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Selected papers from the latest issue:
High concentration capacity sample preparation techniques to improve the informative potential of two-dimensional comprehensive gas chromatography–mass spectrometry: Application to sensomics
07 November 2013,
09:10:03
Publication date: 29 November
2013
Source:Journal of Chromatography A, Volume 1318
Author(s): Chiara Cordero , Cecilia Cagliero , Erica Liberto , Luca Nicolotti , Patrizia Rubiolo , Barbara Sgorbini , Carlo Bicchi
This study reports and critically discusses the results of a systematic investigation on the effectiveness of different and complementary sampling approaches, based on either sorption and adsorption, treated as a further dimension of a two-dimensional comprehensive gas chromatography–mass spectrometry analytical platform for sensomics. The focus is on the potentials of a group of high concentration capacity (HCC) sample preparation (Solid Phase Microextraction, SPME, Stir Bar Sorptive Extraction, SBSE and Headspace Sorptive Extraction, HSSE) and Dynamic Headspace (D-HS) techniques investigated to provide information useful for fingerprinting and profiling studies of food aroma. Volatiles and semi-volatiles contributing to define whole and nonfat dry milk aroma have been successfully characterized thanks to the combination of effective and selective sampling by HCC and D-HS techniques, high separation and detection power of GC×GC–MS and suitable data elaboration (i.e., Comprehensive Template Matching Fingerprinting – CTMF). Out of the sample preparation techniques investigated, HSSE and SBSE have shown to be really effective for sensomics studies because of their high concentration factors, providing highly representative profiles as well as analyte recovery suitable for GC-Olfactometry even with high odor threshold (OT) markers or potent odorants in sub-trace amounts.
Source:Journal of Chromatography A, Volume 1318
Author(s): Chiara Cordero , Cecilia Cagliero , Erica Liberto , Luca Nicolotti , Patrizia Rubiolo , Barbara Sgorbini , Carlo Bicchi
This study reports and critically discusses the results of a systematic investigation on the effectiveness of different and complementary sampling approaches, based on either sorption and adsorption, treated as a further dimension of a two-dimensional comprehensive gas chromatography–mass spectrometry analytical platform for sensomics. The focus is on the potentials of a group of high concentration capacity (HCC) sample preparation (Solid Phase Microextraction, SPME, Stir Bar Sorptive Extraction, SBSE and Headspace Sorptive Extraction, HSSE) and Dynamic Headspace (D-HS) techniques investigated to provide information useful for fingerprinting and profiling studies of food aroma. Volatiles and semi-volatiles contributing to define whole and nonfat dry milk aroma have been successfully characterized thanks to the combination of effective and selective sampling by HCC and D-HS techniques, high separation and detection power of GC×GC–MS and suitable data elaboration (i.e., Comprehensive Template Matching Fingerprinting – CTMF). Out of the sample preparation techniques investigated, HSSE and SBSE have shown to be really effective for sensomics studies because of their high concentration factors, providing highly representative profiles as well as analyte recovery suitable for GC-Olfactometry even with high odor threshold (OT) markers or potent odorants in sub-trace amounts.
A new strategy to eliminate sample mixing during in-tube solid phase microextraction
07 November 2013,
09:10:03
Publication date: 29 November
2013
Source:Journal of Chromatography A, Volume 1318
Author(s): Yang Yang , Heather Lord , Janusz Pawliszyn
During in-tube solid phase microextraction, sample mixing with mobile phase contained in the autosampler tubing during extraction may result in some amount of sample becoming entrained in the mobile phase rather than returning to the sample vial or being directed to waste after extraction. In cases where target analytes have relatively low affinity for the sorbent on the wall of the capillary, mixing can impact data quality. Where the sample contains components that may interfere with either the separation (e.g. proteins) or detection (e.g. ions with MS detection), additional difficulties can arise. In the current research, the magnitude of the sample mixing effect was illustrated by analyzing ranitidine and a series of polycyclic aromatic hydrocarbons (PAH). The sample volume equivalent of mixing was calculated as 37μL for ranitidine and 20μL for PAHs using the same inner diameter of capillary. To address this issue, a novel approach involving adding a switching valve located between the metering pump and the capillary was developed. Capillary flush conditions, draw/eject speed and extraction time were optimized for ranitidine with the result that in the final method, no mixing of sample with mobile phase was apparent in the detected amounts. To provide information on a compound class with intermediate polarity, two β-blockers were also extracted using the optimized washing conditions respectively. The results indicated that the issue of sample mixing had been resolved for these as well. Finally, in-tube SPME calibration of these three analyte classes was shown to be highly linear, providing further indication that sample mixing was not impacting data quality. Available literature on the subject was surveyed, and a discussion on the rational selection of conditions to guide method development was also provided.
Source:Journal of Chromatography A, Volume 1318
Author(s): Yang Yang , Heather Lord , Janusz Pawliszyn
During in-tube solid phase microextraction, sample mixing with mobile phase contained in the autosampler tubing during extraction may result in some amount of sample becoming entrained in the mobile phase rather than returning to the sample vial or being directed to waste after extraction. In cases where target analytes have relatively low affinity for the sorbent on the wall of the capillary, mixing can impact data quality. Where the sample contains components that may interfere with either the separation (e.g. proteins) or detection (e.g. ions with MS detection), additional difficulties can arise. In the current research, the magnitude of the sample mixing effect was illustrated by analyzing ranitidine and a series of polycyclic aromatic hydrocarbons (PAH). The sample volume equivalent of mixing was calculated as 37μL for ranitidine and 20μL for PAHs using the same inner diameter of capillary. To address this issue, a novel approach involving adding a switching valve located between the metering pump and the capillary was developed. Capillary flush conditions, draw/eject speed and extraction time were optimized for ranitidine with the result that in the final method, no mixing of sample with mobile phase was apparent in the detected amounts. To provide information on a compound class with intermediate polarity, two β-blockers were also extracted using the optimized washing conditions respectively. The results indicated that the issue of sample mixing had been resolved for these as well. Finally, in-tube SPME calibration of these three analyte classes was shown to be highly linear, providing further indication that sample mixing was not impacting data quality. Available literature on the subject was surveyed, and a discussion on the rational selection of conditions to guide method development was also provided.
Trace analysis of biocidal oligoguanidines in environmental water samples
07 November 2013,
09:10:03
Publication date: 29 November
2013
Source:Journal of Chromatography A, Volume 1318
Author(s): Tamara Buchberger , Markus Himmelsbach , Wolfgang Buchberger
This paper demonstrates the determination of residues of biocidal oligoguanidines manufactured by polycondensation of guanidine hydrochloride and 2,2′-(ethylenedioxy)bis(ethylamine) in environmental water samples. The analytes were preconcentrated from samples adjusted to pH 4 by solid-phase extraction using a mixed-mode sorbent with weak cation exchange functionalities. Elution from the sorbent was achieved by 2M hydrochloric acid in methanol. After evaporation and reconstitution in water, the extract was analyzed by reversed-phase high performance liquid chromatography combined with quadrupole/time-of-flight or triple quadrupole mass spectrometry employing electrospray ionization. A preconcentration factor of 10,000 could be achieved and detection limits were in the sub-μgL−1 range. This method looks promising to monitor the fate of these biocides released into the aquatic environment during different applications.
