A Method of Correcting for Flow-Rate Fluctuations in Size Exclusion Chromatography Calculations: Applications to Methylene Chloride/Hexafluoroisopropanol Solvent System

Abstract

The mixture of methylene chloride/hexafluoroisopropanol (70/30, v/v) is an excellent polyester solvent, but its low boiling point causes unstable flow when it is used for size exclusion chromatography (SEC). In high-performance SEC experiments, retention time is normally used to measure elution volume; however, unstable flow makes it difficult to calibrate an SEC column set or calculate molecular weight parameters from a chromatogram. We have devised a simple and inexpensive method to compensate for the effect of unstable flow in SEC calculations. A calibration marker injected along with each sample is used to indicate flow-rate variations. The ratio of the sample retention time to the marker retention time is invariant to flow-rate changes and is used in place of retention time as a measure of elution volume in the universal calibration technique. Calibrating a column set and analyzing chromatograms by this method result in a large improvement in the accuracy and precision of calculated molecular weight parameters.     

一种新的标定体积排除色谱（SEC）的方法

基于体积排除色谱中测得的淋出体积和动态激光光散射中测得的平动扩散系数都直接依赖于高分子的流体力学体积这一事实，本文在理论上提出了一种把淋出体积分布和平动扩散系数分布二者结合起来标定体积排除色谱的新方法，并且在实验上通过对宽分布的聚苯乙烯标准样品的测试证实了该方法的可行性．     

Simulation of size-exclusion chromatography distribution coefficients of comb-shaped molecules in spherical pores comparison of simulation and experiment

Simulations of the distribution coefficients of linear polymers and regular combs with various spacings between the arms have been performed. The distribution coefficients were plotted as a function of the number of segments in order to compare the size exclusion chromatography (SEC)-elution behavior of combs relative to linear molecules. By comparing the simulated SEC-calibration curves it is possible to predict the elution behavior of comb-shaped polymers relative to linear ones. In order to compare the results obtained by computer simulations with experimental data, a variety of comb-shaped polymers varying in side chain length, spacing between the side chains and molecular weights of the backbone were analyzed by SEC with light-scattering detection. It was found that the computer simulations could predict the molecular weights of linear molecules having the same retention volume with an accuracy of about 10%, i.e. the error in the molecular weight obtained by calculating the molecular weight of the comb-polymer based on a calibration curve constructed using linear standards and the results of the computer simulations are of the same magnitude as the experimental error of absolute molecular weight determination.     

The size exclusion chromatography calibration of ‘Mixed-A’ and ‘Mixed-D’ columns using various polymers and compounds: An application to coal-derived materials

This work attempts to obtain the calibration curves of two different size exclusion chromatography (SEC) columns operating with 1-methyl-2-pyrrolidinone (NMP) as eluent by using various standards. Polystyrene (PS) and polymethylmethacrylate (PMMA) standards were used for obtaining calibration curves, and checked against polysaccharide (PSAC) standards, some small aromatic polycyclic standards and miscellaneous polymers. Polystyrenes and polymethylmethacrylates gave identical calibrations while polysaccharides and miscellaneous polymers lay within 1 or 2 min of the polystyrene calibration. Small molecules of mass less than 1000 units lay on or near to the polystyrene calibration lines, with a shift to late elution for the smallest molecules. This shift may be caused by the interaction with the column packing. A sample has been examined by analytical size exclusion chromatography, which was calibrated using polystyrene and polymethylmethacrylate standards. Molecular mass (MM) distributions of the sample have been examined in terms of these calibrations.     

Size-exclusion chromatography using deuterated mobile phases

The effect of deuterated solvents in size-exclusion chromatography (SEC) was studied by comparing intrinsic viscosity measurements, SEC calibration curves, and column efficiency using water-soluble polymers. For aqueous SEC, the use of deuterium oxide slightly increases the SEC elution volume. To verify that adsorption onto the packing was absent, data from exclusion experiments were compared at 35 and 50 degrees C. Our results indicate that adsorption is not occurring for pullulan or polyethylene glycol (PEG)/poly(ethylene oxide) (PEO); for the latter, however, the elution volume increased using both D2O and H2O, indicative of slight hydrodynamic volume contraction of PEG/PEO at higher temperatures. A moderate increase in band broadening (moderate decrease in column efficiency) was observed using D2O. Finally, the effects of chloroform versus deuterated chloroform were evaluated, but no hydrodynamic volume changes were observed.     

