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papers from the latest issue:
An improved hollow fiber solvent-stir bar microextraction for the preconcentration of anabolic steroids in biological matrix with determination by gas chromatography–mass spectrometry
22 March 2012,
11:49:56
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
22 March 2012,
11:49:56
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
22 March 2012,
11:49:56
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
22 March 2012,
11:49:56
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
22 March 2012,
11:49:56
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
22 March 2012,
11:49:56
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
22 March 2012,
11:49:56
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
22 March 2012,
11:49:56
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
22 March 2012,
11:49:56
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.
Determination of pharmaceutically related compounds by suppressed ion chromatography: III. Role of electrolytic suppressor design
22 March 2012,
11:49:56
Publication year:
2012
Source:Journal of Chromatography A, Volume 1233
Naama Karu, Greg W. Dicinoski, Melissa Hanna-Brown, Kannan Srinivasan, Christopher A. Pohl, Paul R. Haddad
For the hyphenation of ion chromatography to nebulising detectors or mass spectrometry, suppression of the non-volatile ionic eluent to water is a required step to avoid elevated detector baselines. Presented here is a study of three new designs of electrolytic suppressors, incorporating high ion-exchange capacity screens and high ion-exchange capacity membranes in different thickness and compositions. These designs aim to minimise hydrophobic interactions of the suppressor with organic analytes and to provide higher compatibility with eluents containing acetonitrile. In comparison with a commercially available electrolytic suppressor and also a commercially available chemical suppressor, the new high-capacity suppressor showed superior performance, exhibiting minimal interactions with a test set of analytes under the examined conditions. This led to the attainment of high recoveries of the analytes after suppression (93–99% recovery) and significantly reduced band broadening during suppression. The new suppressor has been shown to perform well under both isocratic and gradient elution conditions.
Source:Journal of Chromatography A, Volume 1233
Naama Karu, Greg W. Dicinoski, Melissa Hanna-Brown, Kannan Srinivasan, Christopher A. Pohl, Paul R. Haddad
For the hyphenation of ion chromatography to nebulising detectors or mass spectrometry, suppression of the non-volatile ionic eluent to water is a required step to avoid elevated detector baselines. Presented here is a study of three new designs of electrolytic suppressors, incorporating high ion-exchange capacity screens and high ion-exchange capacity membranes in different thickness and compositions. These designs aim to minimise hydrophobic interactions of the suppressor with organic analytes and to provide higher compatibility with eluents containing acetonitrile. In comparison with a commercially available electrolytic suppressor and also a commercially available chemical suppressor, the new high-capacity suppressor showed superior performance, exhibiting minimal interactions with a test set of analytes under the examined conditions. This led to the attainment of high recoveries of the analytes after suppression (93–99% recovery) and significantly reduced band broadening during suppression. The new suppressor has been shown to perform well under both isocratic and gradient elution conditions.
Fast immobilized liposome chromatography based on penetrable silica microspheres for screening and analysis of permeable compounds
22 March 2012,
11:49:56
Publication year:
2012
Source:Journal of Chromatography A, Volume 1233
Cong Zhang, Jian Li, Li Xu, Zhi-Guo Shi
In the present study, an immobilized liposome chromatography (ILC) stationary phase based on penetrable silica microspheres was prepared. The silica possessed mesopores and penetrable macropores, which afforded sufficient surface area and fast mass transfer, respectively. Compared with the ILC column based on common porous silica gels, the penetrable silica had larger capacity for liposomes’ immobilization, lower backpressure and higher separation efficiency. Twenty-two kinds of drugs were used as tested analytes and their retention behaviors on newly prepared ILC column were investigated in detail. Column temperature and pH of mobile phase were both key factors affecting the retention of solutes. The retention of these drugs on ILC column reflected their permeability in vivo. Furthermore, the methanolic aqueous extracts of three traditional Chinese medicines (TCMs), Radix Liquiritiae, Scutellaria Baicalensis and Flos Sophorae, were screened on this novel ILC column. Effects of column temperature and eluent pH on chromatographic behaviors of components of the TCM methanolic extracts were also studied. Several permeable components can be found, indicating that they are potentially active components in the TCMs. It is also found that the as-prepared ILC column remained stable in at least 1 month and the relative standard deviations of adjusted retention times for solutes were lower than 9.0%. The proposed ILC based on the penetrable silica microspheres is promising for the fast and low-pressure separation, especially for fast screening of permeable compounds and modeling the drug–membrane interaction in vitro. It would be a useful approach to predict the permeability of potential active drugs.
