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Selected papers from the latest issue:
Role of chromatography in the development of Standard Reference Materials for organic analysis
05 October 2012,
08:32:43
Publication year:
2012
Source:Journal of Chromatography A, Volume 1261
Stephen A. Wise, Karen W. Phinney, Lane C. Sander, Michele M. Schantz
The certification of chemical constituents in natural-matrix Standard Reference Materials (SRMs) at the National Institute of Standards and Technology (NIST) can require the use of two or more independent analytical methods. The independence among the methods is generally achieved by taking advantage of differences in extraction, separation, and detection selectivity. This review describes the development of the independent analytical methods approach at NIST, and its implementation in the measurement of organic constituents such as contaminants in environmental materials, nutrients and marker compounds in food and dietary supplement matrices, and health diagnostic and nutritional assessment markers in human serum. The focus of this review is the important and critical role that separation science techniques play in achieving the necessary independence of the analytical steps in the measurement of trace-level organic constituents in natural matrix SRMs.
Source:Journal of Chromatography A, Volume 1261
Stephen A. Wise, Karen W. Phinney, Lane C. Sander, Michele M. Schantz
The certification of chemical constituents in natural-matrix Standard Reference Materials (SRMs) at the National Institute of Standards and Technology (NIST) can require the use of two or more independent analytical methods. The independence among the methods is generally achieved by taking advantage of differences in extraction, separation, and detection selectivity. This review describes the development of the independent analytical methods approach at NIST, and its implementation in the measurement of organic constituents such as contaminants in environmental materials, nutrients and marker compounds in food and dietary supplement matrices, and health diagnostic and nutritional assessment markers in human serum. The focus of this review is the important and critical role that separation science techniques play in achieving the necessary independence of the analytical steps in the measurement of trace-level organic constituents in natural matrix SRMs.
Highlights
► Key to independent method for SRM certification is separation science. ► For SRMs, the goal is to maximize the differences in extraction, separation and detection. ► Differences in stationary phase selectivity in LC and GC provide independence. ► SRMs for environmental, food, and dietary supplement analysis are described.Current-day employment of the micro-bore open-tubular capillary column in the gas chromatography field
05 October 2012,
08:32:43
Publication year:
2012
Source:Journal of Chromatography A, Volume 1261
Peter Quinto Tranchida, Luigi Mondello
The present contribution is focused on the exploitation of the micro-bore (MB) open-tubular column (ID≤0.18mm), in current-day gas chromatography. The vast majority of GC methods involve the use of MB columns, from a variety of one-dimensional techniques (conventional and high speed), to different forms of heart-cutting and comprehensive multidimensional methodologies. Various aspects related to the use of MB open-tubular capillaries will be described and critically discussed, along with a series of pertinent applications. A series of works from the past have also been cited, essentially because many approaches today-applied, derive from the ideas of the past. Obviously, the present contribution does not review the entire history of the micro-bore capillary (more than one book would be necessary); rather, it intends to portray the great contribution that such analytical tools provide in enhancing two fundamental GC features, namely separation power and speed.
Source:Journal of Chromatography A, Volume 1261
Peter Quinto Tranchida, Luigi Mondello
The present contribution is focused on the exploitation of the micro-bore (MB) open-tubular column (ID≤0.18mm), in current-day gas chromatography. The vast majority of GC methods involve the use of MB columns, from a variety of one-dimensional techniques (conventional and high speed), to different forms of heart-cutting and comprehensive multidimensional methodologies. Various aspects related to the use of MB open-tubular capillaries will be described and critically discussed, along with a series of pertinent applications. A series of works from the past have also been cited, essentially because many approaches today-applied, derive from the ideas of the past. Obviously, the present contribution does not review the entire history of the micro-bore capillary (more than one book would be necessary); rather, it intends to portray the great contribution that such analytical tools provide in enhancing two fundamental GC features, namely separation power and speed.
