Behavior of Reversibly Associating Systems in Size Exclusion Chromatography. Interpretation of Experimental Data Based on Theoretical Model

Abstract

The shape of the SEC chromatogram of a reversibly associating system in the SEC is a result of several competing processes: (a) SEC mechanism, (b) re-equilibration of the perturbed association equilibrium, and (c) the axial dispersion of individual components in the mobile phase. In some associating (micellizing) block copolymer systems in selective solvents, other secondary processes (e.g. temporary trapping of a polymer in pores of the stationary phase) may influence the chromatogram shape and complicate the analysis of experimental data. In this paper, general aspects of the interpretation of experimental data is discussed on the basis of theoretical models.     

Semimicro Size Exclusion Chromatography For Oligomers. I. Column Packing Procedure

Abstract

Polystyrene gels of a particle diameter 10 ± 2 μm for the use in oligomer separation were packed into 1.5 mm i.d. × 25 cm length columns by the balanced density slurry-packing technique under a constant flow rate of 500 μL/min. The slurry solvent was a mixture of toluene and chloroform (50.5/49.5, v/v). The example of the number of theoretical plates (N) of these columns was 8600 plates/25 cm (HETP = 29.1 μm) at flow rate of 40 μL/min by injecting 1 μL of 0.5% benzene solution. Sixteen columns were connected and the overall value of N was 103000 plates/4 m. A typical example of oligomer separation was demonstrated. A constant-flow technique is preferable to a constant-pressure technique. When two or three column blanks were packed together, the columns located at the outlet of the packer-column assembly had higher values of N. Optimum flow rate of the slurry solvent when three column blanks were packed together lay between 400 and 500 μL/min. The packing efficiency, that is, the probability of getting valid columns was about 60%. Viscous slurry solvents were not effective to get efficient columns. To pack gels in the less swollen state gave sometimes efficient columns. Pressure monitoring in progress of packing was very effective to foresee the column efficiency.     

Size Exclusion Chromatography of Poly(vinylpyrrolidone): I. The Chromatographic Method

Abstract

The optimum size exclusion chromatographic (SEC) method for poly-(vinylpyrrolidone) (PVP) was found to be based upon a stationary phase of diol derivatized silica of pore sizes 3000, 500, and 75Å and a mobile phase of 50:50 (v/v) MeOH/H₂O containing 0.1M-LiNO₃. Sample recovery under identical conditions varied for the commercial packings investigated and was found to be inversely related to molecular weight. The latter phenomenon was rationalized on the basis of a limited number of active substrate sites available for binding. Methanol was found to be a more effective mobile phase modifier than either dimethyl formamide or acetonitrile apparently due to its ability to function as a proton donor in hydrogen bonding with PVP. Chromatographic evidence for the existence of semipolyampholyte character in PVP is presented. A procedure for the construction of a column set log-linear in calibration and of extended dynamic range is described and is based upon hydrodynamic volume theory.     

Size Exclusion Chromatography of Polyamides

Abstract

We report here a new chromatographic method for characterization of polyamides and polyether-block-amide copolymers by Size Exclusion Chromatography. First, it concerns absolute characterization of polyamide probes by classical light scattering and Ubbelohde viscometry measurements and the choice of narrow distribution standards to calibrate the GPC system. The GPC analysis of polyamides 6, 11, 12 and copolymers is based upon the dual detection refractometer/on-line continuous viscometer at 130°C using columns packed with cross-linked polystyrene gels, benzyl alcohol as mobile phase and polytetrahydrofuran standards for calibration. The main results are absolute average molecular weights and distribution curve, intrinsic viscosity and viscosity law. In addition, viscometric data produced evidence of long-chain branching occuring beyond a limiting molecular weight in some polyamide 12 samples.     

Quenched Instationary Polymerization Systems, 6. The Direct Determination of Axial Dispersion in Size Exclusion Chromatography by Comparison of Theoretical and Ideal Peak Widths

For the direct determination of axial dispersion in size exclusion chromatography a simple method is presented which makes use of the measured and ideal peak widths. The peak width can be defined in two ways: either absolute as the difference of successive points of inflection or relative as the ratio of these points. If the absolute peak width is invariant for the number, molar mass and hyper distribution then this distribution can unambiguously be classified as Poissonian. The relative peak width for such distributions is strictly determined by the experimental parameters. It is demonstrated that axial dispersion only leads to an additive increase in the peak variances for peaks with a relative peak width smaller than 1.25. Thus, it is possible to determine directly the axial dispersion of an experimental size exclusion chromatography set‐up by the use of Poisson distributions prepared by quenched instationary polymerization techniques or any other technique leading to ideal Poisson distributions.     

