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On-line coupling of a clean-up device with supported liquid membrane to capillary electrophoresis for direct injection and analysis of serum and plasma samples
27 March 2012,
09:18:54
Publication year:
2012
Source:Journal of Chromatography A, Volume 1234
Pavel Kubáň, Petr Boček
A simple sample clean-up device with planar supported liquid membrane (SLM) was developed and coupled on-line to capillary electrophoresis (CE) for direct injection of human body fluids. Donor and acceptor compartments of the device were filled with diluted body fluid and deionized water, respectively, and the two solutions were separated by a thin SLM. Analytes of interest were selectively transported from the donor solution through the SLM into the acceptor solution by diffusion whereas interfering matrix components were efficiently retained on the SLM. Equilibrium between the concentrations of analytes at the SLM was obtained typically in 5min. Then a CE separation capillary was inserted into the acceptor compartment to firmly touch the SLM and the pretreated sample was hydrodynamically injected into the capillary. The analytical procedure was demonstrated by rapid pretreatment, on-line injection, and CE determination of selected amino acids in human serum and plasma samples. 1-Ethyl-2-nitrobenezene and bis(2-ethylhexyl) phosphate (15%, v/v) was used as the selective SLM for clean-up of the body fluids and 0.5M acetic acid was used as a background electrolyte solution for CE analysis of the pretreated amino acids. Concentrations of amino acids on acceptor side of the SLM reached 40–58% of their original concentrations in donor solution after 5min equilibration time and then remained constant proving that equilibrium was achieved at the SLM. Injection of the pretreated samples was highly repeatable with RSD values of peak areas 2.4–8.4% and 3.4–10.5% for standard solutions and real samples, respectively. Limits of detection between 0.75 and 2.5μM were achieved, corresponding to 3.75–12.5μM in 1:4 diluted real samples, which ensure sensitive determination of most amino acids in the body fluids. The developed method is fast, simple, efficient, cheap and selective and may be applied to determination of a wide range of analytes in various samples with complex matrices.
Source:Journal of Chromatography A, Volume 1234
Pavel Kubáň, Petr Boček
A simple sample clean-up device with planar supported liquid membrane (SLM) was developed and coupled on-line to capillary electrophoresis (CE) for direct injection of human body fluids. Donor and acceptor compartments of the device were filled with diluted body fluid and deionized water, respectively, and the two solutions were separated by a thin SLM. Analytes of interest were selectively transported from the donor solution through the SLM into the acceptor solution by diffusion whereas interfering matrix components were efficiently retained on the SLM. Equilibrium between the concentrations of analytes at the SLM was obtained typically in 5min. Then a CE separation capillary was inserted into the acceptor compartment to firmly touch the SLM and the pretreated sample was hydrodynamically injected into the capillary. The analytical procedure was demonstrated by rapid pretreatment, on-line injection, and CE determination of selected amino acids in human serum and plasma samples. 1-Ethyl-2-nitrobenezene and bis(2-ethylhexyl) phosphate (15%, v/v) was used as the selective SLM for clean-up of the body fluids and 0.5M acetic acid was used as a background electrolyte solution for CE analysis of the pretreated amino acids. Concentrations of amino acids on acceptor side of the SLM reached 40–58% of their original concentrations in donor solution after 5min equilibration time and then remained constant proving that equilibrium was achieved at the SLM. Injection of the pretreated samples was highly repeatable with RSD values of peak areas 2.4–8.4% and 3.4–10.5% for standard solutions and real samples, respectively. Limits of detection between 0.75 and 2.5μM were achieved, corresponding to 3.75–12.5μM in 1:4 diluted real samples, which ensure sensitive determination of most amino acids in the body fluids. The developed method is fast, simple, efficient, cheap and selective and may be applied to determination of a wide range of analytes in various samples with complex matrices.
Electrowetting-on-dielectric actuation of droplets with capillary electrophoretic zones for off-line mass spectrometric analysis
27 March 2012,
09:18:54
Publication year:
2012
Source:Journal of Chromatography A, Volume 1234
Jelena Gorbatsova, Maria Borissova, Mihkel Kaljurand
Present article describes a novel technique based on digital microfluidics that allows collecting fractions of interest after electrophoretic separation and detection for further ESI-MS investigation. In this technique, a mixture is injected into a capillary electrophoresis (CE) apparatus, and microliter droplets are generated at the CE outlet at a frequency high enough to fraction each compound into several droplets, compartmentalizing the CE zones into a sequence of droplets. The droplets are transported from the CE outlet to a storage tube inlet using electrowetting-on-dielectric (EWOD) for droplet actuation. By applying a vacuum at the other end of the storage tube, the droplets form a sequence of plugs separated by air gaps. The plugs stored in the tubing are later analyzed using a standalone spectrometric device. Off-line electrospray ionization mass spectrometry (ESI-MS) was used to characterize the corresponding vitamin and was performed by pumping the segmented plugs directly into a spray emitter tip. The technique could be of interest to laboratories without access to well-equipped facilities (e.g. clean-rooms or lab robots).
Source:Journal of Chromatography A, Volume 1234
Jelena Gorbatsova, Maria Borissova, Mihkel Kaljurand
Present article describes a novel technique based on digital microfluidics that allows collecting fractions of interest after electrophoretic separation and detection for further ESI-MS investigation. In this technique, a mixture is injected into a capillary electrophoresis (CE) apparatus, and microliter droplets are generated at the CE outlet at a frequency high enough to fraction each compound into several droplets, compartmentalizing the CE zones into a sequence of droplets. The droplets are transported from the CE outlet to a storage tube inlet using electrowetting-on-dielectric (EWOD) for droplet actuation. By applying a vacuum at the other end of the storage tube, the droplets form a sequence of plugs separated by air gaps. The plugs stored in the tubing are later analyzed using a standalone spectrometric device. Off-line electrospray ionization mass spectrometry (ESI-MS) was used to characterize the corresponding vitamin and was performed by pumping the segmented plugs directly into a spray emitter tip. The technique could be of interest to laboratories without access to well-equipped facilities (e.g. clean-rooms or lab robots).
Characterization of carboxylate-terminated carbosilane dendrimers and their evaluation as nanoadditives in capillary electrophoresis for vegetable protein profiling
27 March 2012,
09:18:54
Publication year:
2012
Source:Journal of Chromatography A, Volume 1234
C. Montealegre, B. Rasines, R. Gómez, F.J. de la Mata, C. García-Ruiz, M.L. Marina
Protein profiles are becoming an important tool to differentiate and classify varieties of several cultivars and to obtain a specific fingerprint for them. The use of protein profiles for these purposes needs to achieve high separation efficiencies to obtain a high number of well resolved peaks. In this work, carbosilane dendrimers with interior carbon–silicon bonds and negatively charged in the dendrimer surface with carboxylic acid as functional groups were employed as nanoadditives to separate soybean and olive seeds proteins. First, these dendrimers were characterized using CE to evaluate their possible impurities. A potentiometric titration was later carried out to determine their pK a values. Afterwards, the characterized dendrimers were used to improve the protein profiles obtained by EKC for vegetable proteins. Different dendrimer generations (G1, G2, and G3) and concentrations (0.01–1% m/v) were tested. The highest dendrimer generation G3 at 0.1% (m/v) allowed observing the best protein profiles for soybean and olive seeds. These results demonstrate that carboxylate-terminated carbosilane dendrimers are attractive nanoadditives in EKC for the effective separation of vegetable proteins.
