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.     

Reduced sample recovery in liquid chromatography at critical adsorption point of high molar mass polystyrene

Low sample recovery may represent an important drawback in liquid chromatography at the critical adsorption point (LC-CAP) if the critical eluent is not carefully fitted to the system. So far, this problem was often overlooked and only few experimental examples can be found in literature. We showed that in the case of polystyrene (PS) in a tetrahydrofuran (THF)/n-hexane critical mixed eluent, PS with molar masses higher than 100 kg mol⁻¹ were not eluted from a tandem of two columns packed by bare silica gels with 30 nm and 100 nm pore size, respectively. The polymer trapped within the columns was well recovered after injection of a small volume of pure THF as demonstrated using 2D chromatography. We studied PS conformations by means of small angle neutron scattering and found that the THF/n-hexane critical eluent is in fact a theta solvent for PS. By replacing it by a CH₂Cl₂/n-hexane critical mixture, which is a good solvent for PS, the limits of reduced sample recovery was displaced towards far higher molar masses. Thus, thermodynamic quality of eluent - theta or good solvent - plays an important role on the phenomenon of sample recovery.     

Liquid chromatography of polymers under limiting conditions of adsorption: III. Role of experimental variables

LC of polymers under limiting conditions of adsorption (LC LCA) is a novel method based on different mobility of (pore excluded) macromolecules compared to (pore permeating) solvent molecules. Polymer sample is injected in a solvent preventing its adsorption within the column. Eluent promotes sample adsorption. Under these conditions, macromolecules cannot leave its initial solvent and elute from the column independently of their molar mass. In contrast, a less interactive simultaneously injected polymer leaves its initial solvent zone and is eluted in the size exclusion mode. As a result, chemically different polymer species can be discriminated. The effect of selected experimental conditions was studied on the LC LCA behavior of poly(methyl methacrylate)s eluted from bare silica gel columns. The parameters were packing pore diameter, injected sample volume and concentration, as well as column temperature. The size independent elution was only little affected by the above parameters and LC LCA produced well-focused peaks. The LC LCA mechanism was operative even at a very large sample of both volume and concentration. This makes LC LCA a robust and user-friendly method, likely suitable also for characterization of minor components of polymer mixtures.     

Liquid chromatography of polyoxyethylenes under critical conditions: a thermodynamic study

Polyethylene glycols (PEG), fatty alcohol ethoxylates (FAE) and fatty acid polyglycol esters were analyzed by liquid chromatography under critical conditions (LCCC) on typical reversed phase columns in mobile phases consisting of methanol-water and acetone-water in the region of the critical adsorption point (CAP) for the oxyethylene unit. Critical conditions can be adjusted by varying the mobile phase composition or temperature. In methanol-water the temperature dependence is almost negligible, while it is much more pronounced in acetone-water. Critical conditions for the oxyethylene unit were realized on octadecyl phases in methanol-water containing 85.8 wt.% methanol and in acetone-water at 89.6 wt.% acetone. In the van't Hoff plots the logarithm of the distribution coefficient was plotted versus the reciprocal temperature. In all plots straight lines were found, from slope and intercept of which the entropy and enthalpy changes were calculated. The relation of the thermodynamic parameters -deltaG/RT, -deltaH/RT, and deltaS/R to the numbers of A and B chain units in block copolymers AB and BAB at the CAP for A is studied both theoretically and experimentally. Various approaches for obtaining the critical temperature are discussed.     

Separation of polyethylene glycols and maleimide‐terminated polyethylene glycols by reversed‐phase liquid chromatography under critical conditions