Source:Journal of Chromatography A, Volume 1318
Author(s): Tamara Buchberger , Markus Himmelsbach , Wolfgang Buchberger
This paper demonstrates the determination of residues of biocidal oligoguanidines manufactured by polycondensation of guanidine hydrochloride and 2,2′-(ethylenedioxy)bis(ethylamine) in environmental water samples. The analytes were preconcentrated from samples adjusted to pH 4 by solid-phase extraction using a mixed-mode sorbent with weak cation exchange functionalities. Elution from the sorbent was achieved by 2M hydrochloric acid in methanol. After evaporation and reconstitution in water, the extract was analyzed by reversed-phase high performance liquid chromatography combined with quadrupole/time-of-flight or triple quadrupole mass spectrometry employing electrospray ionization. A preconcentration factor of 10,000 could be achieved and detection limits were in the sub-μgL−1 range. This method looks promising to monitor the fate of these biocides released into the aquatic environment during different applications.
Graphene oxide: An adsorbent for the extraction and quantification of aflatoxins in peanuts by high-performance liquid chromatography
07 November 2013,
09:10:03
Publication date: 29 November
2013
Source:Journal of Chromatography A, Volume 1318
Author(s): Li Yu , Peiwu Li , Qi Zhang , Wen Zhang , Xiaoxia Ding , Xiupin Wang
In this paper, graphene oxide (GO) was synthesized and specifically selected by centrifugation to extract four aflatoxins (B1, B2, G1, and G2) as an effective adsorbent. Then, the amount of aflatoxins was quantitatively measured by high-performance liquid chromatography (HPLC). The GO was characterized by X-ray diffraction (XRD), atomic force microscopy (AFM), and ultraviolet (UV) spectrophotometer. Several parameters that could affect the extraction efficiency, including the GO amount, methanol concentration in the extraction solvent, spiked amount, extraction time, and elution cycle, were also investigated and optimized in this work. Under optimal conditions, good linear relationships were achieved with the correlation coefficient (r) ranging from 0.99217 to 0.99995. The detection limit of this method for the four aflatoxins ranged from 0.08 to 0.65ng/g. Finally, the proposed method has been successfully applied to determine aflatoxins in peanut samples. The results show that the recoveries of the four aflatoxins range from 85.1% to 100.8% with the relative standard deviations between 2.1% and 7.9%.
Source:Journal of Chromatography A, Volume 1318
Author(s): Li Yu , Peiwu Li , Qi Zhang , Wen Zhang , Xiaoxia Ding , Xiupin Wang
In this paper, graphene oxide (GO) was synthesized and specifically selected by centrifugation to extract four aflatoxins (B1, B2, G1, and G2) as an effective adsorbent. Then, the amount of aflatoxins was quantitatively measured by high-performance liquid chromatography (HPLC). The GO was characterized by X-ray diffraction (XRD), atomic force microscopy (AFM), and ultraviolet (UV) spectrophotometer. Several parameters that could affect the extraction efficiency, including the GO amount, methanol concentration in the extraction solvent, spiked amount, extraction time, and elution cycle, were also investigated and optimized in this work. Under optimal conditions, good linear relationships were achieved with the correlation coefficient (r) ranging from 0.99217 to 0.99995. The detection limit of this method for the four aflatoxins ranged from 0.08 to 0.65ng/g. Finally, the proposed method has been successfully applied to determine aflatoxins in peanut samples. The results show that the recoveries of the four aflatoxins range from 85.1% to 100.8% with the relative standard deviations between 2.1% and 7.9%.
In situ derivatization coupled to microextraction by packed sorbent and gas chromatography for the automated determination of haloacetic acids in chlorinated water
07 November 2013,
09:10:03
Publication date: 29 November
2013
Source:Journal of Chromatography A, Volume 1318
Author(s): Ana María Casas Ferreira , María Esther Fernández Laespada , José Luis Pérez Pavón , Bernardo Moreno Cordero
A novel analytical method is reported for the determination of monochloroacetic acid, dichloroacetic acid, trichloroacetic acid, monobromoacetic acid, and dibromoacetic acid. These are the five haloacetic acids (HAAs) for which the U.S. Environmental Protection Agency (US EPA) has regulated a maximum contamination level (MCL) of 0.060mgL−1 for the sum of their concentrations in drinking waters. The method uses in situ aqueous derivatization, followed by microextraction by packed sorbent (MEPS) prior to gas chromatography–mass spectrometry (GC–MS). The parameters affecting derivatization and extraction were optimized with a view to obtaining maximum sensitivity. The HAAs were derivatized with 2,2,2-trifluroethylamine (TFEA), using N-ethyl-N′-(3-dimethylaminopropyl) carbodiimide (EDC) as a condensation agent. The reaction occurred in aqueous medium and was carried out in 10min in the vial of an autosampler used to perform microextraction by packed sorbent. The whole process, from the mixing of the reagents for the derivatization, was automated. Precision values varied from 4.2 to 9.8% (as intra-day relative standard deviation, RSD) and 9.4 to 14% (as inter-day RSD). The recoveries from spiked concentrations ranged from 83 to 117%, revealing the accuracy of the method. The detection limits ranged from 0.36 to 1.2μgL−1, such that it is possible to measure the US EPA MCL in drinking waters. The method developed was applied to the analysis of HAAs in drinking and swimming pool water from Salamanca (North West of Spain).
Source:Journal of Chromatography A, Volume 1318
Author(s): Ana María Casas Ferreira , María Esther Fernández Laespada , José Luis Pérez Pavón , Bernardo Moreno Cordero
A novel analytical method is reported for the determination of monochloroacetic acid, dichloroacetic acid, trichloroacetic acid, monobromoacetic acid, and dibromoacetic acid. These are the five haloacetic acids (HAAs) for which the U.S. Environmental Protection Agency (US EPA) has regulated a maximum contamination level (MCL) of 0.060mgL−1 for the sum of their concentrations in drinking waters. The method uses in situ aqueous derivatization, followed by microextraction by packed sorbent (MEPS) prior to gas chromatography–mass spectrometry (GC–MS). The parameters affecting derivatization and extraction were optimized with a view to obtaining maximum sensitivity. The HAAs were derivatized with 2,2,2-trifluroethylamine (TFEA), using N-ethyl-N′-(3-dimethylaminopropyl) carbodiimide (EDC) as a condensation agent. The reaction occurred in aqueous medium and was carried out in 10min in the vial of an autosampler used to perform microextraction by packed sorbent. The whole process, from the mixing of the reagents for the derivatization, was automated. Precision values varied from 4.2 to 9.8% (as intra-day relative standard deviation, RSD) and 9.4 to 14% (as inter-day RSD). The recoveries from spiked concentrations ranged from 83 to 117%, revealing the accuracy of the method. The detection limits ranged from 0.36 to 1.2μgL−1, such that it is possible to measure the US EPA MCL in drinking waters. The method developed was applied to the analysis of HAAs in drinking and swimming pool water from Salamanca (North West of Spain).
