Characterization of polyoxyalkylene block copolymers by combination of different chromatographic techniques and MALDI-TOF-MS

Polyoxyalkylene diblock copolymers (consisting of PEO as hydrophilic block and PBO or PHO as hydrophobic block) are characterized by combination of two dimensional liquid chromatography and MALDI-TOF-MS. Liquid chromatography under critical conditions (LCCC) is used as first dimension and fractions are collected, mobile phase evaporated and diluted in the mobile phase used in the second dimension (SEC, LCCC or LAC). This two-dimensional chromatography in combination of MALDI-TOF-MS gives information about purity of reaction products, presence of the byproducts, chemical composition and molar mass distribution of all the products.     

Composition analysis of poly(ethylene glycol)-poly(L-lactide) diblock copolymer studied by two-dimensional column chromatography

We used two-dimensional column chromatography to analyze the composition of a sample of presumably a diblock copolymer of poly(ethylene glycol) (PEG) and poly(L-lactide) synthesized from monomethoxy-terminated PEG. The first dimension of the separation is phase fluctuation chromatography to prepare fractions that contain various components of the copolymer in different ratios. The second dimension is size-exclusion chromatography, NMR, and HPLC at the critical condition of PEG. The PEG initiator has small amounts of diol-terminated dimeric components. We found that the copolymer sample contains a triblock copolymer and low-molecular-mass components in addition to the main part of the diblock copolymer. The SEC chromatograms show that the main part consists of two components with distinct peak lengths for the PLLA block. The low-molecular-mass components have a broad distribution in chemical composition. Phase fluctuation chromatography enriched the triblock copolymer and the diblock copolymer with the longer PLLA block in early fractions when the column was packed with carboxymethyl-modified porous silica. When the porous medium was PLLA-grafted silica, size exclusion dominated, but the low-molecular-mass components were separated according to their chemical composition.     

Characterization of ethylene oxide–propylene oxide block copolymers by combination of different chromatographic techniques and matrix-assisted laser desorption ionization time-of-flight mass spectroscopy

Block copolymers of ethylene oxide (EO) and propylene oxide (PO) are characterized by combination of two-dimensional chromatography and MALDI-TOF-MS. Liquid chromatography under critical conditions (LCCC) is used as first dimension and fractions are collected, mobile phase evaporated and diluted in the mobile phase used in second dimension (SEC or LAC). This two-dimensional chromatography in combination of MALDI-TOF-MS gives information about purity of reaction products, presence of the byproducts, chemical composition and molar mass distribution of all the products.     

A novel software tool for copolymer characterization by coupling of liquid chromatography with matrix-assisted laser desorption/ionization time-of-flight mass spectrometry

The results of copolymer characterization by coupling of chromatography and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) techniques and subsequent calculation of copolymer composition using a novel software tool 'MassChrom2D' are presented. For high-resolution mass analysis copolymer samples were fractionated by means of liquid adsorption chromatography (LAC). These fractions were investigated off-line by MALDI-TOF MS. Various mono-n-butyl ethers of polyethylene oxide-polypropylene oxide copolymers (PEO-co-PPO) were investigated. As well as the copolymer composition presented in two-dimensional plots, the applied approach can give additional hints on specific structure-dependent separation conditions in chromatography.     

Characterization of complex copolymers by two-dimensional Liquid Chromatography

Complex polymers were characterized by combinations of different chromatographic separation mechanisms: liquid adsorption chromatography (LAC), liquid chromatography under critical conditions (LCCC), and liquid exclusion-adsorption chromatography (LEAC). These techniques were combined off-line and on-line in two-dimensional separations. Fatty acid ethoxylates, fatty esters of polyethylene glycol (PEG) and polysorbates were analyzed by two-dimensional liquid chromatography with normal phase LAC as the first and liquid chromatography at critical conditions (LCCC) or liquid exclusion adsorption chromatography (LEAC) as the second dimension. A full separation of all oligomers to the baseline could be achieved in both dimensions. In two-dimensional separations, the offline approach is compared to comprehensive chromatography, and the scope and limitations of both techniques are discussed.     

