal intensities. Finally, the performance of the whole system was tested at high temperature by separating PE reference materials of high molar mass.     

Chromatographic behavior of IgM:DNA complexes

This study documents the presence of stable complexes between monoclonal IgM and genomic DNA in freshly harvested mammalian cell culture supernatants. 75% of the complex population elutes from size exclusion chromatography with the same retention volume as IgM. DNA comprises 24% of the complex mass, corresponding to an average of 347 base pairs per IgM molecule, distributed among fragments smaller than about 115 base pairs. Electrostatic interactions appear to provide most of the binding energy, with secondary stabilization by hydrogen bonding and metal affinity. DNA-dominant complexes are unretained by bioaffinity chromatography, while IgM-dominant complexes are retained and coelute with IgM. DNA-dominant complexes are repelled from cation exchangers, while IgM-dominant complexes are retained and partially dissociated. Partially dissociated forms elute in order of decreasing DNA content. The same pattern is observed with hydrophobic interaction chromatography. All complex compositions bind to anion exchangers and elute in order of increasing DNA content. A porous particle anion exchanger was unable to dissociate DNA from IgM. Monolithic anion exchangers, offering up to 15-fold higher charge density, achieved nearly complete complex dissociation. The charge-dense monolith surface appears to outcompete IgM for the DNA. Monoliths also exhibit more than double the IgM dynamic binding capacity of the porous particle anion exchanger, apparently due to better surface accessibility and more efficient mass transfer.