In-tube solid-phase microextraction with molecularly imprinted polymer to determine interferon alpha 2a in plasma sample by high performance liquid chromatography
07 November 2013,
09:10:03
Publication date: 29 November
2013
Source:Journal of Chromatography A, Volume 1318
Author(s): Andréa Rodrigues Chaves , Maria Eugênia Costa Queiroz
A molecularly imprinted sol–gel polymer (MIP) based on protein (biopharmaceutical) template with a mild template removal condition using protease was synthetized and evaluated as stationary phase for in-tube solid phase microextraction (in-tube SPME) of the interferon alpha 2a from plasma samples, followed by high performance liquid chromatography analysis with fluorescence detection (HPLC-FD). The developed MIP exhibited high selectivity for the analyte in a complex matrix. The in-tube SPME variables such as draw/eject cycles, draw/eject volume, and desorption conditions were optimized to establish the equilibrium conditions in a short time. The MIP in-tube SPME/HPLC-FD method presented linear response over a dynamic range of 8–300ngmL−1, with a correlation coefficient of 0.997. The inter-assay precision presented coefficient of variation lower than 9.2%, and accuracy values between 92% and 98%. The developed MIP performed as well as other selective interferon alpha 2a stationary phases (e.g., immunosorbent and restricted access material), with the advantage that it is robust, easy to handle and cheap to synthesize, in a addition to requiring smaller sample volume (50μL). Based on the analytical validation results, the proposed method (MIP in-tube SPME/HPLC-FD) can be a useful tool to determine interferon alpha 2a in plasma samples from patients receiving therapeutic dosages.
Source:Journal of Chromatography A, Volume 1318
Author(s): Andréa Rodrigues Chaves , Maria Eugênia Costa Queiroz
A molecularly imprinted sol–gel polymer (MIP) based on protein (biopharmaceutical) template with a mild template removal condition using protease was synthetized and evaluated as stationary phase for in-tube solid phase microextraction (in-tube SPME) of the interferon alpha 2a from plasma samples, followed by high performance liquid chromatography analysis with fluorescence detection (HPLC-FD). The developed MIP exhibited high selectivity for the analyte in a complex matrix. The in-tube SPME variables such as draw/eject cycles, draw/eject volume, and desorption conditions were optimized to establish the equilibrium conditions in a short time. The MIP in-tube SPME/HPLC-FD method presented linear response over a dynamic range of 8–300ngmL−1, with a correlation coefficient of 0.997. The inter-assay precision presented coefficient of variation lower than 9.2%, and accuracy values between 92% and 98%. The developed MIP performed as well as other selective interferon alpha 2a stationary phases (e.g., immunosorbent and restricted access material), with the advantage that it is robust, easy to handle and cheap to synthesize, in a addition to requiring smaller sample volume (50μL). Based on the analytical validation results, the proposed method (MIP in-tube SPME/HPLC-FD) can be a useful tool to determine interferon alpha 2a in plasma samples from patients receiving therapeutic dosages.
Stir bar sorptive extraction combined with high performance liquid chromatography-ultraviolet/inductively coupled plasma mass spectrometry for analysis of thyroxine in urine samples
07 November 2013,
09:10:03
Publication date: 29 November
2013
Source:Journal of Chromatography A, Volume 1318
Author(s): Wenying Fan , Xiangju Mao , Man He , Beibei Chen , Bin Hu
In this work, polyethyleneglycol (PEG)/hydroxyl polydimethylsiloxane (OH-PDMS)/γ-mercaptopropyl trimethoxysilane (γ-MPTS) coated stir bar was prepared by sol–gel process and its extraction performance for the extraction of amphoteric thyroxines (3,3′,5,5′-tetraiodothyronin, T4; 3,3′,5-triiodothyronine, T3; reversed-3,3′,5-triiodothyronine, rT3) and their metabolite (3,5-diiodothyronine, T2) was studied. The preparation reproducibility of PEG/OH-PDMS/γ-MPTS coated stir bar was investigated, and the relative standard deviations (RSDs) in the same batch and among different batches were 3.3–14.3% (n =5) and 7.7–16.6% (n =3), respectively. The prepared PEG/OH-PDMS/γ-MPTS coated stir bar could be reused for more than 20 times. Based on this fact, a novel method of stir bar sorptive extraction (SBSE) combined with high performance liquid chromatography (HPLC)-ultraviolet (UV) and HPLC-inductively coupled plasma mass spectrometry (ICP-MS) for the analysis of target thyroxines in human urine samples was developed. The influencing factors of SBSE, such as sample pH, extraction time, stirring rate, salt effect, desorption solution and desorption time, were studied in detail, and the analytical performance of the proposed method was evaluated under the optimized conditions. The enrichment factors (EFs) of the developed method for four target thyroxines were in the range of 14.9–70.4 (theoretical enrichment factor was 100). The RSDs were ranging from 4.0% to 13.8% for SBSE-HPLC-UV (c =25μg/L, n =6) and from 3.7% to 6.1% for SBSE-HPLC-ICP-MS (c =0.5μg/L, n =5). The linear range obtained by SBSE-HPLC-UV was 2–500μg/L for T2 and 5–500μg/L for rT3, T3 and T4, with correlation coefficients (r) ranging from 0.9957 to 0.9998, respectively, while the linear range obtained by SBSE-HPLC-ICP-MS was 0.05–500μg/L for T2 and rT3, 0.10–200μg/L for T3 and 0.05–200μg/L for T4 with r ranging from 0.9979 to 0.9998, respectively. The limits of detection (LODs) for the target thyroxines were 0.60–2.20μg/L for SBSE-HPLC-UV and 0.0071–0.0355μg/L SBSE-HPLC-ICP-MS, respectively. The developed method was applied for the determination of target thyroxines in urine samples, and the recovery for the spiking samples obtained by SBSE-HPLC-UV was in the range of 81.6–137.6% for human urine, while the recovery for the spiking urine samples obtained by SBSE-HPLC-ICP-MS were in the range of 72.0–121.5%.
