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Stir bar sorptive extraction and high performance liquid chromatographic determination of carvedilol in human serum using two different polymeric phases and an ionic liquid as desorption solvent
10 April 2012,
09:29:39
Publication year:
2012
Source:Journal of Chromatography A, Volume 1236
Zahra Talebpour, Maryam Taraji, Nuoshin Adib
This article presents a method employing stir bar coated with a film of poly (methyl methacrylate/ethyleneglycol dimethacrylate) (PA-EG) and polydimethylsiloxane (PDMS) in combination with liquid desorption (LD) using ionic liquid, followed by high performance liquid chromatography (HPLC) equipped with ultraviolet (UV) detection for the determination of carvedilol in human serum samples. Stir bar sorptive extraction (SBSE) variables, such as desorption and extraction time and temperature, desorption solvent and pH of the matrix were optimized, in order to achieve suitable analytical sensitivity in a short period of time. Also, the concentration effect of 1-methyl-3-octylimidazolium tetrafluoroborate [Omim][BF4] ionic liquid on the efficiency of LD was investigated. A comparison between PA-EG/SBSE and PDMS/SBSE was made by calculating the experimental recovery and partition coefficient (K), where PA-EG phase demonstrated to be an excellent alternative for the enrichment of the carvedilol from serum samples. The effect of [Omim][BF4] on carryover was studied and no carryover was observed. Under optimized experimental conditions, the analytical performance showed excellent linear dynamic range, with correlation coefficients higher than 0.999 and limits of detection and quantification of 0.3 and 1.0ngmL−1, respectively. Intra- and inter-day recovery ranged from 94 to 103% and the coefficients of variations were less than 3.2%. The proposed method was shown to be simple, highly sensitive and suitable for the measurement of trace concentration levels of carvedilol in biological fluid media.
Source:Journal of Chromatography A, Volume 1236
Zahra Talebpour, Maryam Taraji, Nuoshin Adib
This article presents a method employing stir bar coated with a film of poly (methyl methacrylate/ethyleneglycol dimethacrylate) (PA-EG) and polydimethylsiloxane (PDMS) in combination with liquid desorption (LD) using ionic liquid, followed by high performance liquid chromatography (HPLC) equipped with ultraviolet (UV) detection for the determination of carvedilol in human serum samples. Stir bar sorptive extraction (SBSE) variables, such as desorption and extraction time and temperature, desorption solvent and pH of the matrix were optimized, in order to achieve suitable analytical sensitivity in a short period of time. Also, the concentration effect of 1-methyl-3-octylimidazolium tetrafluoroborate [Omim][BF4] ionic liquid on the efficiency of LD was investigated. A comparison between PA-EG/SBSE and PDMS/SBSE was made by calculating the experimental recovery and partition coefficient (K), where PA-EG phase demonstrated to be an excellent alternative for the enrichment of the carvedilol from serum samples. The effect of [Omim][BF4] on carryover was studied and no carryover was observed. Under optimized experimental conditions, the analytical performance showed excellent linear dynamic range, with correlation coefficients higher than 0.999 and limits of detection and quantification of 0.3 and 1.0ngmL−1, respectively. Intra- and inter-day recovery ranged from 94 to 103% and the coefficients of variations were less than 3.2%. The proposed method was shown to be simple, highly sensitive and suitable for the measurement of trace concentration levels of carvedilol in biological fluid media.
Recovery of crocins from saffron stigmas (Crocus sativus) in aqueous two-phase systems
10 April 2012,
09:29:39
Publication year:
2012
Source:Journal of Chromatography A, Volume 1236
Bertha Montalvo-Hernández, Marco Rito-Palomares, Jorge Benavides
Crocins are carotenoid derivates that have recently attracted the interest of the scientific community due to their nutraceutical properties. Saffron (dry Crocus sativus stigmas) is one of the main known sources of crocins. In this study the potential use of aqueous two-phase system (ATPS) for the extraction of crocins from C. sativus stigmas was evaluated. The partitioning behavior of crocins in different types of ATPS (polymer–polymer, polymer–salt, alcohol–salt and ionic liquid–salt) was evaluated. Ethanol–potassium phosphate ATPS were selected based on their high top phase recovery yield and low cost of system constituents. The evaluation and optimization of system parameters rendered conditions (V R =3.2, ethanol 19.8% (w/w), potassium phosphate 16.5% (w/w), TLL of 25% (w/w), 0.1M NaCl and 2% (w/w) of sample load) under which more than 75% of total crocins were recovered in the top (ethanol rich) phase, whereas the wasted stigmas accumulated in the bottom phase. Lastly, a comparison between an optimized solid–liquid extraction using ethanol:water as solvent and ATPS was conducted demonstrating that similar yields are achieved with both strategies (76.89±1.8% and 79.27±1.6%, respectively). However, ATPS rendered a higher extraction selectivity of 1.3±0.04mg of crocins for each mg of phenolic compound, whereas ethanolic extraction showed a selectivity of 0.87±0.01. The results reported herein demonstrate the potential application of ATPS, particularly ethanol–potassium phosphate systems, for the recovery of crocins from C. sativus stigmas.
Source:Journal of Chromatography A, Volume 1236
Bertha Montalvo-Hernández, Marco Rito-Palomares, Jorge Benavides
Crocins are carotenoid derivates that have recently attracted the interest of the scientific community due to their nutraceutical properties. Saffron (dry Crocus sativus stigmas) is one of the main known sources of crocins. In this study the potential use of aqueous two-phase system (ATPS) for the extraction of crocins from C. sativus stigmas was evaluated. The partitioning behavior of crocins in different types of ATPS (polymer–polymer, polymer–salt, alcohol–salt and ionic liquid–salt) was evaluated. Ethanol–potassium phosphate ATPS were selected based on their high top phase recovery yield and low cost of system constituents. The evaluation and optimization of system parameters rendered conditions (V R =3.2, ethanol 19.8% (w/w), potassium phosphate 16.5% (w/w), TLL of 25% (w/w), 0.1M NaCl and 2% (w/w) of sample load) under which more than 75% of total crocins were recovered in the top (ethanol rich) phase, whereas the wasted stigmas accumulated in the bottom phase. Lastly, a comparison between an optimized solid–liquid extraction using ethanol:water as solvent and ATPS was conducted demonstrating that similar yields are achieved with both strategies (76.89±1.8% and 79.27±1.6%, respectively). However, ATPS rendered a higher extraction selectivity of 1.3±0.04mg of crocins for each mg of phenolic compound, whereas ethanolic extraction showed a selectivity of 0.87±0.01. The results reported herein demonstrate the potential application of ATPS, particularly ethanol–potassium phosphate systems, for the recovery of crocins from C. sativus stigmas.
Liquid chromatography–flame ionisation detection using a nebuliser/spray chamber interface. Part 1. Design and testing
10 April 2012,
09:29:39
Publication year:
2012
Source:Journal of Chromatography A, Volume 1236
Erepamowei Young, Roger M. Smith, Barry L. Sharp, Joanne R. Bone
A nebuliser and spray chamber have been used to link a flow injection analyser to a flame ionisation detector, with the potential for the combination to be used as a universal detector for liquid chromatography. The hydrogen and air flows were adjusted to achieve a stable system. The detector responded to both volatile and involatile analytes and to compounds with and without chromophores, including alkanes, alkanols, aromatic amides and acids, phenols, amino-acids and carbohydrates and gave a linear response for many analytes. However, for involatile polar analytes it was necessary to add traces of acid or salt to the carrier stream to obtain a linear response.
Source:Journal of Chromatography A, Volume 1236
Erepamowei Young, Roger M. Smith, Barry L. Sharp, Joanne R. Bone
A nebuliser and spray chamber have been used to link a flow injection analyser to a flame ionisation detector, with the potential for the combination to be used as a universal detector for liquid chromatography. The hydrogen and air flows were adjusted to achieve a stable system. The detector responded to both volatile and involatile analytes and to compounds with and without chromophores, including alkanes, alkanols, aromatic amides and acids, phenols, amino-acids and carbohydrates and gave a linear response for many analytes. However, for involatile polar analytes it was necessary to add traces of acid or salt to the carrier stream to obtain a linear response.
Liquid chromatography-flame ionisation detection using a nebuliser/spray chamber interface. Part 2. Comparison of functional group responses
10 April 2012,
09:29:39
Publication year:
2012
Source:Journal of Chromatography A, Volume 1236
Erepamowei Young, Roger M. Smith, Barry L. Sharp, Joanne R. Bone
The application of a LC-nebuliser/spray chamber interface-flame ionisation detection has been demonstrated for the superheated water liquid chromatography of a wide range of aliphatic and aromatic analytes. The linearity and sensitivity of the response of volatile and involatile analytes have been compared. The response of the detector toward different analytes is similar to that in GC-FID and for volatile analytes was comparable to UV detection. However, the responses from involatile analytes, such as amino acids and carbohydrates, were poor and often lower than for a refractive index detector.
Source:Journal of Chromatography A, Volume 1236
Erepamowei Young, Roger M. Smith, Barry L. Sharp, Joanne R. Bone
The application of a LC-nebuliser/spray chamber interface-flame ionisation detection has been demonstrated for the superheated water liquid chromatography of a wide range of aliphatic and aromatic analytes. The linearity and sensitivity of the response of volatile and involatile analytes have been compared. The response of the detector toward different analytes is similar to that in GC-FID and for volatile analytes was comparable to UV detection. However, the responses from involatile analytes, such as amino acids and carbohydrates, were poor and often lower than for a refractive index detector.
Comparison of the fast gradient performance of new prototype silica monolithic columns and columns packed with fully porous and core–shell particles
10 April 2012,
09:29:39
Publication year:
2012
Source:Journal of Chromatography A, Volume 1236
Fabrice Gritti, Nobuo Tanaka, Georges Guiochon
The gradient elution performance of narrow-bore 2.3mm×50mm (N733) and wider bore 3.2mm×50mm (N648 and N655) prototype silica monolithic columns was investigated and compared to the performance of commercially available columns packed with sub-2μm fully porous particles (2.1mm×50mm, 1.7μm BEH-C18, Waters) and sub-3μm superficially porous particles (2.1mm×50mm, 2.7μm Halo-ES-Peptide-C18 (AMT), 1.7 and 2.6μm Kinetex-C18, Phenomenex). Results show that the two wide monolithic columns show peak capacities similar to the one measured for the Kinetex column. In contrast, the narrow-bore monolithic column delivers a lower performance (−30%) than the BEH, the Halo and the Kinetex columns. This work stresses out the importance of reducing the extra-column band broadening contribution of HPLC instruments when short 2.1mm I.D. columns are used. The part of the instrument contribution originating downstream the column is important for all compounds; the one originating upstream the column is significant only for weakly retained compounds.