Characterization of Ethylene methyl methacrylate and Ethylene butylacrylate Copolymers with Interactive Liquid Chromatography

Summary: Copolymers of ethylene with methyl methacrylate (EMMA) and butyl acrylate (EBA), which are of different average chemical composition and block lengths according to NMR analysis, were analyzed by size exclusion chromatography (SEC), differential scanning calorimetry (DSC), Crystallization Analysis Fractionation (CRYSTAF), and high performance liquid chromatography at high temperature (HT-HPLC). With CRYSTAF and DSC crystallizing fractions were detected only in some samples. HT-HPLC fractionated all the samples irrespective of their crystallinity. Homopolymers, PMMA and PE were also found in the copolymer samples of EMMA. EMMA and EBA were separated in HPLC according to the content of polar comonomer. A linear correlation between the MMA content and elution volume could not be established due to the presence of homopolymers as admixtures. In such a case the average chemical composition obtained by NMR does not correspond to the real chemical composition of the copolymers. Unlike EMMA the EBA samples eluted in single peaks, which was used for evaluation of their chemical composition distribution. The comparison of results obtained by fractionation via CRYSTAF and HT-HPLC clearly demonstrates the advantages of the chromatographic approach to study the chemical heterogeneity of olefin based copolymers.     

Analysis of polystyrene-b-polyisoprene copolymers by coupling of liquid chromatography at critical conditions to NMR at critical conditions of polystyrene and polyisoprene

For the investigation of the molecular heterogeneity of polystyrene-b-polyisoprene block copolymers, a chromatographic separation method, namely liquid chromatography at critical conditions was developed. This method was coupled on-line with (1)H-NMR(where NMR stands for nuclear magnetic resonance) for the comprehensive analysis of the polystyrene-b-polyisoprene copolymers. The copolymers were synthesized by two different methods: sequential living anionic polymerization and coupling of living precursor blocks. While (1)H-NMR allows just for the analysis of the bulk chemical composition of the block copolymers, the coupling with liquid chromatography at critical conditions provides selective molar mass information on the polystyrene and polyisoprene blocks within the copolymers. The polyisoprene block molar mass is determined by operating at chromatographic conditions corresponding to the critical point of adsorption of polystyrene and size exclusion chromatography mode for polyisoprene. The molar mass of the polystyrene block is determined by operating at the critical conditions of polyisoprene. In addition to the molar mass of each block of the copolymers, the chemical composition distribution of the block copolymers was determined. By using the coupling of liquid chromatography at critical conditions to (1)H-NMR, one can also detect the homopolymers formed during synthesis. Finally the microstructure of the polyisoprene block in the copolymers was evaluated as a function of molar mass.     

Liquid exclusion-adsorption chromatography,a new technique for isocratic separation of nonionic surfactants. IV. Two-dimensional separation of fatty alcohol ethoxylates with focusing of fractions

Fatty alcohol ethoxylates can be analyzed using a combination of liquid chromatography under critical conditions as the first dimension and liquid exclusion-adsorption chromatography as the second dimension. Transfer of fractions from the first to the second dimension is achieved using the full adsorption-desorption (FAD) technique. The peaks of interest in the first dimension are trapped on a short precolumn before injecting them into the second dimension. Full adsorption is achieved by increasing the water content in the mobile phase before the FAD column. When the fractions are desorbed by switching to the mobile phase of the second dimension, they are focused and reconcentrated. In this way, a full resolution of oligomers is achieved. As both dimensions are run in isocratic mode, density and refractive index detection can be applied, which allows an accurate quantitation.     