Highlights
► An overview of the use of the open-tubular micro-bore column in one-dimensional GC applications. ► An overview of the use of the open-tubular micro-bore column in multidimensional GC-based methods. ► Requirements, advantages, and disadvantages related to the use of the open-tubular micro-bore column in the GC field.Person-portable gas chromatography: Rapid temperature program operation through resistive heating of columns with inherently low thermal mass properties
05 October 2012,
08:32:43
Publication year:
2012
Source:Journal of Chromatography A, Volume 1261
Philip A. Smith
As open tubular gas chromatography was becoming widely adopted, the potential to rapidly heat and cool the low thermal mass of an open tubular fused silica column was recognized. Numerous resistive column heating approaches were subsequently described and demonstrated, often with a common objective to focus heating efforts on the column alone, rather than on a large convection oven. Low thermal mass column bundles have been commercially available for about ten years, where insulated wires in close proximity to a coiled open tubular capillary column provide resistive heating. Before this, person-portable gas chromatographs either operated isothermally at relatively low temperatures or at ambient temperature to lessen power demands, but several person-portable gas chromatography–mass spectrometry (GC–MS) instruments capable of temperature program operation have become available in the past ten years based on this heating method. When low thermal mass heated zones are used, and with a direct GC–MS interface, analysis times of less than 5min are possible for target compounds having a wide range of volatilities. Previous capabilities in transportable and person-portable gas chromatography instrumentation are reviewed to demonstrate the scale of advancement made possible by the adoption of open tubular columns and low power heating techniques now becoming routinely available. Microcomachined columns which are usually etched in a silicon wafer represent a radical break from the traditional fused silica open tubular column design, and increasing efforts to use this column construction approach are also examined. The developments discussed have introduced the potential to rapidly analyze compounds with a wide volatility range in the field to protect deployed military forces, the health of workers, and the health of the general public.
Source:Journal of Chromatography A, Volume 1261
Philip A. Smith
As open tubular gas chromatography was becoming widely adopted, the potential to rapidly heat and cool the low thermal mass of an open tubular fused silica column was recognized. Numerous resistive column heating approaches were subsequently described and demonstrated, often with a common objective to focus heating efforts on the column alone, rather than on a large convection oven. Low thermal mass column bundles have been commercially available for about ten years, where insulated wires in close proximity to a coiled open tubular capillary column provide resistive heating. Before this, person-portable gas chromatographs either operated isothermally at relatively low temperatures or at ambient temperature to lessen power demands, but several person-portable gas chromatography–mass spectrometry (GC–MS) instruments capable of temperature program operation have become available in the past ten years based on this heating method. When low thermal mass heated zones are used, and with a direct GC–MS interface, analysis times of less than 5min are possible for target compounds having a wide range of volatilities. Previous capabilities in transportable and person-portable gas chromatography instrumentation are reviewed to demonstrate the scale of advancement made possible by the adoption of open tubular columns and low power heating techniques now becoming routinely available. Microcomachined columns which are usually etched in a silicon wafer represent a radical break from the traditional fused silica open tubular column design, and increasing efforts to use this column construction approach are also examined. The developments discussed have introduced the potential to rapidly analyze compounds with a wide volatility range in the field to protect deployed military forces, the health of workers, and the health of the general public.
Highlights
► Early portable gas chromatographs operated at ambient or low isothermal temperature. ► Rapid resistive heating of low thermal mass columns is possible using little power. ► Resistive temperature programming is now used for person-portable GC instruments. ► Fast GC analysis in the field is now possible, even for heavy compounds such as PCBs. ► Better instrumentation answers exposure questions quickly to protect human health.Gas chromatography using resistive heating technology
05 October 2012,
08:32:43
Publication year:
2012
Source:Journal of Chromatography A, Volume 1261
Anzi Wang, H. Dennis Tolley, Milton L. Lee
Air bath ovens are standard in conventional gas chromatography (GC) instruments because of their simplicity and reliability for column temperature control. However, their low heating rates, high power consumption and bulky size are in conflict with the increasing demands for fast separation and portable instrumentation. The deficiencies of air bath ovens can be eliminated using resistive heating technology, as the column is conductively heated by compact resistive heaters with low thermal mass. Resistive heating methods were employed in the early years of GC history, and they are emerging again as instrumentation is becoming more compact and sophisticated. Numerous designs have been tested and some have been successfully commercialized. Development of portable GC systems, including lab-on-a-chip devices, greatly benefits from the use of small, low-power resistive heating hardware. High speed GC separations using conventional instruments also can be best achieved with resistive heating modules. Despite some of its own inherent disadvantages, including efficiency loss, complex manufacturing and inconvenient column maintenance, resistive heating is expected to rapidly become a mature technology and even replace oven heating in the not-to-distant future.