Source:Journal of Chromatography A, Volume 1236
Fabrice Gritti, Nobuo Tanaka, Georges Guiochon
The gradient elution performance of narrow-bore 2.3mm×50mm (N733) and wider bore 3.2mm×50mm (N648 and N655) prototype silica monolithic columns was investigated and compared to the performance of commercially available columns packed with sub-2μm fully porous particles (2.1mm×50mm, 1.7μm BEH-C18, Waters) and sub-3μm superficially porous particles (2.1mm×50mm, 2.7μm Halo-ES-Peptide-C18 (AMT), 1.7 and 2.6μm Kinetex-C18, Phenomenex). Results show that the two wide monolithic columns show peak capacities similar to the one measured for the Kinetex column. In contrast, the narrow-bore monolithic column delivers a lower performance (−30%) than the BEH, the Halo and the Kinetex columns. This work stresses out the importance of reducing the extra-column band broadening contribution of HPLC instruments when short 2.1mm I.D. columns are used. The part of the instrument contribution originating downstream the column is important for all compounds; the one originating upstream the column is significant only for weakly retained compounds.
Preparation and characterization of bonded silica hydride intermediate from triethoxysilane and dimethylmethoxysilane using supercritical carbon dioxide and dioxane as reaction medium
10 April 2012,
09:29:39
Publication year:
2012
Source:Journal of Chromatography A, Volume 1236
Benjamin A. Ashu-Arrah, Jeremy D. Glennon, Klaus Albert
This research examines bonding methodology, surface coverage and silanol conversion efficiencies on the preparation of silica hydride (SiH) intermediate from triethoxysilane (TES) and dimethylmethoxysilane (DMMS) using sc-CO2 and dioxane as reaction solvent. Under sc-CO2 reaction conditions (at temperature and pressure of 100°C, 414bar, respectively and 3h reaction time), the surface coverages of SiH (evaluated from %C obtained from elemental analysis) prepared with DMMS (3.39μmol/m2) and TES (4.46μmol/m2) increased by 2- and 4-folds respectively, when compared to reaction performed in dioxane (2.66μmol/m2, SiH, DMMS and 0.69μmol/m2, SiH, TES). The relatively higher surface coverage of SiH from TES over DMMS generated in sc-CO2 is due to the inherent trialkoxy moiety of the TES that favours siloxane crosslinkage, forming polymeric surface attachments to yield a higher ligand density than the monomeric DMMS ligand. A conversion efficiency of ∼84.4% of SiH prepared from TES in sc-CO2 estimated from 29Si CP/MAS NMR analysis is comparable to TES silanization in dioxane or toluene. Moreover, silica hydride (SiH) conversion efficiency of ca. 42.4% achieved for the hydride intermediate prepared from DMMS in sc-CO2 is more superior to 33.3% efficiency obtained in dioxane. The differences in conversion efficiencies is attributed to the ability of sc-CO2 being able to access silica pores that are inaccessible in organic solvents. Bonded silica hydride from TES, DMMS prepared in sc-CO2 were characterized using elemental analysis, thermogravimetric analysis (TGA), BET surface area, Fourier transform infrared (FI-IR) and solid state NMR spectroscopy. Silica hydride technology/chemical functionalization of silica in sc-CO2 avoid extended purification steps (i.e. filtration and washing), generation of waste organic solvent and the need of costly or energy consuming drying processing with improved modification efficiency.
Source:Journal of Chromatography A, Volume 1236
Benjamin A. Ashu-Arrah, Jeremy D. Glennon, Klaus Albert
This research examines bonding methodology, surface coverage and silanol conversion efficiencies on the preparation of silica hydride (SiH) intermediate from triethoxysilane (TES) and dimethylmethoxysilane (DMMS) using sc-CO2 and dioxane as reaction solvent. Under sc-CO2 reaction conditions (at temperature and pressure of 100°C, 414bar, respectively and 3h reaction time), the surface coverages of SiH (evaluated from %C obtained from elemental analysis) prepared with DMMS (3.39μmol/m2) and TES (4.46μmol/m2) increased by 2- and 4-folds respectively, when compared to reaction performed in dioxane (2.66μmol/m2, SiH, DMMS and 0.69μmol/m2, SiH, TES). The relatively higher surface coverage of SiH from TES over DMMS generated in sc-CO2 is due to the inherent trialkoxy moiety of the TES that favours siloxane crosslinkage, forming polymeric surface attachments to yield a higher ligand density than the monomeric DMMS ligand. A conversion efficiency of ∼84.4% of SiH prepared from TES in sc-CO2 estimated from 29Si CP/MAS NMR analysis is comparable to TES silanization in dioxane or toluene. Moreover, silica hydride (SiH) conversion efficiency of ca. 42.4% achieved for the hydride intermediate prepared from DMMS in sc-CO2 is more superior to 33.3% efficiency obtained in dioxane. The differences in conversion efficiencies is attributed to the ability of sc-CO2 being able to access silica pores that are inaccessible in organic solvents. Bonded silica hydride from TES, DMMS prepared in sc-CO2 were characterized using elemental analysis, thermogravimetric analysis (TGA), BET surface area, Fourier transform infrared (FI-IR) and solid state NMR spectroscopy. Silica hydride technology/chemical functionalization of silica in sc-CO2 avoid extended purification steps (i.e. filtration and washing), generation of waste organic solvent and the need of costly or energy consuming drying processing with improved modification efficiency.
Multiple, simultaneous, independent gradients for versatile multidimensional liquid chromatography. Part I: Theory
10 April 2012,
09:29:39
Publication year:
2012
Source:Journal of Chromatography A, Volume 1236
Allen G. Hirsh, Latchezar I. Tsonev
The general method for constructing coupled dual gradients in liquid chromatography (LC) is to begin by filling a reservoir A with a solution of one mobile phase (MP) component at concentration [c 1(A)] and a second MP component at concentration [c 2(A)], followed by filling a reservoir B with a solution containing MP component one at concentration [c 1(B)] and the second MP component at concentration [c 2(B)]. In another scenario the reservoirs A and B are filled with solutions of only one MP component at different concentrations [c 1(A)] and [c 1(B)] and the two solutions are titrated to a different pH value: pH (A) for the reservoir A and pH (B) for the reservoir B respectively. In either case, mixing of flows from the two reservoirs varies the concentrations of the two MP components (MP solutes) or the concentration of one MP component and pH along a particular compositional curve producing an eluent with two compositionally coupled gradients. This is a kind of a two dimensional LC utilizing dual simultaneous dependent gradients (DSDGs) wherein two parameters affecting the binding free energy of an analyte to a stationary phase (SP) are being altered simultaneously. Such a DSDG suffers from a significant limitation in that the gradient concentration of the two solutes or the concentration of one MP component and the pH cannot be varied independently. The only way to attain an optimal multigradient LC system, that promises a remarkable increase in chromatographic resolution of complex analyte mixtures, is to uncouple the multiple (dual) gradients, making each independent of the other(s). In this paper the theory of uncoupling of n such gradients, n ≥2 is developed. It is shown that for n solutes 2 n reservoirs are required in concert with an LC eluent delivery system capable of freely apportioning the flows among the reservoirs according to equations we develop here. We go on to predict a substantial increase in chromatographic resolution when applying dual simultaneous independent gradients (DSIGs) of salt and pH to fractionate difficult to separate proteins. This prediction is naturally explained by the electrostatic interaction theory of protein binding to an ion exchanger. In subsequent experimental papers it will be shown that the algorithms presented here properly instruct a quad pump HPLC system to produce well controlled independent simultaneous gradients of pH and non-buffering solutes with attendant significant gain in chromatographic resolution of complex mixtures of protein isoforms.
Source:Journal of Chromatography A, Volume 1236
Allen G. Hirsh, Latchezar I. Tsonev
The general method for constructing coupled dual gradients in liquid chromatography (LC) is to begin by filling a reservoir A with a solution of one mobile phase (MP) component at concentration [c 1(A)] and a second MP component at concentration [c 2(A)], followed by filling a reservoir B with a solution containing MP component one at concentration [c 1(B)] and the second MP component at concentration [c 2(B)]. In another scenario the reservoirs A and B are filled with solutions of only one MP component at different concentrations [c 1(A)] and [c 1(B)] and the two solutions are titrated to a different pH value: pH (A) for the reservoir A and pH (B) for the reservoir B respectively. In either case, mixing of flows from the two reservoirs varies the concentrations of the two MP components (MP solutes) or the concentration of one MP component and pH along a particular compositional curve producing an eluent with two compositionally coupled gradients. This is a kind of a two dimensional LC utilizing dual simultaneous dependent gradients (DSDGs) wherein two parameters affecting the binding free energy of an analyte to a stationary phase (SP) are being altered simultaneously. Such a DSDG suffers from a significant limitation in that the gradient concentration of the two solutes or the concentration of one MP component and the pH cannot be varied independently. The only way to attain an optimal multigradient LC system, that promises a remarkable increase in chromatographic resolution of complex analyte mixtures, is to uncouple the multiple (dual) gradients, making each independent of the other(s). In this paper the theory of uncoupling of n such gradients, n ≥2 is developed. It is shown that for n solutes 2 n reservoirs are required in concert with an LC eluent delivery system capable of freely apportioning the flows among the reservoirs according to equations we develop here. We go on to predict a substantial increase in chromatographic resolution when applying dual simultaneous independent gradients (DSIGs) of salt and pH to fractionate difficult to separate proteins. This prediction is naturally explained by the electrostatic interaction theory of protein binding to an ion exchanger. In subsequent experimental papers it will be shown that the algorithms presented here properly instruct a quad pump HPLC system to produce well controlled independent simultaneous gradients of pH and non-buffering solutes with attendant significant gain in chromatographic resolution of complex mixtures of protein isoforms.
