Size-exclusion chromatography—from high-performance to ultra-performance

Size-exclusion chromatography (SEC) enables measurement of the average molecular weights and molecular-weight distributions of polymers. Because these characteristics may, in turn, be correlated with important performance characteristics of plastics, SEC is an essential analytical technique for characterization of macromolecules. Although SEC is one of the oldest instrumental chromatographic techniques, it is still under continuous development, as a result of the great demand for increased resolution and faster analysis in SEC. Ultra-high-pressure size-exclusion chromatography (UHPSEC) was recently introduced to satisfy the growing demands of analytical chemists. Using instrumentation capable of generating very high pressures and columns packed with small particles, this technique enables greater separation efficiency and faster analysis than are achieved with conventional SEC. UHPSEC is especially advantageous for high-resolution analysis of oligomers, for very rapid polymer separations, and as a second dimension in comprehensive two-dimensional liquid chromatography of polymers. In this paper we discuss the benefits of UHPSEC for separation of macromolecules, with examples from the literature.     

Comprehensive two-dimensional liquid chromatography×gas chromatography: evaluation of the applicability for the analysis of edible oils and fats

Edible fats and oils are complex mixtures containing a wide range of (classes of) compounds. The most important group of compounds are the triglycerides (triacylglycerides, TAGs). Because of the large number of possible fatty acid combinations, an enormous number of TAGs is possible. In the present feasibility study, the applicability of different modes of comprehensive two-dimensional LC×GC for detailed oil and fat analysis is evaluated. Comprehensive LC×GC was found to be an extremely powerful analytical method for the analysis of complex TAG samples. Using the new comprehensive set-ups, TAGs can be separated according to two independent parameters: carbon number vs. number of double bonds, or fatty acid composition vs. number of double bonds. The information content of comprehensive separations by far exceeds that of the current generation of analytical methods. The quantitative results of the separations show a good agreement with data obtained from standard analytical methods. The comprehensive methods studied can also be used for fingerprinting of oil samples, as well as for the analysis of target compounds or compound groups. Highly detailed separations of olive oil samples were obtained. Zooming in on one region of the chromatogram allowed reliable analysis of wax esters without interferences of sterol esters.     

Branched-polymer separations using comprehensive two-dimensional molecular-topology fractionation x size-exclusion chromatography

Branching has a strong influence on the processability and properties of polymers. However, the accurate characterization of branched polymers is genuinely difficult. Branched molecules of a certain molecular weight exhibit the same hydrodynamic volumes as linear molecules of substantially lower weights. Therefore, separation by size-exclusion chromatography (SEC), will result in the co-elution of molecules with different molecular weights and branching characteristics. Chromatographic separation of the polymer molecules in sub-microm channels, known as molecular-topology fractionation (MTF), may provide a better separation based on topological differences among sample molecules. MTF elution volumes depend on both the topology and molar mass. Therefore co-elution of branched molecules with linear molecules of lower molar mass may also occur in this separation. Because SEC and MTF exhibit significantly different selectivity, the best and clearest separations can be achieved by combining the two techniques in a comprehensive two-dimensional (MTFxSEC) separation system. In this work such a system has been used to demonstrate branching-selective separations of star branched polymers and of randomly long-chain-branched polymers. Star-shaped polymers were separated from linear polymers above a column-dependent molecular weight or size.     

Rapid determination of molecular parameters of synthetic polymers by precipitation/redissolution high‐performance liquid chromatography using “molded” monolithic column