Source:Journal of Chromatography A, Volume 1261
Anzi Wang, H. Dennis Tolley, Milton L. Lee
Air bath ovens are standard in conventional gas chromatography (GC) instruments because of their simplicity and reliability for column temperature control. However, their low heating rates, high power consumption and bulky size are in conflict with the increasing demands for fast separation and portable instrumentation. The deficiencies of air bath ovens can be eliminated using resistive heating technology, as the column is conductively heated by compact resistive heaters with low thermal mass. Resistive heating methods were employed in the early years of GC history, and they are emerging again as instrumentation is becoming more compact and sophisticated. Numerous designs have been tested and some have been successfully commercialized. Development of portable GC systems, including lab-on-a-chip devices, greatly benefits from the use of small, low-power resistive heating hardware. High speed GC separations using conventional instruments also can be best achieved with resistive heating modules. Despite some of its own inherent disadvantages, including efficiency loss, complex manufacturing and inconvenient column maintenance, resistive heating is expected to rapidly become a mature technology and even replace oven heating in the not-to-distant future.
Highlights
► Resistive heating methods for gas chromatography are described. ► Fast heating and cooling allow high-speed separations and high analytical throughput. ► Low power consumption and small size are critical for portable/microfabricated GC. ► Resistive heating technology benefits both laboratory analysis and field detection.Qualitative and quantitative approaches in comprehensive two-dimensional gas chromatography
05 October 2012,
08:32:43
Publication year:
2012
Source:Journal of Chromatography A, Volume 1261
Jacolin A. Murray
Comprehensive two-dimensional gas chromatography (GC×GC) offers advantages over one-dimensional gas chromatography (GC) including, high peak capacity, signal enhancement, and structured chromatograms. These advantages have been exploited to solve several analytical problems that are difficult to achieve in GC. In this review, qualitative and quantitative approaches of GC×GC are explored, including targeted, non-targeted, group, and fingerprinting analysis.
Source:Journal of Chromatography A, Volume 1261
Jacolin A. Murray
Comprehensive two-dimensional gas chromatography (GC×GC) offers advantages over one-dimensional gas chromatography (GC) including, high peak capacity, signal enhancement, and structured chromatograms. These advantages have been exploited to solve several analytical problems that are difficult to achieve in GC. In this review, qualitative and quantitative approaches of GC×GC are explored, including targeted, non-targeted, group, and fingerprinting analysis.
Highlights
► Three main advantages of two-dimensional gas chromatography are explored. ► Quantitation methods including traditional and chemometric methods are reviewed. ► Targeted, non-targeted, group, and fingerprinting analysis methods are reviewed.Maximizing the speed of separations for industrial problems
05 October 2012,
08:32:43
Publication year:
2012
Source:Journal of Chromatography A, Volume 1261
T.L. Chester
Recent improvement efforts in chromatography have provided great improvements in the rate of plate production, but less attention has been spent on optimizing the kinds of problems that are most often encountered in industry. When factors are not independent in their effects on the responses of a chromatographic separation, all adjustable factors must be considered in concert in seeking the best or optimum condition that solves the problem. This requires careful attention to specifying the goals, the adjustable factors, and the constraints required to make sure the outcome can actually be implemented. Strategies for optimizing assay and screening methods in the context of industrial needs are presented. Expanding the factor space of the system being investigated can lead to better outcomes. The prospect of adding column-outlet pressure control and expanding the mobile phase composition to include condensed gases or supercritical fluids is explored. Reversed-phase liquid chromatography, hydrophilic interaction chromatography, electrostatic repulsion hydrophilic interaction chromatography, and supercritical fluid chromatography are contiguous with regard to mobile phase characteristics. Adjustment of selectivity through instrument-controlled factors can benefit method development. Opportunities obtained by blending modifiers, varying temperature and pressure with compressible mobile phases, and controlling pH are discussed in the context of optimizing methods.