Source:Journal of Chromatography A, Volume 1318
Author(s): Wenying Fan , Xiangju Mao , Man He , Beibei Chen , Bin Hu
In this work, polyethyleneglycol (PEG)/hydroxyl polydimethylsiloxane (OH-PDMS)/γ-mercaptopropyl trimethoxysilane (γ-MPTS) coated stir bar was prepared by sol–gel process and its extraction performance for the extraction of amphoteric thyroxines (3,3′,5,5′-tetraiodothyronin, T4; 3,3′,5-triiodothyronine, T3; reversed-3,3′,5-triiodothyronine, rT3) and their metabolite (3,5-diiodothyronine, T2) was studied. The preparation reproducibility of PEG/OH-PDMS/γ-MPTS coated stir bar was investigated, and the relative standard deviations (RSDs) in the same batch and among different batches were 3.3–14.3% (n =5) and 7.7–16.6% (n =3), respectively. The prepared PEG/OH-PDMS/γ-MPTS coated stir bar could be reused for more than 20 times. Based on this fact, a novel method of stir bar sorptive extraction (SBSE) combined with high performance liquid chromatography (HPLC)-ultraviolet (UV) and HPLC-inductively coupled plasma mass spectrometry (ICP-MS) for the analysis of target thyroxines in human urine samples was developed. The influencing factors of SBSE, such as sample pH, extraction time, stirring rate, salt effect, desorption solution and desorption time, were studied in detail, and the analytical performance of the proposed method was evaluated under the optimized conditions. The enrichment factors (EFs) of the developed method for four target thyroxines were in the range of 14.9–70.4 (theoretical enrichment factor was 100). The RSDs were ranging from 4.0% to 13.8% for SBSE-HPLC-UV (c =25μg/L, n =6) and from 3.7% to 6.1% for SBSE-HPLC-ICP-MS (c =0.5μg/L, n =5). The linear range obtained by SBSE-HPLC-UV was 2–500μg/L for T2 and 5–500μg/L for rT3, T3 and T4, with correlation coefficients (r) ranging from 0.9957 to 0.9998, respectively, while the linear range obtained by SBSE-HPLC-ICP-MS was 0.05–500μg/L for T2 and rT3, 0.10–200μg/L for T3 and 0.05–200μg/L for T4 with r ranging from 0.9979 to 0.9998, respectively. The limits of detection (LODs) for the target thyroxines were 0.60–2.20μg/L for SBSE-HPLC-UV and 0.0071–0.0355μg/L SBSE-HPLC-ICP-MS, respectively. The developed method was applied for the determination of target thyroxines in urine samples, and the recovery for the spiking samples obtained by SBSE-HPLC-UV was in the range of 81.6–137.6% for human urine, while the recovery for the spiking urine samples obtained by SBSE-HPLC-ICP-MS were in the range of 72.0–121.5%.
Grafting the sol–gel based sorbents by diazonium salts: A novel approach toward unbreakable capillary microextraction
07 November 2013,
09:10:03
Publication date: 29 November
2013
Source:Journal of Chromatography A, Volume 1318
Author(s): Habib Bagheri , Parisa Bayat , Hamed Piri-Moghadam
The present work deals with a novel approach for grafting a sol–gel based sorbent, using diazonium salts for preparation of an unbreakable capillary microextraction (CME) device in on-line combination with high performance liquid chromatography (HPLC). The use of diazonium salts modifier allowed all types of metallic and non-metallic substrates to be used without any limitation. Substrates including copper, brass, stainless steel and polytetrafluoroethylene (PTFE) were chosen to be functionalized by chemical or electrochemical reduction of 4-amino phenyl acetic acid. Then, 3-(trimethoxysilyl)propylamine (3TMSPA) was selected as the precursor and the only reagent for preparation of the desired surface chemical bonded sorbent. The presence of chemical bond between substrate, diazonium salts and 3TMSPA is more probably responsible for thermal and solvent stability and long lifetime of the prepared sorbent. Characterization of the aryl group formation on the various substrates along with the prepared sorbents was thoroughly investigated by scanning electron microscopy (SEM), Fourier transform infrared spectroscopy (FTIR) and thermogravimetry analysis (TGA). Typically, one of the prepared sorbents, deposited on the inner surface of the copper tube, was selected for assessing the developed method. The CME device was used for on-line extraction of atrazine, ametryn and terbutryn, as model compounds, from the aquatic media. After extraction, the HPLC mobile phase was used for on-line desorption and elution of the extracted analytes from the CME loop, containing the grafted sol–gel based sorbent, through the HPLC column. Figures of merit of the developed method were also obtained in which the linearity for the analytes was in the range of 30–1000μgL−1. The value of LOD (S/N=3) for all analytes was 10μgL−1 and the RSD% values (n =5) were all below 9.4% at the 500μgL−1 level. Applicability of the developed method was examined by analyzing some real water samples in which the relative recovery percentage ranged from 75 to 95%.
Source:Journal of Chromatography A, Volume 1318
Author(s): Habib Bagheri , Parisa Bayat , Hamed Piri-Moghadam
The present work deals with a novel approach for grafting a sol–gel based sorbent, using diazonium salts for preparation of an unbreakable capillary microextraction (CME) device in on-line combination with high performance liquid chromatography (HPLC). The use of diazonium salts modifier allowed all types of metallic and non-metallic substrates to be used without any limitation. Substrates including copper, brass, stainless steel and polytetrafluoroethylene (PTFE) were chosen to be functionalized by chemical or electrochemical reduction of 4-amino phenyl acetic acid. Then, 3-(trimethoxysilyl)propylamine (3TMSPA) was selected as the precursor and the only reagent for preparation of the desired surface chemical bonded sorbent. The presence of chemical bond between substrate, diazonium salts and 3TMSPA is more probably responsible for thermal and solvent stability and long lifetime of the prepared sorbent. Characterization of the aryl group formation on the various substrates along with the prepared sorbents was thoroughly investigated by scanning electron microscopy (SEM), Fourier transform infrared spectroscopy (FTIR) and thermogravimetry analysis (TGA). Typically, one of the prepared sorbents, deposited on the inner surface of the copper tube, was selected for assessing the developed method. The CME device was used for on-line extraction of atrazine, ametryn and terbutryn, as model compounds, from the aquatic media. After extraction, the HPLC mobile phase was used for on-line desorption and elution of the extracted analytes from the CME loop, containing the grafted sol–gel based sorbent, through the HPLC column. Figures of merit of the developed method were also obtained in which the linearity for the analytes was in the range of 30–1000μgL−1. The value of LOD (S/N=3) for all analytes was 10μgL−1 and the RSD% values (n =5) were all below 9.4% at the 500μgL−1 level. Applicability of the developed method was examined by analyzing some real water samples in which the relative recovery percentage ranged from 75 to 95%.
