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).     

Separation of nonionic surfactants according to functionality by hydrophilic interaction chromatography and comprehensive two-dimensional liquid chromatography

It is shown, that amphiphilic polymers--such as polysorbates and fatty esters of polyethylene glycol can be separated by comprehensive two-dimensional liquid chromatography using a reversed phase column (under critical conditions for the polyoxyethylene chain) and a HILIC column, which may arranged in different order. The mobile phases in both dimensions can be 93-97 wt% acetone water. As the retention of higher esters on the reversed phase column is very strong, this column should be used as the first dimension. On the HILIC column all fractions elute within a reasonably short time (at a flow rate of 2.5 ml/min within 2 min). With a flow rate of 0.1 ml/min in the first dimension, a full separation can be achieved in 90 min.     

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.     

Comprehensive two‐dimensional monolithic liquid chromatography of polar compounds

Two‐dimensional liquid chromatography largely increases the number of separated compounds in a single run, theoretically up to the product of the peaks separated in each dimension on the columns with different selectivities. On‐line coupling of a reversed‐phase column with an aqueous normal‐phase (hydrophilic interaction liquid chromatography) column yields orthogonal systems with high peak capacities. Fast on‐line two‐dimensional liquid chromatography needs a capillary or micro‐bore column providing low‐volume effluent fractions transferred to a short efficient second‐dimension column for separation at a high mobile phase flow rate. We prepared polymethacrylate zwitterionic monolithic micro‐columns in fused silica capillaries with structurally different dimethacrylate cross‐linkers. The columns provide dual retention mechanism (hydrophilic interaction and reversed‐phase). Setting the mobile phase composition allows adjusting the separation selectivity for various polar substance classes. Coupling on‐line an organic polymer monolithic capillary column in the first dimension with a short silica‐based monolithic column in the second dimension provides two‐dimensional liquid chromatography systems with high peak capacities. The silica monolithic C₁₈ columns provide higher separation efficiency than the particle‐packed columns at the flow rates as high as 5 mL/min used in the second dimension. Decreasing the diameter of the silica monolithic columns allows using a higher flow rate at the maximum operation pressure and lower fraction volumes transferred from the first, hydrophilic interaction dimension, into the second, reversed‐phase mode, avoiding the mobile phase compatibility issues, improving the resolution, increasing the peak capacity, and the peak production rate.     

Two-dimensional and serial column reversed-phase separation of phenolic antioxidants on octadecyl-, polyethyleneglycol-, and pentafluorophenylpropyl-silica columns

The separation selectivity of octadecyl-silica (C18) and of bonded pentafluorophenylpropyl-silica (F5) and PEG-silica columns was compared for natural phenolic antioxidants. The separation selectivities for phenolic antioxidants on C18 and F5 columns are strongly correlated, but low selectivity correlation indicating strong differences in the retention mechanism was observed between the C18 and PEG columns. Hence, the combination of a C18 and a PEG column is useful for separation of phenolic antioxidants that are not fully separated on single columns. Two-dimensional comprehensive liquid chromatography using a short PEG-silica column in the first dimension and a conventional C18-silica in the second dimension has the advantage of on-column focusing of the fractions transferred onto the C18 column in the second dimension, as a weaker mobile phase is used in the first dimension than in the second dimension. However, a stop-flow set-up in the first dimension system is necessary after the transfer of each fraction to the second dimension. Peak capacity is considerably larger but the separation time is much longer than with serially coupled PEG and C18 columns, which were employed for separation of beer and hop extract samples in connection with coulometric detection.     

高温二维液相色谱系统的构建及其评价

在二维液相色谱中, 第二维的分离速度是制约其发展的重要因素. 升高色谱柱温度可以有效降低流动相粘度, 加快溶质在两相间的传质速率, 有效加快分析速度. 以离子交换色谱法(WAX)为第一维分离模式和反相色谱法(RP)为第二维分离模式, 十通阀和两个捕集柱为接口, 通过将第二维色谱柱温度升高到80 ℃和提高流量到3 mL/min, 构建了高温WAX/RP二维液相色谱系统. 以4种标准蛋白的酶解物为样品评价系统的分离性能, 第一维共有33个馏分被捕集并导入到第二维分析, 高温二维液相色谱系统识别出187个色谱峰.     