Source:Journal of Chromatography A, Volume 1261
T.L. Chester
Recent improvement efforts in chromatography have provided great improvements in the rate of plate production, but less attention has been spent on optimizing the kinds of problems that are most often encountered in industry. When factors are not independent in their effects on the responses of a chromatographic separation, all adjustable factors must be considered in concert in seeking the best or optimum condition that solves the problem. This requires careful attention to specifying the goals, the adjustable factors, and the constraints required to make sure the outcome can actually be implemented. Strategies for optimizing assay and screening methods in the context of industrial needs are presented. Expanding the factor space of the system being investigated can lead to better outcomes. The prospect of adding column-outlet pressure control and expanding the mobile phase composition to include condensed gases or supercritical fluids is explored. Reversed-phase liquid chromatography, hydrophilic interaction chromatography, electrostatic repulsion hydrophilic interaction chromatography, and supercritical fluid chromatography are contiguous with regard to mobile phase characteristics. Adjustment of selectivity through instrument-controlled factors can benefit method development. Opportunities obtained by blending modifiers, varying temperature and pressure with compressible mobile phases, and controlling pH are discussed in the context of optimizing methods.
Highlights
► Goal setting depends on the separation needs and the capabilities of the system. ► A multi-dimensional space describes the adjustable factors. ► Expanding the factor space adds potential improvement. ► Several means of adjusting selectivity without system changes are reviewed. ► We summarize a method development approach utilizing modeling and optimization.Advanced proteomic liquid chromatography
05 October 2012,
08:32:43
Publication year:
2012
Source:Journal of Chromatography A, Volume 1261
Fang Xie, Richard D. Smith, Yufeng Shen
Liquid chromatography coupled with mass spectrometry is the predominant platform used to analyze proteomics samples consisting of large numbers of proteins and their proteolytic products (e.g., truncated polypeptides) and spanning a wide range of relative concentrations. This review provides an overview of advanced capillary liquid chromatography techniques and methodologies that greatly improve separation resolving power and proteomics analysis coverage, sensitivity, and throughput.
Source:Journal of Chromatography A, Volume 1261
Fang Xie, Richard D. Smith, Yufeng Shen
Liquid chromatography coupled with mass spectrometry is the predominant platform used to analyze proteomics samples consisting of large numbers of proteins and their proteolytic products (e.g., truncated polypeptides) and spanning a wide range of relative concentrations. This review provides an overview of advanced capillary liquid chromatography techniques and methodologies that greatly improve separation resolving power and proteomics analysis coverage, sensitivity, and throughput.
Highlights
► Reviewed the advances in separation power, sensitivity and throughput in 1D LC. ► Reviewed the different combinations of 2D LC. ► Compared the pros and cons of 1D and 2D LC in proteomics.Development of coatings for automated 96-blade solid phase microextraction-liquid chromatography–tandem mass spectrometry system, capable of extracting a wide polarity range of analytes from biological fluids
05 October 2012,
08:32:43
Publication year:
2012
Source:Journal of Chromatography A, Volume 1261
Fatemeh S. Mirnaghi, Janusz Pawliszyn
This work presents the development and evaluation of biocompatible polyacrylonitrile–polystyrene–divinylbenzene (PAN–PS–DVB) and polyacrylonitrile–phenylboronic acid (PAN–PBA) coatings for automated 96-blades (thin-film) solid phase microextraction (SPME) system, using high performance liquid chromatography (HPLC) coupled with tandem mass spectrometry (MS/MS). The SPME condition was optimized for 60min equilibrium extraction and 40min desorption for PAN–PS–DVB, and 120min equilibrium extraction and 60min desorption for PAN–PBA for parallel sample preparation of up to 96 samples. The thin film geometry of the SPME blades provided good extraction efficiency due to the larger surface area of the coating, and simultaneous sample preparation provided fast and accurate analysis. The PAN–PS–DVB and PAN–PBA 96-blade SPME coatings were evaluated for extraction of analytes in a wide range of polarity (log P =2.8 to −3.7), and they demonstrated efficient extraction recovery (3.5–98.9% for PAN–PS–DVB and 4.0–74.1% for PAN–PBA) for both polar and non-polar groups of compounds. Reusability, reproducibility, and reliability of the system were evaluated. The results demonstrated that both coatings presented chemical and mechanical stability and long-lasting extraction efficiency for more than 100 usages in phosphate-buffered saline (PBS) and human plasma.