Semiautomated solid-phase extraction followed by derivatisation and gas chromatography–mass spectrometry for determination of perfluoroalkyl acids in water
07 November 2013,
09:10:03
Publication date: 29 November
2013
Source:Journal of Chromatography A, Volume 1318
Author(s): Beatriz Jurado-Sánchez , Evaristo Ballesteros , Mercedes Gallego
This paper describes a sensitive approach for the determination of 6 perfluoroalkyl carboxylic acids and perfluorooctane sulfonic acid in water. Samples were preconcentrated using an automatic solid-phase extraction module and then manually derivatised and determined by gas chromatography–mass spectrometry. The analytes were derivatised with a isobutyl chloroformate/isobutanol mixture, using 3% N,N-dicyclohexylcarbodiimide in pyridine as the catalyst. From a systematic comparison of several reversed-phase and anion-exchange sorbent materials for the retention of perfluoroalkyl acids, the highest retention efficiencies (∼100%) were achieved with LiChrolut EN and Discovery DSC-SAX columns. LiChrolut EN was the sorbent selected due to several advantages (sample pH ∼1; sample flow rate, 5.5mL/min; breakthrough volume, 300mL) over Discovery DSC-SAX (sample pH ∼6; sample flow rate, 3.0mL/min; breakthrough volume, 45mL), for the retention of the studied compounds. Detection and quantification limits within the range of 0.1–0.5ng/L and 0.4–1.7ng/L, respectively, were obtained for a sorbent column of 70mg of LiChrolut EN and 250mL of sample, the relative standard deviation being lower than 7%. The method was applied both to the analysis of water collected at the intake (raw) and at the exit (treated) of two drinking water treatment plants, as well as to various types of water. Few samples were positive for perfluoroalkyl acids and only one acid (perfluoroheptanoic or perfluorooctanoic) was found in each treatment plant. The highest number and concentration of analytes (perfluoroheptanoic, perfluorooctanoic and perfluorodecanoic acid) were found in one wastewater.
Source:Journal of Chromatography A, Volume 1318
Author(s): Beatriz Jurado-Sánchez , Evaristo Ballesteros , Mercedes Gallego
This paper describes a sensitive approach for the determination of 6 perfluoroalkyl carboxylic acids and perfluorooctane sulfonic acid in water. Samples were preconcentrated using an automatic solid-phase extraction module and then manually derivatised and determined by gas chromatography–mass spectrometry. The analytes were derivatised with a isobutyl chloroformate/isobutanol mixture, using 3% N,N-dicyclohexylcarbodiimide in pyridine as the catalyst. From a systematic comparison of several reversed-phase and anion-exchange sorbent materials for the retention of perfluoroalkyl acids, the highest retention efficiencies (∼100%) were achieved with LiChrolut EN and Discovery DSC-SAX columns. LiChrolut EN was the sorbent selected due to several advantages (sample pH ∼1; sample flow rate, 5.5mL/min; breakthrough volume, 300mL) over Discovery DSC-SAX (sample pH ∼6; sample flow rate, 3.0mL/min; breakthrough volume, 45mL), for the retention of the studied compounds. Detection and quantification limits within the range of 0.1–0.5ng/L and 0.4–1.7ng/L, respectively, were obtained for a sorbent column of 70mg of LiChrolut EN and 250mL of sample, the relative standard deviation being lower than 7%. The method was applied both to the analysis of water collected at the intake (raw) and at the exit (treated) of two drinking water treatment plants, as well as to various types of water. Few samples were positive for perfluoroalkyl acids and only one acid (perfluoroheptanoic or perfluorooctanoic) was found in each treatment plant. The highest number and concentration of analytes (perfluoroheptanoic, perfluorooctanoic and perfluorodecanoic acid) were found in one wastewater.
Adsorption of cations onto positively charged surface mesopores
07 November 2013,
09:10:03
Publication date: 29 November
2013
Source:Journal of Chromatography A, Volume 1318
Author(s): Uwe Neue , Pamela Iraneta , Fabrice Gritti , Georges Guiochon
Uwe Neue developed a theoretical treatment to account for the adsorption of ions on mesopores of packing materials the walls of which are bonded to ionic ligands but left this work unfinished. We elaborated upon this treatment and refined it, based on the equivalence that he suggested between charged surface particles and a membrane that separates two ionic solutions but is impermeable to one specification. He had written that the electro-chemical potentials in both ionic solutions are equal (Donnan equilibrium). The equilibrium between the surface and the pore concentrations is accounted for by an homogeneous electrostatically modified Langmuir (EML) isotherm model. The theoretical results are presented for four different charge surface concentrations σ 0 =0, 0.001, 0.002, and 0.003C/m2, using a phosphate buffer ( W S pH = 2.65 ) of ionic strength I =10mM. The average pore size, the specific surface area, and the specific pore volume of the stationary phase were D p =140Å, S p =182m2/g, and V p =0.70cm3/g, respectively. The theoretical results provide the quantitative difference between the ionic strength, the pH, and the concentrations of all the ions in the pores and in the bulk eluent. The theory predicts (1) that the retention times of cations under linear conditions is lower and (2) that their band widths under overloaded conditions for a given retention factor shrinks when the surface charge density σ 0 is increased. These theoretical results are in good agreement with experimental results published previously and explain them.
Source:Journal of Chromatography A, Volume 1318
Author(s): Uwe Neue , Pamela Iraneta , Fabrice Gritti , Georges Guiochon
Uwe Neue developed a theoretical treatment to account for the adsorption of ions on mesopores of packing materials the walls of which are bonded to ionic ligands but left this work unfinished. We elaborated upon this treatment and refined it, based on the equivalence that he suggested between charged surface particles and a membrane that separates two ionic solutions but is impermeable to one specification. He had written that the electro-chemical potentials in both ionic solutions are equal (Donnan equilibrium). The equilibrium between the surface and the pore concentrations is accounted for by an homogeneous electrostatically modified Langmuir (EML) isotherm model. The theoretical results are presented for four different charge surface concentrations σ 0 =0, 0.001, 0.002, and 0.003C/m2, using a phosphate buffer ( W S pH = 2.65 ) of ionic strength I =10mM. The average pore size, the specific surface area, and the specific pore volume of the stationary phase were D p =140Å, S p =182m2/g, and V p =0.70cm3/g, respectively. The theoretical results provide the quantitative difference between the ionic strength, the pH, and the concentrations of all the ions in the pores and in the bulk eluent. The theory predicts (1) that the retention times of cations under linear conditions is lower and (2) that their band widths under overloaded conditions for a given retention factor shrinks when the surface charge density σ 0 is increased. These theoretical results are in good agreement with experimental results published previously and explain them.
