Co-reporter:Xiaofeng Zhao;Xiaofeng Xie;Sonika Sharma;Luke T. Tolley;Alex Plistil;Hal E. Barnett;Martin P. Brisbin;Adam C. Swensen;John C. Price;Paul B. Farnsworth;H. Dennis Tolley;Stanley D. Stearns
Analytical Chemistry January 3, 2017 Volume 89(Issue 1) pp:807-812
Publication Date(Web):November 29, 2016
DOI:10.1021/acs.analchem.6b03575
A compact ultrahigh-pressure nanoflow liquid chromatograph (LC) was developed with the purpose in mind of creating a portable system that could be easily moved to various testing locations or placed in close proximity to other instruments for optimal coupling, such as with mass spectrometry (MS). The system utilized innovative nanoflow pumps integrated with a very low volume stop-flow injector and mixing tee. The system weighed only 5.9 kg (13 lbs) or 4.5 kg (10 lbs) without a controller and could hold up to 1100 bar (16000 psi) of pressure. The total volume pump capacity was 60 μL. In this study, the sample injection volume was determined by either a 60 nL internal sample groove machined in a high-pressure valve rotor or by a 1 μL external sample loop, although other sample grooves or loops could be selected. The gradient dwell volume was approximately 640 nL, which allowed significant reduction in sample analysis time. Gradient performance was evaluated by determining the gradient step accuracy. A low RSD (0.6%, n = 4) was obtained for day-to-day experiments. Linear gradient reproducibility was evaluated by separating a three-component polycyclic aromatic hydrocarbon mixture on a commercial 150 μm inner diameter capillary column packed with 1.7 μm particles. Good retention-time reproducibility (RSD < 0.17%) demonstrated that the pumping system could successfully generate ultrahigh pressures for use in capillary LC. The system was successfully coupled to an LTQ Orbitrap MS in a simple and efficient way; LC−MS of a trypsin-digested bovine serum albumin (BSA) sample provided narrow peaks, short dwell time, and good peptide coverage.
Co-reporter:Xiaofeng Xie, Luke T. Tolley, Thy X. Truong, H. Dennis Tolley, Paul B. Farnsworth, Milton L. Lee
Journal of Chromatography A 2017 Volume 1523(Volume 1523) pp:
Publication Date(Web):10 November 2017
DOI:10.1016/j.chroma.2017.07.097
•A miniaturized detector incorporates two LED-based UV absorption detectors operating at different wavelengths in series.•The detector housing provided automatic alignment of optical components.•Ray tracing modeling allowed the optimization of optical component positions.•Absorbance ratios from the two detectors add a level of specificity not available in a single wavelength system.•The serial arrangement allows for accurate flow rate measurements based on time lag and spacing between detectors.The design of a miniaturized LED-based UV-absorption detector was significantly improved for on-column nanoflow LC. The detector measures approximately 27 mm × 24 mm × 10 mm and weighs only 30 g. Detection limits down to the nanomolar range and linearity across 3 orders of magnitude were obtained using sodium anthraquinone-2-sulfonate as a test analyte. Using two miniaturized detectors, a dual-detector system was assembled containing 255 nm and 275 nm LEDs with only 216 nL volume between the detectors A 100 μm slit was used for on-column detection with a 150 μm i.d. packed capillary column. Chromatographic separation of a phenol mixture was demonstrated using the dual-detector system, with each detector producing a unique chromatogram. Less than 6% variation in the ratios of absorbances measured at the two wavelengths for specific analytes was obtained across 3 orders of magnitude concentration, which demonstrates the potential of using absorption ratio measurements for target analyte detection. The dual-detector system was used for simple, but accurate, mobile phase flow rate measurement at the exit of the column. With a flow rate range from 200 to 2000 nL/min, less than 3% variation was observed.
Co-reporter:Xiaofeng Xie, H. Dennis Tolley, Milton L. Lee
Journal of Chromatography A 2017 Volume 1502(Volume 1502) pp:
Publication Date(Web):16 June 2017
DOI:10.1016/j.chroma.2017.04.020
•A device for sampling trace volatile BTEX compounds in air was developed.•High flow rate air sampling over 5 L/min could be achieved.•Low ppt levels of target analytes could be detected in less than 15 min sampling.•Analytes were transferred into a needle trap for introduction into the GC–MS.Rapid determination of trace level (parts-per-trillion) volatile organic compounds cannot always be achieved with conventional analytical techniques. In this study, a device was developed to sample a large volume of air in a short time period. The basic design involves packing sorbent layers concentrically around an empty permeable tube. Single digit parts-per-trillion detection limits were reached in less than 25 min with this sampling system using gas chromatography–mass spectrometry for analysis. The concentric packed trap can sample at high flow rates (>10 L/min) because it has a large sampling surface cross-section and short combined sorbent bed. Additionally, the large sorbent amount (>1 g) provides large breakthrough volume (>100 L) required to achieve low detection limits. The trapped analytes were thermally desorbed and transferred into a needle trap device for final analysis. This high flow sampling system was explored for detection of low ppt benzene, toluene, ethylbenzene and xylenes (BTEX) in air.
Co-reporter:Abhijit Ghosh, Jacob E. Johnson, Johnathan G. Nuss, Brittany A. Stark, Aaron R. Hawkins, Luke T. Tolley, Brian D. Iverson, H. Dennis Tolley, Milton L. Lee
Journal of Chromatography A 2017 Volume 1517(Volume 1517) pp:
Publication Date(Web):29 September 2017
DOI:10.1016/j.chroma.2017.08.036
•A simple clamp for connecting microchip columns to GC instrumentation was reported.•The new microchip column clamp allowed for leak-free, high temperature GC analyses.•Microchip column seals were successfully applied up to 375 °C for short time periods.•Applications were demonstrated using the thermal gradient GC mode of operation.Miniaturization of gas chromatography (GC) instrumentation is of interest because it addresses current and future issues relating to compactness, portability and field application. While incremental advancements continue to be reported in GC with columns fabricated in microchips (referred to in this paper as “microchip columns”), the current performance is far from acceptable. This lower performance compared to conventional GC is due to factors such as pooling of the stationary phase in corners of non-cylindrical channels, adsorption of sensitive compounds on incompletely deactivated surfaces, shorter column lengths and less than optimum interfacing to injector and detector. In this work, a GC system utilizing microchip columns was developed that solves the latter challenge, i.e., microchip interfacing to injector and detector. A microchip compression clamp was constructed to heat the microchip (i.e., primary heater), and seal the injector and detector fused silica interface tubing to the inlet and outlet ports of the microchip channels with minimum extra-column dead volume. This clamp allowed occasional operation up to 375 °C and routine operation up to 300 °C. The compression clamp was constructed of a low expansion alloy, Kovar™, to minimize leaking due to thermal expansion mismatch at the interface during repeated thermal cycling, and it was tested over several months for more than one hundred injections without forming leaks. A 5.9 m long microcolumn with rectangular cross section of 158 μm × 80 μm, which approximately matches a 100 μm i.d. cylindrical fused silica column, was fabricated in a silicon wafer using deep reactive ion etching (DRIE) and high temperature fusion bonding; finally, the channel was coated statically with a 1% vinyl, 5% phenyl, 94% methylpolysiloxane stationary phase. High temperature separations of C10-C40 n-alkanes and a commercial diesel sample were demonstrated using the system under both temperature programmed GC (TPGC) and thermal gradient GC (TGGC) conditions. TGGC analysis of a complex essential oil sample was also demonstrated. Addition of a secondary heater and polyimide insulation proved to be helpful in achieving the desired elution temperature without having to raise the primary heater temperature above 300 °C for high boiling point compounds.