Source:Journal of Chromatography A, Volume 1261
Fatemeh S. Mirnaghi, Janusz Pawliszyn
This work presents the development and evaluation of biocompatible polyacrylonitrile–polystyrene–divinylbenzene (PAN–PS–DVB) and polyacrylonitrile–phenylboronic acid (PAN–PBA) coatings for automated 96-blades (thin-film) solid phase microextraction (SPME) system, using high performance liquid chromatography (HPLC) coupled with tandem mass spectrometry (MS/MS). The SPME condition was optimized for 60min equilibrium extraction and 40min desorption for PAN–PS–DVB, and 120min equilibrium extraction and 60min desorption for PAN–PBA for parallel sample preparation of up to 96 samples. The thin film geometry of the SPME blades provided good extraction efficiency due to the larger surface area of the coating, and simultaneous sample preparation provided fast and accurate analysis. The PAN–PS–DVB and PAN–PBA 96-blade SPME coatings were evaluated for extraction of analytes in a wide range of polarity (log P =2.8 to −3.7), and they demonstrated efficient extraction recovery (3.5–98.9% for PAN–PS–DVB and 4.0–74.1% for PAN–PBA) for both polar and non-polar groups of compounds. Reusability, reproducibility, and reliability of the system were evaluated. The results demonstrated that both coatings presented chemical and mechanical stability and long-lasting extraction efficiency for more than 100 usages in phosphate-buffered saline (PBS) and human plasma.
Highlights
► Developments and evaluation of two new stationary phases for 96-blade SPME system. ► Coatings were capable of extracting compounds with wide a range of polarity. ► The coatings demonstrated biocompatibility, reusability, and reproducibility for analysis of biological samples.Optimization of solid phase microextraction for non-lethal in vivo determination of selected pharmaceuticals in fish muscle using liquid chromatography–mass spectrometry
05 October 2012,
08:32:43
Publication year:
2012
Source:Journal of Chromatography A, Volume 1261
Oluranti P. Togunde, Ken D. Oakes, Mark R. Servos, Janusz Pawliszyn
A new thin film microextraction (TFME) configuration based on C18 thin film was optimized to improve solid phase microextraction (SPME) sensitivity and extraction kinetics for in vivo determinations of trace pharmaceuticals in fish tissue. Optimization of SPME involved increasing sampler surface area and volume of extraction phase to improve performance within in vitro applications such as agarose gel matrix and spiked fish tissue, as well as in vivo determinations of pharmaceuticals in fish exposed to municipal wastewater. Rainbow trout (Oncorhynchus mykiss) were exposed for 4d to effluents from a municipal wastewater plant that was treated under three different configurations of a pilot plant. Fathead minnow (Pimephales promelas) were caged for 14d upstream and downstream of municipal wastewater effluent discharges along Grand River at near Kitchener, ON. TFME and regular C18 fibers were inserted into the dorsal-epaxial muscle of rainbow trout and fathead minnow, respectively, for 30min sampling intervals. Sample extracts obtained from fibers were desorbed in methanol:water (3:2) for 90min under 1500rpm agitation and analyzed using liquid chromatography coupled with tandem mass spectrometry (LC/MS–MS) to determine pharmaceutical bioconcentration. Most target pharmaceuticals were detected in the wastewater with the exception of norfluoxetine and paroxetine. C18 TFME phase successfully quantified fluoxetine, venlafaxine, sertraline, paroxetine, and carbamazapine in muscle of living fish at concentrations ranging from 1.7 to 259ng/g. Reproducibility of the method in spiked fish muscle was 9–18% RSD with limits of detection and quantification ranging from 0.08 to 0.21ng/g and 0.09 to 0.64ng/g (respectively) for the analytes examined. Ibuprofen and gemfibrozil were not detected in fish tissues, likely due to rapid excretion leading to low rates of bioconcentration. This study demonstrates improved in vivo SPME sensitivity (recovery) and extraction rates during pre-equilibrium sampling of target pharmaceuticals in fish tissue due to the improved C18 extraction phase geometry.