A high-throughput 2D-analytical technique to obtain single protein parameters from complex cell lysates for in silico process development of ion exchange chromatography
07 November 2013,
09:10:03
Publication date: 29 November
2013
Source:Journal of Chromatography A, Volume 1318
Author(s): Frieder Kröner , Dennis Elsäßer , Jürgen Hubbuch
The accelerating growth of the market for biopharmaceutical proteins, the market entry of biosimilars and the growing interest in new, more complex molecules constantly pose new challenges for bioseparation process development. In the presented work we demonstrate the application of a multidimensional, analytical separation approach to obtain the relevant physicochemical parameters of single proteins in a complex mixture for in silico chromatographic process development. A complete cell lysate containing a low titre target protein was first fractionated by multiple linear salt gradient anion exchange chromatography (AEC) with varying gradient length. The collected fractions were subsequently analysed by high-throughput capillary gel electrophoresis (HT-CGE) after being desalted and concentrated. From the obtained data of the 2D-separation the retention-volumes and the concentration of the single proteins were determined. The retention-volumes of the single proteins were used to calculate the related steric-mass action model parameters. In a final evaluation experiment the received parameters were successfully applied to predict the retention behaviour of the single proteins in salt gradient AEC.
Source:Journal of Chromatography A, Volume 1318
Author(s): Frieder Kröner , Dennis Elsäßer , Jürgen Hubbuch
The accelerating growth of the market for biopharmaceutical proteins, the market entry of biosimilars and the growing interest in new, more complex molecules constantly pose new challenges for bioseparation process development. In the presented work we demonstrate the application of a multidimensional, analytical separation approach to obtain the relevant physicochemical parameters of single proteins in a complex mixture for in silico chromatographic process development. A complete cell lysate containing a low titre target protein was first fractionated by multiple linear salt gradient anion exchange chromatography (AEC) with varying gradient length. The collected fractions were subsequently analysed by high-throughput capillary gel electrophoresis (HT-CGE) after being desalted and concentrated. From the obtained data of the 2D-separation the retention-volumes and the concentration of the single proteins were determined. The retention-volumes of the single proteins were used to calculate the related steric-mass action model parameters. In a final evaluation experiment the received parameters were successfully applied to predict the retention behaviour of the single proteins in salt gradient AEC.
Reversed-phase liquid chromatography with octadecylsilyl, immobilized artificial membrane and cholesterol columns in correlation studies with in silico biological descriptors of newly synthesized antiproliferative and analgesic active compounds
07 November 2013,
09:10:03
Publication date: 29 November
2013
Source:Journal of Chromatography A, Volume 1318
Author(s): Małgorzata Janicka , Małgorzata Sztanke , Krzysztof Sztanke
Reversed-phase liquid chromatography (RPLC) with different stationary phases, i.e., octadecylsilyl, immobilized artificial membrane and immobilized cholesterol, was used to study lipophilicity of 56 newly-designed 7,8-dihydroimidazo[2,1-c][1,2,4]triazin-4(6H)-ones and 2,6,7,8-tetrahydroimidazo[2,1-c][1,2,4]triazine-3,4-diones with potential anti-proliferative, anti-metastatic and analgesic activities. Extrapolated retention parameters that correspond to pure buffer as the mobile phase, i.e., log k w values are used as chromatographic lipophilicities. The lipophilic properties of compounds also are characterized by computed log P values and basic pharmacokinetic descriptors calculated in silico with the use of ACD/Percepta software according to Abraham's linear solvation energy relationship. Chromatographic and partitioning parameters are compared with biological descriptors using principal component analysis (PCA), and similarities and dissimilarities between variables and compounds are described. Highly significant, predictive relationships between biological descriptors and chromatographic parameters are obtained. Reversed parabolic relationships, which have very good statistical quality between various biological descriptors, i.e., log K sc , log K p , log BB, and log K hsa , and the log k w values, indicate the advantages of a cholesterol column in comparison with immobilized artificial membrane and octadecylsilyl stationary phase.
Source:Journal of Chromatography A, Volume 1318
Author(s): Małgorzata Janicka , Małgorzata Sztanke , Krzysztof Sztanke
Reversed-phase liquid chromatography (RPLC) with different stationary phases, i.e., octadecylsilyl, immobilized artificial membrane and immobilized cholesterol, was used to study lipophilicity of 56 newly-designed 7,8-dihydroimidazo[2,1-c][1,2,4]triazin-4(6H)-ones and 2,6,7,8-tetrahydroimidazo[2,1-c][1,2,4]triazine-3,4-diones with potential anti-proliferative, anti-metastatic and analgesic activities. Extrapolated retention parameters that correspond to pure buffer as the mobile phase, i.e., log k w values are used as chromatographic lipophilicities. The lipophilic properties of compounds also are characterized by computed log P values and basic pharmacokinetic descriptors calculated in silico with the use of ACD/Percepta software according to Abraham's linear solvation energy relationship. Chromatographic and partitioning parameters are compared with biological descriptors using principal component analysis (PCA), and similarities and dissimilarities between variables and compounds are described. Highly significant, predictive relationships between biological descriptors and chromatographic parameters are obtained. Reversed parabolic relationships, which have very good statistical quality between various biological descriptors, i.e., log K sc , log K p , log BB, and log K hsa , and the log k w values, indicate the advantages of a cholesterol column in comparison with immobilized artificial membrane and octadecylsilyl stationary phase.
Evaluation between ultrahigh pressure liquid chromatography and high-performance liquid chromatography analytical methods for characterizing natural dyestuffs
07 November 2013,
09:10:03
Publication date: 29 November
2013
Source:Journal of Chromatography A, Volume 1318
Author(s): Ana Serrano , Maarten van Bommel , Jessica Hallett
An evaluation was undertaken of ultrahigh pressure liquid chromatography (UHPLC) in comparison to high-performance liquid chromatography (HPLC) for characterizing natural dyes in cultural heritage objects. A new UHPLC method was optimized by testing several analytical parameters adapted from prior UHPLC studies developed in diverse fields of research. Different gradient elution programs were tested on seven UHPLC columns with different dimensions and stationary phase compositions by applying several mobile phases, flow rates, temperatures, and runtimes. The UHPLC method successfully provided more improved data than that achieved by the HPLC method. Indeed, even though carminic acid has shown circa 146% higher resolution with HPLC, UHPLC resulted in an increase of 41–61% resolution and a decrease of 91–422% limit of detection, depending on the dye compound. The optimized method was subsequently assigned to analyse 59 natural reference materials, in which 85 different components were ascribed with different physicochemical properties, in order to create a spectral database for future characterization of dyes in cultural heritage objects. The majority of these reference samples could be successfully distinguished with one single method through the examination of these compounds’ retention times and their spectra acquired with a photodiode array detector. These results demonstrate that UHPLC analyses are extremely valuable for the acquisition of more precise chromatographic information concerning natural dyes with complex mixtures of different and/or closely related physicochemical properties, essential for distinguishing similar species of plants and animals used to colour cultural heritage objects.