Characterization of Xylansulfate by Size Exclusion Chromatography

Abstract

A sulfated, low molecular weight beech xylan was fractionated preparatively on Sephadex G-50 into ten fractions with molecular weights from 17 000 to 1 800. The degree of sulfation, refractive index increment, molecular weight, and specific optical rotation of each fraction was determined. The fractions were then used to calibrate a Sephacryl S–200 gel column for the molecular weight distribution analysis of xylansulfate.

The molecular weights, M _W, obtained by SEC for samples of xylansulfate were in good agreement (within 4 ± 1%) to the values derived by low angle light scattering.     

Temperature effects in Microcolumn Size Exclusion Chromatography

Abstract

Effects of column temperature on the column efficiency, retention time and stability of analytes were studied in microcolumn size-exclusion chromatography. Larger theoretical plates were achieved at column temperatures betwen 70 and 100 °C. In the constant-flow mode the retention time of analytes decreased with increasing column temperature, which was due mainly to thermal expansion of the mobile phase. When the column temperature was around or higher than the critical temperature of the mobile phase, the retention times of the analytes observed under supercritical pressure conditions still indicated dominancy of the size-exclusion mechanism, while another retention mechanism was involved under subcritical pressure conditions. In the case of the analysis of saccharides, the column temperature should be lower than 100 °C because oligosaccharides were decomposed at higher temperatures.     

A Calibration Technique for Size Exclusion Chromatography

Abstract

A modified Weibull distribution function is shown to be useful in calibrating the molecular size separation capabilities of both rigid and swellable gel packing materials. Two parameters are used in this function which are related to a packing material's micropore volume distribution. The calibration curves of a set of different packing materials connected in series were predicted from the Weibull calibration functions obtained for each individual packing material.     

Continuous Viscometric Detection in Size Exclusion Chromatography

Abstract

Continuous viscometric detection is based on the measurement of pressure drop in an on-line small capillary tube in which chromatographic eluents flow at constant flow rate. This detector is always coupled with a concentration detector (usually refractometer) and located before it to avoid back pressure in the refractometer. In order to obtain reliable information for polymer samples, it is generally necessary to connect these two detectors to a computer which performs data acquisition and treatment.

First, we discuss the problem of shape, geometry and dimensions of the viscometer. The typical characteristics are the result of a compromise between contradictory targets, mainly small internal volume, low shear rate and low pressure drop. It is shown that Poiseuille's laminar flow is only obtained when coiling radius of the measurement tube is greater than 6 cm, which is not the case inside the refractometer. Accordingly, two pressure transducers are necessary to eliminate pressure drop data coming from refractometer.

In a second part, we show how to extract information from pressure variation data. By using concentration data, pure solvent pressure and sample pressure it is possible to calculate intrinsic viscosity extrapolated to zero concentration at each point of the chromatogram. By comparison with intrinsic viscosity of the polymer used for calibration, a correction of hydrodynamic volume according to Benoit's universal calibration leads to absolute molecular weights.

In addition, for a linear polymer, the knowledge of log [η] versus log M leads to the determination of Mark-Houwink relationship coefficients. For branched polymers, viscosity laws are curved and the comparison between the linear law corresponding to the linear equivalent polymer and the experimental law allows the determination of the g' branching parameter distribution.     

Size Exclusion Chromatography of Poly(vinylpyrrolidone)

Abstract

Poly(vinylpyrrolidone) and poly(ethylene oxide) separate by hydrodynamic volume on Toyo Soda TSK-PW columns in a mixed solvent mobile phase of 50:50 (v/v) MeOH/H₂O containing 0.1M LiNO₃. From this separation a single universal calibration curve based on hydrodynamic volume [η]M can be obtained. Accurate weight average molecular weights of PVP were obtained by both SEC/LALLS and universal calibration showing good agreement between the two methods. SEC/LALLS overestimates the number average molecular weight for broad distribution polymers due largely to the lack of sensitivity of the LALLS detector to the low molecular weight portion of the polymers, while the universal calibration method slightly underestimates the number average molecular weight as compared to osmometric values.     