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.     

Quantitative aspects of gradient HPLC of copolymers from styrene and ethyl methacrylate

Summary Prerequisite of quantitative evaluation in chromatography is equivalence of sample composition and detector signal. This includes complete retention and proper elution of all sample constituents. In polymer HPLC, complete retention requires a poor starting eluent, a sufficiently active column, and a low ratio of injection volume to column volume. On small pore columns, insufficient retention caused the polymer to elute either in the interstitial volume (sample exclusion), together with the sample solvent, or immediately after the solvent plug.Stat-copoly(styrene/ethyl methacrylate) samples are more difficultly retained thanstat-copoly(styrene/acrylonitrile) specimes. With the former copolymer it could be shown that incomplete retention did not cause sample demixing. In order to gain complete retention, non-exclusion HPLC of polymers should be performed with columns whose solvent volume is at least 50 times as large as the injection volume. This consequence is of practical importance in chromatographic cross-fractionation where rather large volumes of SEC eluate are injected into the apparatus for gradient HPLC.     

Statistical properties of the band-broadening function

Statistical properties of the band-broadening function calculated from the equilibrium and kinetic theories of Giddings and Eyring are compared with real liquid-chromatography elution curves of low-molecular-weight substances and with light-scattering elution curves of a high-molecular-weight polystyrene standard injected at several concentrations. The curves obtained by liquid chromatography agree in a large span of elution times with the theoretical ones and can be described by one value of the plate-height. The size exclusion chromatography (SEC) elution curves seem to approach the predicted statistical properties with decreasing concentration of injected polymer but, in general, a reliable prediction of band-broadening (BB) functions in SEC of polymers, especially of non-symmetric BB functions near the exclusion limit, seems not possible at this point. The relation of the theory with the characteristic-function approach developed by Dondi is also discussed.     

Characterization of copolymers by high performance liquid chromatography

Size exclusion chromatography (SEC) is capable of evaluating the molecular weight distribution (MWD) of a sample. Information about the chemical composition distribution can be gained by gradient high performance liquid chromatography (gradient HPLC), where a poor starting eluent is, in the course of the separation, substituted by another one of increasing elution strength. Both normal-phase and reversed-phase systems can be employed. The combination of SEC and gradient HPLC enables chromatographic cross-fractionation to be performed efficiently.     

The application of the size exclusion chromatography/ viscometry technique to the determination of molar-mass and long-chain branching in polychloroprene

The coupling of a low-pressure size exclusion chromatography (SEC) with a modified Ubbelohde capillary viscometer is described. This SEC/viscometry system measures the flow time of aliquot fractions (5ml) of the SEC effluent along with the refractive index change. This dual detection leads to the determination of the intrinsic viscosity as a function of the elution volume, thus allowing a precise use of Benoit's universal calibration. Moreover information of the branching factors (degree of long-chain branching, long-chain branching frequency) can be calculated under certain assumptions. As an example the changes in molar-mass distribution and branching factors during mechanical shear degradation (mastication) of special polychloroprene samples were investigated. It is shown that the SEC/viscometry system is especially suitable for the characterization of polymers with broad molar-mass distribution and extremely high molecular tails. The data provided by this method are useful for the investigation of the viscoelastic behaviour of concentated polychloroprene solutions and for quality control of polymers in the rubber and adhesives industries.     