Source:Journal of Chromatography A, Volume 1261
Oluranti P. Togunde, Ken D. Oakes, Mark R. Servos, Janusz Pawliszyn
A new thin film microextraction (TFME) configuration based on C18 thin film was optimized to improve solid phase microextraction (SPME) sensitivity and extraction kinetics for in vivo determinations of trace pharmaceuticals in fish tissue. Optimization of SPME involved increasing sampler surface area and volume of extraction phase to improve performance within in vitro applications such as agarose gel matrix and spiked fish tissue, as well as in vivo determinations of pharmaceuticals in fish exposed to municipal wastewater. Rainbow trout (Oncorhynchus mykiss) were exposed for 4d to effluents from a municipal wastewater plant that was treated under three different configurations of a pilot plant. Fathead minnow (Pimephales promelas) were caged for 14d upstream and downstream of municipal wastewater effluent discharges along Grand River at near Kitchener, ON. TFME and regular C18 fibers were inserted into the dorsal-epaxial muscle of rainbow trout and fathead minnow, respectively, for 30min sampling intervals. Sample extracts obtained from fibers were desorbed in methanol:water (3:2) for 90min under 1500rpm agitation and analyzed using liquid chromatography coupled with tandem mass spectrometry (LC/MS–MS) to determine pharmaceutical bioconcentration. Most target pharmaceuticals were detected in the wastewater with the exception of norfluoxetine and paroxetine. C18 TFME phase successfully quantified fluoxetine, venlafaxine, sertraline, paroxetine, and carbamazapine in muscle of living fish at concentrations ranging from 1.7 to 259ng/g. Reproducibility of the method in spiked fish muscle was 9–18% RSD with limits of detection and quantification ranging from 0.08 to 0.21ng/g and 0.09 to 0.64ng/g (respectively) for the analytes examined. Ibuprofen and gemfibrozil were not detected in fish tissues, likely due to rapid excretion leading to low rates of bioconcentration. This study demonstrates improved in vivo SPME sensitivity (recovery) and extraction rates during pre-equilibrium sampling of target pharmaceuticals in fish tissue due to the improved C18 extraction phase geometry.
Highlights
► A new in vivo SPME technique based on thin film geometry is developed. ► Strategies to improve sensitivity and extraction kinetics are demonstrated. ► Application of the method is demonstrated in live fish. ► Bioconcentration of pharmaceuticals in fish is successfully determined.Kinetic performance comparison of a capillary monolithic and a fused-core column in micro-scale liquid chromatography
05 October 2012,
08:32:43
Publication year:
2012
Source:Journal of Chromatography A, Volume 1261
Tünde A. Diószegi, Douglas E. Raynie
The kinetic performance of monolithic and fused-core capillary C18 columns was compared in isocratic-elution liquid chromatography. Heptanophenone was chosen as a test compound. The (u 0, H) couple obtained in a column with permeability K v0 was transferred into a plate number, dead time (N, t 0) couple and the kinetic plots were achieved by using a Kinetic Method Plot Analyzer template. The u 0,opt (0.98mm/s for the monolith and 1.05mm/s for the fused-core column) and H min (17.58μm for the monolith and 18.08μm for the fused-core column) values determined from the van Deemter curve were found to be almost identical, whereas the N opt (the plate number for which the support achieves its best analysis time/pressure cost ratio) (150,000 for monolith and 41,000 for fused-core column), maximal plate number, N max (the maximum plate number that can ever be reached with a support) (550,000 for monolith and 120,000 for the fused-core column) and optimal analysis time, t opt (the time needed to obtain N opt plates) (18,583s for the monolith and 4,068s for the fused-core column) performance numbers determined from the kinetic plots were found to be significantly different.
Source:Journal of Chromatography A, Volume 1261
Tünde A. Diószegi, Douglas E. Raynie
The kinetic performance of monolithic and fused-core capillary C18 columns was compared in isocratic-elution liquid chromatography. Heptanophenone was chosen as a test compound. The (u 0, H) couple obtained in a column with permeability K v0 was transferred into a plate number, dead time (N, t 0) couple and the kinetic plots were achieved by using a Kinetic Method Plot Analyzer template. The u 0,opt (0.98mm/s for the monolith and 1.05mm/s for the fused-core column) and H min (17.58μm for the monolith and 18.08μm for the fused-core column) values determined from the van Deemter curve were found to be almost identical, whereas the N opt (the plate number for which the support achieves its best analysis time/pressure cost ratio) (150,000 for monolith and 41,000 for fused-core column), maximal plate number, N max (the maximum plate number that can ever be reached with a support) (550,000 for monolith and 120,000 for the fused-core column) and optimal analysis time, t opt (the time needed to obtain N opt plates) (18,583s for the monolith and 4,068s for the fused-core column) performance numbers determined from the kinetic plots were found to be significantly different.
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