Co-reporter:Sonika Sharma, Alex Plistil, Hal E. Barnett, H. Dennis Tolley, Paul B. Farnsworth, Stanley D. Stearns, and Milton L. Lee
Analytical Chemistry 2015 Volume 87(Issue 20) pp:10457
Publication Date(Web):September 17, 2015
DOI:10.1021/acs.analchem.5b02583
In this work, a novel splitless nanoflow gradient generator integrated with a stop-flow injector was developed and evaluated using an on-column UV-absorption detector. The gradient pumping system consisted of two nanoflow pumps controlled by micro stepper motors, a mixer connected to a serpentine tube, and a high-pressure valve. The gradient system weighed only 4 kg (9 lbs) and could generate up to 55 MPa (8000 psi) pressure. The system could operate using a 24 V DC battery and required 1.2 A for operation. The total volume capacity of the pump was 74 μL, and a sample volume of 60 nL could be injected. The system provided accurate nanoflow rates as low as 10 nL/min without employing a splitter, making it ideal for capillary column use. The gradient dwell volume was calculated to be 1.3 μL, which created a delay of approximately 4 min with a typical flow rate of 350 nL/min. Gradient performance was evaluated for gradient step accuracy, and excellent reproducibility was obtained in day-to-day experiments (RSD < 1.2%, n = 4). Linear gradient reproducibility was tested by separating a three-component pesticide mixture on a poly(ethylene glycol) diacrylate (PEGDA) monolithic column. The retention time reproducibility was very good in run-to-run experiments (RSD < 1.42%, n = 4). Finally, excellent separation of five phenols was demonstrated using the nanoflow gradient system.
Co-reporter:Sonika Sharma, H. Dennis Tolley, Paul B. Farnsworth, and Milton L. Lee
Analytical Chemistry 2015 Volume 87(Issue 2) pp:1381
Publication Date(Web):December 12, 2014
DOI:10.1021/ac504275m
A 260 nm deep UV LED-based absorption detector with low detection limits was developed and integrated with a small nanoflow pumping system. The detector is small in size (5.2 × 3.0 cm) and weighs only 85 g (without electronics). This detector was specifically designed and optimized for on-column detection to minimize extra-column band broadening. No optical reference was included due to the low drift in the signal. Two ball lenses, one of which was integrated with the LED, were used to increase light throughput through the capillary column. Stray light was minimized by the use of a band-pass filter and an adjustable slit. Signals down to the parts per billion level (nanomolar) were easily detected with a short-term noise level of 4.4 μAU, confirming a low limit of detection and low noise. The detection limit for adenosine-5′-monophosphate was 230 times lower than any previously reported values. Good linearities (3 orders of magnitude) were obtained using sodium anthraquinone-2-sulfonate, adenosine-5′-monophosphate, dl-tryptophan, and phenol. The LC system was demonstrated by performing isocratic separation of phenolic compounds using a monolithic capillary column (16.5 cm × 150 μm i.d.) synthesized from poly(ethylene glycol) diacrylate.
Co-reporter:Sonika Sharma, Luke T. Tolley, H. Dennis Tolley, Alex Plistil, Stanley D. Stearns, Milton L. Lee
Journal of Chromatography A 2015 Volume 1421() pp:38-47
Publication Date(Web):20 November 2015
DOI:10.1016/j.chroma.2015.07.119
•Requirements for portable liquid chromatography (LC) instrumentation are outlined.•Advancements in capillary LC columns are briefly reviewed.•Developments made in low-flow-rate pumps and miniaturized detectors are emphasized.•Finally, integrated portable LC, IC and microchip LC systems are reviewed.Over the last four decades, liquid chromatography (LC) has experienced an evolution to smaller columns and particles, new stationary phases and low flow rate instrumentation. However, the development of person-portable LC has not followed, mainly due to difficulties encountered in miniaturizing pumps and detectors, and in reducing solvent consumption. The recent introduction of small, non-splitting pumping systems and UV-absorption detectors for use with capillary columns has finally provided miniaturized instrumentation suitable for high-performance hand-portable LC. Fully integrated microfabricated LC still remains a significant challenge. Ion chromatography (IC) has been successfully miniaturized and applied for field analysis; however, applications are mostly limited to inorganic and small organic ions. This review covers advancements that make possible more rapid expansion of portable forms of LC and IC.
Co-reporter:Pankaj Aggarwal, Kun Liu, Sonika Sharma, John S. Lawson, H. Dennis Tolley, Milton L. Lee
Journal of Chromatography A 2015 1380() pp: 38-44
Publication Date(Web):
DOI:10.1016/j.chroma.2014.12.017
Co-reporter:Pankaj Aggarwal, Vikas Asthana, John S. Lawson, H. Dennis Tolley, Dean R. Wheeler, Brian A. Mazzeo, Milton L. Lee
Journal of Chromatography A 2014 Volume 1334() pp:20-29
Publication Date(Web):21 March 2014
DOI:10.1016/j.chroma.2014.01.056
•Organic monoliths were characterized using 3D scanning electron microscopy and conductivity measurements.•Chromatographic performance improved with decreasing pore size, heterogeneity and tortuosity.•Chromatographic performance can be predicted from parameters obtained from these characterization techniques.Monoliths are considered to be a low pressure alternative to particle packed columns for liquid chromatography (LC). However, the chromatographic performance of organic monoliths, in particular, has still not reached the level of particle packed columns. Since chromatographic performance can be attributed to morphological features of the monoliths, in-situ characterization of the monolith structure in three dimensions would provide valuable information that could be used to help improve performance. In this work, serial sectioning and imaging were performed with a dual-beam scanning electron microscope for reconstruction and quantitative characterization of poly(ethylene glycol) diacrylate (PEGDA) monoliths inside a capillary column. Chord lengths, homogeneity factors, porosities and tortuosities were calculated from three-dimensional (3D) reconstructions of two PEGDA monoliths. Chromatographic efficiency was better for the monolith with smaller mean chord length (i.e., 5.23 μm), porosity (i.e., 0.49) and tortuosity (i.e., 1.50) compared to values of 5.90 μm, 0.59 and 2.34, respectively, for the other monolithic column. Computational prediction of tortuosity (2.32) was found to be in agreement with the experimentally measured value (2.34) for the same column. The monoliths were found to have significant radial heterogeneity since the homogeneity factor decreased from 5.39 to 4.89 (from center to edge) along the column radius.