Enthalpic Partition-Assisted Size Exclusion Chromatography: 1. Principle of Method

A novel high performance liquid chromatographic (HPLC) method viz. “enthalpic partition assisted size exclusion chromatography” deliberately combines entropic and enthalpic partition mechanisms. It enables separation of homopolymers according to their molar mass with increased selectivity, as well as discrimination of polymer species differing in their nature/composition. Enthalpic partition of macromolecules takes place between the mobile phase and the stationary “liquid” of a different chemical nature, which is immobilized within pores of an appropriate carrier (a bonded phase). The extent of enthalpic partition depends on the accessibility of bonded phase for macromolecules and on the difference of polymer solubility in the mobile phase and in the solvated bonded phase. The enthalpic partition in favor of column packing arises from better solubility of polymer solutes in the solvated stationary phase compared to the mobile phase. Macromolecules are “pushed” into the solvated stationary phase and their retention volumes (V_R) increase. In the area of high molar masses, the extent of enthalpic partition as rule raises with the increasing size of macromolecules. However, under properly chosen experimental conditions the enthalpic partition may rapidly diminish with the sample molar mass (M), likely due to the solubility changes and/or due to partial exclusion of macromolecules from the pores. As result, the corresponding retention volumes sharply drop within a narrow range of M with the increasing size of macromolecules. This results in the log M vs. V_R dependences, which resemble in their form that for size exclusion chromatography but are much more flat indicating highly selective separations of homopolymers according to their molar masses. In this way, enthalpic partition “assists” entropic partition (size exclusion). Polymer species, which do not undergo enthalpic partition, elute from the HPLC column in the conventional size exclusion mode and can be discriminated from the partitioning species. Enthalpic partition assisted size exclusion chromatography can be utilized in separation and characterization of various homopolymers, and polymer blends.     

Quantitative Size Exclusion Chromatography: Assessing New Developments

Abstract

Methods of assessing new technology are achieving increased importance because rapid technological changes cause obsolescence of evaluations before they are completed. Users now must often evaluate the specific version of the new technology that they receive. Four major areas are used here to provide examples of assessment methods: high resolution columns, coupled concentration-molecular weight-differential viscometer detectors, flow rate monitoring using a thermal pulse flowmeter and determination of Mark-Houwink constants from polydisperse standards. Specific methods of assessment examined centre about error analysis and sensitivity analysis. Several of the methods use the conventional calibration curve. The idea of correction priority (i.e. thoroughly examining the most fundamental significant corrections first) is emphasized.     

Size Exclusion Chromatography of Polymers With Random Tetrafunctional Branching

Abstract

The behaviour of polydisperse branched copolymers of methyl methacrylate with a small amount of randomly situated tetrafunctional ethylenedimethacrylate units was investigated by means of size exclusion chromatography (SEC). A procedure has been suggested for the conversion of apparent values of molecular parameters of real polymer branched systems (M_{n, app}; M_{w, app} obtained from SEC data by calibration of the separation system using a linear polymer) into actual values. This was made possible by off-line coupling of SEC and viscometry. The branching was characterized by the weight average number of branching sites in the macro-molecule, m_w, and the branching index, y.     

Characterization of Polymer Architectures by Multidetection Size Exclusion Chromatography

Summary: This paper discusses the analysis of star-branched and dendritic polyisobutylenes (PIBs) using branching parameters based on radii of gyration R_g,z and hydrodynamic radii R_h,z. R_g,z and R_h,z were measured by Size Exclusion Chromatography equipped with a Wyatt Technology Viscostar viscometer (VIS), a Wyatt Optilab DSP RI detector, a Wyatt DAWN EOS 18 angle Multiangle Light Scattering (MALS) detector and a Wyatt Quasi-elastic Light Scattering QELS detector. Branching parameters were calculated from these measurements and compared with those computed for these architectures.     