Source:Journal of Chromatography A, Volume 1234
C. Montealegre, B. Rasines, R. Gómez, F.J. de la Mata, C. García-Ruiz, M.L. Marina
Protein profiles are becoming an important tool to differentiate and classify varieties of several cultivars and to obtain a specific fingerprint for them. The use of protein profiles for these purposes needs to achieve high separation efficiencies to obtain a high number of well resolved peaks. In this work, carbosilane dendrimers with interior carbon–silicon bonds and negatively charged in the dendrimer surface with carboxylic acid as functional groups were employed as nanoadditives to separate soybean and olive seeds proteins. First, these dendrimers were characterized using CE to evaluate their possible impurities. A potentiometric titration was later carried out to determine their pK a values. Afterwards, the characterized dendrimers were used to improve the protein profiles obtained by EKC for vegetable proteins. Different dendrimer generations (G1, G2, and G3) and concentrations (0.01–1% m/v) were tested. The highest dendrimer generation G3 at 0.1% (m/v) allowed observing the best protein profiles for soybean and olive seeds. These results demonstrate that carboxylate-terminated carbosilane dendrimers are attractive nanoadditives in EKC for the effective separation of vegetable proteins.
Evaluation of new cellulose-based chiral stationary phases Sepapak-2 and Sepapak-4 for the enantiomeric separation of pesticides by nano liquid chromatography and capillary electrochromatography
27 March 2012,
09:18:54
Publication year:
2012
Source:Journal of Chromatography A, Volume 1234
Virginia Pérez-Fernández, Elena Dominguez-Vega, Bezhan Chankvetadze, Antonio L. Crego, Maria Ángeles García, Maria Luisa Marina
Two novel polysaccharide-based chiral stationary phases (CSPs), known as Sepapak-2 (cellulose tris(3-chloro-4-methylphenylcarbamate)) and Sepapak-4 (cellulose tris(4-chloro-3-methylphenylcarbamate)), have been evaluated in this work for the chiral separation of a group of 16 pesticides including herbicides, insecticides and fungicides. The optimization of the mobile phase employed in nano-liquid chromatography (nano-LC) enabled the chiral separation of seven pesticides on Sepapak-2 and of nine pesticides on Sepapak-4. Due to the fact that Sepapak-4 gave better results, this column was selected to compare nano-LC and capillary electrochromatography (CEC) under the same conditions that consisted in the use of a 90/9/1 (v/v/v) ACN/H2O/ammonium formate (pH 2.5) background electrolyte (BGE). As expected, both the efficiency and the chiral resolution obtained in CEC experiments were higher than in nano-LC for all the analyzed compounds. The analytical characteristics of the CEC developed methodology were evaluated in terms of linearity, LODs, LOQs, precision, selectivity, and accuracy allowing its application to the quantitation of metalaxyl and its enantiomeric impurity in a commercial fungicide product marketed as enantiomerically pure (metalaxyl-M) and in soil and tap water samples after solid phase extraction (SPE). The determined amount of metalaxyl-M was found to be a 26% above the labeled content and it contained an enantiomeric impurity of a 3.7% of S-metalaxyl was determined.
Source:Journal of Chromatography A, Volume 1234
Virginia Pérez-Fernández, Elena Dominguez-Vega, Bezhan Chankvetadze, Antonio L. Crego, Maria Ángeles García, Maria Luisa Marina
Two novel polysaccharide-based chiral stationary phases (CSPs), known as Sepapak-2 (cellulose tris(3-chloro-4-methylphenylcarbamate)) and Sepapak-4 (cellulose tris(4-chloro-3-methylphenylcarbamate)), have been evaluated in this work for the chiral separation of a group of 16 pesticides including herbicides, insecticides and fungicides. The optimization of the mobile phase employed in nano-liquid chromatography (nano-LC) enabled the chiral separation of seven pesticides on Sepapak-2 and of nine pesticides on Sepapak-4. Due to the fact that Sepapak-4 gave better results, this column was selected to compare nano-LC and capillary electrochromatography (CEC) under the same conditions that consisted in the use of a 90/9/1 (v/v/v) ACN/H2O/ammonium formate (pH 2.5) background electrolyte (BGE). As expected, both the efficiency and the chiral resolution obtained in CEC experiments were higher than in nano-LC for all the analyzed compounds. The analytical characteristics of the CEC developed methodology were evaluated in terms of linearity, LODs, LOQs, precision, selectivity, and accuracy allowing its application to the quantitation of metalaxyl and its enantiomeric impurity in a commercial fungicide product marketed as enantiomerically pure (metalaxyl-M) and in soil and tap water samples after solid phase extraction (SPE). The determined amount of metalaxyl-M was found to be a 26% above the labeled content and it contained an enantiomeric impurity of a 3.7% of S-metalaxyl was determined.
Electromembrane extraction using stabilized constant d.c. electric current—A simple tool for improvement of extraction performance
27 March 2012,
09:18:54
Publication year:
2012
Source:Journal of Chromatography A, Volume 1234
Andrea Šlampová, Pavel Kubáň, Petr Boček
This contribution presents an experimental approach for improvement of analytical performance of electromembrane extraction (EME), which is based on the use of stabilized constant d.c. electric current. Extractions were performed using a high voltage power supply, which provided stabilized constant d.c. current down to 1μA and facilitated current-controlled transfer of ions of interest from a donor solution through a supported liquid membrane (SLM) into an acceptor solution. Repeatability of the extraction process has significantly improved for EME at constant electric current compared to EME at constant voltage. The improved repeatability of the extraction process was demonstrated on EME-capillary electrophoresis (EME-CE) analyses of selected basic drugs and amino acids in standard solutions and in human urine and serum samples. RSD values of peak areas of the analytes for EME-CE analyses were about two-fold better for EME at constant electric current (2.8–8.9%) compared to EME at constant voltage (3.6–17.8%). Other analytical parameters of the EME-CE methods, such as limits of detection, linear ranges and correlation coefficients were not statistically different for the two EME modes. Moreover, EME at constant electric current did not suffer from SLM instabilities frequently observed for EME at constant voltage.
Source:Journal of Chromatography A, Volume 1234
Andrea Šlampová, Pavel Kubáň, Petr Boček
This contribution presents an experimental approach for improvement of analytical performance of electromembrane extraction (EME), which is based on the use of stabilized constant d.c. electric current. Extractions were performed using a high voltage power supply, which provided stabilized constant d.c. current down to 1μA and facilitated current-controlled transfer of ions of interest from a donor solution through a supported liquid membrane (SLM) into an acceptor solution. Repeatability of the extraction process has significantly improved for EME at constant electric current compared to EME at constant voltage. The improved repeatability of the extraction process was demonstrated on EME-capillary electrophoresis (EME-CE) analyses of selected basic drugs and amino acids in standard solutions and in human urine and serum samples. RSD values of peak areas of the analytes for EME-CE analyses were about two-fold better for EME at constant electric current (2.8–8.9%) compared to EME at constant voltage (3.6–17.8%). Other analytical parameters of the EME-CE methods, such as limits of detection, linear ranges and correlation coefficients were not statistically different for the two EME modes. Moreover, EME at constant electric current did not suffer from SLM instabilities frequently observed for EME at constant voltage.