Co-reporter:H. Dennis Tolley, Samuel E. Tolley, Anzi Wang, Milton L. Lee
Journal of Chromatography A 2014 Volume 1374() pp:189-198
Publication Date(Web):29 December 2014
DOI:10.1016/j.chroma.2014.10.090
Highlights•Model predicts GC solute band widths narrow with increases in thermal gradient slope.•Predicted injection/stationary phase band broadening reduced with moving gradients.•Predicted thermal gradient effects realized within the first 40–70 cm of the column.This paper examines the separation effects of a moving thermal gradient on a chromatographic column in gas chromatography. This movement of the gradient has a focusing effect on the analyte bands, limiting band broadening in the column. Here we examine the relationship between the slope of this gradient, the velocity of the gradient and the resulting band width. Additionally we examine how transport of analytes along the column at their analyte specific constant temperatures, determined by the gradient slope and velocity, affects resolution. This examination is based primarily on a theoretical model of partitioning and transport of analyte under low concentration conditions. Preliminary predictions indicate that analytes reach near constant temperatures, relative positions and resolutions in less than 100 cm of column transport. Use of longer columns produces very little improvement in resolution for any fixed slope. Properties of the thermal gradient determine a fixed solute band width for each analyte. These widths are nearly reached within the first 40–70 cm, after which little broadening or narrowing of the bands occur. The focusing effect of the thermal gradient corrects for broad injections, reduces effects of irregular stationary phase coatings and can be used with short columns for fast analysis. Thermal gradient gas chromatographic instrumentation was constructed and used to illustrate some characteristics predicted from the theoretical results.
Co-reporter:Anzi Wang, Sampo Hynynen, Aaron R. Hawkins, Samuel E. Tolley, H. Dennis Tolley, Milton L. Lee
Journal of Chromatography A 2014 Volume 1374() pp:216-223
Publication Date(Web):29 December 2014
DOI:10.1016/j.chroma.2014.11.035
•Axial thermal gradients were generated along microfabricated gas chromatography columns.•A thermal gradient reduced separation time by focusing the injection band during separation.•Signal-to-noise ratio, peak symmetry and resolution were improved by the focusing effect of the gradient.Fabrication technologies for microelectromechanical systems (MEMS) allow miniaturization of conventional benchtop gas chromatography (GC) to portable, palm-sized microfabricated GC (μGC) devices, which are suitable for on-site chemical analysis and remote sensing. The separation performance of μGC systems, however, has not been on par with conventional GC. Column efficiency, peak symmetry and resolution are often compromised by column defects and non-ideal injections. The relatively low performance of μGC devices has impeded their further commercialization and broader application. In this work, the separation performance of μGC columns was improved by incorporating thermal gradient gas chromatography (TGGC). The analysis time was ∼20% shorter for TGGC separations compared to conventional temperature-programmed GC (TPGC) when a wide sample band was introduced into the column. Up to 50% reduction in peak tailing was observed for polar analytes, which improved their resolution. The signal-to-noise ratios (S/N) of late-eluting peaks were increased by 3–4 fold. The unique focusing effect of TGGC overcomes many of the previous shortcomings inherent in μGC analyses.
Co-reporter:Kun Liu, Pankaj Aggarwal, H. Dennis Tolley, John S. Lawson, Milton L. Lee
Journal of Chromatography A 2014 Volume 1367() pp:90-98
Publication Date(Web):7 November 2014
DOI:10.1016/j.chroma.2014.09.046
•Improved control of polymer monolith morphology was obtained for capillary LC.•Tellurium-mediated living radical polymerization was used for the first time.•High plate numbers were obtained for reversed-phase separations of alkylbenzenes.New monolithic reversed-phase liquid chromatography (RPLC) stationary phases based on single multi-acrylate/methacrylate-containing monomers [i.e., 1,12-dodecanediol dimethacrylate (1,12-DoDDMA), trimethylolpropane trimethacrylate (TRIM) and pentaerythritol tetraacrylate (PETA)] were synthesized using organotellurium-mediated living radical polymerization (TERP), which was expected to produce more efficient monolithic columns than conventional free-radical polymerization. The rationale behind selection of porogens, relative concentrations of reagents and polymerization conditions are described. The new monolithic columns were applied to the separation of small molecules (i.e., alkylbenzenes) under isocratic conditions. Chromatographic efficiencies as high as 60,200 plates/m (71,300 plates/m when corrected for extra-column variance) were obtained, showing a general improvement over previous RPLC monoliths.
Co-reporter:Pankaj Aggarwal, John S. Lawson, H. Dennis Tolley, Milton L. Lee
Journal of Chromatography A 2014 Volume 1364() pp:96-106
Publication Date(Web):17 October 2014
DOI:10.1016/j.chroma.2014.08.056
•Efficient monoliths were synthesized in capillary columns from polyethylene glycol diacrylates.•A new statistical model was found to aid porogen selection and morphology optimization.•Correlation was established between chromatographic efficiency and physical/chemical properties of reagents.•A corrected chromatographic efficiency of 186,000 plates/m was obtained for a non-retained compound.•The retention mechanism was primarily reversed-phase with additional hydrogen bonding interaction.Highly cross-linked monolithic networks (i.e., polyethylene glycol diacrylate, PEGDA) synthesized from monomers containing varying ethylene oxide chain lengths were fabricated inside fused silica capillary columns for use in liquid chromatography (LC) of small molecules. Tergitol was used as a surfactant porogen in combination with other typical organic liquid porogens. Column performance was correlated with quantitative descriptors of the physical/chemical properties of the monomers and porogens using a statistical model. Solubility and viscosity values of the components were identified as important predictors of monolith morphology and efficiency. The chromatographic retention mechanism was determined to be principally reversed-phase (RP) with additional hydrogen bonding between the polar groups of the analytes and the ethylene oxide groups embedded in the monolith structure. The fabricated monolithic columns were evaluated under RPLC conditions using phenols, hydroxy benzoic acids, and alkyl parabens as test compounds. Isocratic elution of hydroxy benzoic acids at a linear velocity of 0.04 cm/s using a PEGDA-700 monolith gave chromatographic peaks with little tailing (i.e., tailing factor < 1.28). The chromatographic efficiency measured for a non-retained compound (uracil) using this column was 186,000 plates/m when corrected for injector dead volume. High resolution gradient separations of selected pharmaceutical compounds and phenylurea herbicides were achieved in less than 18 min. Optimized monoliths synthesized from all four crosslinking monomers exhibited high permeability and demonstrated little swelling or shrinking in different polarity solvents. Column preparation was highly reproducible, with relative standard deviation (RSD) values less than 2.1%, based on retention times of the phenol standards (3 different columns).