Source:Journal of Chromatography A, Volume 1233
Cong Zhang, Jian Li, Li Xu, Zhi-Guo Shi
In the present study, an immobilized liposome chromatography (ILC) stationary phase based on penetrable silica microspheres was prepared. The silica possessed mesopores and penetrable macropores, which afforded sufficient surface area and fast mass transfer, respectively. Compared with the ILC column based on common porous silica gels, the penetrable silica had larger capacity for liposomes’ immobilization, lower backpressure and higher separation efficiency. Twenty-two kinds of drugs were used as tested analytes and their retention behaviors on newly prepared ILC column were investigated in detail. Column temperature and pH of mobile phase were both key factors affecting the retention of solutes. The retention of these drugs on ILC column reflected their permeability in vivo. Furthermore, the methanolic aqueous extracts of three traditional Chinese medicines (TCMs), Radix Liquiritiae, Scutellaria Baicalensis and Flos Sophorae, were screened on this novel ILC column. Effects of column temperature and eluent pH on chromatographic behaviors of components of the TCM methanolic extracts were also studied. Several permeable components can be found, indicating that they are potentially active components in the TCMs. It is also found that the as-prepared ILC column remained stable in at least 1 month and the relative standard deviations of adjusted retention times for solutes were lower than 9.0%. The proposed ILC based on the penetrable silica microspheres is promising for the fast and low-pressure separation, especially for fast screening of permeable compounds and modeling the drug–membrane interaction in vitro. It would be a useful approach to predict the permeability of potential active drugs.
Supercritical fluid chromatographic resolution of water soluble isomeric carboxyl/amine terminated peptides facilitated via mobile phase water and ion pair formation
22 March 2012,
11:49:56
Publication year:
2012
Source:Journal of Chromatography A, Volume 1233
M.A. Patel, F. Riley, M. Ashraf-Khorassani, L.T. Taylor
Both analytical scale and preparative scale packed column supercritical fluid chromatography (SFC) have found widespread applicability for chiral separations of multiple polar pharmaceutical candidates. However, SFC is rapidly becoming an achiral technique. More specifically, ion pair SFC is finding greater utility for separation of ionic analytes such as amine salts and organic sulfonates. The key to this success is, in part, the incorporation of additives such as trifluoroacetic acid and ammonium acetate into the mobile phase in association with a wide variety of both bonded silica stationary phases and high purity bare silica. Ion pairing SFC coupled with evaporative light scattering detection and mass spectrometric detection is presented here for the separation of water soluble, uncapped, isomeric peptide pairs that differ in amino acid arrangement. The separation is best achieved on either diol-bonded silica or bare silica with 1–5% (w/w) water as a significant ingredient in the mobile phase. Nitrogenous stationary phases such as 2-ethylpyridine, which had been very successful for the separation of capped peptides failed to yield the desired separation regardless of the mobile phase composition. A HILIC type retention mechanism is postulated for the separation of both isomeric uncapped peptide pairs.