Purification of alkaloids from Corydalis yanhusuo W. T. Wang using preparative 2‐D HPLC

Two‐dimensional preparative multi‐channel parallel high performance liquid chromatography was successfully applied for the first time to isolate and purify alkaloids from Corydalis yanhusuo. The experiments were performed in off‐line mode using the same preparative chromatographic column with pH 3.5 in the first and pH 10.0 in the second separation dimension. In the preparative process, UV‐triggered fraction collection was used in the first dimension while UV and MS‐triggered collection were used in the second dimension for reasons of sensitivity and complementarity. Two pure compounds and nine fractions were obtained in the first dimension. Then two representative fractions were further purified in the second dimension and six pure compounds were obtained. The results demonstrated that this procedure is an effective approach for the preparative isolation and purification of alkaloids from Corydalis yanhusuo. Based on the different pH values of the mobile phase in this method, it is also suitable for the preparative isolation and purification of other compounds from TCMs which are sensitive to the pH of the solutions. Moreover, this method will be a promising tool for the purification of low content compounds from natural products.     

正相模式/反相模式的二维液相色谱系统的构建与应用

以4.6mm×50 mm i. d.的Hypersil SiO2正相色谱柱为第一维,4.6mm×250 mm i. d.的Kromasil C18反相色谱柱为第二维,通过升高第二维色谱温度的方法增加两维流动相间互溶性的方法构建了定量环-阀切换接口的二维液相色谱系统(NPLC×RPLC)。根据有机溶剂的特征,在第一维正相色谱流动相中加入二氧六环;第二维反相色谱流动相中加入异丙醇,在改善流动相兼容性的同时,有效调整分离选择性。采用此系统对正天丸样品进行分离分析,达到1120的峰容量。     

Online high‐pH reversed‐phase liquid chromatography × low‐pH reversed‐phase liquid chromatography tandem electrospray ionization mass spectrometry combined with pulse elution gradient in the first dimension for the analysis of alkaloids in Macleaya cordata (willd.) R. Br

An online high‐pH reversed‐phase liquid chromatography× low‐pH reversed‐phase liquid chromatography tandem electrospray ionization mass spectrometry combined with pulse elution gradient in the first dimension was constructed to separate and identify alkaloids from Macleaya cordata (willd.) R. Br. The modulation was performed by using a dual second dimensional columns interface combined with a make‐up dilution pump, which is responsible for dilution and neutralization of the first dimensional effluent, and the dual second dimensional columns integrated the trapping and the separation function to reduce the second dimension system dead volume. Taking advantage of the dissociable characteristics of alkaloids, mobile phases with different pH values were applied in the first dimension (pH 9.0) and the second dimension (pH 2.6) to improve the orthogonality of two‐dimension separation. Besides, the pulse elution gradient in first dimension and second dimensional gradient were carefully optimized and much better separation was achieved compared to the separation with the traditional two‐dimensional liquid chromatography approach. Finally, mass measurement was performed for alkaloids in M. cordata (willd.) R. Br. by coupling proposed two‐dimensional liquid chromatography system with triple quadrupole mass spectrometry, and 39 alkaloids were successfully identified by comparing the obtained result with the former reported results.     

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.     

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.     

Improvement of the sensitivity of 2D LC-MEKC separation of phenolic acids and flavonoids natural antioxidants using the on-line preconcentration step

A 2D method was developed for separation of phenolic acids and flavonoids natural antioxidants combining LC with MEKC. The in-capillary preconcentration step was applied for the improvement of the sensitivity of 2D method before the second dimension MEKC analysis. The influence of first dimension LC mobile phase composition on migration times in the second MEKC dimension was evaluated. When gradient elution is applied in the first dimension of 2D LC-MEKC system, increasing concentration of organic solvent in the mobile phase and in fractions transferred from LC influences the electroosmotic flow, partitioning equilibria of samples in micelles and properties of the micelles, which results in shifts of migration times during the consecutive runs in the second MEKC separation dimension. The shifts of migration times caused by the influence of increasing concentration of ACN on MEKC separation in second dimension of 2D LC-MEKC system were compensated by aligning the time axis using electroosmotic flow and micellar marker migration times. The optimized LC-MEKC method was applied on the separation of natural antioxidants in the plant extracts samples.     