Analysis of polyphenols and methylxantines in tea samples by means of nano-liquid chromatography utilizing capillary columns packed with core–shell particles
27 March 2012,
09:18:54
Publication year:
2012
Source:Journal of Chromatography A, Volume 1234
Chiara Fanali, Anna Rocco, Zeineb Aturki, Luigi Mondello, Salvatore Fanali
In this study, a rapid separation of eleven polyphenols and three methylxanthines was obtained by means of nano-liquid chromatography (nano-LC), employing a 100μm I.D. capillary column packed with C18 core–shell particles (2.7μm) for 10cm. All compounds were baseline resolved with a step gradient elution in less than 15min. The developed analytical method was validated and the resulting RSD% for intra-day and inter-day repeatability, related to retention time, retention factor and peak area, were below 5.1 and 5.7%. LOD and LOQ values corresponded to 0.300 and 0.625μg/mL, while linearity range assessed gave R 2 no lower than 0.990. Then, the method was used to determine studied compounds in tea extracts. Further, the nano-LC system was coupled with a mass spectrometer to confirm the components present in real samples. Finally the results were compared with those obtained using a capillary column (100μm I.D.×10cm) packed with C18 sub-2μm particles, applying the same nano-LC experimental conditions.
Source:Journal of Chromatography A, Volume 1234
Chiara Fanali, Anna Rocco, Zeineb Aturki, Luigi Mondello, Salvatore Fanali
In this study, a rapid separation of eleven polyphenols and three methylxanthines was obtained by means of nano-liquid chromatography (nano-LC), employing a 100μm I.D. capillary column packed with C18 core–shell particles (2.7μm) for 10cm. All compounds were baseline resolved with a step gradient elution in less than 15min. The developed analytical method was validated and the resulting RSD% for intra-day and inter-day repeatability, related to retention time, retention factor and peak area, were below 5.1 and 5.7%. LOD and LOQ values corresponded to 0.300 and 0.625μg/mL, while linearity range assessed gave R 2 no lower than 0.990. Then, the method was used to determine studied compounds in tea extracts. Further, the nano-LC system was coupled with a mass spectrometer to confirm the components present in real samples. Finally the results were compared with those obtained using a capillary column (100μm I.D.×10cm) packed with C18 sub-2μm particles, applying the same nano-LC experimental conditions.
Chiral capillary liquid chromatography based on penicillin G acylase immobilized on monolithic epoxy silica column
27 March 2012,
09:18:54
Publication year:
2012
Source:Journal of Chromatography A, Volume 1234
Roberto Gotti, Jessica Fiori, Enrica Calleri, Caterina Temporini, Dieter Lubda, Gabriella Massolini
An epoxy derivatized monolithic silica capillary column (100μm i.d.) was used as a support for immobilization of penicillin G acylase (PGA), an enzyme used in the production of semisynthetic antibiotics. In order to allow for sensitive UV detection, the PGA-based monolithic capillary column was coupled to an open fused-silica capillary via a TFE (Teflon®) shrink tube sleeve (1cm long, 300μm i.d.), which proved to be a robust, dead-volume free and easily replaceable connector. This configuration resulted in a duplex fritless column for capillary liquid chromatography (CLC) and electrically assisted CLC (eCLC). In particular, using the driving pressure (2–12bar) supplied by the commercial CE instruments, CLC separations could be obtained in short time due to the low column backpressure of the monolith. In particular, the developed stationary phase characterized by the chiral recognition ability of PGA, was successfully applied in enantioseparation of arylpropionic acids of pharmaceutical interest (i.e., profens). As an example, by using a 7cm long monolith capillary column, the enantioresolution (Rs>3.0) of rac-ketoprofen was achieved in less than 2min (pressure 12bar) with a minimum plate height in the order of 20μm and using as a mobile phase a 50mM phosphate buffer pH 7.0. Validation data such as repeatability of retention time (intraday<0.62, n =6; interday<1.62, n =9; and column-to-column<10.5, n =2), linearity (r 2 =0.999), and sensitivity (LOQ 0.25% (w/w) of (R)-ketoprofen with respect to (S)-ketoprofen) showed good method performance. The method was successfully applied to the determination of (S)-ketoprofen in pharmaceutical samples (tablets).
Source:Journal of Chromatography A, Volume 1234
Roberto Gotti, Jessica Fiori, Enrica Calleri, Caterina Temporini, Dieter Lubda, Gabriella Massolini
An epoxy derivatized monolithic silica capillary column (100μm i.d.) was used as a support for immobilization of penicillin G acylase (PGA), an enzyme used in the production of semisynthetic antibiotics. In order to allow for sensitive UV detection, the PGA-based monolithic capillary column was coupled to an open fused-silica capillary via a TFE (Teflon®) shrink tube sleeve (1cm long, 300μm i.d.), which proved to be a robust, dead-volume free and easily replaceable connector. This configuration resulted in a duplex fritless column for capillary liquid chromatography (CLC) and electrically assisted CLC (eCLC). In particular, using the driving pressure (2–12bar) supplied by the commercial CE instruments, CLC separations could be obtained in short time due to the low column backpressure of the monolith. In particular, the developed stationary phase characterized by the chiral recognition ability of PGA, was successfully applied in enantioseparation of arylpropionic acids of pharmaceutical interest (i.e., profens). As an example, by using a 7cm long monolith capillary column, the enantioresolution (Rs>3.0) of rac-ketoprofen was achieved in less than 2min (pressure 12bar) with a minimum plate height in the order of 20μm and using as a mobile phase a 50mM phosphate buffer pH 7.0. Validation data such as repeatability of retention time (intraday<0.62, n =6; interday<1.62, n =9; and column-to-column<10.5, n =2), linearity (r 2 =0.999), and sensitivity (LOQ 0.25% (w/w) of (R)-ketoprofen with respect to (S)-ketoprofen) showed good method performance. The method was successfully applied to the determination of (S)-ketoprofen in pharmaceutical samples (tablets).
Comparative high-performance liquid chromatography enantioseparations on polysaccharide based chiral stationary phases prepared by coating totally porous and core–shell silica particles
27 March 2012,
09:18:54
Publication year:
2012
Source:Journal of Chromatography A, Volume 1234
Ketevan Lomsadze, George Jibuti, Tivadar Farkas, Bezhan Chankvetadze
This article reports comparative high-performance liquid chromatographic separations of enantiomers with chiral stationary phases (CSPs) prepared by coating cellulose tris(4-chloro-3-methylphenylcarbamate) on totally porous and on core–shell type silica of comparable particle diameter. Several interesting observations were made: (1) the selectivity of separation was higher on core–shell type CSP compared to totally porous CSP at comparable content of chiral selector (polysaccharide derivative); (2) much flatter dependence of plate height on the mobile phase flow rate was observed for columns packet with CSP prepared with core–shell silica compared to the ones packed with CSPs prepared with totally porous particles; (3) at low mobile phase flow rates core–shell CSP provided lower resolving ability compared to a commercially available CSP having four times higher content of chiral selector along with higher retention of chiral analytes. However, at high flow rates core–shell type CSP performed similarly or better than the commercial column in regards of plate count (N) and peak resolution (R s) per column length and within a given total analysis time. The advantage of CSP prepared with core–shell silica is obvious from the viewpoint of plate numbers and resolution calculated per unit time (i.e. speed of analysis).