体积排阻色谱法测定胸腺肽分子量校准曲线条件的优化

在体积排阻色谱(SEC)法测定胸腺肽分子量校准曲线过程中,流动相中乙腈的比例对核糖核酸酶A、人胰岛素、胸腺肽α1和生长激素释放抑制因子4种蛋白的保留时间有重要影响,进而影响校准曲线的线性关系。当乙腈比例为75%时,胸腺肽分子量校准曲线线性最好,此时分子量校准方程为y=-3.138 6x+21.724,线性相关系数r2=0.988 5。4种蛋白的理论塔板数在45 783～63 345之间,拖尾因子在0.96～1.18之间,分离度在3.52～8.82之间。SEC法测定胸腺肽分子量校准曲线的液相色谱条件对4种蛋白的分离效果优异,分子量校准曲线线性良好,可用于胸腺肽制剂中高分子量物质的检测。     

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.     

Trace element speciation in largemouth bass ( Micropterus salmoides) from a fly ash settling basin by liquid chromatography-ICP-MS

Analytical techniques used to examine the chemical speciation of multiple trace elements are important for the investigation of biological systems. Size exclusion chromatography (SEC) coupled to ICP-MS was used to investigate the speciation of Se, As, Cu, Cd and Zn in tissue extracts from a largemouth bass (Micropterus salmoides) collected from a coal fly ash basin and results were compared to a largemouth bass collected at a reference site. Using a Biosil SEC column, with an effective separation range of 100-7 KDa, Cu, Zn and Cd were shown to be bound to metallothionein (MT) in the liver, gill and, to a lesser extent, gonad tissue extract. In liver, muscle and gill of the ash basin bass, Se was predominantly present as low molecular weight species. Only in the gonad extract was the major fraction of Se associated with high molecular weight species. For the liver and gill extracts, further SEC-ICP-MS on a column with an effective separation range of 7000-500 Da was performed, but Se species still eluted near the total volume of the column suggesting a low molecular weight organic or inorganic species. Ion chromatography (IC)-ICP-MS using an AS7 column and HNO(3) gradient elution indicated that the Se and As species in the liver and gill extracts had similar retention times but these retention times did not correspond to retention times for As(III), As(V), dimethylarsenate, arsenobetaine, Se(IV), Se(VI), seleno-methionine, or seleno-cystine.     

Hexafluoroisopropanol as size exclusion chromatography mobile phase for polyamide 6

The present study deals with the use of hexafluoroisopropanol (HFIP) as size exclusion chromatography (SEC) mobile phase for polyamide 6 (PA6). Contradictory conclusions relating to the use of HFIP as SEC mobile phase for polyamides are found in the literature. By using a multi-detector SEC apparatus equipped with on-line viscometer and multi-angle light scattering we have studied the chromatographic artifacts and the validity of the universal calibration (UC) in HFIP for different PA6 samples (hydrolytic and anionic, monofunctional or bifunctional activator). Appropriate SEC columns and optimized experimental conditions allow most of the chromatographic artifacts to be avoided, even in neat HFIP. The use of a salt in the mobile phase, namely 0.01 M tetraethylammonium nitrate (TEAN), slightly increases the elution volume for both PA6 and PMMA polymers. Under the right conditions, the UC substantially holds for PA6. The validity of the UC is not linked to the presence of TEAN in the mobile phase. With some PA6 samples, namely those anionically synthesized using a bifunctional activator, aggregation becomes a problem and the molar mass in neat HFIP is overestimated. Addition of TEAN prevents any aggregation of the above anionically synthesized PA6. In contrast, the use of a different salt, namely potassium trifluoroacetate, increases the extent of aggregation.     

Separation of complex branched polymers by size-exclusion chromatography probed with multiple detection

Size-exclusion chromatography (SEC) separates polymers by hydrodynamic volume (the universal calibration principle). Molecular weights can be determined using viscometry (relying on universal calibration) and light scattering (independent of universal calibration). In the case of complex branched polyacrylates with tetrahydrofuran as eluent, universal calibration is valid, although the separation in term of molecular weight is incomplete: a given elution slice contains a range of molecular weights, described in terms of a 'local polydispersity'. The local polydispersity index decreases when the number of branches per chain increases and complete separation is reached for highly branched chains.     