Co-reporter:Sonika Sharma, Alex Plistil, Robert S. Simpson, Kun Liu, Paul B. Farnsworth, Stanley D. Stearns, Milton L. Lee
Journal of Chromatography A 2014 Volume 1327() pp:80-89
Publication Date(Web):31 January 2014
DOI:10.1016/j.chroma.2013.12.059
•Components for a hand-portable LC system were miniaturized and evaluated.•The nano-flow pumping system demonstrated excellent flow rate reproducibility.•The injector gave high precision and low carry-over.•The UV-absorption detector gave good linearity and sensitivity.•Reversed-phase separations were performed using the new prototype system.Liquid chromatography (LC) has lagged behind gas chromatography (GC) in developments related to hand-portable instrumentation. In this work, a new battery-operated (24 V DC) nano-flow pumping system with a stop-flow injector was developed and integrated with an on-column UV-absorption detector (254 nm) that was reduced in size to an acceptable weight and power usage for field operation. The pumping system, which includes nano-flow pump, stepper motor and high-pressure valve weighs only 1.372 kg (3 lbs) and can generate up to 110.32 MPa (16,000 psi) pressure. A major advantage of this pump is that it does not employ a splitter, since it was specifically designed for capillary column use. The volume capacity of the pump is 24 μL, and a sample volume as low as 10 nL can be injected. Flow rate calibration (300 nL to 6.12 μL per min) was performed, and an accuracy >99.94% was obtained. The percent injection carry-over was found to be low (RSD 0.31%), which makes it practical for quantitative analysis. The detector linear range and limit of detection (LOD) were determined using sodium anthraquinone-2-sulfonate. A linear regression coefficient (R) of 0.9996 was obtained for a plot of log peak area versus log concentration over the range of 3.2 μM to 6.5 mM, and the LOD (S/N = 3) was found to be 7.8 fmol (0.13 μM). The short term noise of the detector is comparable to commercially available detectors (∼10−5 AU). In this work, the system was tested in the laboratory using regular line power (120 V AC) with an AC to DC adapter. Reversed-phase isocratic separations were performed using a 15.5 cm × 75 μm i.d. fused silica capillary column containing a monolithic stationary phase synthesized from 1,6-hexanediol dimethacrylate. Good retention time repeatability (RSD 0.09–0.74%) was obtained for a mixture containing an unretained marker (i.e., uracil) and a homologous series of alkyl benzenes.
Co-reporter:Jie Xuan and Milton L. Lee
Analytical Methods 2014 vol. 6(Issue 1) pp:27-37
Publication Date(Web):28 Nov 2013
DOI:10.1039/C3AY41364K
Reports of novel micro/nanostructures designed to separate biomacromolecules and bioparticles are increasing in number, and these studies have greatly advanced our understanding of nanoscale fluidics and nanoparticle behavior in confined channels. This review is aimed at summarizing previous developments in micro/nanofabricated systems for nanoparticle separations. These are discussed in three groups based on architecture, namely, micro/nanopillar array structures, nanoplane gap structures and artificial nanoporous membranes.
Co-reporter:Dan Li, Anthony D. Rands, Scott C. Losee, Brian C. Holt, John R. Williams, Stephen A. Lammert, Richard A. Robison, H. Dennis Tolley, Milton L. Lee
Analytica Chimica Acta 2013 Volume 775() pp:67-74
Publication Date(Web):2 May 2013
DOI:10.1016/j.aca.2013.03.011
•An automated sample preparation system for Bacillus anthracis endospores was developed.•A thermochemolysis method was applied to produce and derivatize biomarkers for Bacillus anthracis detection.•The autoreactor controlled the precise delivery of reagents, and TCM reaction times and temperatures.•Solid phase microextraction was used to extract biomarkers, and GC–MS was used for final identification.•This autoreactor was successfully applied to the identification of Bacillus anthracis endospores.An automated sample preparation system was developed and tested for the rapid detection of Bacillus anthracis endospores by gas chromatography–mass spectrometry (GC–MS) for eventual use in the field. This reactor is capable of automatically processing suspected bio-threat agents to release and derivatize unique chemical biomarkers by thermochemolysis (TCM). The system automatically controls the movement of sample vials from one position to another, crimping of septum caps onto the vials, precise delivery of reagents, and TCM reaction times and temperatures. The specific operations of introduction of sample vials, solid phase microextraction (SPME) sampling, injection into the GC–MS system, and ejection of used vials from the system were performed manually in this study, although they can be integrated into the automated system. Manual SPME sampling is performed by following visual and audible signal prompts for inserting the fiber into and retracting it from the sampling port. A rotating carousel design allows for simultaneous sample collection, reaction, biomarker extraction and analysis of sequential samples. Dipicolinic acid methyl ester (DPAME), 3-methyl-2-butenoic acid methyl ester (a fragment of anthrose) and two methylated sugars were used to compare the performance of the autoreactor with manual TCM. Statistical algorithms were used to construct reliable bacterial endospore signatures, and 24 out of 25 (96%) endospore-forming Bacillus species were correctly identified in a statistically designed test.
Co-reporter:Kun Liu, H. Dennis Tolley, John S. Lawson, Milton L. Lee
Journal of Chromatography A 2013 Volume 1321() pp:80-87
Publication Date(Web):20 December 2013
DOI:10.1016/j.chroma.2013.10.071
•Efficient monoliths were synthesized for reversed-phase LC of small molecules.•Monoliths were based on single monomers containing C6 functional groups.•The linear alkyl functional group provided the best efficiency.•A chromatographic efficiency of 86,000 plates/m was obtained.Three crosslinking monomers, i.e., 1,6-hexanediol dimethacrylate (HDDMA), cyclohexanediol dimethacrylate (CHDDMA) and 1,4-phenylene diacrylate (PHDA), were used to synthesize highly cross-linked monolithic capillary columns for reversed-phase liquid chromatography (RPLC) of small molecules. Selection of porogen type and concentration was investigated in detail. Isocratic elution of alkylbenzenes at a flow rate of 300 nL/min was performed using HDDMA and CHDDMA monolithic columns. Gradient elution of alkylbenzenes using all three monolithic columns showed good separations. Optimized monoliths synthesized from all three crosslinking monomers possessed high permeabilities. Poly(HDDMA) monoliths demonstrated column efficiencies up to 86,000 plates/m. Column preparation of poly(HDDMA) monolithic columns was highly reproducible; the relative standard deviation (RSD) values (n = 3) for run-to-run and column-to-column were less than 0.26% and 0.70%, respectively, based on retention times of alkylbenzenes.
Co-reporter:Jesse A. Contreras, Alan L. Rockwood, H. Dennis Tolley, Milton L. Lee
Journal of Chromatography A 2013 Volume 1278() pp:160-165
Publication Date(Web):22 February 2013
DOI:10.1016/j.chroma.2013.01.010
When axial temperature gradients are applied in gas chromatography (GC), i.e., “thermal gradient GC” (TGGC), the temperature changes both in time and position, T(t,L), along the column, allowing unique control of the movement and elution of sample components. One method of performing TGGC involves introducing a sample into a column with a preset decreasing temperature gradient along its length, waiting for a short time until the sample separates along the gradient, and then raising the temperature to sweep all of the compounds out of the column and into the detector (i.e., “peak sweeping”). This method of operation is demonstrated here using a simple laboratory apparatus based on simultaneous resistive heating and convective cooling. An experimental comparison between isothermal GC (ITGC), temperature programmed GC (TPGC) and TGGC shows that TGGC is essentially equivalent in performance to TPGC operation when using the same column length (peak capacity production rate of 106, 381 and 469 min−1, respectively); however, narrower peaks and higher signal-to-noise are achieved in TGGC. Furthermore, TGGC helps to minimize band broadening and peak tailing that arise from column adsorption and less than perfect sample injection. The low thermal mass of the TGGC system allows rapid column heating (4000 °C/min) and cooling (3500 °C/min) for selective separation (i.e., “peak gating”) of compounds in a mixture without sacrificing the resolution of earlier or later eluting compounds.Highlights► A simple laboratory apparatus was used to evaluate axial temperature gradients in GC. ► Direct comparison of thermal gradient GC (TGGC), ITGC and TPGC was performed. ► TGGC separations were equivalent to TPGC for the same column length. ► “Peak sweeping” was used to rapidly transfer separated compounds to the detector. ► A novel method to selectively isolate compounds, called “peak gating,” was introduced.