Source:Journal of Chromatography A, Volume 1233
M.A. Patel, F. Riley, M. Ashraf-Khorassani, L.T. Taylor
Both analytical scale and preparative scale packed column supercritical fluid chromatography (SFC) have found widespread applicability for chiral separations of multiple polar pharmaceutical candidates. However, SFC is rapidly becoming an achiral technique. More specifically, ion pair SFC is finding greater utility for separation of ionic analytes such as amine salts and organic sulfonates. The key to this success is, in part, the incorporation of additives such as trifluoroacetic acid and ammonium acetate into the mobile phase in association with a wide variety of both bonded silica stationary phases and high purity bare silica. Ion pairing SFC coupled with evaporative light scattering detection and mass spectrometric detection is presented here for the separation of water soluble, uncapped, isomeric peptide pairs that differ in amino acid arrangement. The separation is best achieved on either diol-bonded silica or bare silica with 1–5% (w/w) water as a significant ingredient in the mobile phase. Nitrogenous stationary phases such as 2-ethylpyridine, which had been very successful for the separation of capped peptides failed to yield the desired separation regardless of the mobile phase composition. A HILIC type retention mechanism is postulated for the separation of both isomeric uncapped peptide pairs.
Synthesis of sulfo/vinyl biphasic silica hybrid monolithic capillary column and its application to on-column preconcentration for capillary electrochromatography
22 March 2012,
11:49:56
Publication year:
2012
Source:Journal of Chromatography A, Volume 1233
Yingzhuang Chen, Keyi Wang, Huihui Yang, Yixuan Liu, Shouzhuo Yao, Bo Chen, Lihua Nie, Guangming Xu
A novel method to synthesize sulfo/vinyl biphasic silica hybrid monolithic column in one step was developed for on-column preconcentration. In this method, sulfo-based segment is located at the inlet of capillary column, which acts as preconcentration column. It is synthesized by polymerization of 3-sulfopropyl methacrylate potassium salt (SPMA) and vinyltrimethoxysilane (VTMS) with tetramethoxysilane (TOMS). Close to the preconcentration column, a vinyl functionalized segment is formed and serves as separation column. It is synthesized by polymerization of only VTMS with TOMS. Vinyl groups on vinyl functionalized segment are modified with ligand containing sulfhydryl group, such as octadecanethiol (C18-SH for short), 6-mercapto-1-hexanol (HO-C6-SH for short), via thiol-ene click reaction. The interface between the two segments is seamless and without any dead volume. The applicability of this system is demonstrated by successful separation of closely related amines including p-phenylenediamine, aniline, p-toluidine, N-methyl aniline, N,N′-dimethylaniline, and diphenylamine. Good separation and enrichment are obtained. The proposed system is also successfully applied to complex biological samples, such as peptide, diluted BSA hydrolysate, and the results indicate that the system has a capability for preconcentration of low abundance peptides.
Source:Journal of Chromatography A, Volume 1233
Yingzhuang Chen, Keyi Wang, Huihui Yang, Yixuan Liu, Shouzhuo Yao, Bo Chen, Lihua Nie, Guangming Xu
A novel method to synthesize sulfo/vinyl biphasic silica hybrid monolithic column in one step was developed for on-column preconcentration. In this method, sulfo-based segment is located at the inlet of capillary column, which acts as preconcentration column. It is synthesized by polymerization of 3-sulfopropyl methacrylate potassium salt (SPMA) and vinyltrimethoxysilane (VTMS) with tetramethoxysilane (TOMS). Close to the preconcentration column, a vinyl functionalized segment is formed and serves as separation column. It is synthesized by polymerization of only VTMS with TOMS. Vinyl groups on vinyl functionalized segment are modified with ligand containing sulfhydryl group, such as octadecanethiol (C18-SH for short), 6-mercapto-1-hexanol (HO-C6-SH for short), via thiol-ene click reaction. The interface between the two segments is seamless and without any dead volume. The applicability of this system is demonstrated by successful separation of closely related amines including p-phenylenediamine, aniline, p-toluidine, N-methyl aniline, N,N′-dimethylaniline, and diphenylamine. Good separation and enrichment are obtained. The proposed system is also successfully applied to complex biological samples, such as peptide, diluted BSA hydrolysate, and the results indicate that the system has a capability for preconcentration of low abundance peptides.
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