Kinetic optimisation of the reversed phase liquid chromatographic separation of proanthocyanidins on sub-2μm and superficially porous phases
10 April 2012,
09:29:39
Publication year:
2012
Source:Journal of Chromatography A, Volume 1236
Kathithileni M. Kalili, Deirdre Cabooter, Gert Desmet, André de Villiers
Phenolic compounds, and proanthocyanidins in particular, are important natural molecules which are of significant importance due to their sensory and biological activities. The analysis of proanthocyanidins in natural products is very challenging due to their complex nature. In this study, the kinetic performance of a range of recently developed C18 columns, including sub-2μm fully porous and 2.6μm superficially porous particle-packed columns, was evaluated for improved proanthocyanidin analysis. The kinetic plot method was employed to compare the ultimate performance limits of each column in terms of efficiency and speed for different maximum pressures and temperatures using representative proanthocyanidins comprising a range of molecular weights and functionalities as test analytes. By combining plate height data with relevant parameters such as column permeability and mobile phase viscosity, plots of practically attainable efficiencies as a function of analysis time for specific experimental configurations were obtained and performance limits for all investigated supports could accurately be compared. Both fully- and superficially porous particles provided significant speed and/or efficiency gains compared to conventional 5μm particle packed columns. Analyte properties, particle size and packing quality as well as analysis temperature were all found to have a significant influence on the performance of the presently investigated chromatographic supports. For smaller compounds, higher optimal linear velocities and better performance in the low-efficiency range were observed, while the lower diffusion coefficients of larger procyanidins resulted in lower optimal linear velocities and better performance in the high-efficiency regime. Faster analyses become possible at higher temperatures due to decreased eluent viscosity and faster mass transfer, which was especially beneficial for larger compounds and resulted in dramatic improvement in efficiency. A possible explanation of the abnormal behaviour of oligomeric procyanidins is presented. Our findings indicate that new column formats, when used under optimal conditions, significantly improve the speed and/or efficiency of reversed phase liquid chromatographic analyses of proanthocyanidins.
Source:Journal of Chromatography A, Volume 1236
Kathithileni M. Kalili, Deirdre Cabooter, Gert Desmet, André de Villiers
Phenolic compounds, and proanthocyanidins in particular, are important natural molecules which are of significant importance due to their sensory and biological activities. The analysis of proanthocyanidins in natural products is very challenging due to their complex nature. In this study, the kinetic performance of a range of recently developed C18 columns, including sub-2μm fully porous and 2.6μm superficially porous particle-packed columns, was evaluated for improved proanthocyanidin analysis. The kinetic plot method was employed to compare the ultimate performance limits of each column in terms of efficiency and speed for different maximum pressures and temperatures using representative proanthocyanidins comprising a range of molecular weights and functionalities as test analytes. By combining plate height data with relevant parameters such as column permeability and mobile phase viscosity, plots of practically attainable efficiencies as a function of analysis time for specific experimental configurations were obtained and performance limits for all investigated supports could accurately be compared. Both fully- and superficially porous particles provided significant speed and/or efficiency gains compared to conventional 5μm particle packed columns. Analyte properties, particle size and packing quality as well as analysis temperature were all found to have a significant influence on the performance of the presently investigated chromatographic supports. For smaller compounds, higher optimal linear velocities and better performance in the low-efficiency range were observed, while the lower diffusion coefficients of larger procyanidins resulted in lower optimal linear velocities and better performance in the high-efficiency regime. Faster analyses become possible at higher temperatures due to decreased eluent viscosity and faster mass transfer, which was especially beneficial for larger compounds and resulted in dramatic improvement in efficiency. A possible explanation of the abnormal behaviour of oligomeric procyanidins is presented. Our findings indicate that new column formats, when used under optimal conditions, significantly improve the speed and/or efficiency of reversed phase liquid chromatographic analyses of proanthocyanidins.
Quantifying injection solvent effects in reversed-phase liquid chromatography
10 April 2012,
09:29:39
Publication year:
2012
Source:Journal of Chromatography A, Volume 1236
Bradley J. VanMiddlesworth, John G. Dorsey
Peak distortion due to the injection was measured as a function of injection solvent strength, volume, mass, retention factor, and column selectivity. The concept of a method's sensitivity (s) to injection solvent strength was mathematically defined as a vector of theoretical plate counts compared to an ideal vector that does not change with injection solvent strength. Near ideal sensitivity (s >0.90) was measured on all columns with all analytes in low volume injections of 1.25μL. Increasing the injection volume reduces the measured sensitivity from ideality to a greater extent than increasing the injection mass, with differing values for each column. Using column parameters measured from the hydrophobic-subtraction model and fitting parameters from the acetonitrile excess adsorption isotherm, differences among the columns studied are explained. Decreased ligand density and increased silanol activity provide a consistent peak shape with changes in injection volume or solvent strength. For method development, a quick test is suggested with the ratio of hydrophobic-subtraction column parameters, H/A, to predict the injection solvent sensitivity of a column. As H/A decreases, the sensitivity to injection solvent worsens. Sensitivity to organic modifiers other than acetonitrile are predicted with cited sorbed layer thickness, such that MeOH>EtOH>IPA≈THF≈MeCN, i.e., a strong MeOH diluent is more ideal (better) than a strong MeCN diluent.
Source:Journal of Chromatography A, Volume 1236
Bradley J. VanMiddlesworth, John G. Dorsey
Peak distortion due to the injection was measured as a function of injection solvent strength, volume, mass, retention factor, and column selectivity. The concept of a method's sensitivity (s) to injection solvent strength was mathematically defined as a vector of theoretical plate counts compared to an ideal vector that does not change with injection solvent strength. Near ideal sensitivity (s >0.90) was measured on all columns with all analytes in low volume injections of 1.25μL. Increasing the injection volume reduces the measured sensitivity from ideality to a greater extent than increasing the injection mass, with differing values for each column. Using column parameters measured from the hydrophobic-subtraction model and fitting parameters from the acetonitrile excess adsorption isotherm, differences among the columns studied are explained. Decreased ligand density and increased silanol activity provide a consistent peak shape with changes in injection volume or solvent strength. For method development, a quick test is suggested with the ratio of hydrophobic-subtraction column parameters, H/A, to predict the injection solvent sensitivity of a column. As H/A decreases, the sensitivity to injection solvent worsens. Sensitivity to organic modifiers other than acetonitrile are predicted with cited sorbed layer thickness, such that MeOH>EtOH>IPA≈THF≈MeCN, i.e., a strong MeOH diluent is more ideal (better) than a strong MeCN diluent.
Purification of a PEGylated single chain Fv
10 April 2012,
09:29:39
Publication year:
2012
Source:Journal of Chromatography A, Volume 1236
Anna Moosmann, Elke Gerlach, Robert Lindner, Heiner Böttinger
In this manuscript we describe the two-step purification of a mono-PEGylated anti-epidermal growth factor receptor (EGFR) single-chain Fv. A weak cation exchanger was used for capture. Elution using arginine suppressed protein aggregation and allowed a very good resolution with purity and product-recovery was above 90%. Free PEG was removed completely. The use of hydrophobic interaction chromatography (HIC) increased purity to 98%. Increasing the size of PEG from 5 to 30kDa increased retention on HIC and reduced it on cation exchangers. Bioactivity of PEGylated scFv was confirmed by 125I based cell tests. Proteins modified with 5kDa PEG showed higher bioactivity than proteins modified with larger PEGs. The combination of cation exchange and HIC provides a rational and effective basis for PEGylated scFv purification.
Source:Journal of Chromatography A, Volume 1236
Anna Moosmann, Elke Gerlach, Robert Lindner, Heiner Böttinger
In this manuscript we describe the two-step purification of a mono-PEGylated anti-epidermal growth factor receptor (EGFR) single-chain Fv. A weak cation exchanger was used for capture. Elution using arginine suppressed protein aggregation and allowed a very good resolution with purity and product-recovery was above 90%. Free PEG was removed completely. The use of hydrophobic interaction chromatography (HIC) increased purity to 98%. Increasing the size of PEG from 5 to 30kDa increased retention on HIC and reduced it on cation exchangers. Bioactivity of PEGylated scFv was confirmed by 125I based cell tests. Proteins modified with 5kDa PEG showed higher bioactivity than proteins modified with larger PEGs. The combination of cation exchange and HIC provides a rational and effective basis for PEGylated scFv purification.
Simultaneous determination of fluoxetine and norfluoxetine enantiomers using isotope discrimination mass spectroscopy solution method and its application in the CYP2C9-mediated stereoselective interactions
10 April 2012,
09:29:39
Publication year:
2012
Source:Journal of Chromatography A, Volume 1236
Lushan Yu, Shengjia Wang, Huidi Jiang, Hui Zhou, Su Zeng
In this study, we developed an LC–MS/MS method based on an isotope discrimination mass spectroscopy solution (IDMSS) technology to simultaneously quantify enantiomers of fluoxetine (FLX) and norfluoxetine (NFLX) in a CYP2C9 incubation mixture. S-FLX and S-NFLX were labeled to form S-FLX-d5 and S-NFLX-d5. The method has several advantages over conventional chiral separation methods, in terms of the analysis period, resolution, and lower limit of quantification. The primary advantage of the method is that the two enantiomers can always be simultaneously determined by mass spectroscopy regardless if they are separated on column or not, owing to which it has high throughput and high sensitivity. The lower limit of quantification (amount on column) is 12.5 and 1.25pg for FLX and NFLX, respectively. The retention time of FLX, NFLX, and the internal standard is only 1.9min. The calibration curves were linear over the concentration range of 0.1–100ng/ml for NFLX and 1–1000ng/ml for FLX with an accepted reproducible (RSD<10%) and accurate (CV<10%). No significant kinetic isotope effect was found in the metabolism of S-FLX-d5 catalyzed by CYP2C9*1 and CYP2C9*2. The half-maximal inhibitory concentration values between R-FLX and S-FLX catalyzed by CYP2C9*1 and CYP2C9*2 were determined in this study. The inhibitory effects of R- to S-FLX were stronger than those of S- to R-FLX in both CYP2C9*1 and CYP2C9*2. The IDMSS technology is useful for stereoselective study of chiral compound in vitro.
