SEC-Gradients, an alternative approach to polymer gradient chromatography: 1. Proof of the concept

A new approach to gradient chromatography of polymers is presented, in which the sample is introduced at the end of the gradient and elutes within the elution volume range typical for size exclusion chromatography (SEC). Due to the gradient the samples are retarded and elute nearly independent of molar mass at the adsorption threshold. The concept was proven for a series of narrowly distributed poly(methy methacrylate)s (PMMA) in a chloroform-tetrahydrofuran (THF) SEC-gradient. The application of the SEC-gradient to a blend of PMMA and polystyrene standards of similar molar masses, which could not be separated by SEC due to their similar hydrodynamic sizes, resulted in a clear separation according to chemical composition. Since SEC-gradients allow dissolving the sample in strong eluents, which might result in breakthrough peaks in conventional gradients, the new approach is a valuable alternative to conventional gradient chromatography.     

Characterization of branched polymers by size exclusion chromatography coupled with multiangle light scattering detector. I. Size exclusion chromatography elution behavior of branched polymers

The elution behavior of branched macromolecules during their separation by size exclusion chromatography (SEC) was studied. The elution behavior of branched polymers was investigated using samples of randomly branched polystyrene and star branched poly(benzyl methacrylate) of different levels of branching by means of a SEC chromatograph coupled with a multiangle light scattering detector. Abnormal SEC elution behavior was found to be typical for highly branched polymers. After a normal elution at small elution volumes the molar mass and root mean square radius of the eluting molecules increased with increasing elution volume. Several SEC experiments were carried out to find explanation for this effect and SEC separation was compared with the separation by thermal field flow fractionation. © 2001 John Wiley & Sons, Inc. J Appl Polym Sci 81: 1588–1594, 2001     

Chromatographic investigations of macromolecules in the critical range of liquid chromatography,XIII. Separation of blends of styrene-butadiene rubber and butyl rubber

The separation of blends of styrene-butadiene rubber and butyl rubber is accomplished by liquid chromatography at the critical point of adsorption. Using a non-polar stationary phase and methyl ethyl ketone-cyclohexane as the eluent, the critical point of adsorption of polybutadiene is established. Under these chromatographic conditions, the blends are separated regardless of the molar masses of the components. The exact chemical structure of the blend components can be analysed by coupling the chromatographic separation to FTIR detection. The FTIR spectra of the components reveal information on the styrene and butadiene content and the conformation of the butadiene units (1,2-, 1,4-cis, 1,4-trans units). Poorly soluble high molar mass samples are separated by combining critical separation with a gradient elution technique.     

Use of gradient,critical, and two‐dimensional chromatography in the analysis of styrene‐ and methyl methacrylate‐grafted epoxidized natural rubber

The growing number of heterogeneous polymeric species that are being synthesized places increasing demands on existing analytical techniques. Although size‐exclusion chromatography (SEC) has established itself as a powerful analytical tool, it has its limits when complex polymers, e.g., graft copolymers, must be analyzed. In this case, complementary techniques such as gradient HPLC and liquid chromatography at critical conditions (LCCC) are more favorable. The present study describes the synthesis and analysis of methyl methacrylate‐ and styrene‐grafted epoxidized natural rubber by different chromatographic techniques. The grafting efficiency was evaluated by gradient HPLC under normal and reversed phase conditions. Methyl methacrylate‐grafted ENR50 was further analyzed by LCCC, where separation of the rubber and grafted rubber occurred according to chemical composition but was independent of the molar mass of the methyl methacrylate homopolymers. This was followed by the combination of LCCC and SEC, where separation was achieved in two dimensions. Relevant deductions were made of both the chemical composition distribution and the molar mass distribution of the functional groups of methyl methacrylate‐grafted ENR50. © 2003 Wiley Periodicals, Inc. J Appl Polym Sci 88: 2530–2538, 2003     

Liquid chromatography at critical conditions of polyethylene

Olefin copolymers are of increasing scientific interest due to their important application potential. Liquid chromatography can deliver important information, especially on their molecular structure. In particular, liquid chromatography at critical conditions (LCCC) is of interest because, in this chromatographic mode, the elution is molar mass independent for a given repeat unit. LCCC is conducted at conditions where the entropic effect of size exclusion is equal to the enthalpic effect of interaction between the macromolecules and the stationary phase and can be used to separate copolymers as well as some homopolymers containing one single repeat unit but having different end groups. For the first time, critical conditions for linear PE were experimentally identified by using porous graphite (Hypercarb™) as the stationary phase, and either 1-decanol/ortho-dichlorobenzene (ODCB), 1-decanol/1,2,4-trichlorobenzene (TCB), n-decane/ODCB, or n-decane/TCB as the mobile phase at 160 °C. The identified critical conditions for PE using the above approach in the above solvents have been verified to be correct, barring slight deviations, by two different techniques which were previously used to determine critical conditions for polymers soluble at ambient temperature. The critical conditions for polyethylene were applied to separate statistical copolymers of ethylene/1-octene with similar molar mass.     