The separation of polyethylene glycols and maleimide‐substituted polyethylene glycol derivatives based on the number of maleimide end‐groups under critical liquid chromatography conditions has been investigated on a reversed‐phase column. The critical solvent compositions for nonfunctional polyethylene glycols and bifunctional maleimide‐substituted polyethylene glycols were determined to be identical at about 40% acetonitrile in water on a reversed‐phase octadecyl carbon chain‐bonded silica column using mixtures of acetonitrile and water of varying composition as the mobile phase at 25°C. The maleimide‐functionalized polyethylene glycols were successfully separated according to maleimide functionality (with zero, one, two, or three maleimide end‐groups, respectively) under the critical isocratic elution conditions without obvious effect of molar mass. The separation was mainly due to the hydrophobic interaction between the maleimide end‐groups and the column packing. Off‐line matrix‐assisted laser desorption/ionization time of flight mass spectrometry was used to identify the repeating units and, especially, the end‐groups of the maleimide‐substituted polyethylene glycols. Liquid chromatography analysis at critical conditions could provide useful information to optimize the synthesis of functional polyethylene glycols. To our knowledge, this is the first report of the baseline separation of maleimide‐functionalized polyethylene glycols based on the functionality independent of the molar mass without derivatization by isocratic elution.     

Liquid Chromatography of Synthetic Polymers under Limiting Conditions of Insolubility III

Summary Performance was evaluated of silica based commercial monolithic rod-like columns in liquid chromatography of synthetic polymers under limiting conditions of enthalpic interactions (LC LC). LC LC employs the barrier effect of the pore permeating and therefore slowly eluting small molecules toward the pore excluded, fast eluting macromolecules. Phase separation (precipitation) barrier action was applied in present study. The barrier was created either by the narrow pulse of an appropriate nonsolvent injected into the column just before the sample solution (LC LC of insolubility – LC LCI) or by the eluent itself. In the latter case, the polymer sample was dissolved and injected in a good solvent (LC LC of solubility – LC LCS). In LC LCI, polymer species cannot break thru the nonsolvent zone while in LC LCS they cannot enter eluent, which is their precipitant. Therefore, polymer species keep moving in the zone of their original solvent. Macromolecules eluting under the LC LC mechanism leave the column in the retention volume (V_R) roughly corresponding to V_R of the low molar mass substances and can be efficiently separated from the polymer species non-hindered by the barrier action. The known advantages of monoliths were confirmed. From the point of view of LC LCI and LC LCS the most important quality of monolithic columns represents their excellent permeability, which allows both working at high flow rates and injecting very high (in the range of 5%) sample concentrations. Monolithic column tolerate also extremely high molar mass samples (M>10,000 kg · mol⁻¹). On the other hand, the mesopores (separation pores) of the tested monoliths exhibited rather small volume and wide size distribution. These shortcomings partially impair the permeability advantage of monoliths because in order to obtain high LC LC separation selectivity a tandem of several monolithic columns must be applied. Presence of large mesopores also reduces applicability of monolithic columns for molar masses below about 50 kg · mol⁻¹ because V_Rs of polymers eluted behind the barrier are similar to that of freely eluting species. The non- negligible break-thru phenomenon was observed for the very high polymer molar masses largely eluting behind the barrier. It is assumed that the fraction of very large mesopores present in the monoliths or association/microphase separation of macromolecules may be responsible for this phenomenon. This is why the presently marketed SiO₂ monolithic columns are mainly suitable for the fast purification of the LC LC eluting macromolecules from the polymeric admixtures non-hindered by the barrier-forming liquid. Still, monolithic columns have large potential in the LC LCI and LC LCS procedures provided size (effective diameter) of the mesopores can be reduced and their volume increased.     

Liquid chromatography of synthetic polymers under limiting conditions of insolubility. I. Principle of the method

A novel liquid chromatographic procedure is presented. It is based on differences in the transport velocities of the fast-moving, pore-excluded macromolecules and slow-progressing, pore-permeating small molecules of an auxiliary liquid. A barrier of small molecules selectively decelerates certain kind of macromolecules while other kind remains unhindered. As a result, polymers of different nature are efficiently separated. In this new approach, the barrier is formed by a zone of a non-solvent injected immediately before the sample solution. The resulting method is denoted liquid chromatography under limiting conditions of insolubility.     

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.     