Source:Journal of Chromatography A, Volume 1234
Ketevan Lomsadze, George Jibuti, Tivadar Farkas, Bezhan Chankvetadze
This article reports comparative high-performance liquid chromatographic separations of enantiomers with chiral stationary phases (CSPs) prepared by coating cellulose tris(4-chloro-3-methylphenylcarbamate) on totally porous and on core–shell type silica of comparable particle diameter. Several interesting observations were made: (1) the selectivity of separation was higher on core–shell type CSP compared to totally porous CSP at comparable content of chiral selector (polysaccharide derivative); (2) much flatter dependence of plate height on the mobile phase flow rate was observed for columns packet with CSP prepared with core–shell silica compared to the ones packed with CSPs prepared with totally porous particles; (3) at low mobile phase flow rates core–shell CSP provided lower resolving ability compared to a commercially available CSP having four times higher content of chiral selector along with higher retention of chiral analytes. However, at high flow rates core–shell type CSP performed similarly or better than the commercial column in regards of plate count (N) and peak resolution (R s) per column length and within a given total analysis time. The advantage of CSP prepared with core–shell silica is obvious from the viewpoint of plate numbers and resolution calculated per unit time (i.e. speed of analysis).
Optimization of the liquid chromatography enantioseparation of chiral acidic compounds using cellulose tris(3-chloro-4-methylphenylcarbamate) as chiral selector and polar organic mobile phases
27 March 2012,
09:18:54
Publication year:
2012
Source:Journal of Chromatography A, Volume 1234
K.S.S. Dossou, E. Farcas, A.-C. Servais, P. Chiap, B. Chankvetadze, J. Crommen, M. Fillet
The LC enantioseparation of chiral acidic and zwitterionic drugs selected as model compounds was optimized using chlorine containing cellulose based chiral stationary phases and polar organic mobile phases. The main solvent of the mobile phase was acetonitrile, the temperature was settled at 25°C and a stationary phase with cellulose tris(3-chloro-4-methylphenylcarbamate) as chiral selector (3-Cl-4-Me-PC) was selected. In the screening step, the nature and concentration of both acidic and basic additives were found to have a significant effect on retention, selectivity and resolution. Acetic acid (AcA) was selected as acidic additive for the optimization step since it could lead to the enantioseparation of more acidic compounds than trifluoroacetic acid (TFA) and formic acid (FA), while among the three basic additives tested, diethylamine (DEA) most often gave better results with respect to enantioresolution and selectivity than butylamine (BuA) and triethylamine (TEA). The optimization was performed using a central composite face-centered design with two factors, namely the concentration of acetic acid (0.1–0.3%) and the concentration of DEA (0.01–0.1%) in the mobile phase. On the basis of the results obtained in the screening and optimization steps, a strategy for the rapid development of methods for the enantioseparation of acidic or neutral compounds was proposed.
Source:Journal of Chromatography A, Volume 1234
K.S.S. Dossou, E. Farcas, A.-C. Servais, P. Chiap, B. Chankvetadze, J. Crommen, M. Fillet
The LC enantioseparation of chiral acidic and zwitterionic drugs selected as model compounds was optimized using chlorine containing cellulose based chiral stationary phases and polar organic mobile phases. The main solvent of the mobile phase was acetonitrile, the temperature was settled at 25°C and a stationary phase with cellulose tris(3-chloro-4-methylphenylcarbamate) as chiral selector (3-Cl-4-Me-PC) was selected. In the screening step, the nature and concentration of both acidic and basic additives were found to have a significant effect on retention, selectivity and resolution. Acetic acid (AcA) was selected as acidic additive for the optimization step since it could lead to the enantioseparation of more acidic compounds than trifluoroacetic acid (TFA) and formic acid (FA), while among the three basic additives tested, diethylamine (DEA) most often gave better results with respect to enantioresolution and selectivity than butylamine (BuA) and triethylamine (TEA). The optimization was performed using a central composite face-centered design with two factors, namely the concentration of acetic acid (0.1–0.3%) and the concentration of DEA (0.01–0.1%) in the mobile phase. On the basis of the results obtained in the screening and optimization steps, a strategy for the rapid development of methods for the enantioseparation of acidic or neutral compounds was proposed.
Development of a reversed-phase high-performance liquid chromatography analytical methodology for the determination of antihypertensive peptides in maize crops
27 March 2012,
09:18:54
Publication year:
2012
Source:Journal of Chromatography A, Volume 1234
Patrycja Puchalska, M. Luisa Marina, M. Concepción García
The aim of this work was to estimate the content of three highly antihypertensive peptides (LQP, LSP, and LRP) in different maize crops. For that purpose, a method consisting of the extraction of the protein containing these peptides (α-zeins), releasing of peptides by thermolysin digestion, and separation and detection of peptides was designed. The rapid and efficient ultrasound assisted extraction of α-zeins proteins from whole maize kernels was achieved using 70% of ethanol followed by precipitation with acetone. A 10mM Tris–HCl (pH 8.0) buffer containing 8M urea enabled to dissolve the precipitated α-zeins. This buffer was diluted to reach a 6M urea concentration before digestion to keep active the enzyme. Other digestion parameters that were optimized were: enzyme to substrate ratio (5:100 was selected), digestion temperature (50°C) and digestion time (6h). The RP-HPLC separation in a fused-core column was also optimized allowing the separation of the three peptides extracted from maize kernels in 6min. The presence of the three antihypertensive peptides in the digested extract was confirmed using HPLC–Q-TOF-MS analysis and by comparison with peptide standards. Clear differences were observed in the content of the three antihypertensive peptides and, thus, in the antihypertensive activity of the analyzed crops. The content of LRP peptide was very low regardless of the maize variety while the content of LQP and LSP significantly varied among studied maize lines.
Source:Journal of Chromatography A, Volume 1234
Patrycja Puchalska, M. Luisa Marina, M. Concepción García
The aim of this work was to estimate the content of three highly antihypertensive peptides (LQP, LSP, and LRP) in different maize crops. For that purpose, a method consisting of the extraction of the protein containing these peptides (α-zeins), releasing of peptides by thermolysin digestion, and separation and detection of peptides was designed. The rapid and efficient ultrasound assisted extraction of α-zeins proteins from whole maize kernels was achieved using 70% of ethanol followed by precipitation with acetone. A 10mM Tris–HCl (pH 8.0) buffer containing 8M urea enabled to dissolve the precipitated α-zeins. This buffer was diluted to reach a 6M urea concentration before digestion to keep active the enzyme. Other digestion parameters that were optimized were: enzyme to substrate ratio (5:100 was selected), digestion temperature (50°C) and digestion time (6h). The RP-HPLC separation in a fused-core column was also optimized allowing the separation of the three peptides extracted from maize kernels in 6min. The presence of the three antihypertensive peptides in the digested extract was confirmed using HPLC–Q-TOF-MS analysis and by comparison with peptide standards. Clear differences were observed in the content of the three antihypertensive peptides and, thus, in the antihypertensive activity of the analyzed crops. The content of LRP peptide was very low regardless of the maize variety while the content of LQP and LSP significantly varied among studied maize lines.