Source:Journal of Chromatography A, Volume 1236
Lushan Yu, Shengjia Wang, Huidi Jiang, Hui Zhou, Su Zeng
In this study, we developed an LC–MS/MS method based on an isotope discrimination mass spectroscopy solution (IDMSS) technology to simultaneously quantify enantiomers of fluoxetine (FLX) and norfluoxetine (NFLX) in a CYP2C9 incubation mixture. S-FLX and S-NFLX were labeled to form S-FLX-d5 and S-NFLX-d5. The method has several advantages over conventional chiral separation methods, in terms of the analysis period, resolution, and lower limit of quantification. The primary advantage of the method is that the two enantiomers can always be simultaneously determined by mass spectroscopy regardless if they are separated on column or not, owing to which it has high throughput and high sensitivity. The lower limit of quantification (amount on column) is 12.5 and 1.25pg for FLX and NFLX, respectively. The retention time of FLX, NFLX, and the internal standard is only 1.9min. The calibration curves were linear over the concentration range of 0.1–100ng/ml for NFLX and 1–1000ng/ml for FLX with an accepted reproducible (RSD<10%) and accurate (CV<10%). No significant kinetic isotope effect was found in the metabolism of S-FLX-d5 catalyzed by CYP2C9*1 and CYP2C9*2. The half-maximal inhibitory concentration values between R-FLX and S-FLX catalyzed by CYP2C9*1 and CYP2C9*2 were determined in this study. The inhibitory effects of R- to S-FLX were stronger than those of S- to R-FLX in both CYP2C9*1 and CYP2C9*2. The IDMSS technology is useful for stereoselective study of chiral compound in vitro.
Kinetic performance of narrow-bore columns on a micro-system for high performance liquid chromatography
10 April 2012,
09:29:39
Publication year:
2012
Source:Journal of Chromatography A, Volume 1236
Fabrice Gritti, Georges Guiochon
The kinetic performance of 0.5mm×50mm columns packed with 2.7μm Halo-C18 core–shell particles and 3μm EP-120-C18 fully porous particles fitted on an Eksigent LC-Express Ultra μHPLC system were measured. The instrument contribution to band broadening was obtained by directly connecting the injection valve and the detector cell with a short, narrow PEEKSIL tube. The connections between the column and the connecting tubes, the column endfittings and its frits contribute to band spreading and are responsible for a significant rear peak tailing, even for retained compounds, resulting in a significant loss of efficiency. Our results show that the μHPLC system could outperform the current VHPLC systems using 2.1mm I.D. columns packed with 1.7μm particles if it were using 0.5mm I.D. columns packed with 1μm particles, if it could operate at a few kbar pressure drop, and if the sum of the contributions of the instrument, column endfittings and the column frits to band dispersion were three times smaller than it is at present.
Source:Journal of Chromatography A, Volume 1236
Fabrice Gritti, Georges Guiochon
The kinetic performance of 0.5mm×50mm columns packed with 2.7μm Halo-C18 core–shell particles and 3μm EP-120-C18 fully porous particles fitted on an Eksigent LC-Express Ultra μHPLC system were measured. The instrument contribution to band broadening was obtained by directly connecting the injection valve and the detector cell with a short, narrow PEEKSIL tube. The connections between the column and the connecting tubes, the column endfittings and its frits contribute to band spreading and are responsible for a significant rear peak tailing, even for retained compounds, resulting in a significant loss of efficiency. Our results show that the μHPLC system could outperform the current VHPLC systems using 2.1mm I.D. columns packed with 1.7μm particles if it were using 0.5mm I.D. columns packed with 1μm particles, if it could operate at a few kbar pressure drop, and if the sum of the contributions of the instrument, column endfittings and the column frits to band dispersion were three times smaller than it is at present.
Ion-exchange centrifugal partition chromatography: A methodological approach for peptide separation
10 April 2012,
09:29:39
Publication year:
2012
Source:Journal of Chromatography A, Volume 1236
Leslie Boudesocque, Pedro Lameiras, Nassima Amarouche, Matthieu Giraud, Francesca Quattrini, John Mc Garrity, Jean-Marc Nuzillard, Jean-Hugues Renault
This article presents the scope and optimization strategies employed in ion-exchange centrifugal partition chromatography (IXCPC). Both the weak and the strong modes were used to separate the constituents of a model mixture of dipeptides. Thus, the combined use of the quaternary biphasic solvent system, methyl-tert-butylether/acetonitrile/n-butanol/water (2:1:2:5, v/v) in the descending mode, of the lipophilic di(2-ethylhexyl)phosphoric acid (DEHPA) cation-exchanger, and of two displacers: calcium chloride and hydrochloric acid, has proven to be efficient for the preparative separation of the model mixture of five dipeptides (GG, GY, AY, LV and LY, in the order they were collected). The separation was optimized by splitting the stationary phase into two sections that differed by their triethylamine concentration. Moreover, the chemical nature of the exchanger/analyte entities that were involved in the chromatographic process was determined by 31P and 1H DOSY NMR experiments.
Source:Journal of Chromatography A, Volume 1236
Leslie Boudesocque, Pedro Lameiras, Nassima Amarouche, Matthieu Giraud, Francesca Quattrini, John Mc Garrity, Jean-Marc Nuzillard, Jean-Hugues Renault
This article presents the scope and optimization strategies employed in ion-exchange centrifugal partition chromatography (IXCPC). Both the weak and the strong modes were used to separate the constituents of a model mixture of dipeptides. Thus, the combined use of the quaternary biphasic solvent system, methyl-tert-butylether/acetonitrile/n-butanol/water (2:1:2:5, v/v) in the descending mode, of the lipophilic di(2-ethylhexyl)phosphoric acid (DEHPA) cation-exchanger, and of two displacers: calcium chloride and hydrochloric acid, has proven to be efficient for the preparative separation of the model mixture of five dipeptides (GG, GY, AY, LV and LY, in the order they were collected). The separation was optimized by splitting the stationary phase into two sections that differed by their triethylamine concentration. Moreover, the chemical nature of the exchanger/analyte entities that were involved in the chromatographic process was determined by 31P and 1H DOSY NMR experiments.
Simulated moving bed enantioseparation of amino acids employing memory effect-constrained chromatography columns
10 April 2012,
09:29:39
Publication year:
2012
Source:Journal of Chromatography A, Volume 1236
Markus Fuereder, Sven Panke, Matthias Bechtold
Teicoplanin aglycone-based chromatography columns (Chirobiotic TAG) enable amino acid enantioseparation with aqueous mobile phases, which perfectly accommodates the distinct hydrophilicity of most amino acids. Therefore, this stationary phase constitutes a promising option in particular for preparative-scale separations that require high feed concentrations for economic operation. However, detailed studies revealed a solute-related memory effect when this column is subjected to high loadings of amino acids, conditions that prevail in SMB operation. High loadings yield an activation of the column as indicated by increased retention times when comparing finite injection chromatograms obtained before and after the column had been subjected to a concentrated amino acid feed. This effect can be slowly reversed by flushing the column with solvent devoid of amino acid. Obviously, the activation of the stationary phase needs to be accounted for in the determination of adsorption isotherms that are used for SMB design. In this work we introduce a perturbation method adapted specifically to capture the stationary phase behaviour at SMB-like conditions. The adsorption isotherms obtained from this method indeed allowed for accurate SMB design of a methionine enantioseparation as judged by the very good agreement of experimentally obtained and model-predicted purities. Furthermore, SMB operation over 3 days with constant purities (besides deviations originating from a dip in temperature) was accomplished indicating that the adsorption behaviour in the activated state is indeed time invariant and stable long-term SMB operation with these columns is principally feasible.
Source:Journal of Chromatography A, Volume 1236
Markus Fuereder, Sven Panke, Matthias Bechtold
Teicoplanin aglycone-based chromatography columns (Chirobiotic TAG) enable amino acid enantioseparation with aqueous mobile phases, which perfectly accommodates the distinct hydrophilicity of most amino acids. Therefore, this stationary phase constitutes a promising option in particular for preparative-scale separations that require high feed concentrations for economic operation. However, detailed studies revealed a solute-related memory effect when this column is subjected to high loadings of amino acids, conditions that prevail in SMB operation. High loadings yield an activation of the column as indicated by increased retention times when comparing finite injection chromatograms obtained before and after the column had been subjected to a concentrated amino acid feed. This effect can be slowly reversed by flushing the column with solvent devoid of amino acid. Obviously, the activation of the stationary phase needs to be accounted for in the determination of adsorption isotherms that are used for SMB design. In this work we introduce a perturbation method adapted specifically to capture the stationary phase behaviour at SMB-like conditions. The adsorption isotherms obtained from this method indeed allowed for accurate SMB design of a methionine enantioseparation as judged by the very good agreement of experimentally obtained and model-predicted purities. Furthermore, SMB operation over 3 days with constant purities (besides deviations originating from a dip in temperature) was accomplished indicating that the adsorption behaviour in the activated state is indeed time invariant and stable long-term SMB operation with these columns is principally feasible.
Bioactivity-guided fractionation of the volatile oil of Angelica sinensis radix designed to preserve the synergistic effects of the mixture followed by identification of the active principles
10 April 2012,
09:29:39
Publication year:
2012
Source:Journal of Chromatography A, Volume 1236
Ju-Ching Yeh, Ian J. Garrard, Chin-Wen Chantal Cho, S.W. Annie Bligh, Guang-hua Lu, Tai-Ping Fan, Derek Fisher
In natural product research, it is a common experience that fractionation of biologically-active crude extracts can lead to the loss of their original activity. This is attributed to synergistic effects, where two or more components are required to be present together for full activity of the sample. Our previous study showed that a volatile oil of Angelica sinensis radix (VOAS) inhibited endothelial cell proliferation in culture. Here we have used a bioactivity-guided fractionation method to preserve any synergistic effects of VOAS combining countercurrent chromatography (CCC), the MTS cell viability assay and gas chromatography (GC). Using a two-phase CCC solvent system (heptane–ethyl acetate–methanol–water at a volume ratio of 27:23:27:23%), forty-five fractions were isolated, nine of which exhibited anti-endothelial properties. GC analysis showed two bioactive alkylphthalides, Z-ligustilide and n-butylidenephthalide (BP) were the major compounds detected in the bioactive fractions, and were absent in non-bioactive fractions. Our results indicate that Z-ligustilide and BP are the main constituents responsible for the anti-endothelial properties of VOAS. This rapid and reliable approach in preserving sample activity while isolating and identifying its active compounds suggests that this protocol can be a powerful tool for drug discovery from natural products.