Rapid high‐performance liquid chromatography (HPLC) of polystyrenes, poly(methyl methacrylates), poly(vinyl acetates), and polybutadienes using a monolithic 50 × 4.6 mm i.d. poly(styrene‐co‐divinylbenzene) column have been carried out. The separation process involves precipitation of the macromolecules on the macroporous monolithic column followed by progressive elution utilizing a gradient of the mobile phase. Depending on the character of the separated polymer, solvent gradients were composed of a poor solvent such as water, methanol, or hexane and increasing amounts of a good solvent such as THF or dichloromethane. Monolithic columns are ideally suited for this technique because convection through the large pores of the monolith enhances the mass transport of large polymer molecules and accelerates the separation process. Separation conditions including the selection of a specific pair of solvent and precipitant, flow rate, and gradient steepness were optimized for the rapid HPLC separations of various polymers that differed broadly in their molecular weights. Excellent separations were obtained demonstrating that the precipitation‐redissolution technique is a suitable alternative to size‐exclusion chromatography (SEC). The molecular weight parameters calculated from the HPLC data match well those obtained by SEC. However, compared to SEC, the determination of molecular parameters using gradient elution could be achieved at comparable flow rates in a much shorter period of time, typically in about 1 min. © 2000 John Wiley & Sons, Inc. J Polym Sci A: Polym Chem 38: 2767–2778, 2000     

高效液相色谱表征高聚物*

最常用的测试高聚物的分子量和分子量分布的体积排除色谱(SEC)是高效液相色谱 (HPLC)的一个重要分支,HPLC的另一个重要分支是相互作用液相色谱, 它是20世纪90年代开始用于高分子分离和表征的研究领域。相互作用液相色谱可以根据高分子的化学结构（如共混物组成、共聚物组成、端基）来分离,它比SEC 有更高的分离效率。本文介绍了高聚物液相色谱的分离模式,并就高聚物体积排除色谱、相互作用液相色谱、临界液相色谱和全二维液相色谱用于分离和表征高聚物的研究进展进行了较系统的综述,并对该技术目前存在的问题和今后可能的发展前景进行了探讨。     

Separation of water-soluble polymers using capillary-channeled polymer fiber stationary phases

Chromatographic separations of synthetic and natural polymers are usually affected by a size exclusion chromatography (SEC) mechanism. Although SEC is a proven method of separation based on hydrodynamic size, a chromatographic method based solely on chemical interactions would present certain advantages. This laboratory has been investigating the use of capillary-channeled polymer (C-CP) fibers as stationary phases in HPLC for the separation of biomacromolecules. C-CP fibers allow highly efficient fluid transport and an amorphous surface structure, minimizing mass transfer effects commonly associated with porous, packed-bed technologies. Choice of the base fiber identity allows flexibility in the potential types of solute-surface interactions. Two water-soluble polymers, glycolic acid ethoxylate 4-nonylphenyl ether, and poly(4-vinylpyridine hydrochloride), were used as test solutes because of their similarities to polymers of interest in the consumer products industry. SEC separation of this pair was not possible due to the similarities in hydrodynamic size. Poly(ethylene terephthalate), polyester and nylon-6 C-CP fibers were evaluated as stationary phase materials. The former was found to offer superior chromatographic separations and recoveries when operating under what would be considered to be typical RP separation conditions: a flow rate of 1?mL/min and gradient of 0-100% H(2)O/ACN with 0.06% TFA over 5?min.     

Size-Exclusion Chromatography: A Twenty-First Century Perspective

Now in its sixth decade, size-exclusion chromatography (SEC) remains the premier method by which to determine the molar mass averages and distributions of natural and synthetic macromolecules. Aided by its coupling to a variety and multiplicity of detectors, it has also shown its ability to characterize a host of other physicochemical properties, such as branching, chemical, and sequence length heterogeneity size distribution; chain rigidity; fractal dimension and its change as a function of molar mass; etc. SEC is also an integral part of most macromolecular two-dimensional separations, providing a second-dimension size-based technique for determining the molar mass of the components separated in the first dimension according to chemical composition, thus yielding the combined chemical composition and molar mass distributions of a sample. While the potential of SEC remains strong, our awareness of the pitfalls and challenges inherent to it and to its practice must also be ever-present. This Perspective aims to highlight some of the advantages and applications of SEC, to bring to the fore these caveats with regard to its practice, and to provide an outlook as to potential areas for expansion and growth.