Co-reporter:Jesse A. Contreras, Anzi Wang, Alan L. Rockwood, H. Dennis Tolley, Milton L. Lee
Journal of Chromatography A 2013 Volume 1302() pp:143-151
Publication Date(Web):9 August 2013
DOI:10.1016/j.chroma.2013.06.008
•A dynamic thermal gradient gas chromatography instrument was developed.•The system is able to rapidly and repetitively generate customized thermal gradients.•Band broadening and peak tailing are reduced by the focusing effect of the gradient.•Elution of analytes can be easily adjusted by changing the gradient profile.The use of negative axial thermal gradients in gas chromatography (TGGC) has intrigued chromatographers since the early 1950s because of the dramatic narrowing of analyte bands and concomitant raised expectations for improving resolving power. However, technical difficulties experienced in construction of TGGC instrumentation and control of the temperature along the column have made its implementation and, hence, detailed study difficult. In this work, we describe a TGGC system capable of rapidly producing and varying thermal gradient profiles by simultaneous use of resistive heating and convective cooling. Heating and cooling rates as high as 1200 and 2500 °C/min, respectively, allowed the creation of dynamic temperature gradients. The separation characteristics of TGGC with dynamically changing temperature gradients are demonstrated. A gradient velocity of 2.22 cm/s provided repetitive separations every 45 s, and injection band widths of 45 s duration were transformed into approximately 1-s peak widths. Peak tailing for basic compounds was nearly eliminated. Dynamic TGGC allows unique control over separations, oftentimes improving resolution and detection signal-to-noise. Thermally controlled elution in TGGC holds great promise for performing smart separations in which the separation time window is most efficiently utilized, and optimized separations can be quickly achieved. Rapid adjustment of relative compound elution can be used to greatly reduce GC method development time.
Co-reporter:Kun Liu;Pankaj Aggarwal;John S. Lawson;H. Dennis Tolley
Journal of Separation Science 2013 Volume 36( Issue 17) pp:2767-2781
Publication Date(Web):
DOI:10.1002/jssc.201300431
RPLC is the most common mode of LC. It is widely used for separations of both small and large molecules. Monolithic columns, which are currently under intensive study by many groups, have the potential of becoming attractive alternatives to particle-packed columns. They are generally easier and faster to fabricate, and they demonstrate a lower pressure drop, less nonspecific adsorption, and richer chemistry (in the case of organic polymer monoliths) for providing broad selectivity. Silica monoliths, as is also true for columns packed with silica particles, are best applied to small-molecule separations. Organic polymer monoliths, on the other hand, have shown advantages for large-molecule separations. Recently, improvements in organic monoliths have led to efficiencies for small molecules that are approaching and even surpassing 100 000 plates/m. While this performance is still far short of what is currently available using modern small particles and silica monoliths in RPLC, steady progress is being made. This review describes recent developments in the synthesis and performance of organic polymer RPLC monoliths, and identifies areas where additional work is needed to significantly improve their performance for both small- and large-molecule separations.
Co-reporter:Xiaofeng Xie, Tai V. Truong, Jacolin A. Murray, Jesse A. Contreras, H. Dennis Tolley and Milton L. Lee
Analytical Methods 2013 vol. 5(Issue 22) pp:6312-6318
Publication Date(Web):20 Sep 2013
DOI:10.1039/C3AY41393D
A simple approach for preparing standard mixtures of volatile and semi-volatile organic compounds is reported. When placed in a closed container, standard mixture components partition between a polymeric material such as poly(dimethylsiloxane) (PDMS) and headspace to provide constant vapor concentrations. The granular form of heat-conditioned PDMS provides rapid equilibration with the headspace vapor and serves as a standard reservoir. Solid phase micro extraction (SPME) or gas-tight syringe can be used to deliver sample from the headspace to the analytical instrument. Quantitative calibration can be achieved with either active temperature control or by using a previously constructed look-up table. The effects of PDMS form and temperature on equilibrium distribution, initial equilibrium time, and re-equilibrium time after sampling were investigated. With respect to long term use and stability, analytes introduced onto 2.0 g of PDMS in a 7.4 mL vial were sampled more than 114 times during a test period of 43 days, giving chromatographic peak area %RSD values below 4.5% for all compounds. This device was designed to be solventless, quantitative, reproducible, environmentally friendly, and robust for routine evaluation and calibration of gas chromatography-mass spectrometry (GC-MS) systems.
Co-reporter:Dan Li, Tai V. Truong, Teri M. Bills, Brian C. Holt, Douglas N. VanDerwerken, John R. Williams, Abhilasha Acharya, Richard A. Robison, H. Dennis Tolley, and Milton L. Lee
Analytical Chemistry 2012 Volume 84(Issue 3) pp:1637
Publication Date(Web):December 21, 2011
DOI:10.1021/ac202606x
A simple method was developed for detection of Bacillus anthracis (BA) endospores and for differentiation of them from other species in the Bacillus cereus group. Chemical profiles that include lipids (i.e., fatty acids), carbohydrates (i.e., sugars), and the spore-specific biomarker, dipicolinic acid, were generated by one-step thermochemolysis (TCM) at 140 °C in 5 min to provide specific biomarker signatures. Anthrose, which is a biomarker characteristic of the B. cereus group of bacteria, was determined from a fragment produced by TCM. Surprisingly, several virulent BA strains contained very low levels of anthrose, which confounded their detection. A statistical discrimination algorithm was constructed using a combination of biomarkers, which was robust against different growth conditions (medium and temperature). Fifteen endospore-forming Bacillus species were confirmed in a statistically designed test (∼90%) using the algorithm, including six BA strains (four virulent isolates), five B. thuringiensis (BT) isolates, and one isolate each for B. cereus (BC), B. mycoides (BM), B. atrophaeus (BG), and B. subtilis (BS). The detection limit for B. anthracis was found to be 50 000 endospores, on the basis of the GC/MS detection limits for 3-methyl-2-butenoic acid methyl ester, which is the biomarker derived from TCM of anthrose.