Combined use of isopropylamine and trifluoroacetic acid in methanol-containing mobile phases for chiral supercritical fluid chromatography
27 March 2012,
09:18:54
Publication year:
2012
Source:Journal of Chromatography A, Volume 1234
Katrijn De Klerck, Debby Mangelings, David Clicq, Filip De Boever, Yvan Vander Heyden
In chiral supercritical fluid chromatography (SFC), mobile-phase additives are often used to improve enantioseparations and peak shapes. An acidic or basic additive is chosen, depending on the nature of the compound. This work highlights the simultaneous use of the acidic additive trifluoroacetic acid (TFA) and the basic additive isopropylamine (IPA) in supercritical fluid chromatography for enantioseparations. To evaluate the combination of TFA and IPA, 59 chiral pharmaceutical compounds were analyzed on four polysaccharide-based chiral stationary phases (CSPs): Lux® Cellulose-1, Lux® Cellulose-2, Lux® Cellulose-4 and Lux® Amylose-2. The results show that an important increase in enantioselectivity of the chromatographic system can occur when combining trifluoroacetic acid and isopropylamine in the mobile phase (MP), compared to the individual use of these additives. However, the combination of isopropylamine and trifluoroacetic acid in a supercritical methanol-containing mobile phase can also lead to problems as a result of the formation of salt complexes between the two additives. Combining the additives trifluoroacetic acid and isopropylamine and taking the appropriate measures to avoid salt formation, i.e. reducing the additives’ concentrations, can lead to simpler chiral SFC screening conditions that display even broader enantioselectivity.
Source:Journal of Chromatography A, Volume 1234
Katrijn De Klerck, Debby Mangelings, David Clicq, Filip De Boever, Yvan Vander Heyden
In chiral supercritical fluid chromatography (SFC), mobile-phase additives are often used to improve enantioseparations and peak shapes. An acidic or basic additive is chosen, depending on the nature of the compound. This work highlights the simultaneous use of the acidic additive trifluoroacetic acid (TFA) and the basic additive isopropylamine (IPA) in supercritical fluid chromatography for enantioseparations. To evaluate the combination of TFA and IPA, 59 chiral pharmaceutical compounds were analyzed on four polysaccharide-based chiral stationary phases (CSPs): Lux® Cellulose-1, Lux® Cellulose-2, Lux® Cellulose-4 and Lux® Amylose-2. The results show that an important increase in enantioselectivity of the chromatographic system can occur when combining trifluoroacetic acid and isopropylamine in the mobile phase (MP), compared to the individual use of these additives. However, the combination of isopropylamine and trifluoroacetic acid in a supercritical methanol-containing mobile phase can also lead to problems as a result of the formation of salt complexes between the two additives. Combining the additives trifluoroacetic acid and isopropylamine and taking the appropriate measures to avoid salt formation, i.e. reducing the additives’ concentrations, can lead to simpler chiral SFC screening conditions that display even broader enantioselectivity.
27 March 2012,
09:18:54
Publication year:
2012
Source:Journal of Chromatography A, Volume 1233
Wei Liu, Lan Zhang, Liangbiao Fan, Zian Lin, Yimin Cai, Zhenyi Wei, Guonan Chen
In this paper, a convenient and self-assembled hollow fiber solvent-stir bar microextraction (HF-SSBME) device was developed, which could stir by itself. In the extraction process, the proposed device made the solvent “bar” not floating at the sample solution and exposing to air while organic solvents outside hollow fiber always wrapped with donor phase solvent, which reduced the vaporization of organic solvents. This design could improve the precisions and recoveries of experiments. For evaluating the device, seven anabolic steroids (prasterone, 5α-androstane-3α, 17β-diol, methandriol, 19-norandrostenediol, androstenediol, methyltestosterone and methandienone) were used as model analytes and extraction conditions such as type and volume of organic solvents, agitation speed, extraction time, extraction temperature and salt addition were studied in detail. Under the optimum conditions (15μL toluene, 40°C, stirring at 750rpm for 30min with 1.5g sodium chloride addition in 20.0mL donor phase), the linear ranges of anabolic steroids were 0.25–200ngmL−1 with gas chromatography–mass spectrometry. The limits of detection were lower than 0.10ngmL−1. The recoveries and precisions in spiked urine and hair samples were between 73.97–93.56% and 2.18–4.47% (n =5). HF-SSBME method combined the intrinsical merits of hollow fiber with the superiority of the proposed self-stirring device which can be developed to two-phase, three-phase and in situ derivatization modes with wide prospect of application. Besides, the pedestal of this proposed device can be converted to fix stir bar in stir bar sorptive extraction (SBSE) method.
Source:Journal of Chromatography A, Volume 1233
Wei Liu, Lan Zhang, Liangbiao Fan, Zian Lin, Yimin Cai, Zhenyi Wei, Guonan Chen
In this paper, a convenient and self-assembled hollow fiber solvent-stir bar microextraction (HF-SSBME) device was developed, which could stir by itself. In the extraction process, the proposed device made the solvent “bar” not floating at the sample solution and exposing to air while organic solvents outside hollow fiber always wrapped with donor phase solvent, which reduced the vaporization of organic solvents. This design could improve the precisions and recoveries of experiments. For evaluating the device, seven anabolic steroids (prasterone, 5α-androstane-3α, 17β-diol, methandriol, 19-norandrostenediol, androstenediol, methyltestosterone and methandienone) were used as model analytes and extraction conditions such as type and volume of organic solvents, agitation speed, extraction time, extraction temperature and salt addition were studied in detail. Under the optimum conditions (15μL toluene, 40°C, stirring at 750rpm for 30min with 1.5g sodium chloride addition in 20.0mL donor phase), the linear ranges of anabolic steroids were 0.25–200ngmL−1 with gas chromatography–mass spectrometry. The limits of detection were lower than 0.10ngmL−1. The recoveries and precisions in spiked urine and hair samples were between 73.97–93.56% and 2.18–4.47% (n =5). HF-SSBME method combined the intrinsical merits of hollow fiber with the superiority of the proposed self-stirring device which can be developed to two-phase, three-phase and in situ derivatization modes with wide prospect of application. Besides, the pedestal of this proposed device can be converted to fix stir bar in stir bar sorptive extraction (SBSE) method.
Comparative evaluation of post-column free radical scavenging and ferric reducing antioxidant power assays for screening of antioxidants in strawberries
27 March 2012,
09:18:54
Publication year:
2012
Source:Journal of Chromatography A, Volume 1233
Raimondas Raudonis, Lina Raudone, Valdas Jakstas, Valdimaras Janulis
ABTS and FRAP post-column techniques evaluate the antioxidant characteristics of HPLC separated compounds with specific reagents. ABTS characterize their ability to scavenge free radicals by electron-donating antioxidants, resulting in the absorbance decrease of the chromophoric radical. FRAP – is based on the reduction of Fe(III)–tripyridyltriazine complex to Fe(II)–tripyridyltriazine at low pH by electron-donating antioxidants, resulting in an absorbance increase. Both post-column assays were evaluated and compared according to the following validation parameters: specificity, precision, limit of detection (LoD), limit of quantitation (LoQ) and linearity. ABTS and FRAP post-column assays were specific, repeatable and sensitive and thus can be used for the evaluation of antioxidant active compounds. Antioxidant active compounds were quantified according to TEAC for each assay and ABTS/FRAP ratio was derived. No previous records of antioxidative activity of leaves and fruits of strawberries (Fragaria viridis, Fragaria moschata) research have been found. The research results confirm the reliability of ABTS and FRAP post-column assays for screening of antioxidants in complex mixtures and the determination of radical scavenging and ferric reducing ability by their TEAC values.