Liquid exclusion-adsorption chromatography: a new technique for isocratic separation of non-ionic surfactants. V. Two-dimensional separation of fatty acid polyglycol ethers

Fatty acid polyglycol esters can be fully characterized using two-dimensional liquid chromatography with liquid chromatography under critical conditions (LCCC) as the first and liquid exclusion-adsorption chromatography (LEAC) as the second dimension. LEAC is run under isocratic conditions, which allows the use of the refractive index detector, and thus accurate quantitation. Fractions from LCCC are transferred to LEAC using the full adsorption-desorption technique, by which they are focussed and reconcentrated before injection into the second dimension. This is achieved by increasing the water content of the mobile phase behind the LCCC column. Monoester oligomers of up to 20 oxyethylene units can be resolved to the baseline. Diester oligomers are partially separated in the first dimension (LCCC).     

Two‐dimensional liquid chromatography with active solvent modulation for studying monomer incorporation in copolymer dispersants

A pseudo‐comprehensive two‐dimensional liquid chromatography approach with size exclusion chromatography in the first dimension and gradient reversed‐phase liquid chromatography in the second dimension was successfully developed for the characterization of vinyl acetate/acrylic acid copolymers and vinyl acetate/itaconic acid/acrylic acid terpolymers. Active solvent modulation was exploited to prevent the polymer breakthrough in the second dimension separation caused by the strong solvent used in the first dimension. The conditions of the active solvent modulation valve were optimized to achieve sufficient on‐line dilution and to completely prevent polymer breakthrough without adding excessive time to the modulation cycle. Using this approach, copolymers made with different monomer ratios and processes were studied. Heterogeneous composition distribution due to insufficient monomer incorporation was detected in some of the copolymer samples. We demonstrated that with active solvent modulation, the two‐dimensional liquid chromatography approach is no longer limited to water‐soluble polymers and can be used for a broader range of polymers and copolymers.     

全二维液相色谱(NPLC×RPLC)接口及其应用

用内径为0.53 mm的填充毛细管正相液相色谱为第一维, 用4.6 mm(i.d.)×50 mm RP-18e整体柱反相色谱为第二维, 建立了定量环-阀切换接口的全二维液相色谱系统(NPLC×RPLC). 第一维色谱分离洗脱出的组分交替存储在十通阀上的两个定量环中, 同时定量环中前一个组分被转移到第二维进行反相分离. 因为第一维的流动相流量仅是第二维的1/500, 自然解决了流动相兼容问题. 采用芳香族化合物的混合物和中药丹参正己烷提取液对该全二维液相系统的分离能力进行了评价.     

Characterization of poly(ethylene glycol)‐b‐ poly(ε‐caprolactone) by two‐dimensional liquid chromatography

Block copolymers of ethylene oxide and ε‐caprolactone were synthesized by microwave‐assisted polymerization of ε‐caprolactone with polyethylene glycol monomethyl ethers as initiator. The samples thus obtained were characterized by two‐dimensional liquid chromatography with liquid chromatography at critical conditions as the first and liquid exclusion adsorption chromatography as the second dimension. A full baseline separation of all oligomers could be achieved in both dimensions.     

Comprehensive two-dimensional liquid chromatography (NPLCxRPLC) with vacuum-evaporation interface

A comprehensive two-dimensional liquid chromatographic system incorporating a vacuum-evaporation interface was developed. Normal-phase liquid chromatography with a CN microcolumn was used as the first dimension (1(st)-D), and reversed-phase liquid chromatography with a C(18) monolithic column was used as the second dimension (2(nd)-D). An electronically controlled dual-position, ten-port valve with two identical storage loops served as the interface and the analysis time in the 2(nd)-D was 1.5 min. The solvent in the loops of the interface was evaporated at 25 degrees C under vacuum conditions, leaving the analytes on the inner wall of the loops. The mobile phase of the 2(nd)-D dissolved the analytes in the loop and injected them onto the second column, allowing an on-line solvent exchange of the fractions from the 1(st)-D to the 2(nd)-D. The chromatographic resolution of analytes on the two dimensions was evaluated. Sample loss due to evaporation in the interface was investigated with standard samples having different boiling points. The usefulness of the comprehensive 2-DLC system was demonstrated in the analysis of a traditional Chinese medicine Radix salviae miltiorrhiza bage extract.     

