The obstruction factor in size-exclusion chromatography. 1. The intraparticle obstruction factor

We report the first of a series of studies on the obstruction factor γ in size-exclusion chromatography (SEC). Here, using narrow dispersity polymer standards we examine how the intraparticle obstruction factor γ(p) depends individually on a number of analyte properties, column characteristics, and user-defined parameters. Far from being constant, γ(p) is seen to vary with analyte molar mass and solvent, as well as with the pore size and particle size of the column packing material, sometimes in seemingly counterintuitive manner. Over the limited temperature range accessible to our equipment, however, no statistically significant change in γ(p) with temperature was discovered. The results presented should be applicable to forms of packed column chromatography other than SEC. The latter technique, however, presents a convenient test bed for quantitative determination of the obstruction factor, due to minimized sorptive mass transfer and longitudinal diffusion contributions to band broadening in most forms of SEC.     

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.     

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.     

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.     

Thin-layer chromatography of pitch and a petroleum vacuum residue. Relation between mobility and molecular size shown by size-exclusion chromatography

A coal tar pitch and a petroleum vacuum residue have been separated by TLC using pyridine, acetonitrile, toluene and pentane to develop the chromatograms. The bands of material detected were recovered in 1-methyl-2-pyrrolidinone (NMP) solvent and examined by size-exclusion chromatography (SEC) in NMP eluent. The relation between elution time in SEC and mobility on the TLC plate indicated that molecular size increased steadily with increasing immobility on the plate. This relation was reinforced by UV fluorescence spectroscopy in that the fluorescence moved to longer wavelengths with increasing immobility. The molecular size of the material excluded from the porosity of the SEC column remains undefined; some excluded material was found in all of the fractions from both samples. The valley of zero intensity separating the retained material from the excluded material may suggest a change of structure from near-planar in the retained region to three-dimensional in the excluded region.     

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.     

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.     

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.     

Liquid chromatography of polymers under limiting conditions of desorption II. Tandem injection and quantitative molar mass determination

Liquid chromatography under limiting conditions of desorption (LC LCD) is a method which allows molar mass independent elution of various synthetic polymers. A narrow, slowly moving zone of small molecules, which promotes full adsorption of one kind of polymer species within column (an adsorli) acts as an impermeable barrier for the fast moving macromolecules. The latter accumulate on the barrier edge and elute nearly in total volume of liquid within column. At the same time, transport of less adsorptive macromolecules is not hampered so that these are eluted in the size exclusion (SEC) mode. As result, polymers differing in their polarity and adsorptivity can be easily separated without molar mass interference. Three methods of barrier creation are discussed and compared. It is shown that a fraction of sample may elute unretained if the adsorli sample solvent is used as a barrier in connection with a narrow-pore column packing. One part of excluded macromolecules likely breaks-out from the adsorli zone and this results in partial loss of sample and distortion of the LC LCD peaks. This problem can be avoided if the adsorli zone is injected immediately before sample solution. Applicability of the LC LCD method for polymer separation has been demonstrated with a model mixture of poly(methyl methacrylate) (adsorbing polymer) and polystyrene (non adsorbing polymer) using bare silica gel as a column packing with a combination of tetrahydrofuran (a desorption promoting liquid -a desorli) and toluene (adsorli). It has been shown that the LC LCD procedure with tandem injection allows simple and fast discrimination of polymer blend components with good repeatability and high sample recovery. For quantitative determination of molar masses of both LC LCD and SEC eluted polymers, an additional size exclusion chromatographic column can be applied either in a conventional way or in combination with a multi-angle light scattering detector. A single eluent is used in the latter column, which separates the mixed mobile phase, system peaks and the desorli zone from the polymer peaks so that measurements are free from disturbances caused by the changing eluent composition. The resulting LC LCD x SEC procedure has been successfully applied to poly(methyl methacrylate) samples.     

Molar mass characterization of cationic methyl methacrylate-ethyl acrylate copolymers using size-exclusion chromatography with online multi-angle light scattering and refractometric detection

Size-exclusion chromatography (SEC) combined with online multi-angle light scattering (MALS) and refractometric (RI) detection has been employed for the molar mass characterisation of water-insoluble cationic methyl methacrylate-ethyl acrylate copolymers (Eudragit RS and RL). Due to their positive charge, cationic polymers are particularly difficult to separate on a SEC column, in worst cases being completely adsorbed on the oppositely charged packing material. This work has examined how a careful addition of salt (LiCl) to the copolymer solution in ethanol decreases the electrostatic interactions, clearly seen as a decrease in elution volume from the SEC column as well as an improved recovery. At a certain level of ionic strength, typically about 50 mM, the copolymer recovery from the SEC column reached 100% and molar mass distributions corresponding to the complete sample could be obtained. The combined MALS/RI detection gives the opportunity to measure the absolute molar mass independent of recovery and retention. Thus, in this study, it turned out to be a favourable tool for tracing the changes in elution behaviour of the charged copolymer as the ionic strength was increased.     