Co-reporter:Pankaj Aggarwal, H. Dennis Tolley, and Milton L. Lee
Analytical Chemistry 2012 Volume 84(Issue 1) pp:247
Publication Date(Web):November 19, 2011
DOI:10.1021/ac203010r
Polyethylene glycol diacrylate monoliths prepared using different amounts of monomer, porogen ratio, and capillary dimensions were characterized using capillary flow porometry (CFP) and scanning electron microscopy (SEM). Our results reveal good agreement between SEM and CFP measurements for through-pore size distribution. The CFP measurements for monoliths prepared by the same procedure in capillaries with different diameters (i.e., 75, 150, and 250 μm) clearly confirmed a change in through-pore size distribution with capillary diameter, thus, certifying the need for in-column measurement techniques over bulk measurements (e.g., mercury intrusion porosimetry). The mean through-pore size varied from 3.52 to 1.50 μm with a change in capillary diameter from 75 to 250 μm. Consistent mean through-pore size distribution for capillary columns with the same internal diameter but with different lengths (1.5, 2, and 3 cm) confirms the high interconnectivity of the pores and independence of CFP measurements with respect to capillary length. CFP and SEM measurements not only allow pore structure analysis but also prediction of relative column performance. Monoliths with narrow through-pore size distribution (0.8–1.2 μm), small mean through-pore size, and thin skeletal size (0.55 μm) gave the best performance in terms of efficiency for polyethylene glycol diacrylate monoliths.
Co-reporter:Anzi Wang, H. Dennis Tolley, Milton L. Lee
Journal of Chromatography A 2012 Volume 1261() pp:46-57
Publication Date(Web):26 October 2012
DOI:10.1016/j.chroma.2012.05.021
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.
Co-reporter:Pankaj Aggarwal, H. Dennis Tolley, Milton L. Lee
Journal of Chromatography A 2012 Volume 1219() pp:1-14
Publication Date(Web):6 January 2012
DOI:10.1016/j.chroma.2011.10.083
Monolithic stationary phases show promise for LC as a result of their good permeability, ease of preparation and broad selectivity. Inorganic silica monoliths have been extensively studied and applied for separation of small molecules. The presence of a large number of through pores and small skeletal structure allows the chromatographic efficiencies of silica monoliths to be comparable to columns packed with 5 μm silica particles, at much lower back pressure. In comparison, organic polymeric monoliths have been mostly used for separation of bio-molecules; however, recently, applications are expanding to small molecules as well. Organic monoliths with high surface areas and fused morphology rather than conventional globular morphology have shown good performance for small molecule separations. Factors such as domain size, through-pore size and mesopore size of the monolithic structures have been found to govern the efficiency of monolithic columns. The structure and performance of monolithic columns are reviewed in comparison to particle packed columns. Studying and characterizing the bed structures of organic monolithic columns can provide great insights into their performance, and aid in structure-directed synthesis of new and improved monoliths.Highlights► Monolithic column technology and morphology for capillary LC are reviewed. ► Morphologies of monolithic and packed capillary columns are compared. ► Performances of capillary columns are related to their morphologies.
Co-reporter:Kun Liu, H. Dennis Tolley, Milton L. Lee
Journal of Chromatography A 2012 Volume 1227() pp:96-104
Publication Date(Web):2 March 2012
DOI:10.1016/j.chroma.2011.12.081
Seven crosslinking monomers, i.e., 1,3-butanediol dimethacrylate (1,3-BDDMA), 1,4-butanediol dimethacrylate (1,4-BDDMA), neopentyl glycol dimethacrylate (NPGDMA), 1,5-pentanediol dimethacrylate (1,5-PDDMA), 1,6-hexanediol dimethacrylate (1,6-HDDMA), 1,10-decanediol dimethacrylate (1,10-DDDMA), and 1,12-dodecanediol dimethacrylate (1,12-DoDDMA), were used to synthesize highly cross-linked monolithic capillary columns for reversed-phase liquid chromatography (RPLC) of small molecules. Dodecanol and methanol were chosen as “good” and “poor” porogenic solvents, respectively, for these monoliths, and were investigated in detail to provide insight into the selection of porogen concentration using 1,12-DoDDMA. Isocratic elution of alkylbenzenes at a flow rate of 300 nL/min was conducted for all of the monoliths. Gradient elution of alkylbenzenes and alkylparabens provided high resolution separations. Optimized monoliths synthesized from all seven crosslinking monomers showed high permeability. Several of the monoliths demonstrated column efficiencies in excess of 50,000 plates/m. Monoliths with longer alkyl-bridging chains showed very little shrinking or swelling in solvents of different polarities. Column preparation was highly reproducible; the relative standard deviation (RSD) values (n = 3) for run-to-run and column-to-column were less than 0.25% and 1.20%, respectively, based on retention times of alkylbenzenes.Highlights► Monoliths were synthesized in fused silica capillaries from alkyl dimethacrylates. ► All provided good resolution and peak shapes for alkylbenzenes and alkylparabens. ► Alkyl branching groups gave higher efficiencies compared to linear functionalities.
Co-reporter:Xin Chen, H. Dennis Tolley, Milton L. Lee
Journal of Chromatography A 2011 Volume 1218(Issue 28) pp:4322-4331
Publication Date(Web):15 July 2011
DOI:10.1016/j.chroma.2011.04.074
Monoliths containing phosphoric acid functional groups were synthesized from only one monomer, bis[2-(methacryloyloxy)ethyl] phosphate (BMEP), in 75-μm i.d. UV transparent fused-silica capillaries by photo-initiated polymerization for cation exchange chromatography of peptides and proteins. Various synthetic conditions, including porogen solvents, monomer concentration, and polymerization time, were studied. The hydrophobicities of the resulting monoliths were evaluated using propyl paraben under reversed-phase conditions and synthetic peptides under ion-exchange conditions. These monoliths exhibited low hydrophobicities and relatively low porosities due to their highly cross-linked structures. A dynamic binding capacity (lysozyme) of 73 mg/mL of column volume was measured using the best performing monolith. Synthetic peptides were eluted in approximately 30 min without addition of acetonitrile to the mobile phase, yielding a peak capacity of 28. Efficiencies of 52,900 plates/m for peptides and 71,000 plates/m for proteins were obtained under isocratic conditions. The effects of separation conditions, i.e., mobile phase pH and salt gradient rate, were studied. Good run-to-run reproducibility was achieved with a relative standard deviation (RSD) less than 1.5% for retention times of proteins. The column-to-column retention time reproducibility for peptides was less than 3.5% RSD. A monolithic column was used to follow the deamidation of ribonuclease A. The kinetics of deamidation were founded to be first order with a half life of 195 h. A cytochrome C digest was also separated using a linear gradient of sodium chloride.