     

The measurement of mass and size distributions,conformation, and branching of important food polymers by mals following sample fractionation

Three distinctive food polymers (ultra high molar mass amylopectin, guar gum, and hemi-cellulose) were chosen as model samples to illustrate the use of multi angle light scattering (MALS) detection in conjunction with size exclusion chromatography (SEC) separations for the characterization of such macromolecules. By combining SEC and MALS, absolute molar mass, rms radius and their distributions can be measured readily without reference to any molar mass standards and without the need to make structural assumptions. In addition, the conformation and branching of those polymers can be derived also.     

Fast and efficient size-based separations of polymers using ultra-high-pressure liquid chromatography

Ultra-high-pressure liquid chromatography (UHPLC) has great potential for the separations of both small molecules and polymers. However, the implementation of UHPLC for the analysis of macromolecules invokes several problems. First, to provide information on the molecular-weight distribution of a polymer, size-exclusion (SEC) columns with specific pore sizes are needed. Development of packing materials with large pore diameters and pore volumes which are mechanically stable at ultra-high-pressures is a technological challenge. Additionally, narrow-bore columns are typically used in UHPLC to minimize the problem of heat dissipation. Such columns pose stringent requirements on the extra-column dispersion, especially for large (slowly diffusing) molecules. Finally, UHPLC conditions generate high shear rates, which may affect polymer chains. The possibilities and limitations of UHPLC for size-based separations of polymers are addressed in the present study. We demonstrate the feasibility of conducting efficient and very fast size-based separations of polymers using conventional and wide-bore (4.6 mm I.D.) UHPLC columns. The wider columns allow minimization of the extra-column contribution to the observed peak widths down to an insignificant level. Reliable SEC separations of polymers with molecular weights up to ca. 50 kDa are achieved within less than 1 min at pressures of about 66 MPa. Due to the small particles used in UHPLC it is possible to separate high-molecular-weight polymers (50 kDa ≤ M(r) ≤ 1-3 MDa, upper limit depends on the flow rate) in the hydrodynamic-chromatography (HDC) mode. Very fast and efficient HDC separations are presented. For very large polymer molecules (typically larger than several MDa, depending on the flow rate) two chromatographic peaks are observed. This is attributed to the onset of molecular deformation at high shear rates and the simultaneous actions of hydrodynamic and slalom chromatography.     

Two-dimensional liquid chromatography analysis of synthetic polymers using fast size exclusion chromatography at high column temperature

In recent years, two-dimensional liquid chromatography (2D-LC) has been used increasingly for the analysis of synthetic polymers. A 2D-LC analysis provides richer information than a single chromatography analysis at the cost of longer analysis time. The time required for a comprehensive 2D-LC analysis is essentially proportional to the analysis time of the second dimension separation. Many of 2D-LC analyses of synthetic polymers have employed size exclusion chromatography (SEC) for the second-dimension analysis due to the relatively short analysis time in addition to the wide use in the polymer analysis. Nonetheless, short SEC columns are often used for 2D-LC analyses to reduce the separation time, which inevitably deteriorates the resolution. In this study, we demonstrated that high temperature SEC can be employed as an efficient second-LC in the 2D-LC separation of synthetic polymers. By virtue of high temperature operation (low solvent viscosity and high diffusivity of the polymer molecules), a normal length SEC column can be used at high flow rate with little loss in resolution.     

High-performance size-exclusion chromatography with crosslinked polystyrene. Separation of polymers and oligomers using LiChrogel PS

Summary A new series of polystyrene-divinyl benzene PS/DVB-based microparticulate packings for size-exclusion chromatography [SEC] is presented. These gel-based packings are characterized by mechanical stability, minimal interaction with solutes and ability to handle a wide variety and size range of polymers and low relative molecular mass samples. Combinations of different pore sizes are discussed and separations of standard polymers and commercial products are shown.     