Source:Journal of Chromatography A, Volume 1318
Author(s): Ana Serrano , Maarten van Bommel , Jessica Hallett
An evaluation was undertaken of ultrahigh pressure liquid chromatography (UHPLC) in comparison to high-performance liquid chromatography (HPLC) for characterizing natural dyes in cultural heritage objects. A new UHPLC method was optimized by testing several analytical parameters adapted from prior UHPLC studies developed in diverse fields of research. Different gradient elution programs were tested on seven UHPLC columns with different dimensions and stationary phase compositions by applying several mobile phases, flow rates, temperatures, and runtimes. The UHPLC method successfully provided more improved data than that achieved by the HPLC method. Indeed, even though carminic acid has shown circa 146% higher resolution with HPLC, UHPLC resulted in an increase of 41–61% resolution and a decrease of 91–422% limit of detection, depending on the dye compound. The optimized method was subsequently assigned to analyse 59 natural reference materials, in which 85 different components were ascribed with different physicochemical properties, in order to create a spectral database for future characterization of dyes in cultural heritage objects. The majority of these reference samples could be successfully distinguished with one single method through the examination of these compounds’ retention times and their spectra acquired with a photodiode array detector. These results demonstrate that UHPLC analyses are extremely valuable for the acquisition of more precise chromatographic information concerning natural dyes with complex mixtures of different and/or closely related physicochemical properties, essential for distinguishing similar species of plants and animals used to colour cultural heritage objects.
Effect of pressure on secondary structure of proteins under ultra high pressure liquid chromatographic conditions
07 November 2013,
09:10:03
Publication date: 29 November
2013
Source:Journal of Chromatography A, Volume 1318
Author(s): Alexey Makarov , Rosario LoBrutto , Paul Karpinski
There are several spectroscopic techniques such as IR and CD, that allow for analyzing protein secondary structure in solution. However, a majority of these techniques require using purified protein, concentrated enough in the solution, to produce a relevant spectrum. Fundamental principles for the usage of reversed-phase ultra high pressure liquid chromatography (UHPLC) as an alternative technique to study protein secondary structures in solution were investigated. Several “model” proteins, as well as several small ionizable and neutral molecules, were used for these studies. The studies were conducted with UHPLC in isocratic mode, using premixed mobile phases at constant flow rate and temperature. The pressure was modified by a backpressure regulator from about 6000psi to about 12,000psi. It was found that when using a mobile phase composition at which proteins were fully denatured (loss of alpha-helix secondary structure), the retention factors of the proteins increased upon pressure increase in the same manner as non-proteins. When using a mobile phase composition in which proteins were not fully denatured, it was observed that the retention factors of the proteins displayed a much steeper (by one order of magnitude) increase in retention upon pressure increase. It was concluded that in a mobile phase in which the protein is not initially fully denatured, the increase of pressure may facilitate the folding back of the protein to its native state (alpha-helix secondary structure). The impact of different mobile phase compositions on the denaturation of the proteins was studied using CD (Circular Dichroism). Moreover, the effect of flow rate on retention of proteins and small molecules was studied at constant pressure on the different pore size silicas and the impact of internal frictional heating was evaluated.
Source:Journal of Chromatography A, Volume 1318
Author(s): Alexey Makarov , Rosario LoBrutto , Paul Karpinski
There are several spectroscopic techniques such as IR and CD, that allow for analyzing protein secondary structure in solution. However, a majority of these techniques require using purified protein, concentrated enough in the solution, to produce a relevant spectrum. Fundamental principles for the usage of reversed-phase ultra high pressure liquid chromatography (UHPLC) as an alternative technique to study protein secondary structures in solution were investigated. Several “model” proteins, as well as several small ionizable and neutral molecules, were used for these studies. The studies were conducted with UHPLC in isocratic mode, using premixed mobile phases at constant flow rate and temperature. The pressure was modified by a backpressure regulator from about 6000psi to about 12,000psi. It was found that when using a mobile phase composition at which proteins were fully denatured (loss of alpha-helix secondary structure), the retention factors of the proteins increased upon pressure increase in the same manner as non-proteins. When using a mobile phase composition in which proteins were not fully denatured, it was observed that the retention factors of the proteins displayed a much steeper (by one order of magnitude) increase in retention upon pressure increase. It was concluded that in a mobile phase in which the protein is not initially fully denatured, the increase of pressure may facilitate the folding back of the protein to its native state (alpha-helix secondary structure). The impact of different mobile phase compositions on the denaturation of the proteins was studied using CD (Circular Dichroism). Moreover, the effect of flow rate on retention of proteins and small molecules was studied at constant pressure on the different pore size silicas and the impact of internal frictional heating was evaluated.
Fast gradient separation by very high pressure liquid chromatography: Reproducibility of analytical data and influence of delay between successive runs
07 November 2013,
09:10:03
Publication date: 29 November
2013
Source:Journal of Chromatography A, Volume 1318
Author(s): Joseph J. Stankovich , Fabrice Gritti , Lois Ann Beaver , Paul G. Stevenson , Georges Guiochon
Five methods were used to implement fast gradient separations: constant flow rate, constant column-wall temperature, constant inlet pressure at moderate and high pressures (controlled by a pressure controller), and programmed flow constant pressure. For programmed flow constant pressure, the flow rates and gradient compositions are controlled using input into the method instead of the pressure controller. Minor fluctuations in the inlet pressure do not affect the mobile phase flow rate in programmed flow. The reproducibilities of the retention times, the response factors, and the eluted band width of six successive separations of the same sample (9 components) were measured with different equilibration times between 0 and 15min. The influence of the length of the equilibration time on these reproducibilities is discussed. The results show that the average column temperature may increase from one separation to the next and that this contributes to fluctuation of the results.
Source:Journal of Chromatography A, Volume 1318
Author(s): Joseph J. Stankovich , Fabrice Gritti , Lois Ann Beaver , Paul G. Stevenson , Georges Guiochon
Five methods were used to implement fast gradient separations: constant flow rate, constant column-wall temperature, constant inlet pressure at moderate and high pressures (controlled by a pressure controller), and programmed flow constant pressure. For programmed flow constant pressure, the flow rates and gradient compositions are controlled using input into the method instead of the pressure controller. Minor fluctuations in the inlet pressure do not affect the mobile phase flow rate in programmed flow. The reproducibilities of the retention times, the response factors, and the eluted band width of six successive separations of the same sample (9 components) were measured with different equilibration times between 0 and 15min. The influence of the length of the equilibration time on these reproducibilities is discussed. The results show that the average column temperature may increase from one separation to the next and that this contributes to fluctuation of the results.