Theory of chromatography of complex cyclic polymers: eight-shaped and daisy-like macromolecules

A theory of chromatography of eight-shaped, trefoil-shaped and daisy-like polymers is developed. For a model of an ideal chain in a slit-like pore exact equations and a number of approximate formulae for the distribution coefficient K of these polymers are derived. All modes of chromatography of complex macrocycles of arbitrary molar mass in both narrow and wide pores are covered by the theory. It is shown that complex macrocycles always elute after linear polymers and rings of the same contour length. The effective chromatographic radius of eight-shaped and daisy-like macromolecules, which determines retention in size-exclusion chromatography are calculated. The increase in the retention with molar mass is predicted for all types of macrocycles at the critical interaction condition. Non-monotonous molar mass dependences of K are found at pre-critical interaction. We simulate separation of complex cyclic polymers from linear and ring precursors, discuss possibilities to separate symmetric and asymmetric eights, and speculates on the use of chromatography for separating knotted and unknotted polymer rings. According to the theory, the chromatography under the critical and pre-critical interaction conditions is expected to be especially efficient in these and similar problems. Boundary conditions for the theory and its applicability to real systems are discussed.     

RP-HPLC法分离异黄酮中移动相条件的优化

In reversed-phase high performance liquid chromatography （RP-HPLC）, the mobile phase condition for separating eight isoflavones （daidzin, glycitin, genistin, 6＇-o-acetyl daidzin, 6＇-o-malonyl genistin, daidzein, glycitein and genistein） was optimized using the HCI （High-Purity Separation Laboratory, Department of Chemical Engineering, Inha University） program software. The optimum composition of mobile phase for the separation of the eight isoflavones was obtained. The elution profiles were calculated by the plate theory based on the equations of retention factor, In k=A＋BF＋CF2, where F was the volume percentage of acetonitrile with 0.1% acetic acid （AA）. The first mobile phase composition was water with 0.1% AA/acetonitrile with 0.1% AA （88%/12%, by volume）, followed at 9min later by the second composition of mobile phase which was step-changed to 85%/15%, at 19rain by the third composition which was step-changed to 73%/27%, at 30min when it was changed to 65%/35% and finally it was maintained in isocratic mode to the end of the run time at 50rain. Although, using step gradient mode to separate the isoflavones, the calculated and experimented data were not achieved very good agreement, we could estimate the closed retention time before experiment. And the agreement between the experimental data and the calculated values was relatively good using isocratic separation for eight isoflavones, but the retention time is very long.     

Compositional heterogeneity in partially hydrolysed poly(vinyl alcohol) by reversed phase liquid chromatography

Reversed phase high performance liquid chromatography (HPLC) was employed to elucidate the composition distribution of partially hydrolysed samples of poly(vinyl alcohol) (PVOH), demonstrating that elution across a chromatogram proceeded from higher to lower degree of hydrolysis (DoH). Fractions isolated by preparative HPLC fractionation and characterised by ¹H nuclear magnetic resonance spectroscopy (NMR) were used as HPLC standards to construct a calibration curve of retention time versus DoH, allowing for DoH determination of any PVOH sample once its chromatogram was available. Plots of cumulative and differential distributions as a function of DoH were determined, allowing for comparisons of samples having average DoH in the range 70–90 mol%. A second set of fractions originating from a parent polymer having different molar mass was also isolated to confirm that calibration was not influenced by molar mass or size exclusion effects.     

Characterization of bisphenol A–based epoxy resins by HPLC,GPC, GPC–MALLS,VPO, viscometry,and end group analysis: On the identification of 2,3‐dihydroxypropyl‐containing compounds,determination of molar mass distribution,and branching

Reversed‐phase high performance liquid chromatography (HPLC), conventional gel permeation chromatography (GPC), and gel permeation chromatography coupled with a multiangle laser light scattering photometer (GPC–MALLS) were used for the analysis of epoxy resins based on bisphenol A. Compounds containing 2,3‐dihydroxypropyl group were identified in HPLC chromatograms by means of the derivatization of sample by acetone. The presence of branched molecules was proved by GPC–MALLS using a molar mass versus root mean square (RMS) radius plot or molar mass versus elution volume plot. The molar mass distribution determined by HPLC was compared with that obtained by GPC–MALLS. Molar mass averages measured by means of GPC, GPC–MALLS, vapor pressure osmometry (VPO), and end group analysis (EG) were compared and the differences of results obtained by particular methods were discussed. An appropriate GPC calibration was found on the basis of literature data and the comparison of molar mass averages measured by GPC, VPO, and GPC–MALLS. The refractive index increment of epoxy resins was determined. © 1999 John Wiley & Sons, Inc. J Appl Polym Sci 74: 2432–2438, 1999     