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.     

Two-dimensional liquid chromatography normal-phase and reversed-phase separation of (co)oligomers

Many samples contain compounds with various numbers of two or more regular structural groups. Such "multidimensional" samples (according to the Giddings' notation) are best separated in orthogonal chromatographic systems with different selectivities for the individual repeat structural groups, described by separation factors. Correlations between the repeat group selectivities characterize the degree of orthogonality and suitability of chromatographic systems for two-dimensional (2D) separations of two-dimensional samples. The range of the structural units in that can be resolved in a given time can be predicted on the basis of a model describing the repeat group selectivity in the first- and second-dimension systems. Two-dimensional liquid chromatographic system combining reversed-phase (RP) mode in the first dimension and normal-phase (NP) mode in the second dimension were studied with respect to the possibilities of in-line fraction transfer between the two modes. Hydrophilic interaction liquid chromatography (HILIC) with an aminopropyl silica column (APS) is more resistant than classical non-aqueous NP systems against adsorbent desactivation with aqueous solvents transferred in the fractions from the first, RP dimension to the second dimension. Hence, HILIC is useful as a second-dimension separation system for comprehensive RP-NP LCxLC. A comprehensive 2D RP-NP HPLC method was developed for comprehensive 2D separation of ethylene oxide-propylene oxide (EO-PO) (co)oligomers. The first-dimension RP system employed a 120 min gradient of acetonitrile in water on a C18 microbore column at the flow-rate of 10 microL/min. In the second dimension, isocratic HILIC NP with ethanol-dichloromethane-water mobile phase on an aminopropyl silica column at 0.5 mL/min was used. Ten microliter fractions were transferred from the RP to the HILIC NP system at 1 min switching valve cycle frequency.     

Two-dimensional separation of peptides using RP-RP-HPLC system with different pH in first and second separation dimensions

Two-dimensional high performance liquid chromatography is a useful tool for proteome analysis, providing a greater peak capacity than single-dimensional LC. The most popular 2D-HPLC approach used today for proteomic research combines strong cation exchange and reversed-phase HPLC. We have evaluated an alternative mode for 2D-HPLC of peptides, employing reversed-phase columns in both separation dimensions. The orthogonality of 2D separation was investigated for selected types of RP stationary phases, ion-pairing agents and mobile phase pH. The pH appears to have the most significant impact on the RP-LC separation selectivity; the greatest orthogonality was achieved for the system with C18 columns using pH 10 in the first and pH 2.6 in the second LC dimension. Separation was performed in off-line mode with partial fraction evaporation. The achievable peak capacity in RP-RP-HPLC and overall performance compares favorably to SCX-RP-HPLC and holds promise for proteomic analysis.     

Purification of flavonoids from licorice using an off‐line preparative two‐dimensional normal‐phase liquid chromatography/reversed‐phase liquid chromatography method

An orthogonal (71.9%) off‐line preparative two‐dimensional normal‐phase liquid chromatography/reversed‐phase liquid chromatography method coupled with effective sample pretreatment was developed for separation and purification of flavonoids from licorice. Most of the nonflavonoids were firstly removed using a self‐made Click TE‐Cys (60 μm) solid‐phase extraction. In the first dimension, an industrial grade preparative chromatography was employed to purify the crude flavonoids. Click TE‐Cys (10 μm) was selected as the stationary phase that provided an excellent separation with high reproducibility. Ethyl acetate/ethanol was selected as the mobile phase owing to their excellent solubility for flavonoids. Flavonoids co‐eluted in the first dimension were selected for further purification using reversed‐phase liquid chromatography. Multiple compounds could be isolated from one normal‐phase fraction and some compounds with bad resolution in one‐dimensional liquid chromatography could be prepared in this two‐dimensional system owing to the orthogonal separation. Moreover, this two‐dimensional liquid chromatography method was beneficial for the preparation of relatively trace flavonoid compounds, which were enriched in the first dimension and further purified in the second dimension. Totally, 24 flavonoid compounds with high purity were obtained. The results demonstrated that the off‐line two‐dimensional liquid chromatography method was effective for the preparative separation and purification of flavonoids from licorice.     