Co-reporter:Yuanyuan Li, H. Dennis Tolley, Milton L. Lee
Journal of Chromatography A 2011 Volume 1218(Issue 10) pp:1399-1408
Publication Date(Web):11 March 2011
DOI:10.1016/j.chroma.2011.01.028
Highly cross-linked networks resulting from single crosslinking monomers were found to enhance the concentrations of mesopores in, and the surface areas of, polymeric monoliths. Four crosslinking monomers, i.e., bisphenol A dimethacrylate (BADMA), bisphenol A ethoxylate diacrylate (BAEDA, EO/phenol = 2 or 4) and pentaerythritol diacrylate monostearate (PDAM), were used to synthesize monolithic capillary columns for reversed phase liquid chromatography (RPLC) of small molecules. Tetrahydrofuran (THF) and decanol were chosen as good and poor porogenic solvents for BAEDA-2 and BAEDA-4 monoliths. For the formation of the BADMA monolith, THF was replaced with dimethylformamide (DMF) to improve the column reproducibility. Appropriate combinations of THF, isopropyl alcohol and an additional triblock poly(ethylene oxide)–poly(propylene oxide)–poly(ethylene oxide) (PEO–PPO–PEO) or PPO–PEO–PPO porogen were found to be effective in forming rigid PDAM monoliths with the desired porosities. Selection of porogens for the BADMA and PDAM monoliths was investigated in further detail to provide more insight into porogen selection. Isocratic elution of alkyl benzenes at a flow rate of 0.3 μL/min was conducted for BADMA and PDAM monoliths. The peaks showed little tailing on both monoliths without addition of acid to the mobile phase. The column efficiency measured for pentylbenzene using the BADMA monolithic column was 60,208 plates/m (k = 7.9). Gradient elution of alkyl benzenes and alkyl parabens was achieved with high resolution. Optimized monoliths synthesized from all four crosslinking monomers showed high permeability, and demonstrated little swelling or shrinking in different polarity solvents. Column preparation was highly reproducible; relative standard deviation (RSD) values were less than 1.2% and 7.5% based on retention times and peak areas, respectively, of alkyl benzenes.
Co-reporter:Jie Xuan, Mark N. Hamblin, John M. Stout, H. Dennis Tolley, R. Daniel Maynes, Adam T. Woolley, Aaron R. Hawkins, Milton L. Lee
Journal of Chromatography A 2011 Volume 1218(Issue 50) pp:9102-9110
Publication Date(Web):16 December 2011
DOI:10.1016/j.chroma.2011.10.005
An array of parallel planar nanochannels containing two or three segments with varying inner heights was fabricated and used for size fractionation of inorganic and biological nanoparticles. A liquid suspension of the particles was simply drawn through the nanochannels via capillary action. Using fluorescently labeled 30 nm polyacrylonitrile beads, different trapping behaviors were compared using nanochannels with 200–45 nm and 208–54–30 nm height segments. Addition of sodium dodecyl sulfate (SDS) surfactant to the liquid suspension and application of an AC electric field were shown to aid in the prevention of channel clogging. After initial particle trapping at the segment interfaces, significant particle redistribution occurred when applying a sinusoidal 8 V peak-to-peak oscillating voltage with a frequency of 150 Hz and DC offset of 4 V. Using the 208–54–30 nm channels, 30 nm hepatitis B virus (HBV) capsids were divided into three fractions. When the AC electric field was applied to this trapped sample, all of the virus particles passed through the interfaces and accumulated at the channel ends.Highlights► Parallel nanochannel arrays with different height segments were fabricated. ► Nanoparticles were separated at interfaces of different height segments. ► Movement through channels was accomplished primarily by capillary action. ► Polyacrylamide beads and virus capsids of 30 nm diameter were studied. ► Electrophoretic oscillation and addition of surfactant minimized channel clogging.
Co-reporter:Xin Chen;H. Dennis Tolley
Journal of Separation Science 2011 Volume 34( Issue 16-17) pp:2063-2071
Publication Date(Web):
DOI:10.1002/jssc.201100156
Abstract
A stable poly(2-carboxyethyl acrylate-co-poly(ethylene glycol) diacrylate) monolith was synthesized inside a 75-μm id capillary by direct in situ photo-initiated polymerization in a binary porogenic solvent consisting of methanol and ethyl ether. The resulting monolith was evaluated for weak cation-exchange capillary liquid chromatography of peptides and proteins. A high dynamic binding capacity of 72.7 mg lysozyme per cm3 column volume was measured. Fast mass transfer was demonstrated by steep breakthrough curves. The resulting monolith exhibited negligible hydrophobicity, leading to good separation of peptides and proteins. Peak capacities of 11 for peptides with a 10-min salt gradient and 39 for proteins with a 20-min salt gradient were measured. An efficiency of 37 000 plates/m for proteins was obtained under isocratic conditions. The effects of functional group concentration, porogenic solvent composition, mobile phase pH, salt gradient rate, and hydrophobicity on the retention of analytes were investigated. Good run-to-run relative standard deviation (RSD) <1.93% and column-to-column RSD <4.63% were achieved.
Co-reporter:Xin Chen;H. Dennis Tolley
Journal of Separation Science 2011 Volume 34( Issue 16-17) pp:2088-2096
Publication Date(Web):
DOI:10.1002/jssc.201100155
Abstract
Porous zwitterionic monolithic columns based on photo-initiated copolymerization of N,N-dimethyl-N-methacryloxyethyl-N-(3-sulfopropyl)ammonium betaine and poly(ethylene glycol) diacrylate in a binary porogen system comprising isopropanol and decanol were prepared in 75-μm-id fused silica capillaries. The resulting monolith was evaluated by hydrophilic interaction chromatography. Inverse size-exclusion chromatography was used to characterize the pore structure of the resulting monolith. A typical hydrophilic interaction chromatography mechanism was observed when the organic content in the mobile phase was higher than 60%. Good separations of amides, phenols, and benzoic acids were achieved. An efficiency of 75 000 plates/m was obtained. The effects of mobile phase pH, salt concentration, and organic modifier content on retention were investigated. For polar-charged analytes, both hydrophilic interactions and electrostatic interactions contributed to the selectivity.
Co-reporter:Aaron N. Nackos, Tai V. Truong, Trenton C. Pulsipher, Jon A. Kimball, H. Dennis Tolley, Richard A. Robison, Calvin H. Bartholomew and Milton L. Lee
Analytical Methods 2011 vol. 3(Issue 2) pp:245-258
Publication Date(Web):06 Dec 2010
DOI:10.1039/C0AY00270D
Methyl sulfate (MeSO4−) salts were explored as thermochemolysis–methylation (TCM) reagents for gas chromatographic (GC) analysis of dipicolinic acid (DPA) as its dimethyl ester (Me2DPA) from bacterial endospores. The reaction was carried out under non-pyrolytic conditions by inserting a small coiled wire filament coated with the sample and reagents directly inside a GC injection port at 290 °C. Above 10:1 methyl donor/DPA ratios, alkali metal salts of MeSO4− effected 80–90% conversion of DPA to Me2DPA, which was 10–20 times more active than the same amount of tetramethylammonium hydroxide (TMA-OH) at this temperature. A quaternary salt mixture consisting of 1:3:1:3 TMA+/Na+/OH−/MeSO4− methylated spore DPA with an average conversion of 86% (mean conversion by TMA-OH under the same conditions was 4%). Therefore, the sensitivity for detection of bacterial endospores was increased over 20-fold compared to that observed with the more commonly employed TMA-OH methylating reagent. The limit of detection by this method was 9 × 104 total spores. Mechanisms describing the observed behavior are proposed and discussed. This is the first use of MeSO4− as a TCM reagent for GC.