Source:Journal of Chromatography A, Volume 1233
Raimondas Raudonis, Lina Raudone, Valdas Jakstas, Valdimaras Janulis
ABTS and FRAP post-column techniques evaluate the antioxidant characteristics of HPLC separated compounds with specific reagents. ABTS characterize their ability to scavenge free radicals by electron-donating antioxidants, resulting in the absorbance decrease of the chromophoric radical. FRAP – is based on the reduction of Fe(III)–tripyridyltriazine complex to Fe(II)–tripyridyltriazine at low pH by electron-donating antioxidants, resulting in an absorbance increase. Both post-column assays were evaluated and compared according to the following validation parameters: specificity, precision, limit of detection (LoD), limit of quantitation (LoQ) and linearity. ABTS and FRAP post-column assays were specific, repeatable and sensitive and thus can be used for the evaluation of antioxidant active compounds. Antioxidant active compounds were quantified according to TEAC for each assay and ABTS/FRAP ratio was derived. No previous records of antioxidative activity of leaves and fruits of strawberries (Fragaria viridis, Fragaria moschata) research have been found. The research results confirm the reliability of ABTS and FRAP post-column assays for screening of antioxidants in complex mixtures and the determination of radical scavenging and ferric reducing ability by their TEAC values.
Evaluation of sulfonated graphene sheets as sorbent for micro-solid-phase extraction combined with gas chromatography–mass spectrometry
27 March 2012,
09:18:54
Publication year:
2012
Source:Journal of Chromatography A, Volume 1233
Hong Zhang, Wei Ping Low, Hian Kee Lee
This report describes the use of sulfonated graphene sheets as sorbent in micro-solid-phase extraction (μ-SPE), together with gas chromatography–mass spectrometry, for the determination of polycyclic aromatic hydrocarbons (PAHs) in water. In this study, for the first time, graphene sheets were used as a sorbent material for this mode of microextraction. The modified graphene sheets were characterized by transmission electron microscopy, Fourier transform infrared spectroscopy, and elemental analysis. μ-SPE parameters such as extraction time, desorption time and desorption solvent were optimized. The method showed good precision, reproducibility and linear response for PAH analysis over a concentration range of 0.05–100μg/L for naphthalene and 0.01–100μg/L for the remaining PAHs (acenaphthene, fluorene, phenanthrene, anthracene, fluoranthene and pyrene) with coefficient of determination (r 2) of higher than 0.992. Limits of detection of from 0.8 to 3.9ng/L for 7 PAHs were achieved. The developed method was successfully applied to determine PAHs in river water samples.
Source:Journal of Chromatography A, Volume 1233
Hong Zhang, Wei Ping Low, Hian Kee Lee
This report describes the use of sulfonated graphene sheets as sorbent in micro-solid-phase extraction (μ-SPE), together with gas chromatography–mass spectrometry, for the determination of polycyclic aromatic hydrocarbons (PAHs) in water. In this study, for the first time, graphene sheets were used as a sorbent material for this mode of microextraction. The modified graphene sheets were characterized by transmission electron microscopy, Fourier transform infrared spectroscopy, and elemental analysis. μ-SPE parameters such as extraction time, desorption time and desorption solvent were optimized. The method showed good precision, reproducibility and linear response for PAH analysis over a concentration range of 0.05–100μg/L for naphthalene and 0.01–100μg/L for the remaining PAHs (acenaphthene, fluorene, phenanthrene, anthracene, fluoranthene and pyrene) with coefficient of determination (r 2) of higher than 0.992. Limits of detection of from 0.8 to 3.9ng/L for 7 PAHs were achieved. The developed method was successfully applied to determine PAHs in river water samples.
Simultaneous determination of polycyclic aromatic hydrocarbons and benzene, toluene, ethylbenzene and xylene in water samples using a new sampling strategy combining different extraction modes and temperatures in a single extraction solid-phase microextraction-gas chromatography–mass spectrometry procedure
27 March 2012,
09:18:54
Publication year:
2012
Source:Journal of Chromatography A, Volume 1233
Joyce Nunes Bianchin, Giuliana Nardini, Josias Merib, Adriana Neves Dias, Edmar Martendal, Eduardo Carasek
This study proposes a new optimization approach for the simultaneous determination of polycyclic aromatic hydrocarbons (PAHs) and benzene, toluene, ethylbenzene and xylene isomers (BTEX) from water samples using the solid-phase microextraction technique followed by gas chromatography–mass spectrometry (GC–MS) separation and detection. The objective of the study was to achieve compromise extraction conditions, suitable for all semi-volatile and volatile compounds, under which the amount extracted is maximized for all analytes. This was achieved by careful optimization of the fiber coating, salting-out effect, extraction time and temperature and extraction mode (headspace or direct immersion). With the optimized fiber coating – PDMS/DVB 65μm – the other selected factors were optimized using a response surface methodology through central composite designs. As expected, the optimized results for each class of analytes varied significantly, probably due to the differences in their volatility and the equilibrium constants for the analyte/fiber coating. In order to overcome this issue, a new optimization approach was proposed based on a combination of extraction modes and extraction temperatures in a single extraction procedure. The final optimized procedure was: 48min of extraction in direct immersion mode with the sample maintained at 80°C followed by a further 32min of headspace extraction with the sample temperature kept at 10°C. The proposed procedure was compared with conventional methods based on the use of a single extraction mode and temperature (80min of headspace extraction at 60°C or 80min of direct immersion extraction at 50°C). The newly proposed method was shown to be more attractive as it extracted higher amounts of both semi-volatile and volatile compounds in a single extraction procedure compared to the conventional approaches. The optimized method was validated and excellent results were obtained.
Source:Journal of Chromatography A, Volume 1233
Joyce Nunes Bianchin, Giuliana Nardini, Josias Merib, Adriana Neves Dias, Edmar Martendal, Eduardo Carasek
This study proposes a new optimization approach for the simultaneous determination of polycyclic aromatic hydrocarbons (PAHs) and benzene, toluene, ethylbenzene and xylene isomers (BTEX) from water samples using the solid-phase microextraction technique followed by gas chromatography–mass spectrometry (GC–MS) separation and detection. The objective of the study was to achieve compromise extraction conditions, suitable for all semi-volatile and volatile compounds, under which the amount extracted is maximized for all analytes. This was achieved by careful optimization of the fiber coating, salting-out effect, extraction time and temperature and extraction mode (headspace or direct immersion). With the optimized fiber coating – PDMS/DVB 65μm – the other selected factors were optimized using a response surface methodology through central composite designs. As expected, the optimized results for each class of analytes varied significantly, probably due to the differences in their volatility and the equilibrium constants for the analyte/fiber coating. In order to overcome this issue, a new optimization approach was proposed based on a combination of extraction modes and extraction temperatures in a single extraction procedure. The final optimized procedure was: 48min of extraction in direct immersion mode with the sample maintained at 80°C followed by a further 32min of headspace extraction with the sample temperature kept at 10°C. The proposed procedure was compared with conventional methods based on the use of a single extraction mode and temperature (80min of headspace extraction at 60°C or 80min of direct immersion extraction at 50°C). The newly proposed method was shown to be more attractive as it extracted higher amounts of both semi-volatile and volatile compounds in a single extraction procedure compared to the conventional approaches. The optimized method was validated and excellent results were obtained.