Source:Journal of Chromatography A, Volume 1236
Ju-Ching Yeh, Ian J. Garrard, Chin-Wen Chantal Cho, S.W. Annie Bligh, Guang-hua Lu, Tai-Ping Fan, Derek Fisher
In natural product research, it is a common experience that fractionation of biologically-active crude extracts can lead to the loss of their original activity. This is attributed to synergistic effects, where two or more components are required to be present together for full activity of the sample. Our previous study showed that a volatile oil of Angelica sinensis radix (VOAS) inhibited endothelial cell proliferation in culture. Here we have used a bioactivity-guided fractionation method to preserve any synergistic effects of VOAS combining countercurrent chromatography (CCC), the MTS cell viability assay and gas chromatography (GC). Using a two-phase CCC solvent system (heptane–ethyl acetate–methanol–water at a volume ratio of 27:23:27:23%), forty-five fractions were isolated, nine of which exhibited anti-endothelial properties. GC analysis showed two bioactive alkylphthalides, Z-ligustilide and n-butylidenephthalide (BP) were the major compounds detected in the bioactive fractions, and were absent in non-bioactive fractions. Our results indicate that Z-ligustilide and BP are the main constituents responsible for the anti-endothelial properties of VOAS. This rapid and reliable approach in preserving sample activity while isolating and identifying its active compounds suggests that this protocol can be a powerful tool for drug discovery from natural products.
CHROM2—A method to enhance the dynamic binding capacity, yield and productivity of a chromatographic column
10 April 2012,
09:29:39
Publication year:
2012
Source:Journal of Chromatography A, Volume 1236
Mayuratheepan Puthirasigamany, Peter van Beijeren, Peter Kreis
Therapeutic proteins are biotechnological products with a fast-growing market. Despite the rapid development of available process technologies, a bottleneck in production capacities is still present due to limitations in the associated downstream process, particularly within chromatographic purification steps. Membrane chromatography has been introduced as a promising alternative for conventional chromatography because it allows for higher throughputs but it does not deliver comparable dynamic binding capacities. To combine the strengths of the two technologies, the so-called “CHROM2 concepts” are introduced, which merge conventional chromatography with membrane adsorption. The serial connection of a large conventional chromatographic column followed by a small membrane chromatography unit enables to combine the strength of both the individual technologies. The larger column delivers the required high binding capacity, whereas the rapid binding kinetics of membrane chromatography sharpens the breakthrough curve. Furthermore applied higher velocities do not result in poor breakthrough performance since the membrane chromatography is able to compensate for the poor column breakthrough performance. In comparison to column chromatography, the CHROM2 setup exploits the full column capacity and delivers higher productivities and yields.
Source:Journal of Chromatography A, Volume 1236
Mayuratheepan Puthirasigamany, Peter van Beijeren, Peter Kreis
Therapeutic proteins are biotechnological products with a fast-growing market. Despite the rapid development of available process technologies, a bottleneck in production capacities is still present due to limitations in the associated downstream process, particularly within chromatographic purification steps. Membrane chromatography has been introduced as a promising alternative for conventional chromatography because it allows for higher throughputs but it does not deliver comparable dynamic binding capacities. To combine the strengths of the two technologies, the so-called “CHROM2 concepts” are introduced, which merge conventional chromatography with membrane adsorption. The serial connection of a large conventional chromatographic column followed by a small membrane chromatography unit enables to combine the strength of both the individual technologies. The larger column delivers the required high binding capacity, whereas the rapid binding kinetics of membrane chromatography sharpens the breakthrough curve. Furthermore applied higher velocities do not result in poor breakthrough performance since the membrane chromatography is able to compensate for the poor column breakthrough performance. In comparison to column chromatography, the CHROM2 setup exploits the full column capacity and delivers higher productivities and yields.
Sequential determination of anionic-type detergents by complexation with methylene blue using dual high speed counter-current chromatography
10 April 2012,
09:29:39
Publication year:
2012
Source:Journal of Chromatography A, Volume 1236
Eiichi Kitazume, Saki Koikawa, Lu Hui, Syou Sannohe, Yanjun Yang, Yonosuke Maki, Yoichiro Ito
A new dual high-speed counter-current chromatographic system using organic extraction phase and aqueous mobile phase containing methylene blue was applied to the analysis of anionic-type detergents. After selecting appropriate conditions such as flow rate of each mobile phase and sample volume, the new system was successfully applied to the analysis of anionic detergent in river water. As all the analytical procedures can be made in a closed system, the method has no health hazard. The present method is safe, precise, and highly sensitive, and can be applied for sequential determination of multiple samples in a short analysis time.
Source:Journal of Chromatography A, Volume 1236
Eiichi Kitazume, Saki Koikawa, Lu Hui, Syou Sannohe, Yanjun Yang, Yonosuke Maki, Yoichiro Ito
A new dual high-speed counter-current chromatographic system using organic extraction phase and aqueous mobile phase containing methylene blue was applied to the analysis of anionic-type detergents. After selecting appropriate conditions such as flow rate of each mobile phase and sample volume, the new system was successfully applied to the analysis of anionic detergent in river water. As all the analytical procedures can be made in a closed system, the method has no health hazard. The present method is safe, precise, and highly sensitive, and can be applied for sequential determination of multiple samples in a short analysis time.
On the chromatographic efficiency of analytical scale column format porous polymer monoliths: Interplay of morphology and nanoscale gel porosity
10 April 2012,
09:29:39
Publication year:
2012
Source:Journal of Chromatography A, Volume 1236
Ivo Nischang
Porous monolithic poly(styrene-co-divinylbenzene) stationary phases in 4.6mm I.D. analytical-scale column format with varying porosity, globule scale polymer morphology and flow-through pore structure have been investigated with respect to their transport properties toward small retained solutes in isocratic elution, reversed-phase liquid chromatography. The current study was performed under kinetically and thermodynamically relevant conditions comprising retention factors from close to zero up to the order of 50–100 under most extreme conditions, while a linear chromatographic flow velocity up to 4mm/s, in some instances up to 7mm/s, was realized. Carefully designed experiments aimed at resolving issues associated with the monoliths performance, while a particular focus is given on gel porosity, chromatographic retention and band dispersion. Elucidation of three important metric properties gave orthogonal insight. These are: (i) the columns dry-state morphology and surface area, (ii) the gel porosity with tetrahydrofuran as solvent determined by size exclusion chromatography using a range of small subnanometer-sized molecules and polystyrene standards, as well as (iii) the isocratic reversed-phase performance of small molecules at varying binary acetonitrile/water mobile phase solvent compositions, modulating gel porosity. Consistently throughout the study, the adjustable and general retention-factor-dependence of the performance of these monolithic materials is shown. It can also be correlated to the analytes molecular weight and consequently size. Isocratic performance strongly depends on the amount of gel porosity of the scaffold, which can be changed by varying the percentage of organic modifier in the mobile phase and indicates the adjustable chromatographic nature of porous polymer monoliths. This gel porosity which is absent in the dry-state of the polymer monoliths and is characterized by sub-nanometer to nanometer-sized pore space induces, additionally to permanent porosity, stagnant mass transfer zones. The displayed major reason for mass transfer resistance implied by the use of polymeric monolithic columns determines dispersion behavior of small molecules and its varying importance with respect to morphology and size of the globular features containing stagnant mass transfer zones is addressed. This leads to the conclusion, that a reduction in polymer feature size and increase in number of flow-through pores per unit cross-section of the monolith with an improved homogeneity may be an interesting option of tailoring column performance. It is further concluded that dry-state methods (such as nitrogen adsorption analysis and scanning electron microscopy) or solvated-state methods (such as size-exclusion chromatography in tetrahydrofuran) by itself are insufficient measures to explain the adjustable chromatographic performance of porous polymer monoliths.
Source:Journal of Chromatography A, Volume 1236
Ivo Nischang
Porous monolithic poly(styrene-co-divinylbenzene) stationary phases in 4.6mm I.D. analytical-scale column format with varying porosity, globule scale polymer morphology and flow-through pore structure have been investigated with respect to their transport properties toward small retained solutes in isocratic elution, reversed-phase liquid chromatography. The current study was performed under kinetically and thermodynamically relevant conditions comprising retention factors from close to zero up to the order of 50–100 under most extreme conditions, while a linear chromatographic flow velocity up to 4mm/s, in some instances up to 7mm/s, was realized. Carefully designed experiments aimed at resolving issues associated with the monoliths performance, while a particular focus is given on gel porosity, chromatographic retention and band dispersion. Elucidation of three important metric properties gave orthogonal insight. These are: (i) the columns dry-state morphology and surface area, (ii) the gel porosity with tetrahydrofuran as solvent determined by size exclusion chromatography using a range of small subnanometer-sized molecules and polystyrene standards, as well as (iii) the isocratic reversed-phase performance of small molecules at varying binary acetonitrile/water mobile phase solvent compositions, modulating gel porosity. Consistently throughout the study, the adjustable and general retention-factor-dependence of the performance of these monolithic materials is shown. It can also be correlated to the analytes molecular weight and consequently size. Isocratic performance strongly depends on the amount of gel porosity of the scaffold, which can be changed by varying the percentage of organic modifier in the mobile phase and indicates the adjustable chromatographic nature of porous polymer monoliths. This gel porosity which is absent in the dry-state of the polymer monoliths and is characterized by sub-nanometer to nanometer-sized pore space induces, additionally to permanent porosity, stagnant mass transfer zones. The displayed major reason for mass transfer resistance implied by the use of polymeric monolithic columns determines dispersion behavior of small molecules and its varying importance with respect to morphology and size of the globular features containing stagnant mass transfer zones is addressed. This leads to the conclusion, that a reduction in polymer feature size and increase in number of flow-through pores per unit cross-section of the monolith with an improved homogeneity may be an interesting option of tailoring column performance. It is further concluded that dry-state methods (such as nitrogen adsorption analysis and scanning electron microscopy) or solvated-state methods (such as size-exclusion chromatography in tetrahydrofuran) by itself are insufficient measures to explain the adjustable chromatographic performance of porous polymer monoliths.