Molar mass distribution by size exclusion chromatography: Comparison of multi-angle light scattering and universal calibration

Series of polymers of various molar mass, chemical composition, and molecular architecture was analyzed by size exclusion chromatography (SEC) coupled with a multi-angle light scattering (MALS) photometer and an online viscometer. The molar mass averages were determined from the signal of MALS or calculated from the intrinsic viscosity and universal calibration. The comparison of the obtained results showed significant differences between the two methods. The MALS detection was shown to be more accurate for the determination of the weight-average molar mass and less vulnerable to the spreading of polymer peak by band broadening. The universal calibration can yield more accurate estimation of the number-average molar mass of branched polymers. It is also significantly more accurate for the characterization of fluorescent polymers than MALS with a regular laser of 660 nm. © 2019 Wiley Periodicals, Inc. J. Appl. Polym. Sci. 2019 , 136, 47561.     

Control of the Interaction Potential for Improved Resolution in Potential Barrier Chromatography

Abstract

Potential barrier chromatography (PBC) is a recently demonstrated protein separation technique which uses an isocratic elution procedure and exploits the differences in the interaction potentials between the proteins and the adsorbent. The interaction potential is determined by the van der Waals attraction, double-layer, Born, and hydration repulsions between the adsorbent and adsorbates and is very sensitive to the properties of the molecules, such as charge and size. A separation is feasible without any change in the composition of the mobile phase when the interaction potentials have surmountable potential barriers to adsorption and moderately deep adsorption energy wells. To ensure short analysis time and useful resolution, the total interaction potential must be controlled by suitably modifying the van der Waals attraction and the double-layer repulsion. The van der Waals attraction can be controlled by the introduction of small amounts of organic solvents in the aqueous mobile phase. The double-layer repulsion can be modified by changes in pH, ionic strength, or chemical nature of the ions of the mobile phase. Additionally, changes in temperature may be used to improve resolution. Here an updated high performance liquid chromatography version of PBC is reported. Using an isocratic elution procedure and an inexpensive ion-exchange column, the effect of changes in the pH, ionic strength, chemical nature of the ions, and organic solvent content of the mobile phase on the retention times and resolution of two model proteins (ovalbumin and bovine serum albumin) are demonstrated. Improved separations with high resolutions are achieved.     

A theory of topological separation of linear and star-shaped polymers by two-dimensional chromatography

Topology influences the size of macromolecules, but polymers are usually distributed with respect to molar mass, which also results in the size distribution within a polymeric sample. Due to this fact size-exclusion chromatography (SEC) is not able to separate even moderately polydisperse polymers by topology; the same is also true for the adsorption chromatography (AC). The full separation by molar mass and topology is not possible by any single mode of chromatography. These problems can be solved by means of two-dimensional chromatography which combines SEC and AC mechanisms. A theory of interactive chromatography of linear and star-shaped ideal-chain polymers is used to analyze two-dimensional chromatographic separation of polydisperse linear and star polymers. Basing on this theory, we simulate 2D-chromatograms for model mixtures of polydisperse linear and star-shaped polymers of equal average molar mass, and demonstrate that 2D-separation of such polymers by topology is possible. A possibility to separate symmetric and very asymmetric stars by 2D-chromatography is predicted. The influence of the molar-mass heterogeneity, pore size and adsorption interaction parameter on the 2D chromatographic pattern is analysed, and the conditions for a good separation of linear and star polymers are formulated. The theoretical results are in a qualitative agreement with the experimental data, which have been reported previously by Gerber and Radke.     

Topological separation of linear and star-shaped polystyrenes by off-line 2D chromatography. Stars having high molar mass arms and quantification of the star fraction

Off-line 2D separations on mixtures of linear and star-shaped polymers were performed using temperature gradient interaction chromatography (TGIC) as a first and SEC as a second dimension. The experiments resulted in clear separations of the linear and star shaped-structures for arm molar masses up to 42,000 g/mol. The resolution is nearly independent of the molar masses of the arms and depends only on the number of star arms. From the 2D chromatograms it is possible to determine the molar mass of the first branched structure, i.e. the three arm star. The evaluation of the relative peak volume allows a reliable estimation of the amount of branched structures in the complex mixture.     