Two-dimensional chromatography applied to the study of the thermo-oxidative degradation of poly(styrene-b-butadiene) star block copolymers

Two-dimensional chromatography with gradient polymer elution chromatography in the first dimension and gel permeation chromatography in the second dimension was used to characterize a poly(styrene-b-butadiene) star block copolymer. The data evidence several populations that are clues left by the different steps in the sequential reaction employed to make the polymer. The sample was subjected to thermo-oxidative degradation at 180°C and was analyzed at different times during the process. After a relatively long induction period, the two-dimensional chromatograms show how the different populations are progressively degraded via random chain scission of the polybutadiene block to leave essentially polystyrene as the only soluble component. With longer thermal aging times, the polystyrene also degrades via chain scission.     

Development of high temperature comprehensive two-dimensional liquid chromatography hyphenated with infrared and light scattering detectors for characterization of chemical composition and molecular weight heterogeneities in polyolefin copolymers

The application of high temperature comprehensive two-dimensional (2D) liquid chromatography for quantitative characterization of chemical composition and molecular weight (MW) heterogeneities in polyolefins is demonstrated in this study by separating a physical blend of isotactic-polypropylene, ethylene-random-propylene copolymer, and high density polyethylene. The first dimension separation is based on adsorption liquid chromatography that fractionates the blend from low to high ethylene content. The second dimension is size-exclusion chromatography connected with light scattering (LS) and infrared (IR) detectors. The IR detector shows desired sensitivity and linearity for monitoring analyte concentrations in the eluent after 2D separations. In addition, the compositions of the analytes are also determined from the ratio of two IR absorbances at the specified wavelength regions, an absorbance for measuring the level of methyl groups in polyolefins and another absorbance for measuring concentration. The LS detector is used to determine absolute molecular weight of the analytes from the ratio of the light scattering signal to the IR concentration signal. The ability to obtain concentration, chemical composition, and MW of polyolefins after 2D separation provides new opportunities to discover structure-property relationships for polyolefins with complex structures/architectures.     

Chromatographic characterization of amphiphilic di‐ and tri‐block copolymers of poly(ethylene oxide) and poly(ε‐caprolactone)

Amphiphilic di‐ and tri‐block copolymers based on poly(ethylene oxide) as a hydrophilic segment and poly(ε‐caprolactone) as a hydrophobic part are synthesized by the ring‐opening polymerization of ε‐caprolactone while using poly(ethylene glycol)s and methoxy poly(ethylene glycol)s of varying molar masses as macro‐initiators. The synthesized block copolymers are characterized with respect to their total relative molar mass and its distribution by size exclusion chromatography. Liquid chromatography at critical conditions of both blocks is established for the analysis of individual block lengths and tracking presence of unwanted homopolymers of both types in the block copolymer samples. New critical conditions of polycaprolactone on reversed phase column are reported using organic mobile phase. The established critical conditions of polycaprolactone extended the applicable molar mass range significantly compared to already reported critical conditions of polycaprolactone in aqueous mobile phase. Block copolymers are also analyzed at critical conditions of poly(ethylene glycol). Complete analysis of the di‐ and tri‐block copolymers at corresponding critical conditions provided a fair estimate of molar mass of non‐critical block besides information regarding presence of homopolymers of both types in the samples.     

Multidimensional liquid chromatography system with an innovative solvent evaporation interface

An orthogonal two-dimensional liquid chromatographic (2D-LC) system was developed by using a vacuum-evaporation loop-type valve interface. Normal-phase liquid chromatography (NPLC) with a bonded CN phase column was used as the first dimension, and reversed-phase liquid chromatography (RPLC) with a C(18) column was used as the second dimension. All the solvents in the loop of the interface were evaporated at 90 degrees C under vacuum conditions, leaving the analytes on the inner wall of the loop. The mobile phase of the second dimension dissolved the analytes in the loop and injected them onto the secondary column, allowing an on-line solvent exchange of a selected fraction from the first dimension to the second dimension. The chromatographic resolution of analytes on the two dimensions was maintained at their optimal condition. Sample loss due to evaporation in the interface was observed that depended on the boiling point of the compound. Separation of sixteen polycyclic aromatic hydrocarbon mixtures and a traditional Chinese medicine Angelica dahurica was demonstrated.     