Size Exclusion Chromatography of Nonionic and Anionic Copolymers of Vinylpyrrolidone

Abstract

Size exclusion chromatography (SEC) of poly(vinylpyrrolidoneco-vinylacetate), PVPVA, and poly(vinylpyrrolidone-co-dimethylamincethylmethacrylate-co-vinylcaprolactan),PVPDMMAEMAVC, is evaluated in terms of resolution between polymer and solvent peaks using aqueous and nonaqueous mobile phases. A 1:1 (v/v) water/methanol, 0.1M LiNO₂ mobile phase is used with four Waters Ultrahydrogel columns with pore sizes of 120Å, 500Å, 1000Å, and 2000Å. DMF, 0.1M LiNO₃ mobile phase is used with three different two column sets. Two of the column sets are comprised of a mixed bed packing of poly(styrene-co-divinyl benzene), obtained from Shodex (KD80M), followed by either an Ultrahydrogel 120Å or a PLge1 100Å. The third two column set is a PLge1 10⁴Å followed by a PLgel 500Å. Any of the second columns in these sets improve separation between the trailing end of the polymer peak and the leading end of the solvent peak but the column set using an Ultrahydrogel 120Å yields a separation of these peaks whose valley is closer to the baseline. The aqueous mobile phase with the four column set yields a separation between the trailing end of the polymer peak and the leading end of the solvent peak whose valley is closest to the baseline. Recovery of PVPVA and PVPDMAEMAVC from the columns evaluated is 100%. Vinylpyrrolidone compositions of PVPVA ranging from 30 to 70 mole % were studied using both mobile phases. SEC of     

Instrumental Broadening Correction in Size Exclusion Chromatography. Comparison of Several Deconvolution Techniques

Abstract

Several deconvolution techniques (1–7) and a novel method herein presented, are compared in relation to their ability for correcting size exclusion chromatograms for the undesirable effect of instrumental broadening. Such methods are evaluated on the same computer, and through a “synthetic” example of known solution. Methods based on the frequency domain are only applicable to uniform deconvolution problems with stationary statistics. However, in the herein presented heuristic method (based on the Wiener filter in the frequency domain), it is possible to relax this last restriction, and generate solutions that are equivalent to considering signals with time-varying statistics. The evaluated techniques are compared on the basis of quality of results, computational considerations and adjustment facility. Stochastic techniques provide the best (and nearly identical) numerical solutions. This is mainly due to their increased facility to introduce “a priori” information about the expected solution. As a counterpart, stochastic techniques are conceptually more complex, more difficult to implement, and normally inolve more elaborate adjustment procedures.     

Comparing Dichromate Oxidation and Size Exclusion Chromatography of Dissolved Carbohydrates

Standardised methods are used to characterise the pulp or cellulosic material for its alkali resistance and alkali solubility. Today, pulp is usually characterised by its solubility in sodium hydroxide. The characterisation test was based on the treatment of pulp with sodium hydroxide solution of a defined concentration, according to a specified procedure. The dissolved organic matter was then determined by dichromate oxidation. The results obtained by size exclusion chromatography showed that both the amount and the molecular weight of the dissolved carbohydrates vary when investigating solubility and α-cellulose content of pulps. The results show further that S₁₈ values obtained by dichromate oxidation are overestimated when characterising pulps containing higher amounts of hemicellulose and/ or short chain cellulose, compared with results obtained by the SEC method. On the contrary, S₁₀ values are overestimated when determined by dichromate oxidation in higher alpha pulps.     

Characterization of LC Stationary Phases by Inverse Size Exclusion Chromatography

Abstract

The most efficient use of a porous particulate stationary phase can only be made when the material in the column is fully characterized. While complete characterization includes both chemical and physical aspects, the macro structure provides the framework for the microscopic chemical interactions. The physical analysis is therefore critically important for a more complete calibration of the chromatographic effects.     

Size Exclusion Chromatography Calibration Assessment Utilizing Coupled Molecular Weight Detectors

Abstract

This paper shows that the usual method of representing an SEC calibration curve by a single polynomial curve may often be inadequate with new high resolution columns. Data points wind about the fitted line. The significant magnitude and systematic nature of these deviations clearly appear when a plot of residuals is derived from the conventional calibration curve and expressed in terms of the percent error in molecular weight. The deviation of the calibration data from the fitted line was approximately 10% for the conventional molecular weight and intrinsic viscosity calibration curves. It became 20% for the universal calibration curve. LALLS and DV detectors were used together with the DRI detector to provide evidence that the calibration curve deviations were due to the column packings and not due to some other cause (e.g., vendor values of molecular weight). Use of a polynomial fit to a portion of the curve corresponding to the retention volume range of an unknown was used to show the significant improvement in results which occurred when the calibration variations were taken into account. At present, use of many individual narrow standards is necessary to elucidate the effect.