Theory of chromatography of linear and cyclic polymers with functional groups

We discuss the chromatographic behavior of linear polymers and rings having specific (functional) adsorption-active groups. Functionalized eight-shaped, daisy-like and theta-shaped macromolecules are considered as well. By using a model of an ideal chain with point-type defects in a slit-like pore we derive equations for the distribution coefficient covering all modes of chromatography of functionalized polymers of any molar mass in both narrow and wide pores. Additionally simple approximate formulae are obtained for a number of important modes of chromatography; chromatograms are simulated for model mixtures of polydisperse non-functional and functional polymers. By using the theory we analyze separation of polymers by molar mass, functionality and topology. Although there is a principal possibility to use adsorption chromatography or size-exclusion chromatography (SEC), we conclude that the liquid chromatography at the critical condition (LCCC) is especially efficient for separation of polydisperse polymers by both functionality and topology. The theory predicts that functionalized linear and cyclic polymers can be separated from each others by LCCC even better than non-functionalized ones. The LCCC behavior of some other types of polymers such as comb-like and semicyclic ones is discussed as well.     

Separating ethylene-propylene-diene terpolymers according to the content of diene by HT-HPLC and HT 2D-LC

The separation of ethylene-propylene-diene terpolymers (EPDM) according to the three monomer units is an important task to understand the macroscopic properties of these technically important elastomers. In particular a separation with regard to the content of diene is of extreme value because the distribution of the latter along and across the molar mass axis determines the cross-linking behavior. In this study we show that high-temperature liquid chromatography (HT-HPLC) can be used for this purpose: the chromatographic retention of EPDM on porous graphite using a gradient of 1-decanol→trichlorobenzene is a function of both the content of ethylene and diene. The contribution of the diene alone to the chromatographic retention can be quantified by calculating the difference in elution volume between the EPDM and an EP copolymer having an equivalent content of ethylene. The chromatographic separation of fully hydrogenated EPDM indicates that the additional retention due to diene is the result of its geometrical nature. Coupling the HPLC separation according to the chemical composition with size exclusion chromatography (SEC) enables to reveal for the first time the complete molecular heterogeneity, i.e. the relationship between the chemical composition distribution and the molar mass distribution of EPDM.     

Temperature gradient interaction chromatography of low molecular weight polystyrene

Separation of low molecular weight polystyrene (PS) by temperature gradient interaction chromatography (TGIC) is reported. As in the separation of high molecular weight PS, temperature is an efficient variable to control the retention of low molecular weight PS in the reversed phase HPLC separation. However, a right choice of the eluent, typically a marginal solvent for the polymeric solute of interest, is crucial for the temperature to play an effective role in the retention control. For an example, PS oligomers were well separated by TGIC under the isocratic elution condition of C18 bonded silica and methanol as the stationary and the mobile phase, respectively.     

Simultaneous measurement of the molecular weight distribution and 5‐ethylidene‐2‐norbornene content across the molecular weight distribution of ethylene–propylene–diene terpolymer via a new size exclusion chromatography–ultraviolet–refractive index method

A size exclusion chromatography (SEC)–UV–refractive index (RI) method was developed to measure the 5‐ethylidene‐2‐norbornene (ENB) content across the molecular weight distribution (MWD) in ethylene–propylene–diene terpolymer (EPDM) at room temperature. The ratio of the UV and RI signals at the same effective elution volume was converted to ENB content. The feasibility of using this method to measure the ENB content across the MWD in EPDM at high temperature was also demonstrated. Prior understanding was that ENB had insufficient UV absorbance relative to high‐temperature SEC solvents to allow for useful measurements. We demonstrated this by using high‐boiling‐point solvents, such as decalin, with a low UV absorbance in the UV wavelength range of interest for ENB. These solvents also gave rise to a high enough specific RI increment (dn/dc) for EPDM that a suitable RI detector response was obtained. Additionally, this methodology could be readily applied to other polymers soluble at high temperature as long as the polymers contained a UV chromophore. These include polymers containing vinyl, conjugated vinyl, aromatic ring, carbonyl, or halocarbon groups. This UV‐absorption‐based detection concept might also be extended to high‐temperature thermal‐gradient interactive chromatography‐UV, high‐temperature solvent‐gradient interactive chromatography‐UV (high‐temperature liquid chromatography‐UV), temperature‐rising elution fractionation‐UV, crystallization analysis fractionation‐UV, and crystallization elution fractionation‐UV. © 2016 Wiley Periodicals, Inc. J. Appl. Polym. Sci. 2016 , 133, 43911.     