Development of anion-exchange/reversed-phase high performance liquid chromatography–inductively coupled plasma-mass spectrometry methods for the speciation of bio-available iodine and bromine from edible seaweed
10 April 2012,
09:29:39
Publication year:
2012
Source:Journal of Chromatography A, Volume 1236
Vanessa Romarís-Hortas, Pilar Bermejo-Barrera, Antonio Moreda-Piñeiro
Anion exchange high performance liquid chromatography hyphenated with inductively coupled plasma-mass spectrometry has been novelly applied to assess inorganic (iodide and iodate) and organic (3-iodotyrosine – MIT, and 3,5-diiodotyrosine – DIT) iodine species in a single chromatographic run. The optimized operating conditions (Dionex IonPac AS7, gradient elution with 175mM ammonium nitrate plus 15% (v/v) methanol, pH 3.8, as a mobile phase and flow rates within the 0.5–1.5mLmin−1 range) have also been used to perform inorganic bromine speciation analysis (bromide and bromate). The developed method has been applied for determining the bio-available contents of iodine and bromine species in dialyzates from edible seaweed. Reverse phase high performance liquid chromatography (Zorbax Eclipse XDB-C8, gradient elution with 0.2% (m/m) acetic acid, and 0.2% (m/m) acetic acid in methanol, as mobile phases, and a constant flow rate of 0.75mLmin−1) also hyphenated with inductively coupled plasma-mass spectrometry was used to confirm the presence of organic iodine species (MIT and DIT) in the dialyzates. The verification of the presence of iodinated amino acids (MIT and DIT) in the extracts was also performed by reverse phase high performance liquid chromatography–electrospray ionization-mass spectrometry (LTQ Orbitrap). The developed methods have provided good repeatability (RSD values lower than 10% for both anion exchange and reverse phase separations) and analytical recoveries within the 90–105% range for all cases. The in vitro bio-availability method consisted of a simulated gastric and an intestinal digestion/dialysis (10kDa molecular weight cut-off – MWCO) two-stage procedure. Iodide and MIT were the main bio-available species quantified, whereas bromide was the major bromine species found in the extracts.
Source:Journal of Chromatography A, Volume 1236
Vanessa Romarís-Hortas, Pilar Bermejo-Barrera, Antonio Moreda-Piñeiro
Anion exchange high performance liquid chromatography hyphenated with inductively coupled plasma-mass spectrometry has been novelly applied to assess inorganic (iodide and iodate) and organic (3-iodotyrosine – MIT, and 3,5-diiodotyrosine – DIT) iodine species in a single chromatographic run. The optimized operating conditions (Dionex IonPac AS7, gradient elution with 175mM ammonium nitrate plus 15% (v/v) methanol, pH 3.8, as a mobile phase and flow rates within the 0.5–1.5mLmin−1 range) have also been used to perform inorganic bromine speciation analysis (bromide and bromate). The developed method has been applied for determining the bio-available contents of iodine and bromine species in dialyzates from edible seaweed. Reverse phase high performance liquid chromatography (Zorbax Eclipse XDB-C8, gradient elution with 0.2% (m/m) acetic acid, and 0.2% (m/m) acetic acid in methanol, as mobile phases, and a constant flow rate of 0.75mLmin−1) also hyphenated with inductively coupled plasma-mass spectrometry was used to confirm the presence of organic iodine species (MIT and DIT) in the dialyzates. The verification of the presence of iodinated amino acids (MIT and DIT) in the extracts was also performed by reverse phase high performance liquid chromatography–electrospray ionization-mass spectrometry (LTQ Orbitrap). The developed methods have provided good repeatability (RSD values lower than 10% for both anion exchange and reverse phase separations) and analytical recoveries within the 90–105% range for all cases. The in vitro bio-availability method consisted of a simulated gastric and an intestinal digestion/dialysis (10kDa molecular weight cut-off – MWCO) two-stage procedure. Iodide and MIT were the main bio-available species quantified, whereas bromide was the major bromine species found in the extracts.
Evaluation of a new wide pore core–shell material (Aeris™ WIDEPORE) and comparison with other existing stationary phases for the analysis of intact proteins
10 April 2012,
09:29:39
Publication year:
2012
Source:Journal of Chromatography A, Volume 1236
Szabolcs Fekete, Róbert Berky, Jenő Fekete, Jean-Luc Veuthey, Davy Guillarme
The separation of large biomolecules such as proteins or monoclonal antibodies (mAbs) by RPLC can be drastically enhanced thanks to the use of columns packed with wide-pore porous sub-2μm particles or shell particles. In this context, a new wide-pore core–shell material has been recently released under the trademark Aeris WIDEPORE. It is made of a 3.2μm solid inner core surrounded by a 0.2μm porous layer (total particle size of 3.6μm). The aim of this study was to evaluate the performance of this new material, compare it to other recently developed and older conventional wide-pore columns and demonstrate its applicability to real-life separations of proteins and mAbs. At first, the traditional h min values of the Aeris WIDEPORE column were determined for small model compounds. The h min values were equal to 1.7–1.8 and 1.4 for the 2.1 and 4.6mm I.D. columns, respectively, which are in agreement with the values reported for other core–shell materials. In the case of a small protein Insulin (5.7kDa), the achievable lowest h value was below 2 and this impressive result confirms that the Aeris WIDEPORE material should be dedicated to protein analysis. This column was then compared with five other commercially available wide-pore and medium-pore stationary phases, in the gradient elution mode, using various flow rates, gradient steepness and model proteins of MW=5.7–66.8kDa. The Aeris WIDEPORE material often provided the best performance, in terms of peak capacity, peak capacity per time and pressure unit (PPT) and also based on the gradient kinetic plot representation. Finally, real separations of filgrastim (18.8kDa) and its oxidized and reduced forms were performed on the different columns and the Aeris WIDEPORE material provided the most impressive performance (peak capacity>100 for t grad <6min). Last but not least, this new material was also evaluated on digested and reduced mAb and powerful, high-throughput separations were also attained.
Source:Journal of Chromatography A, Volume 1236
Szabolcs Fekete, Róbert Berky, Jenő Fekete, Jean-Luc Veuthey, Davy Guillarme
The separation of large biomolecules such as proteins or monoclonal antibodies (mAbs) by RPLC can be drastically enhanced thanks to the use of columns packed with wide-pore porous sub-2μm particles or shell particles. In this context, a new wide-pore core–shell material has been recently released under the trademark Aeris WIDEPORE. It is made of a 3.2μm solid inner core surrounded by a 0.2μm porous layer (total particle size of 3.6μm). The aim of this study was to evaluate the performance of this new material, compare it to other recently developed and older conventional wide-pore columns and demonstrate its applicability to real-life separations of proteins and mAbs. At first, the traditional h min values of the Aeris WIDEPORE column were determined for small model compounds. The h min values were equal to 1.7–1.8 and 1.4 for the 2.1 and 4.6mm I.D. columns, respectively, which are in agreement with the values reported for other core–shell materials. In the case of a small protein Insulin (5.7kDa), the achievable lowest h value was below 2 and this impressive result confirms that the Aeris WIDEPORE material should be dedicated to protein analysis. This column was then compared with five other commercially available wide-pore and medium-pore stationary phases, in the gradient elution mode, using various flow rates, gradient steepness and model proteins of MW=5.7–66.8kDa. The Aeris WIDEPORE material often provided the best performance, in terms of peak capacity, peak capacity per time and pressure unit (PPT) and also based on the gradient kinetic plot representation. Finally, real separations of filgrastim (18.8kDa) and its oxidized and reduced forms were performed on the different columns and the Aeris WIDEPORE material provided the most impressive performance (peak capacity>100 for t grad <6min). Last but not least, this new material was also evaluated on digested and reduced mAb and powerful, high-throughput separations were also attained.
Comparison of indirect and direct quantification of esters of monochloropropanediol in vegetable oil
10 April 2012,
09:29:39
Publication year:
2012
Source:Journal of Chromatography A, Volume 1236
Mathieu Dubois, Adrienne Tarres, Till Goldmann, Anna Maria Empl, Alfred Donaubauer, Walburga Seefelder
The presence of fatty acid esters of monochloropropanediol (MEs) in food is a recent concern raised due to the carcinogenicity of their hydrolysable moieties 2- and 3-monochloropropanediol (2- and 3-MCPD). Several indirect methods for the quantification of MEs have been developed and are commonly in use until today, however significant discrepancies among analytical results obtained are challenging their reliability. The aim of the present study was therefore to test the trueness of an indirect method by comparing it to a newly developed direct method using palm oil and palm olein as examples. The indirect method was based on ester cleavage under acidic conditions, derivatization of the liberated 2- and 3-MCPD with heptafluorobutyryl imidazole and GC–MS determination. The direct method was comprised of two extraction procedures targeting 2-and 3-MCPD mono esters (co-extracting as well glycidyl esters) by the use of double solid phase extraction (SPE), and 2- and 3-MCPD di-esters by the use of silica gel column, respectively. Detection was carried out by liquid chromatography coupled to time of flight mass spectrometry (LC–ToF-MS). Accurate quantification of the intact compounds was assured by means of matrix matched standard addition on extracts. Analysis of 22 palm oil and 7 palm olein samples (2- plus 3-MCPD contamination ranged from 0.3 to 8.8μg/g) by both methods revealed no significant bias. Both methods were therefore considered as comparable in terms of results; however the indirect method was shown to require less analytical standards, being less tedious and furthermore applicable to all type of different vegetable oils and hence recommended for routine application.