Plasmid DNA partitioning and separation using poly(ethylene glycol)/poly(acrylate)/salt aqueous two-phase systems
27 March 2012,
09:18:54
Publication year:
2012
Source:Journal of Chromatography A, Volume 1233
Hans-Olof Johansson, Tiago Matos, Juliana S. Luz, Eloi Feitosa, Carla C. Oliveira, Adalberto Pessoa, Leif Bülow, Folke Tjerneld
Phase diagrams of poly(ethylene glycol)/polyacrylate/Na2SO4 systems have been investigated with respect to polymer size and pH. Plasmid DNA from Escherichia coli can depending on pH and polymer molecular weight be directed to a poly(ethylene glycol) or to a polyacrylate-rich phase in an aqueous two-phase system formed by these polymers. Bovine serum albumin (BSA) and E. coli homogenate proteins can be directed opposite to the plasmid partitioning in these systems. Two bioseparation processes have been developed where in the final step the pDNA is partitioned to a salt-rich phase giving a total process yield of 60–70%. In one of them the pDNA is partitioned between the polyacrylate and PEG-phases in order to remove proteins. In a more simplified process the plasmid is partitioned to a PEG-phase and back-extracted into a Na2SO4-rich phase. The novel polyacrylate/PEG system allows a strong change of the partitioning between the phases with relatively small changes in composition or pH.
Source:Journal of Chromatography A, Volume 1233
Hans-Olof Johansson, Tiago Matos, Juliana S. Luz, Eloi Feitosa, Carla C. Oliveira, Adalberto Pessoa, Leif Bülow, Folke Tjerneld
Phase diagrams of poly(ethylene glycol)/polyacrylate/Na2SO4 systems have been investigated with respect to polymer size and pH. Plasmid DNA from Escherichia coli can depending on pH and polymer molecular weight be directed to a poly(ethylene glycol) or to a polyacrylate-rich phase in an aqueous two-phase system formed by these polymers. Bovine serum albumin (BSA) and E. coli homogenate proteins can be directed opposite to the plasmid partitioning in these systems. Two bioseparation processes have been developed where in the final step the pDNA is partitioned to a salt-rich phase giving a total process yield of 60–70%. In one of them the pDNA is partitioned between the polyacrylate and PEG-phases in order to remove proteins. In a more simplified process the plasmid is partitioned to a PEG-phase and back-extracted into a Na2SO4-rich phase. The novel polyacrylate/PEG system allows a strong change of the partitioning between the phases with relatively small changes in composition or pH.
Determination of triazine herbicides in cereals using dynamic microwave-assisted extraction with solidification of floating organic drop followed by high-performance liquid chromatography
27 March 2012,
09:18:54
Publication year:
2012
Source:Journal of Chromatography A, Volume 1233
Hui Wang, Guijie Li, Yiqun Zhang, Haiyan Chen, Qi Zhao, Weitao Song, Yang Xu, Haiyan Jin, Lan Ding
A simple and cost-effective method of dynamic microwave-assisted extraction (DMAE) combined with solidification of floating organic drop (SFO) was developed for determining the five triazine herbicides in cereals. The approach combines the advantages of DMAE and SFO technique, and up to 15 samples can be treated simultaneously in 16min. Firstly, triazine herbicides were extracted with 1mL of methanol containing 90μL of 1-dodecanol and following with 10mL of water under the action of microwave energy. After that, 1.5g sodium chloride was added into the obtained extract, and the mixture was centrifuged and cooled. The 1-dodecanol drop which contained the target analytes was solidified and transferred for analysis by HPLC-UV. Limits of detection of the five triazines obtained were in the range of 1.1–1.5ngg−1. Relative standard deviations of intra- and inter-day tests ranging from 5% to 7% were obtained. The method was successfully applied to the analysis of ten cereals and the recoveries of the triazines for the spiked samples were in the range of 80–102%. The proposed method is an alternative approach to the analysis of triazine herbicides in complex solid samples, being more rapid and simpler compared with the traditional extraction method.
Source:Journal of Chromatography A, Volume 1233
Hui Wang, Guijie Li, Yiqun Zhang, Haiyan Chen, Qi Zhao, Weitao Song, Yang Xu, Haiyan Jin, Lan Ding
A simple and cost-effective method of dynamic microwave-assisted extraction (DMAE) combined with solidification of floating organic drop (SFO) was developed for determining the five triazine herbicides in cereals. The approach combines the advantages of DMAE and SFO technique, and up to 15 samples can be treated simultaneously in 16min. Firstly, triazine herbicides were extracted with 1mL of methanol containing 90μL of 1-dodecanol and following with 10mL of water under the action of microwave energy. After that, 1.5g sodium chloride was added into the obtained extract, and the mixture was centrifuged and cooled. The 1-dodecanol drop which contained the target analytes was solidified and transferred for analysis by HPLC-UV. Limits of detection of the five triazines obtained were in the range of 1.1–1.5ngg−1. Relative standard deviations of intra- and inter-day tests ranging from 5% to 7% were obtained. The method was successfully applied to the analysis of ten cereals and the recoveries of the triazines for the spiked samples were in the range of 80–102%. The proposed method is an alternative approach to the analysis of triazine herbicides in complex solid samples, being more rapid and simpler compared with the traditional extraction method.
Separation and quantification of 15 carotenoids by reversed phase high performance liquid chromatography coupled to diode array detection with isosbestic wavelength approach
27 March 2012,
09:18:54
Publication year:
2012
Source:Journal of Chromatography A, Volume 1233
Kamila Mitrowska, Ursula Vincent, Christoph von Holst
The manuscript presents the development of a new reverse phase high performance liquid chromatography (RP-HPLC) photo diode array detection method allowing the separation and quantification of 15 carotenoids (adonirubin, adonixanthin, astaxanthin, astaxanthin dimethyl disuccinate, asteroidenone, beta-apo-8′-carotenal, beta-apo-8′-carotenoic acid ethyl ester, beta-carotene, canthaxanthin, capsanthin, citranaxanthin, echinenone, lutein, lycopene, and zeaxanthin), 10 of which are feed additives authorised within the European Union. The developed method allows for the reliable determination of the total carotenoid content in one run using the corresponding E-isomer as calibration standard while taking into account the E/Z-isomers composition. This is a key criterion for the application of the method, since for most of the analytes included in this study analytical standards are only available for the E-isomers. This goal was achieved by applying the isosbestic concept, in order to identify specific wavelengths, at which the absorption coefficients are identical for all stereoisomers concerned. The second target referred to the optimisation of the LC conditions. By means of an experimental design, an optimised RP-HPLC method was developed allowing for a sufficient chromatographic separation of all carotenoids. The selected method uses a Suplex pKb-100 HPLC column and applying a gradient with a mixture of acetonitrile, tert-butyl-methyl ether and water as mobile phases. The limits of detection and limits of quantification ranged from 0.06mgL−1 to 0.14mgL−1 and from 0.20mgL−1 to 0.48mgL−1, respectively.