Comprehensive two-dimensional liquid chromatography for the characterization of functional acrylate polymers

Comprehensive two-dimensional liquid chromatography-size-exclusion chromatography (LC x SEC) was investigated as a tool for the characterization of functional poly(methyl methacrylate) (PMMA) polymers. Ultraviolet-absorbance and evaporative light-scattering detection (ELSD) were used. A simple method to quantify ELSD data is presented. Each data point from the ELSD chromatogram can be converted into a mass concentration using experimental calibration curves. The qualitative and quantitative information obtained on two representative samples is used to demonstrate the applicability of LC x SEC for determining the mutually dependent molar-mass distributions (MMD) and functionality-type distributions (FTD) of functional polymers. The influence of the molar mass on the retention behavior in LC was investigated using LC x SEC for hydroxyl-functional PMMA polymers. The critical conditions, at which retention is--by definition--independent of molar mass, were not exactly the same for PMMA series with different end-groups. Our observations are in close agreement with theoretical curves reported in the literature. However, for practical applications of LC x SEC it is not strictly necessary to work at the exact critical solvent composition. Near-critical conditions are often sufficient to determine the mutually dependent distributions (MMD and FTD) of functional polymers.     

Size exclusion chromatography of branched polymers: Star and comb polymers

Monte Carlo simulations were conducted to estimate the elution curve of size exclusion chromatography (SEC). The present simulation can be applied to various types of branched polymers, as long as the kinetic mechanism of nonlinear polymer formation is given. We considered two types of detector systems, (1) a detector that measures the polymer concentration in the elution volume to determine the calibrated molecular weights, such as by using the differential refractive index detector (RI), and (2) a detector that determines the weight‐average molecular weight of polymers within the elution volume directly, such as a light scattering photometer (LS). For polydisperse star polymers, both detector systems tend to give a reasonable estimate of the true molecular weight distribution (MWD). On the other hand, for comb‐branched polymers, the RI detector underestimates the molecular weight of branched polymers significantly. The LS detector system improves the measured MWD, but still is not exact. The present simulation technique promises to establish various types of complicated reaction mechanisms for nonlinear polymer formation by using the SEC data quantitatively. In addition, the present technique could be used to reinvestigate a large amount of SEC data obtained up to the present to estimate the true MWD.     

Transfer-volume effects in two-dimensional chromatography: adsorption-phenomena in second-dimension size-exclusion chromatography

Gradient-elution LC × LC is a valuable technique for the characterization of complex biological samples as well as for synthetic polymers. Breakthrough and viscous fingering may yield misleading information on the sample characteristics or deteriorate separation. In LC × SEC another phenomenon may jeopardize the separation. If the analytes adsorb on the SEC column under the injection-plug conditions, peak splitting may occur. In LC × LC the effluent from the first column is the sample solvent for the analytes injected into the second dimension. If a gradient-elution LC × SEC setup is used (i.e. if reversed-phase gradient-elution LC is coupled to organic SEC and if normal-phase gradient-elution LC is coupled to SEC with a polar solvent), the percentage of weak solvent may be significant, especially at short analysis times. In this case adsorption in the first-dimension-effluent zone on the second-dimension SEC column can become an issue and two peaks--the first eluting in size-exclusion mode and the second undergoing adsorption--can be obtained. The work presented in this paper documents peak splitting in LC × SEC of polymers. The adsorption of the polymer on the size-exclusion column was proven in one-dimensional isocratic runs. The observed effects were modeled and visualized through simulation. Studies on the influence of the transfer volume were carried out. Keeping the transfer volume as small as possible helped to minimize peak splitting due to adsorption.     

Two‐dimensional chromatography as a tool for studying band broadening in size‐exclusion chromatography

Comprehensive 2‐D size‐exclusion chromatography (SEC×SEC) has been realized. SEC×SEC is not a useful technique for characterizing complex polymers. However, it is potentially an elegant tool to study band‐broadening phenomena. If narrow fractions can be collected from the first dimension, the band broadening in the second dimension is only due to chromatographic dispersion. This would allow a clear distinction to be made between chromatographic band broadening (column and extra‐column) and SEC selectivity (band broadening due to sample polydispersity). In comparison with MALDI‐MS, SEC×SEC allows the study of polymers across a much broader molar‐mass range.     