Multidimensional high‐temperature liquid chromatography: A new technique to characterize the chemical heterogeneity of Ziegler‐Natta‐based bimodal HDPE

High temperature two‐dimensional liquid chromatography (HT 2D‐LC) was recently introduced as a new technique to analyze the heterogeneities with regard to composition and molar mass present in model blends of polyolefins and various olefin copolymers. The method uses graphite as stationary phase and solvent gradients of 1‐decanol → 1,2,4‐trichlorobenzene as mobile phase for the compositional separation. With the aim to maximize the chromatographic resolution, the influence of the separation's temperature in the first dimension was evaluated: approaching the θ‐temperature of polyethylene (PE) in 1‐decanol selectively enhances the retention of higher molar mass PE standards while that of the lower molar mass ones is hardly affected. A bimodal ethylene/1‐butene copolymer and its temperature rising elution fraction (TREF) fractions were separated by HT 2D‐LC. For the first time, both axes of the contour plot were calibrated with regard to chemical composition and molar mass, respectively. Prefractionation of the bulk sample by TREF enhances the detectability of separated components of the 2D separation. The influence of the separation temperature, that is, working around the θ‐temperature of PE in 1‐decanol, can be used to enhance the chromatographic resolution of the 2D chromatography. © 2012 Wiley Periodicals, Inc. J. Appl. Polym. Sci., 2013     

A determination of the apparent molar absorption coefficients of the cobalt thiocyanate complexes of nonylphenol ethylene oxide adducts

Cobalt thiocyanate is used as a colorimetric reagent to determine traces of polyoxyethylene nonionic surfactants. It has been established that when there is an insufficient number of oxide units per hydrophobe the color intensity is markedly diminished, and with smaller numbers of oxide units the color fails to form altogether. the exact number of oxide units at which the colorimetric procedure fails has not been established previously, due to the unavailability of the pure individual ethylene oxide adducts. The individual ethylene oxide adducts of high purity were obtained by liquid chromatography on silica gel. The mixed solvent used was originally developed for thin-layer chromatography and was applied without change to column chromatography. The composition of the separated isomers has been determined by infrared, ultraviolet, nuclear magnetic resonance, and mass spectroscopy. The apparent molar absorption coefficients have been obtained for the individual cobalt thiocyanate complexes both in benzene and chlorform. For the low molecular weight adducts studied, the efficiency of color development and extraction into the organic phase has been found to be dependent on the concentration of the cobalt thiocyanate reagent. A saturated aqueous solution of cobalt thiocyanate was found to be preferable and benzene was found to be a more reliable extractant than chloroform. The apparent molar absorption coefficients do not vary linearly with chain length at low molecular weights and the minimum number of ethylene oxide units that will form an extractable color with the saturated reagent was found to be 3.     

Field-flow fractionation: New and exciting perspectives in polymer analysis

The development of advanced polymeric materials requires state-of-the-art synthesis and molecular characterization protocols. Only the precise knowledge of molecular structure–property correlations allows achieving optimum performance properties of novel materials. The analysis of the molecular composition of a complex polymeric material requires the determination of its molar mass, chemical composition, functionality and molecular topology among other (less important) parameters.A number of column-based fractionation methods, including size exclusion chromatography (SEC) and high performance interaction chromatography (HPLC) are the standard techniques for the analysis of complex polymers. These methods work well as long as the molar mass is not too high and/or the macromolecules do not exhibit undesired interactions with the stationary phase (column). Certain polymers form large aggregates or other entities (micelles, liposomes) in solution that typically cannot be analyzed by column-based fractionation methods.One alternative for the fractionation of such complex materials is field-flow fractionation (FFF), an open-channel technique which does not use a stationary phase. In FFF, all problems related to the stationary phase such as undesired adsorption, shear degradation of large macromolecules, co-elution of linear and branched macromolecules, can be avoided. Different sub-techniques of FFF render the fractionation of complex polymer systems according to molecular size, chemical composition or molecular topology.In this review article, most recent developments of FFF in polymer analysis are addressed. Natural and synthetic polymers, polyolefins and polymeric nanocomposites are embraced. The most important FFF sub-techniques in polymer analysis include asymmetric flow field-flow fractionation (AF4) and thermal field-flow fractionation (ThFFF). Major developments in these very topics since 2008 are critically discussed following a previous review article that summarized earlier work (see Prog. Polym. Sci. 2009; 34: 351–68). The potentials and limitations of the different FFF sub-techniques for polymer analysis are elaborated and most recent methods of hyphenating FFF with other techniques are highlighted.