Source:Journal of Chromatography A, Volume 1233
Kamila Mitrowska, Ursula Vincent, Christoph von Holst
The manuscript presents the development of a new reverse phase high performance liquid chromatography (RP-HPLC) photo diode array detection method allowing the separation and quantification of 15 carotenoids (adonirubin, adonixanthin, astaxanthin, astaxanthin dimethyl disuccinate, asteroidenone, beta-apo-8′-carotenal, beta-apo-8′-carotenoic acid ethyl ester, beta-carotene, canthaxanthin, capsanthin, citranaxanthin, echinenone, lutein, lycopene, and zeaxanthin), 10 of which are feed additives authorised within the European Union. The developed method allows for the reliable determination of the total carotenoid content in one run using the corresponding E-isomer as calibration standard while taking into account the E/Z-isomers composition. This is a key criterion for the application of the method, since for most of the analytes included in this study analytical standards are only available for the E-isomers. This goal was achieved by applying the isosbestic concept, in order to identify specific wavelengths, at which the absorption coefficients are identical for all stereoisomers concerned. The second target referred to the optimisation of the LC conditions. By means of an experimental design, an optimised RP-HPLC method was developed allowing for a sufficient chromatographic separation of all carotenoids. The selected method uses a Suplex pKb-100 HPLC column and applying a gradient with a mixture of acetonitrile, tert-butyl-methyl ether and water as mobile phases. The limits of detection and limits of quantification ranged from 0.06mgL−1 to 0.14mgL−1 and from 0.20mgL−1 to 0.48mgL−1, respectively.
Determination of parameters for the steric mass action model—A comparison between two approaches
27 March 2012,
09:18:54
Publication year:
2012
Source:Journal of Chromatography A, Volume 1233
A. Osberghaus, S. Hepbildikler, S. Nath, M. Haindl, E. von Lieres, J. Hubbuch
The application of mechanistic modeling for the optimization of chromatographic steps increased recently due to time efficiency of algorithms and rising calculation power. In the modeling of ion exchange chromatography steps, the sorption processes occurring on adsorbent particle surfaces can be simulated with the steric mass action (SMA) model introduced by Brooks and Cramer (1992) [14]. In this paper, two approaches for the determination of SMA parameters will be carried out and discussed concerning their specific experimental effort, quality of results, method differences, reasons for uncertainties and consequences for SMA parameter determination: Approach I: estimation of SMA parameters based on gradient and frontal experiments according to instructions in Brooks and Cramer (1992) [14] and Shukla et al. (1998) [16]. Approach II: application of an inverse method for parameter estimation, resulting in SMA parameters that induce a best fit of chromatographic data to a mechanistic model for column chromatography. These approaches for SMA parameter determination were carried out for three proteins (ribonuclease A, cytochrome c and lysozyme) at pH 5 and pH 7. The results were comparable and the order of parameter values and their relations to the chromatographic data similar. Nevertheless, differences in the complexity and effort of methods as well as the parameter values themselves were observed. The comparison of methods demonstrated that discrepancies depend mainly on model sensitivities and additional parameters influencing the calculations. However, the discrepancies do not affect predictivity; predictivity is high in both approaches. The approach based on an inverse method and the mechanistic model has the advantage that not only retention times but also complete elution profiles can be predicted. Thus, the inverse method based on a mechanistic model for column chromatography is the most comfortable way to establish highly predictive SMA parameters lending themselves for the optimization of chromatography steps and process control.
Source:Journal of Chromatography A, Volume 1233
A. Osberghaus, S. Hepbildikler, S. Nath, M. Haindl, E. von Lieres, J. Hubbuch
The application of mechanistic modeling for the optimization of chromatographic steps increased recently due to time efficiency of algorithms and rising calculation power. In the modeling of ion exchange chromatography steps, the sorption processes occurring on adsorbent particle surfaces can be simulated with the steric mass action (SMA) model introduced by Brooks and Cramer (1992) [14]. In this paper, two approaches for the determination of SMA parameters will be carried out and discussed concerning their specific experimental effort, quality of results, method differences, reasons for uncertainties and consequences for SMA parameter determination: Approach I: estimation of SMA parameters based on gradient and frontal experiments according to instructions in Brooks and Cramer (1992) [14] and Shukla et al. (1998) [16]. Approach II: application of an inverse method for parameter estimation, resulting in SMA parameters that induce a best fit of chromatographic data to a mechanistic model for column chromatography. These approaches for SMA parameter determination were carried out for three proteins (ribonuclease A, cytochrome c and lysozyme) at pH 5 and pH 7. The results were comparable and the order of parameter values and their relations to the chromatographic data similar. Nevertheless, differences in the complexity and effort of methods as well as the parameter values themselves were observed. The comparison of methods demonstrated that discrepancies depend mainly on model sensitivities and additional parameters influencing the calculations. However, the discrepancies do not affect predictivity; predictivity is high in both approaches. The approach based on an inverse method and the mechanistic model has the advantage that not only retention times but also complete elution profiles can be predicted. Thus, the inverse method based on a mechanistic model for column chromatography is the most comfortable way to establish highly predictive SMA parameters lending themselves for the optimization of chromatography steps and process control.
Hydrophilic interaction liquid chromatography of anthranilic acid-labelled oligosaccharides with a 4-aminobenzoic acid ethyl ester-labelled dextran hydrolysate internal standard
27 March 2012,
09:18:54
Publication year:
2012
Source:Journal of Chromatography A, Volume 1233
David C.A. Neville, Dominic S. Alonzi, Terry D. Butters
Hydrophilic interaction liquid chromatography (HILIC) of fluorescently labelled oligosaccharides is used in many laboratories to analyse complex oligosaccharide mixtures. Separations are routinely performed using a TSK gel-Amide 80 HPLC column, and retention times of different oligosaccharide species are converted to glucose unit (GU) values that are determined with reference to an external standard. However, if retention times were to be compared with an internal standard, consistent and more accurate GU values would be obtained. We present a method to perform internal standard-calibrated HILIC of fluorescently labelled oligosaccharides. The method relies on co-injection of 4-aminobenzoic acid ethyl ester (4-ABEE)-labelled internal standard and detection by UV absorption, with 2-AA (2-aminobenzoic acid)-labelled oligosaccharides. 4-ABEE is a UV chromophore and a fluorophore, but there is no overlap of the fluorescent spectrum of 4-ABEE with the commonly used fluorescent reagents. The dual nature of 4-ABEE allows for accurate calculation of the delay between UV and fluorescent signals when determining the GU values of individual oligosaccharides. The GU values obtained are inherently more accurate as slight differences in gradients that can influence retention are negated by use of an internal standard. Therefore, this paper provides the first method for determination of HPLC-derived GU values of fluorescently labelled oligosaccharides using an internal calibrant.
Source:Journal of Chromatography A, Volume 1233
David C.A. Neville, Dominic S. Alonzi, Terry D. Butters
Hydrophilic interaction liquid chromatography (HILIC) of fluorescently labelled oligosaccharides is used in many laboratories to analyse complex oligosaccharide mixtures. Separations are routinely performed using a TSK gel-Amide 80 HPLC column, and retention times of different oligosaccharide species are converted to glucose unit (GU) values that are determined with reference to an external standard. However, if retention times were to be compared with an internal standard, consistent and more accurate GU values would be obtained. We present a method to perform internal standard-calibrated HILIC of fluorescently labelled oligosaccharides. The method relies on co-injection of 4-aminobenzoic acid ethyl ester (4-ABEE)-labelled internal standard and detection by UV absorption, with 2-AA (2-aminobenzoic acid)-labelled oligosaccharides. 4-ABEE is a UV chromophore and a fluorophore, but there is no overlap of the fluorescent spectrum of 4-ABEE with the commonly used fluorescent reagents. The dual nature of 4-ABEE allows for accurate calculation of the delay between UV and fluorescent signals when determining the GU values of individual oligosaccharides. The GU values obtained are inherently more accurate as slight differences in gradients that can influence retention are negated by use of an internal standard. Therefore, this paper provides the first method for determination of HPLC-derived GU values of fluorescently labelled oligosaccharides using an internal calibrant.
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