Size-exclusion chromatography (SEC) of branched polymers and polysaccharides

Branched polymers are among the most important polymers, ranging from polyolefins to polysaccharides. Branching plays a key role in the chain dynamics. It is thus very important for application properties such as mechanical and adhesive properties and digestibility. It also plays a key role in viscous properties, and thus in the mechanism of the separation of these polymers in size-exclusion chromatography (SEC). Critically reviewing the literature, particularly on SEC of polyolefins, polyacrylates and starch, we discuss common pitfalls but also highlight some unexplored possibilities to characterize branched polymers. The presence of a few long-chain branches has been shown to lead to a poor separation in SEC, as evidenced by multiple-detection SEC or multidimensional liquid chromatography. The local dispersity can be large in that case, and the accuracy of molecular weight determination achieved by current methods is poor, although hydrodynamic volume distributions offer alternatives. In contrast, highly branched polymers do not suffer from this extensive incomplete separation in terms of molecular weight.     

Two-dimensional liquid chromatography of diblock copolymers: Simulation at various adsorption interaction conditions

Diblock copolymers, which are heterogeneous in both molar mass and composition, can be fully characterized by using two-dimensional chromatography. Since the size-exclusion, the adsorption, and the critical interaction based modes of chromatography are possible for each of the polymers A and B, this leads to a variety of options for 2D-chromatography of copolymers AB. Using the theory of chromatography of block copolymers, 2D-chromatograms are simulated that correspond to the most interesting of these options. Orthogonal 2D-chromatograms are expected, if in the 1st dimension the critical condition is created for A, while in the 2nd dimension – for B. The situations, where A and B are both adsorbable, as well as those where the conditions of adsorption for A and SEC for B are created, are also considered. In particular, it is shown that the 2nd dimension combination of the critical condition for A and SEC – for B is preferable than that with SEC condition for both A and B. The simulated 2D-chromatograms of low- and high molar mass diblock copolymers, as well as of copolymers with one short block are compared with the reported real ones; it is concluded that the corresponding virtual and real 2D-chromatograms are qualitatively very similar.     

Packed-column hydrodynamic chromatography using 1-μm non-porous silica particles

150×3 mm I.D. columns, packed with 1-μm non-porous spherical silica particles, were used to separate soluble synthetic polymers by hydrodynamic chromatography. The columns exhibited a plate height of about 1.4 μm allowing very fast and efficient separations of polymers in the molecular mass range 10³−2·10⁶ g/mol. The migration behaviour of polymers could be well described by a simple theoretical model. The applicability of packed bed HDC for the fast separation of polymers was illustrated with separations of polystyrene and poly(methyl methacrylate) mixtures.     

Characterization of copolymers of polycarbonate and polydimethylsiloxane by 2D chromatographic separation,MALDI-TOF mass spectrometry,and FTIR spectroscopy

Abstract

The structure and composition of polycarbonate polydimethylsiloxane copolymer (PC-co-PDMS) was investigated by applying various analytical approaches including chromatographic separation methods, spectrometric, and spectroscopic detection techniques. In particular, size exclusion chromatography (SEC) and liquid adsorption chromatography operating at different conditions (e.g. using gradient solvent systems) were used to achieve separations according to molar mass and functionality distribution. The coupling of both techniques resulted in fingerprint two-dimensional plots, which could be used to easily compare different copolymer batches. Matrix-assisted laser desorption/ionization-time-of-flight (MALDI-TOF) mass spectrometry was applied for structural investigations. The different ionization behavior of both comonomers, however, strongly limited the applicability of this technique. In contrast to that, Fourier-transform Infrared (FTIR) spectroscopy could be used to quantify the amount of PDMS in the copolymer at different points in the chromatogram. The resulting methodology was capable of distinguishing PC-co-PDMS copolymer from PC homopolymer chains present in the material.