Study of the abnormal late co-elution phenomenon of low density polyethylene in size exclusion chromatography using high temperature size exclusion chromatography and high temperature asymmetrical flow field-flow fractionation

The elution behaviour of linear and branched polyethylene samples in SEC was studied. For the branched samples an abnormal late co-elution of large and small macromolecules manifests itself as an abnormal re-increase of the molar mass and the radius of gyration values detected with multi angle light scattering at high elution volumes in SEC. The late co-elution of small and large macromolecules cannot be explained by the SEC mechanism alone. The influence of several experimental parameters on the late co-elution was studied. It was found that the type of SEC column and the flow rate have a significant influence. The late eluting part of the sample was fractionated and separated by HT-SEC- and HT-AF4-MALS. The different results of both methods have been discussed with the aim to find possible explanations for the late elution. The experiments indicate that especially large branched structures show an increased tendency for the phenomenon.     

凝胶渗透色谱对多孔物孔结构的测定——柱外测定法

多孔物的孔结构,一般多在干态下用氮吸附法或汞压法测定.I.Halasz等首先用反演凝胶色谱法对硅胶类多孔物作了测定获得成功.D.H.Freeman对有机交联高聚物的孔结构提出了新的数据处理方法,其它在这方而的工作亦很多。     

Sulfoacylated macro-porous polystyrene-divinylbenzene low-capacity cation exchanger selective for amino acids.

A low-capacity cation-exchange column was newly developed for the separation of amino acids. A highly cross-linked macro-porous polystyrene-divinylbenzene co-polymer was functionalized by a sulfoacylation reaction. The exchange capacity was controllable at the acylation step. The capacity between 55 and 60 micromol/column was adequate for the practical separations in acceptable retention times. The 5-microm base polymers having average pore diameters smaller than 3 nm gave satisfactory results, and those having 1.5-nm pore was most favorable. Several isocratic elution conditions at different pH values adjusted by phosphate buffer of mM order with or without acetonitrile could provide good separations for individual classes of amino acids, i.e., acidic, neutral, hydrophobic, and basic groups. The results provided fundamental data for constructing gradient elution systems required for the simultaneous separation of protein amino acids.     

Separation of poly-3-hydroxyvalerate-co-3-hydroxybutyrate through gradient polymer elution chromatography

Polyhydroxyalkanoates are biodegradable polyesters produced by bacteria that can have a wide distribution in molecular weight, composition of monomers, and functionalities. This large distribution often leads to unpredictable physical properties making commercial applications challenging. To improve polymer homogeneity and obtain samples with a clear set of physical characteristics, poly-3-hydroxyvalerate-co-3-hydroxybutyrate copolymers were fractionated using gradient polymer elution chromatography (GPEC) as opposed to extensively used bulk fractionation. Separation was achieved using a reversed-phase column with chloroform and ethanol as the solvent and non-solvent, respectively. A separation was also conducted on a normal-phase column to compare elution patterns between columns of varied polarity. The fractions were analyzed using Size Exclusion Chromatography (SEC) and NMR to determine the percentage of 3-hydroxyvalerate in the copolymer as well as its molecular weight. It was found that as the percentage of "good" solvent was increased in the mobile phase, the polymers eluted with decreasing percentage of 3-hydroxyvalerate and increasing molecular weight which indicates the importance of precipitation/redissolution in the separation. The elution pattern of the polymer remained unchanged when using both a normal- and reversed-phase column which also illustrates the dominance of precipitation/redissolution in GPEC of polyhydroxyalkanoates. As such, GPEC is shown to be an excellent choice to provide polyhydroxyalkanoate samples with a narrower distribution in composition than the original bulk copolymer sample.     

高效液相色谱法检测溴化丁基胶塞中11种抗氧剂及游离硫的浸出量及迁移量

采用Inertsil ODS-SP C18(250×4.6 mm,5μm)色谱柱,以0.1%乙酸水-甲醇-乙腈为流动相,进行梯度洗脱,流速为1.0 mL/min,检测波长为277 nm。胶塞提取溶剂分别为无水乙醇和模拟药液。11种抗氧剂及游离硫的分离度均大于1.5;在2.47~54.51μg/mL浓度范围内,线性关系良好(R^2均大于0.999);检出限为0.021~0.403μg/mL;以无水乙醇为提取溶剂时,加标回收率为82.8%~115.3%,相对标准偏差为1.1%~6.1%;以模拟药液为提取溶剂时,加标回收率为62.9%~103.2%,相对标准偏差为1.4%~5.6%。该方法可用于包材相容性试验中实际样品的检测。     

Effect of pore size of packing materials on molecular-weight separation by temperature gradient interaction chromatography

Temperature gradient interaction chromatography (TGIC) is an interactive polymer chromatography technique varying the column temperature during the elution in a programmed manner to control the solute retention. In the present paper, the effect of the pore size of packing materials on the molecular-weight separation of polystyrene and poly(methyl methacrylate) standard samples by TGIC was studied by using the columns (octadecyl modified silica) with different pore size (100, 300 and 1000 Å) and eluent mixture of CH₂Cl₂/CH₃CN. By rising temperature gradient, both polymers were separated by molecular weight from lower to higher. It became clear that each sample elutes out earlier as the pore size is larger. These experimental results could be explained by the theory based on the scaling concept of Gorbunov and Skvortsov.     

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     

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     

THE MOLECULAR WEIGHT DEPENDENCE OF THE SPREADING FACTOR OF SEC COLUMNS AND THE SURFACE STRUCTURE OF THE PORE OF PACKINGS

The molecular weight separation and column dispersion for a number of SEC columns packedwith various packing materials were calibrated simultaneously using narrow MWD polystyrene stan-dards as probe. The molecular weight dependence of the spreading factor is closely related to thesurface structure in the pore of packings.