Comprehensive two-dimensional liquid chromatography with parallel gradients for separation of phenolic and flavone antioxidants

Various combinations of PEG-silica, phenyl-silica and C18 columns in a single-column or serial (tandem) arrangement in the first dimension and a monolithic Chromolith column in the second dimension were tested for comprehensive two-dimensional (2D) LCxLC separation of phenolic and flavone natural antioxidants. The combinations of different stationary phase chemistries provided low selectivity correlations between the first-dimension and the second-dimension separation systems. Improvement in system orthogonality, bandwidths suppression, more regular band distribution over the whole 2D retention plane and increased peak capacity in different 2D setups was achieved by using gradients with matching profiles running in parallel in the two dimensions over the whole 2D separation time range. Instead of two sampling loops, two alternating trapping XTerra columns were used for sample fraction transfer from the first-dimension column to the second dimension. Stronger retention on the XTerra columns in comparison to PEG-silica or phenyl-silica columns in the first dimension allowed using focusing of sample fractions in narrow zones on the top of a trapping column and back-flushing into the second dimension in a very low volume of the mobile phase. This fraction transfer modulation provided significant bandwidth suppression in the second dimension. 2D systems with optimized stationary phase selectivity, parallel gradients and fraction transfer modulation using two trapping columns were applied for the analysis of natural antioxidants in beer and wine samples.     

Development of different comprehensive two dimensional systems for the separation of phenolic antioxidants

Three different comprehensive 2-D HPLC systems for the separation of phenolic antioxidants have been developed on the basis of different selectivities of a PEG-silica column in the first dimension and a packed or monolithic C18 or a ZR-CARBON column, respectively, in the second dimension. Two-dimensional comprehensive liquid chromatography using a serially connected short PEG-silica column and a conventional C18-silica or a ZR-CARBON column in the second dimension was tested to improve the resolution of the earlier eluting compounds in the first dimension. Various types of interface were used to connect the columns in the first and in the second dimension: i) two injection sampling loops of 100 microL in conventional arrangement; ii) a 10-port 2-position valve equipped with two trapping X-Terra columns instead of loops; and iii) two analytical D2 columns in parallel. The mobile phase in the first dimension has a lower elution strength than in the second dimension, allowing band compression of the solutes transferred from the first to the second dimension. This effect was enhanced using trapping columns instead of sampling loops as the interface between the two dimensions, thus allowing a decrease in the time of analysis. These systems were used for the analysis of beer samples. The relative location of the components in the 2-D retention plane varied in relation to their chemical structure in each instrumental set-up and allowed positive peak identification.     

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.     

Design of a microfluidic device for comprehensive spatial two‐dimensional liquid chromatography

This study discusses the design aspects for the construction of a microfluidic device for comprehensive spatial two‐dimensional liquid chromatography. In spatial two‐dimensional liquid chromatography each peak is characterized by its coordinates in the plane. After completing the first‐dimension separation all fractions are analyzed in parallel second‐dimension separations. Hence, spatial two‐dimensional liquid chromatography potentially provides much higher peak‐production rates than a coupled column multi‐dimensional liquid chromatography approach in which the second‐dimension analyses are performed sequentially. A chip for spatial two‐dimensional liquid chromatography has been manufactured from cyclic olefin copolymer and features a first‐dimension separation channel and 21 parallel second‐dimension separation channels oriented perpendicularly to the former. Compartmentalization of first‐ and second‐dimension developments by physical barriers allowed for a preferential flow path with a minimal dispersion into the second‐dimension separation channels. To generate a homogenous flow across all the parallel second‐dimension channels, a radially interconnected flow distributor containing two zones of diamond‐shaped pillars was integrated on‐chip. A methacrylate ester based monolithic stationary phase with optimized macroporous structure was created in situ in the confines of the microfluidic chip. In addition, the use of a photomask was explored to localize monolith formation in the parallel second‐dimension channels. Finally, to connect the spatial chip to the liquid chromatography instrument, connector ports were integrated allowing the use of Viper fittings. As an alternative, a chip holder with adjustable clasp locks was designed that allows the clamping force to be adjusted.