Source:Journal of Chromatography A, Volume 1236
Mathieu Dubois, Adrienne Tarres, Till Goldmann, Anna Maria Empl, Alfred Donaubauer, Walburga Seefelder
The presence of fatty acid esters of monochloropropanediol (MEs) in food is a recent concern raised due to the carcinogenicity of their hydrolysable moieties 2- and 3-monochloropropanediol (2- and 3-MCPD). Several indirect methods for the quantification of MEs have been developed and are commonly in use until today, however significant discrepancies among analytical results obtained are challenging their reliability. The aim of the present study was therefore to test the trueness of an indirect method by comparing it to a newly developed direct method using palm oil and palm olein as examples. The indirect method was based on ester cleavage under acidic conditions, derivatization of the liberated 2- and 3-MCPD with heptafluorobutyryl imidazole and GC–MS determination. The direct method was comprised of two extraction procedures targeting 2-and 3-MCPD mono esters (co-extracting as well glycidyl esters) by the use of double solid phase extraction (SPE), and 2- and 3-MCPD di-esters by the use of silica gel column, respectively. Detection was carried out by liquid chromatography coupled to time of flight mass spectrometry (LC–ToF-MS). Accurate quantification of the intact compounds was assured by means of matrix matched standard addition on extracts. Analysis of 22 palm oil and 7 palm olein samples (2- plus 3-MCPD contamination ranged from 0.3 to 8.8μg/g) by both methods revealed no significant bias. Both methods were therefore considered as comparable in terms of results; however the indirect method was shown to require less analytical standards, being less tedious and furthermore applicable to all type of different vegetable oils and hence recommended for routine application.
Micellar electrokinetic chromatography of aromatic anions and non-ionic aromatic compounds with stepwise changes of the concentration of cetyltrimethylammonium chloride
10 April 2012,
09:29:39
Publication year:
2012
Source:Journal of Chromatography A, Volume 1236
Yukihiro Esaka, Miki Kobayashi, Hiroya Murakami, Bunji Uno
Micellar electrokinetic chromatography in which the concentration of cetyltrimetylammmonium chloride (CTAC) was sequentially changed in the separation system was investigated using 10 aromatic anions and 11 non-ionic aromatic compounds as model analytes. All separations were performed in the absence of electroosmotic flow (EOF), and thus, analytes were detected in the order of their strength of interaction with micelles in the system. In isocratic elutions without EOF, the model analytes could be separated better with lower concentrations of CTAC but migration times of the analytes possessing relatively higher polarities increased markedly, and thus, long analysis times were required. Therefore, we attempted to increase the concentration of CTAC during a single measurement to reduce the analysis time without hindering the resultant separation of analytes obtained with lower concentrations. Briefly, the present surfactant stepwise elution can be performed by a sequential increase in CTAC concentrations of the running solution in the anodic reservoir from 30 to 50mM for the anions and from 20 to 50mM for the non-ionic compounds. Additionally, to perform expected gradient separations with good reproducibility, each running solution with a different CTAC concentration was treated with tetraethylammmonium chloride as an additive to adjust electric conductivities of each running solution to be equal. Under this condition, CTAC micelles of each zone of different CTAC concentrations would migrate with practically the same velocity. Consequently, by the present stepwise method, both the 10 anionic analytes and the 11 non-ionic analytes were well separated within reasonable periods which corresponded approximately to two-third and less than half of those by the isocratic elutions, respectively.
Source:Journal of Chromatography A, Volume 1236
Yukihiro Esaka, Miki Kobayashi, Hiroya Murakami, Bunji Uno
Micellar electrokinetic chromatography in which the concentration of cetyltrimetylammmonium chloride (CTAC) was sequentially changed in the separation system was investigated using 10 aromatic anions and 11 non-ionic aromatic compounds as model analytes. All separations were performed in the absence of electroosmotic flow (EOF), and thus, analytes were detected in the order of their strength of interaction with micelles in the system. In isocratic elutions without EOF, the model analytes could be separated better with lower concentrations of CTAC but migration times of the analytes possessing relatively higher polarities increased markedly, and thus, long analysis times were required. Therefore, we attempted to increase the concentration of CTAC during a single measurement to reduce the analysis time without hindering the resultant separation of analytes obtained with lower concentrations. Briefly, the present surfactant stepwise elution can be performed by a sequential increase in CTAC concentrations of the running solution in the anodic reservoir from 30 to 50mM for the anions and from 20 to 50mM for the non-ionic compounds. Additionally, to perform expected gradient separations with good reproducibility, each running solution with a different CTAC concentration was treated with tetraethylammmonium chloride as an additive to adjust electric conductivities of each running solution to be equal. Under this condition, CTAC micelles of each zone of different CTAC concentrations would migrate with practically the same velocity. Consequently, by the present stepwise method, both the 10 anionic analytes and the 11 non-ionic analytes were well separated within reasonable periods which corresponded approximately to two-third and less than half of those by the isocratic elutions, respectively.
Study of chemical selectivity of molecular binary mixed micelles of sodium 10-undecenyl sulfate and sodium N-undecenyl leucinate using linear solvation energy relationships model
10 April 2012,
09:29:39
Publication year:
2012
Source:Journal of Chromatography A, Volume 1236
Hamid H. Ahmed, David M. Ahlstrom, Hakan Arslan, Mustafa Guzel, Cevdet Akbay
Poly(sodium 10-undecenyl sulfate) (poly-SUS), poly(sodium N-undecenyl leucinate) (poly-SUL) and their five molecular binary mixed micelles with varied SUS:SUL composition were prepared and used as pseudostationary phases in micellar electrokinetic chromatography (MEKC). Linear solvation energy relationships (LSERs) model and free energy of transfer studies were used to characterize the retention behavior and the selectivity differences among the seven surfactant systems. System constant differences and regression models for varied benzene derivative compounds are used to establish the selectivity differences of the seven pseudostationary phases. The cavity formation and dispersion interaction (the v system constant) and the hydrogen-bonding acidity (the b system constant) of the surfactant systems were found to have the most significant influence on selectivity and MEKC retention. The molecular micelle with sulfate head group, poly-SUS, was found to be more hydrogen-bond acidic than the molecular micelle with leucinate head group, poly-SUL. The other system constants (a, s and e) have modest effect on the retention and selectivity of the benzene derivatives. The model intercept coefficients (c system constants), which are negative for all surfactant systems have unusually large values. The free energy changes of transfer for the functional groups studied have all negative values except phenol and benzyl alcohol. Selectivity differences between pseudostationary phases were also compared by plotting the log k values against each other and were found to agree well with LSER results.
Source:Journal of Chromatography A, Volume 1236
Hamid H. Ahmed, David M. Ahlstrom, Hakan Arslan, Mustafa Guzel, Cevdet Akbay
Poly(sodium 10-undecenyl sulfate) (poly-SUS), poly(sodium N-undecenyl leucinate) (poly-SUL) and their five molecular binary mixed micelles with varied SUS:SUL composition were prepared and used as pseudostationary phases in micellar electrokinetic chromatography (MEKC). Linear solvation energy relationships (LSERs) model and free energy of transfer studies were used to characterize the retention behavior and the selectivity differences among the seven surfactant systems. System constant differences and regression models for varied benzene derivative compounds are used to establish the selectivity differences of the seven pseudostationary phases. The cavity formation and dispersion interaction (the v system constant) and the hydrogen-bonding acidity (the b system constant) of the surfactant systems were found to have the most significant influence on selectivity and MEKC retention. The molecular micelle with sulfate head group, poly-SUS, was found to be more hydrogen-bond acidic than the molecular micelle with leucinate head group, poly-SUL. The other system constants (a, s and e) have modest effect on the retention and selectivity of the benzene derivatives. The model intercept coefficients (c system constants), which are negative for all surfactant systems have unusually large values. The free energy changes of transfer for the functional groups studied have all negative values except phenol and benzyl alcohol. Selectivity differences between pseudostationary phases were also compared by plotting the log k values against each other and were found to agree well with LSER results.
10 April 2012,
09:29:39
Publication year:
2012
Source:Journal of Chromatography A, Volume 1235
Liang Guo, Hian Kee Lee
A fast and efficient method for the determination of trace level of carbamate pesticides using a lower-density-than-water solvent for ultrasound-assisted emulsification microextraction coupled to on-column derivatization and analysis by GC–MS has been developed and studied. In this approach, a soft plastic Pasteur pipette was employed as a convenient extraction device. Fifty microliters of extraction solvent, of lower density than water, was injected into the sample solution held in the pipette. The latter was immediately immersed in an ultrasound water bath to form an emulsion. After 2min extraction, the emulsion was fractionated into two layers by centrifugation. The upper layer (organic extract) could be collected conveniently by squeezing the bulb of the pipette, now held upside down, to move it into the narrow stem of the device, facilitating its retrieval for analysis. The extract was then combined with trimethylphenylammonium hydroxide and directly injected into a gas chromatography–mass spectrometry (GC–MS) system for on-column derivatization and analysis. The on-column derivatization provided an added convenience (since a separate step was not necessary). Parameters affecting the derivatization and extraction were investigated. Under the most favorable conditions, the method demonstrated high extraction efficiency with low limits of detection of between 0.01 and 0.1μg/L, good linearity in the range of 0.05–50μg/L, to 0.5–100μg/L, and good repeatability (RSD below 9.2%, n =5). The proposed method was evaluated by determining carbamate pesticides in river water samples.
Source:Journal of Chromatography A, Volume 1235
Liang Guo, Hian Kee Lee
A fast and efficient method for the determination of trace level of carbamate pesticides using a lower-density-than-water solvent for ultrasound-assisted emulsification microextraction coupled to on-column derivatization and analysis by GC–MS has been developed and studied. In this approach, a soft plastic Pasteur pipette was employed as a convenient extraction device. Fifty microliters of extraction solvent, of lower density than water, was injected into the sample solution held in the pipette. The latter was immediately immersed in an ultrasound water bath to form an emulsion. After 2min extraction, the emulsion was fractionated into two layers by centrifugation. The upper layer (organic extract) could be collected conveniently by squeezing the bulb of the pipette, now held upside down, to move it into the narrow stem of the device, facilitating its retrieval for analysis. The extract was then combined with trimethylphenylammonium hydroxide and directly injected into a gas chromatography–mass spectrometry (GC–MS) system for on-column derivatization and analysis. The on-column derivatization provided an added convenience (since a separate step was not necessary). Parameters affecting the derivatization and extraction were investigated. Under the most favorable conditions, the method demonstrated high extraction efficiency with low limits of detection of between 0.01 and 0.1μg/L, good linearity in the range of 0.05–50μg/L, to 0.5–100μg/L, and good repeatability (RSD below 9.2%, n =5). The proposed method was evaluated by determining carbamate pesticides in river water samples.