Size exclusion chromatography – A blessing and a curse of science and technology of synthetic polymers

Size exclusion chromatography, SEC is one of the most popular methods for the separation of different kinds of macromolecules. This critical review gives concise information about macromolecules and their behavior in solution, basic understanding about principles, instrumentation, and application possibilities of SEC, and more in detail discusses drawbacks and pitfalls of the method with the emphasis on synthetic polymers. Selected practical advices are included to help enhance the quality of SEC results.     

Liquid chromatography under limiting conditions of desorption 6: Separation of a four‐component polymer blend

Baseline separation was achieved of a model four‐component polymer blend of polystyrene‐poly(methyl methacrylate)‐poly(ethylene oxide)‐poly(2‐vinyl pyridine) in a single chromatographic run with help of the unconventional method of liquid chromatography under limiting conditions of desorption. Narrow barriers of liquids were employed, which selectively decelerated elution of particular kinds of macromolecules. Bare silica gel was the column packing, and the eluent was a mixture of dimethylformamide/tetrahydrofuran/toluene 30:50:20 w/w/w. Barrier compositions were neat toluene, B#1, neat tetrahydrofuran, B#2, and dimethylformamide/tetrahydrofuran/toluene 15:55:30, B#3. Minor blend constituents (∼1%) could be identified, as well. The result represents a step toward the separation and molecular characterization of triblock‐copolymers, many of which are expected to contain besides both parent homopolymers also the diblock chains and thus they are in fact four‐component polymer blends.     

Dietary‐flavonoid‐rich flowers of Rumex nervosus Vahl: Liquid chromatography with electrospray ionization tandem mass spectrometry profiling and in vitro anti‐inflammatory effects

Rumex nervosus is a plant species found widely in Eastern Africa and the Arabian Peninsula. In addition to its uses in traditional medicinal, the plant shows various biological activities, such as antiviral, antibacterial, and antioxidant activity. In this study, nine flavonols, six flavones, three flavanones, and one flavanol were characterized from the flowers of R. nervosus using liquid chromatography with electrospray ionization tandem mass spectrometry and literature data. Validation data indicated that the determination coefficients (R²) were ≥ 0.9914. The limits of detection and quantification were in the ranges of 0.15–1.24 and 0.50–4.13 mg/L, respectively. Recoveries at 10 and 50 mg/L were 71.1–110.2 and 65.4–115.1%, with relative standard deviations of 7.4–40.1 and 2.1–13.0%, respectively. Quercetin 3‐O‐rhamnoside ( 10 ) was the dominant component, contributing 30.8% of total flavonoids (1003.0 ± 26.2 mg/kg fresh flower sample), whereas luteolin 6‐C‐glucoside (3) was the lowest yielding compound (0.1%). The 19 flavonoids identified were characterized for the first time. In vitro anti‐inflammatory studies showed that this mixture can suppress the production of inflammatory mediators, including inducible nitric oxide synthase, cyclooxygenase‐2, kappa B inhibitor, and interleukin‐1β, by down‐regulating the nuclear factor‐kappa B and mitogen‐activated protein kinases pathways. The results of this study may provide information for processing R. nervosus as a potential source of functional food.     

Evaluation of the limit of performance of an analytical method based on a statistical calculation of its critical concentrations according to ISO standard 11843: Application to routine control of banned veterinary drug residues in food according to European Decision 657/2002/EC

Traceability of the measurement of analytical parameters capable of evaluating the performance of methods is an important concept for the assessment of quality for the routine control, especially for residue monitoring of non-authorized medicinal substances in food from animal origin. The European Decision no. 657/2002/EC recommends to calculate two statistical limits, CCα and CCβ, which allow to evaluate the critical concentrations above which the method reliably distinguish and quantify a substance taking into account the variability of the method and the statistical risk to take a wrong decision. The calculation, which can be derived from the ISO standard no. 11843 is applied on a routine basis. An example displays a very simple way for evaluating the performance of an LC-MSMS method which has been validated a few years ago and is qualified onto a Micromass Quattro LCZ tandem mass spectrometer to monitor and confirm the nitrofuran metabolite residues in food from animal origin. Community Reference Laboratory for Antimicrobial Veterinary Drug Residue Control in Food from Animal Origin