Analytical techniques for polymers with complex architectures

Complex polymers are distributed in more than one direction of molecular heterogeneity. In addition to the molar mass distribution, they are frequently distributed with respect to chemical composition, functionality, and molecular architecture. For the characterization of the different types of molecular heterogeneity it is necessary to use a wide range of analytical techniques. Preferably, these techniques should be selective towards a specific type of heterogeneity. The combination of two selective analytical techniques is assumed to yield a two-dimensional information on the molecular heterogeneity. For the analysis of complex polymers different liquid chromatographic techniques have been developed, including size exclusion chromatography (SEC) separating with respect to hydrodynamic volume, and liquid adsorption chromatography (LAC) which is used to separate according to chemical composition. Liquid chromatography at the critical point of adsorption (LC-CC) has been shown to be a versatile method for the determination of the functionality type distribution of macromonomers, the molecular architecture of homopolymers and the chemical heterogeneity of block and graft copolymers. The present paper presents the principle ideas of combining different analytical techniques in multidimensional analysis schemes for the analysis of polymers with complex architectures. Branched block and graft copolymers can efficiently be analyzed with respect to chemical composition and molar mass by LC-CC and two-dimensional chromatography. The chemical heterogeneity as a function of molar mass can be determined by combining interaction chromatography and FTIR spectroscopy. For the analysis of star-like polymers LC-CC is shown to be a powerful technique when the molar mass of different segments (blocks, grafts) must be determined.     

Use of combined chromatographic methods including thin-layer chromatography for analysis of complex polymer systems. Determination of the polydispersity of block copolymers of styrene and methyl methacrylate by gel permeation, thin-layer and pyrolysis gas chromatography.

A combination of gel permeation chromatography (GPC), thin-layer chromatography (TLC) and pyrolysis gas chromatography (PGC) has been used for investigations of a polymethyl methacrylate-polystyrene-polymethyl methacrylate block copolymer. Continuous distribution of the polymer (40-mg sample) was attained according to the content of the styrene and methyl methacrylate units and of the block copolymer and according to the composition of the copolymer as functions of the hydrodynamic radius of the macromolecules. The polymer was subjected to a preliminary fractionation with an analytical gel chromatograph. The fractions were investigated by TLC, which permitted the separation of the block copolymer and the homopolymers. The composition of the fractions obtained by GPC and TLC was determined by PGC. As a result, it was possible to establish the composition of the block copolymer and its ratio to polymethyl methacrylate in each fraction. This investigation was based on a combination of highly effective fractionation by chromatographic methods with precise quantitative ratios obtained from Benoit's universal calibration graph and from determinations of the composition of the polymer fractions by PGC. The mechanism of the TLC of polymers, including the appearance of artefacts that distort the results of analysis, is also discussed.     

在线富集二维毛细管电泳分析新体系检测尿样中药物及对映体

将在线富集技术同二维(2D)毛细管电泳(CE)分离相结合同时提高复杂样品中痕量组分的分离度和检测灵敏度.毛细管区带电泳(CZE)作为第一维,分析物根据淌度不同进行分离,第一维流出组分进入第二维毛细管,根据分配系数不同进行胶束电动毛细管色谱(MEKC)分离.采用阳离子选择性耗尽进样(CSEI)在柱预富集,延长进样时间,增大进样量;同时在二维毛细管接口处采用动态pH联接/胶束扫集在线富集技术不仅避免第一维分离组分在接口处扩散,还可进一步压缩样品区带.同常规电动进样CE分离相比,该在线富集二维分离技术的分离能力远远高于一维CZE或MEKC分离,富集倍数达到(0.5～1.2)×104.该法成功应用于人体尿样中四种药物及对映体的分析测定,浓度检出限为0.1～0.3μg/L.进一步研究了人体尿样中四种药物24h内的药代动力学规律.