Determination of egg yolk xanthophylls by isocratic high-performance liquid chromatography
07 November 2013,
09:10:03
Publication date: 29 November
2013
Source:Journal of Chromatography A, Volume 1318
Author(s): Lučka Brulc , Breda Simonovska , Irena Vovk , Vesna Glavnik
An isocratic HPLC method was developed for the determination of eight xanthophylls (lutein, capsanthin, zeaxanthin, canthaxanthin, β-apo-8′-carotenal, ethyl-8′-apo-β-carotene-8′-oate, citranaxanthin and β-cryptoxanthin; registered as additives in poultry feeding) in egg yolks. Optimum separation of all-E-isomers of these xanthophylls was achieved in less than 18min on a ProntoSIL C30 column at 27°C using acetone–methanol–0.5M triethylammonium acetate buffer pH 7 14:5:1 (v/v) as the mobile phase with a flow rate of 1mL/min using spectrophotometric detection at 450nm. Other mobile phases were also found suitable, including acetone–water 93:7 (v/v) and acetone–methanol 1:4 (v/v) and the influences of column temperature on the separation and addition of triethylammonium acetate buffer pH 7 to the mobile phase on enhancement of the peak areas were evaluated. Preparation of test solution from yolks included a short vortexing of 0.5g of yolk in 10mL of acetone, followed by 15min magnetic stirring under nitrogen and centrifugation. The method was validated for 5 analytes. The calibration range was between 0.04 and 2μg/mL and the mean recovery of the analytes (95%) and the intra-day precision of the method (RSD less than 5%) on three levels in triplicates were obtained. Analyses of eggs from four husbandry classes showed the presence of up to four xanthophylls (lutein, zeaxanthin, canthaxanthin and ethyl-8′-apo-β-carotene-8′-oate) and traces of β-cryptoxanthin.
Source:Journal of Chromatography A, Volume 1318
Author(s): Lučka Brulc , Breda Simonovska , Irena Vovk , Vesna Glavnik
An isocratic HPLC method was developed for the determination of eight xanthophylls (lutein, capsanthin, zeaxanthin, canthaxanthin, β-apo-8′-carotenal, ethyl-8′-apo-β-carotene-8′-oate, citranaxanthin and β-cryptoxanthin; registered as additives in poultry feeding) in egg yolks. Optimum separation of all-E-isomers of these xanthophylls was achieved in less than 18min on a ProntoSIL C30 column at 27°C using acetone–methanol–0.5M triethylammonium acetate buffer pH 7 14:5:1 (v/v) as the mobile phase with a flow rate of 1mL/min using spectrophotometric detection at 450nm. Other mobile phases were also found suitable, including acetone–water 93:7 (v/v) and acetone–methanol 1:4 (v/v) and the influences of column temperature on the separation and addition of triethylammonium acetate buffer pH 7 to the mobile phase on enhancement of the peak areas were evaluated. Preparation of test solution from yolks included a short vortexing of 0.5g of yolk in 10mL of acetone, followed by 15min magnetic stirring under nitrogen and centrifugation. The method was validated for 5 analytes. The calibration range was between 0.04 and 2μg/mL and the mean recovery of the analytes (95%) and the intra-day precision of the method (RSD less than 5%) on three levels in triplicates were obtained. Analyses of eggs from four husbandry classes showed the presence of up to four xanthophylls (lutein, zeaxanthin, canthaxanthin and ethyl-8′-apo-β-carotene-8′-oate) and traces of β-cryptoxanthin.
Synchronized gradient elution in capillary liquid chromatography
07 November 2013,
09:10:03
Publication date: 29 November
2013
Source:Journal of Chromatography A, Volume 1318
Author(s): Enrique Javier Carrasco-Correa , Agustín Acquaviva , José Manuel Herrero-Martínez , Guillermo Ramis-Ramos
The synchronization of injection valve operation and gradient elution in capillary liquid chromatography (cHPLC) is studied. Focus is placed on the cHPLC systems which rely on the splitting of a primary flow to provide the much smaller secondary flow required at the injection device and analytical column. Owing to the tiny secondary flow rates, synchronization is necessary to achieve proper optimization of gradient elution methods. Otherwise, there is a risk of having the analytes totally or partially eluted in the initial isocratic conditions, and there is no control on the actual gradient profile reaching the column. Synchronization is first achieved by switching back the valve to bypass after injection. This is important to save time, and to avoid the gradient slope to be reduced by mixing within the internal volume of the injector (a 47% of slope reduction, in the conditions used in this work). Valve switching to bypass should be produced immediately after the arrival of the end of the sample plug to the valve (t V ). Fine system synchronization is further achieved by starting the gradient at the match time (t M ), which is the time required to match the arrival of both the gradient front and the end of the sample plug to the valve, and therefore also to the column inlet. Synchronization of these two events requires starting the gradient either before or even after the injection, thus to prevent a late or an early arrival of the gradient front to the injection valve, respectively. Owing to their dependence with the backpressure, both t V and t M should be measured in the presence of the column at the initial gradient conditions. Simple experiments designed to measure t V and t M are described. With synchronization according to the techniques described in this work, control on the real gradient elution conditions at the column location is maintained, the analysis time is reduced and efficiency improves. The effects of synchronization are illustrated by injecting a mixture of alkylbenzenes. At 1μL min−1, valve switching to bypass reduced analysis time from ca. 36 to 12min (butylbenzene), and improved peak symmetry (from 2.00 to 0.94 for methylbenzene) and efficiency (the average apparent plate count increased approximately 60%). Synchronization according to the match time further improved efficiency (approximately, up to 120%).
Source:Journal of Chromatography A, Volume 1318
Author(s): Enrique Javier Carrasco-Correa , Agustín Acquaviva , José Manuel Herrero-Martínez , Guillermo Ramis-Ramos
The synchronization of injection valve operation and gradient elution in capillary liquid chromatography (cHPLC) is studied. Focus is placed on the cHPLC systems which rely on the splitting of a primary flow to provide the much smaller secondary flow required at the injection device and analytical column. Owing to the tiny secondary flow rates, synchronization is necessary to achieve proper optimization of gradient elution methods. Otherwise, there is a risk of having the analytes totally or partially eluted in the initial isocratic conditions, and there is no control on the actual gradient profile reaching the column. Synchronization is first achieved by switching back the valve to bypass after injection. This is important to save time, and to avoid the gradient slope to be reduced by mixing within the internal volume of the injector (a 47% of slope reduction, in the conditions used in this work). Valve switching to bypass should be produced immediately after the arrival of the end of the sample plug to the valve (t V ). Fine system synchronization is further achieved by starting the gradient at the match time (t M ), which is the time required to match the arrival of both the gradient front and the end of the sample plug to the valve, and therefore also to the column inlet. Synchronization of these two events requires starting the gradient either before or even after the injection, thus to prevent a late or an early arrival of the gradient front to the injection valve, respectively. Owing to their dependence with the backpressure, both t V and t M should be measured in the presence of the column at the initial gradient conditions. Simple experiments designed to measure t V and t M are described. With synchronization according to the techniques described in this work, control on the real gradient elution conditions at the column location is maintained, the analysis time is reduced and efficiency improves. The effects of synchronization are illustrated by injecting a mixture of alkylbenzenes. At 1μL min−1, valve switching to bypass reduced analysis time from ca. 36 to 12min (butylbenzene), and improved peak symmetry (from 2.00 to 0.94 for methylbenzene) and efficiency (the average apparent plate count increased approximately 60%). Synchronization according to the match time further improved efficiency (approximately, up to 120%).
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