Smart polymer mediated purification and recovery of active proteins from inclusion bodies
10 April 2012,
09:29:39
Publication year:
2012
Source:Journal of Chromatography A, Volume 1235
Saurabh Gautam, Priyanka Dubey, Pranveer Singh, S. Kesavardhana, Raghavan Varadarajan, Munishwar N. Gupta
Obtaining correctly folded proteins from inclusion bodies of recombinant proteins expressed in bacterial hosts requires solubilization with denaturants and a refolding step. Aggregation competes with the second step. Refolding of eight different proteins was carried out by precipitation with smart polymers. These proteins have different molecular weights, different number of disulfide bridges and some of these are known to be highly prone to aggregation. A high throughput refolding screen based upon fluorescence emission maximum around 340nm (for correctly folded proteins) was developed to identify the suitable smart polymer. The proteins could be dissociated and recovered after the refolding step. The refolding could be scaled up and high refolding yields in the range of 8mgL−1 (for CD4D12, the first two domains of human CD4) to 58mgL−1 (for malETrx, thioredoxin fused with signal peptide of maltose binding protein) were obtained. Dynamic light scattering (DLS) showed that polymer if chosen correctly acted as a pseudochaperonin and bound to the proteins. It also showed that the time for maximum binding was about 50min which coincided with the time required for incubation (with the polymer) before precipitation for maximum recovery of folded proteins. The refolded proteins were characterized by fluorescence emission spectra, circular dichroism (CD) spectroscopy, melting temperature (T m), and surface hydrophobicity measurement by ANS (8-anilino1-naphthalene sulfonic acid) fluorescence. Biological activity assay for thioredoxin and fluorescence based assay in case of maltose binding protein (MBP) were also carried out to confirm correct refolding.
Source:Journal of Chromatography A, Volume 1235
Saurabh Gautam, Priyanka Dubey, Pranveer Singh, S. Kesavardhana, Raghavan Varadarajan, Munishwar N. Gupta
Obtaining correctly folded proteins from inclusion bodies of recombinant proteins expressed in bacterial hosts requires solubilization with denaturants and a refolding step. Aggregation competes with the second step. Refolding of eight different proteins was carried out by precipitation with smart polymers. These proteins have different molecular weights, different number of disulfide bridges and some of these are known to be highly prone to aggregation. A high throughput refolding screen based upon fluorescence emission maximum around 340nm (for correctly folded proteins) was developed to identify the suitable smart polymer. The proteins could be dissociated and recovered after the refolding step. The refolding could be scaled up and high refolding yields in the range of 8mgL−1 (for CD4D12, the first two domains of human CD4) to 58mgL−1 (for malETrx, thioredoxin fused with signal peptide of maltose binding protein) were obtained. Dynamic light scattering (DLS) showed that polymer if chosen correctly acted as a pseudochaperonin and bound to the proteins. It also showed that the time for maximum binding was about 50min which coincided with the time required for incubation (with the polymer) before precipitation for maximum recovery of folded proteins. The refolded proteins were characterized by fluorescence emission spectra, circular dichroism (CD) spectroscopy, melting temperature (T m), and surface hydrophobicity measurement by ANS (8-anilino1-naphthalene sulfonic acid) fluorescence. Biological activity assay for thioredoxin and fluorescence based assay in case of maltose binding protein (MBP) were also carried out to confirm correct refolding.
One step solvent bar microextraction and derivatization followed by gas chromatography–mass spectrometry for the determination of pharmaceutically active compounds in drain water samples
10 April 2012,
09:29:39
Publication year:
2012
Source:Journal of Chromatography A, Volume 1235
Liang Guo, Hian Kee Lee
For the first time, a simple and novel one-step combined solvent bar microextraction with derivatization with GC–MS analysis, was developed for the determination of pharmaceutically active compounds (PhACs) in water samples. In the procedure, the derivatization reagent was added in the extraction solvent (solvent bar), so that the analytes could be extracted from the aqueous sample and simultaneously derivatized in the solvent bar to enhance their volatility and improve chromatographic performance. After extraction, the derivatized analytes in the extract were directly injected into a GC–MS system for analysis. Six PhACs including naproxen, ibuprofen, ketoprofen, propranolol, diclofenac, and alprenolol were used here to develop and evaluate the method. The parameters affecting the derivatization and extraction efficiency including derivatization time and temperature, the proportion of derivatization reagent, the type of organic solvent, extraction time, extraction temperature, pH of sample solution, effect of ionic strength, and sample agitation speed, were investigated in detail. Under the most favorable conditions, the method provided good limits of detection ranging from 0.006 to 0.022μg/L, linearity (from 0.1–50 to 0.2–50μg/L, depending on analytes) and repeatability of extractions (RSDs below 9.5%, n =5). The proposed method was compared to hollow fiber protected liquid-phase microextraction and solid-phase microextraction, and showed higher extraction efficiency and/or shorter extraction time. The proposed method was applied to the determination of six PhACs in drain water, and was demonstrated to be simple, fast and efficient.
Source:Journal of Chromatography A, Volume 1235
Liang Guo, Hian Kee Lee
For the first time, a simple and novel one-step combined solvent bar microextraction with derivatization with GC–MS analysis, was developed for the determination of pharmaceutically active compounds (PhACs) in water samples. In the procedure, the derivatization reagent was added in the extraction solvent (solvent bar), so that the analytes could be extracted from the aqueous sample and simultaneously derivatized in the solvent bar to enhance their volatility and improve chromatographic performance. After extraction, the derivatized analytes in the extract were directly injected into a GC–MS system for analysis. Six PhACs including naproxen, ibuprofen, ketoprofen, propranolol, diclofenac, and alprenolol were used here to develop and evaluate the method. The parameters affecting the derivatization and extraction efficiency including derivatization time and temperature, the proportion of derivatization reagent, the type of organic solvent, extraction time, extraction temperature, pH of sample solution, effect of ionic strength, and sample agitation speed, were investigated in detail. Under the most favorable conditions, the method provided good limits of detection ranging from 0.006 to 0.022μg/L, linearity (from 0.1–50 to 0.2–50μg/L, depending on analytes) and repeatability of extractions (RSDs below 9.5%, n =5). The proposed method was compared to hollow fiber protected liquid-phase microextraction and solid-phase microextraction, and showed higher extraction efficiency and/or shorter extraction time. The proposed method was applied to the determination of six PhACs in drain water, and was demonstrated to be simple, fast and efficient.
Application of step-wise gradient high-performance counter-current chromatography for rapid preparative separation and purification of diterpene components from Pseudolarix kaempferi Gordon
10 April 2012,
09:29:39
Publication year:
2012
Source:Journal of Chromatography A, Volume 1235
Shichao He, Shucai Li, Jianhong Yang, Haoyu Ye, Shijie Zhong, Hang Song, Yongkui Zhang, Cheng Peng, Aihua Peng, Lijuan Chen
In general, simultaneously separation and purification of components with a broad polarity range from traditional Chinese medicine (TCM) is a challenge by an ordinary high-speed counter-current chromatography (HSCCC) method. In this paper, we describes a rapid and efficient separation method of combining three-step gradient elution and two-step flow-rate gradient elution using high-performance counter-current chromatography (HPCCC) to separate 8 diterpene compounds simultaneously within 80min in a single run from the alcohol extract of Pseudolarix kaempferi Gordon. This separation process produced 166mg pseudolaric acid B O-β-d-glucopyranoside (PABGly), 152mg pseudolaric acid C (PAC), 8mg deacetylpseudolaric acid A (deacetylPAA), 5mg pseudolaric acid A O-β-d-glucopyranoside (PAAGly), 484mg pseudolaric acid B (PAB), 33mg pseudolaric acid B methyl ester (PAB methyl ester), 10mg pseudolaric acid A (PAA) and 18mg pseudolaric acid H (PAH) from 1.0g crude sample with purities of 98.6%, 99.6%, 92.3%, 92.2%, 99.2%, 99.4%, 98.3%, 91.0%, respectively. Our study indicates that the suitable combination of step-wise gradient elution and flow-rate gradient elution using HPCCC is an effective strategy to separate complex components from natural products.
Source:Journal of Chromatography A, Volume 1235
Shichao He, Shucai Li, Jianhong Yang, Haoyu Ye, Shijie Zhong, Hang Song, Yongkui Zhang, Cheng Peng, Aihua Peng, Lijuan Chen
In general, simultaneously separation and purification of components with a broad polarity range from traditional Chinese medicine (TCM) is a challenge by an ordinary high-speed counter-current chromatography (HSCCC) method. In this paper, we describes a rapid and efficient separation method of combining three-step gradient elution and two-step flow-rate gradient elution using high-performance counter-current chromatography (HPCCC) to separate 8 diterpene compounds simultaneously within 80min in a single run from the alcohol extract of Pseudolarix kaempferi Gordon. This separation process produced 166mg pseudolaric acid B O-β-d-glucopyranoside (PABGly), 152mg pseudolaric acid C (PAC), 8mg deacetylpseudolaric acid A (deacetylPAA), 5mg pseudolaric acid A O-β-d-glucopyranoside (PAAGly), 484mg pseudolaric acid B (PAB), 33mg pseudolaric acid B methyl ester (PAB methyl ester), 10mg pseudolaric acid A (PAA) and 18mg pseudolaric acid H (PAH) from 1.0g crude sample with purities of 98.6%, 99.6%, 92.3%, 92.2%, 99.2%, 99.4%, 98.3%, 91.0%, respectively. Our study indicates that the suitable combination of step-wise gradient elution and flow-rate gradient elution using HPCCC is an effective strategy to separate complex components from natural products.
The imidazole ring, shown in Figure 1, is a versatile ionic liquid scaffold. Imidazole-based ionic liquids have been used in a variety of applications including potential water treatment agents because they have the ability to coordinate with metal atoms and are not volatile. Imidazolium ionic liquids
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