Size exclusion and anion exchange high performance liquid chromatography for characterizing metals bound to marine dissolved organic matter

Size exclusion chromatography (SEC) followed by anion exchange chromatography (AEC) hyphenated with inductively coupled plasma-mass spectrometry (ICP-MS) was applied for fractionating metals bound to marine dissolved organic matter (DOM). Surface seawater samples (100 L) were subjected to tangential flow ultrafiltration (10,000 Da cut off) for isolating and pre-concentrating dissolved large molecules. The isolated fraction (retentate) consisted of 1 L, which was further freeze-dried and re-dissolved to 250 mL with ultrapure water. After HI Trap desalting of the re-dissolved retentate, SEC with UV detection showed marine DOM ranging from 6.5 kDa (lower than the permeable volume of the SEC column) to 16 kDa. A further characterization of this fraction by AEC with UV detection revealed the existence of four groups of macromolecules exhibiting retention times of 2.3, 2.8, 4.5 and 14.0 min. AEC hyphenated with ICP-MS showed the presence of strontium and zinc in the first AE fraction isolated from the SEC fraction; while manganese was found to be bound to the second AE fraction. Cobalt was found to be bound to molecules comprising the third AE fraction.     

Direct zonal liquid chromatographic method for the kinetic study of actinomycin-DNA binding

The binding of an anticancer drug (actinomycin D or ACTD) to double-stranded DNA (dsDNA) was studied by means of high-performance liquid chromatography (HPLC). ACTD is an antitumor antibiotic containing one chromophore group and two pentapeptidic lactone cycles that binds dsDNA. Incubations of ACTD with DNA were performed at physiological pH. The complexed and free ligand concentrations of the mixture were quantified at 440 nm from their separation on a size-exclusion chromatographic (SEC) column using the same buffer for the elution and the sample incubation. The DNA and the ACTD-DNA complexes were eluted at the column exclusion volume while the ligand was retained on the support. An apparent binding curve was obtained by plotting the amount emerging at the exclusion column volume against that eluted at free ACTD retention volume. A dissociating effect was evidenced and the binding parameters were significantly different from those obtained at equilibrium by visible absorbance titration. The equilibrium binding parameters determined by absorption spectroscopy were used as starting data in the numerical simulations of the chromatographic process. The results showed a strong dependency of the apparent binding parameters on the reaction kinetics. Finally the comparison of the apparent binding curve obtained from the HPLC experiments and from the numerical simulations permitted an evaluation of the dissociation rate constant (kd = 0.004 s(-1)).     

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.     

Characterization of polyolefins by comprehensive high-temperature two-dimensional liquid chromatography (HT 2D-LC)

Temperature rising elution fractionation hyphenated to size exclusion chromatography (TREF × SEC) is a routine technique to determine the chemical heterogeneity of semicrystalline olefin copolymers. A serious limitation is its applicability to non crystallizing samples. Comprehensive high temperature two-dimensional liquid chromatography (HT 2D-LC) gives an alternative to characterize the chemical heterogeneity of copolymers irrespective of their crystallizability. We have hyphenated interactive HPLC, which separates polyolefins according to their chemical composition, with high-temperature size exclusion chromatography (SEC), which distinguishes polyolefins with regard to their molar mass at 160 °C. The first separation step was based on a selective adsorption of macromolecules on a Hypercarb® column packed with porous graphite particles and subsequent desorption by a gradient 1-decanol → 1,2,4-trichlorobenzene at 160 °C. The SEC column was calibrated with polypropylene (PP) and polyethylene (PE) standards and it turned out that the injection solvent from the first dimension influenced the elution of PP in the SEC column, while the retention of PE was virtually constant. HT 2D-LC was then used to separate a broad variety of polyolefin blends containing PE, PP with different microstructure, ethylene–propylene (EP) and ethylene–propylene–diene (EP(D)M) rubber and ethylene/1-hexene copolymers. For the first time it has been shown that the elution of iPP in the gradient HPLC is molar mass dependent. The results from the HT 2D-LC separation were compared to those from TREF × SEC-experiments. The particular advantage of HT 2D-LC over TREF × SEC is the fact that HT 2D-LC is also applicable to non crystallizing polyolefin samples. The new technique therefore resolves the problem to analyze the chemical heterogeneity of non crystallizing olefin copolymers like EP and EP(D)M copolymers.     

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.