Co-reporter:Miao Wang;Hannah E. Quist;Brett J. Hansen
Journal of The American Society for Mass Spectrometry 2011 Volume 22( Issue 2) pp:369-378
Publication Date(Web):2011 February
DOI:10.1007/s13361-010-0027-2
The halo ion trap (IT) was modified to allow for axial ion ejection through slits machined in the ceramic electrode plates rather than ejecting ions radially to a center hole in the plates. This was done to preserve a more uniform electric field for ion analysis. An in-depth evaluation of the higher-order electric field components in the trap was also performed to improve resolution. The linear, cubic and quintic (5th order) electric field components for each electrode ring inside the IT were calculated using SIMION (SIMION version 8, Scientific Instrument Services, Ringoes, NJ, USA) simulations. The preferred electric fields with higher-order components were implemented experimentally by first calculating the potential on each electrode ring of the halo IT and then soldering appropriate capacitors between rings without changing the original trapping plate design. The performance of the halo IT was evaluated for 1% to 7% cubic electric field (A4/A2) component. A best resolution of 280 (m/Δm) for the 51-Da fragment ion of benzene was observed with 5% cubic electric field component. Confirming results were obtained using toluene, dichloromethane, and heptane as test analytes.
Co-reporter:Xin Chen, H. Dennis Tolley, Milton L. Lee
Journal of Chromatography A 2010 Volume 1217(Issue 24) pp:3844-3854
Publication Date(Web):11 June 2010
DOI:10.1016/j.chroma.2010.04.032
Two different monoliths, both containing phosphoric acid functional groups and polyethylene glycol (PEG) functionalities were synthesized for cation-exchange chromatography of peptides and proteins. Phosphoric acid 2-hydroxyethyl methacrylate (PAHEMA) and bis[2-(methacryloyloxy)ethyl] phosphate (BMEP) were reacted with polyethylene glycol diacrylate (PEGDA) and polyethylene glycol acrylate (PEGA), respectively, in 75-μm i.d. UV-transparent fused-silica capillaries by photo-initiated polymerization. The hydrophobicities of the monoliths were evaluated using propyl paraben under reversed-phase conditions and synthetic peptides under ion-exchange conditions. The resulting monoliths exhibited lower hydrophobicities than strong cation-exchange monoliths previously reported using PEGDA as cross-linker. Dynamic binding capacities of 31.2 and 269 mg/mL were measured for the PAHEMA–PEGDA and BMEP–PEGA monoliths, respectively. Synthetic peptides were eluted from both monoliths in 15 min without addition of acetonitrile to the mobile phase. Peak capacities of 50 and 31 were measured for peptides and proteins, respectively, using a PAHEMA–PEGDA monolith. The BMEP–PEGA monolith showed negligible hydrophobicity. A peak capacity of 31 was measured for the BMEP–PEGA monolith when a 20-min salt gradient rate was used to separate proteins. The effects of functional group concentration, mobile phase pH, salt gradient rate, and hydrophobicity on the retention of analytes were investigated. Good run-to-run [relative standard deviation (RSD) < 1.99%] and column-to-column (RSD < 5.64) reproducibilities were achieved. The performance of the monoliths in ion-exchange separation of peptides and proteins was superior to other polymeric monolithic columns reported previously when organic solvents were not added to the mobile phase.
Co-reporter:Yun Li, H. Dennis Tolley, Milton L. Lee
Journal of Chromatography A 2010 Volume 1217(Issue 52) pp:8181-8185
Publication Date(Web):24 December 2010
DOI:10.1016/j.chroma.2010.10.067
Biocompatible poly(ethylene glycol methyl ether acrylate-co-polyethylene glycol diacrylate) monoliths were prepared for size exclusion chromatography (SEC) of proteins in the capillary format using Brij 58P in a mixture of hexanes and dodecanol as porogens. The monolithic columns provided size separation of four proteins in 20 mM sodium phosphate buffer (pH 7.0) containing 0.15 M NaCl, and there was a linear relationship between the retention times and the logarithmic values of the molecular weights. Compared to SEC monoliths previously synthesized using a triblock copolymer of polyethylene oxide and polypropylene oxide, an increase in mesoporosity was confirmed by inverse size exclusion chromatography. As a result, improved protein separation in the high molecular weight range and reduced column back-pressure were observed.
Co-reporter:Yuanyuan Li, H. Dennis Tolley, Milton L. Lee
Journal of Chromatography A 2010 Volume 1217(Issue 30) pp:4934-4945
Publication Date(Web):23 July 2010
DOI:10.1016/j.chroma.2010.05.048
Rigid monoliths were synthesized solely from poly(ethylene glycol) diacrylates (PEGDA) or poly(ethylene glycol) dimethacrylates (PEGDMA) containing different ethylene glycol chain lengths by one-step UV-initiated polymerization. Methanol/ethyl ether and cyclohexanol/decanol were used as bi-porogen mixtures for the PEGDA and PEGDMA monoliths, respectively. Effects of PEG chain length, bi-porogen ratio and reaction temperature on monolith morphology and back pressure were investigated. For tri- and tetra-ethylene glycol diacrylates (i.e., PEGDA 258 and PEGDA 302), most combinations of methanol and ethyl ether were effective in forming monoliths, while for diacrylates containing longer chain lengths (i.e., PEGDA 575 and PEGDA 700), polymerization became more sensitive to the bi-porogen ratio. A similar tendency was also observed for PEGDMA monomers. Polymerization of monoliths was conducted at approximately 0 °C and room temperature, which produced significant differences in monolith morphology and permeability. Monoliths prepared from PEGDA 258 were found to provide the best chromatographic performance with respect to peak capacity and resolution in hydrophobic interaction chromatography (HIC). Detailed study of these monoliths demonstrated that chromatographic performance was not affected by changing the ratios of the two porogens, but resulted in almost identical retention times and comparable peak capacities. An optimized PEGDA 258 monolithic column was able to separate proteins using a 20-min elution gradient with a peak capacity of 62. Mass recoveries for test proteins were found to be greater than 90, indicating its excellent biocompatibility. All monoliths demonstrated nearly no swelling or shrinking in different polarity solvents, and most of them could be stored dry, indicating excellent stability due to their highly crosslinked networks. The preparation of these in situ polymerized single-monomer monolithic columns was highly reproducible. The relative standard deviation (RSD) values based on retention times of retained proteins were all within 2.2%, and in most cases, less than 1.2%. The RSD values based on peak areas were within 9.5%, and in most cases, less than 7.0%. The single-monomer synthesis approach clearly improves column-to-column reproducibility.
Co-reporter:Yan Fang, H. Dennis Tolley, Milton L. Lee
Journal of Chromatography A 2010 Volume 1217(Issue 41) pp:6405-6412
Publication Date(Web):8 October 2010
DOI:10.1016/j.chroma.2010.08.026
A simple capillary flow porometer (CFP) was assembled for through-pore structure characterization of monolithic capillary liquid chromatography columns in their original chromatographic forms. Determination of differential pressures and flow rates through dry and wet short capillary segments provided necessary information to determine the mean diameters and size distributions of the through-pores. The mean through-pore diameters of three capillary columns packed with 3, 5, and 7 μm spherical silica particles were determined to be 0.5, 1.0 and 1.4 μm, with distributions ranging from 0.1 to 0.7, 0.3 to 1.1 and 0.4 to 2.6 μm, respectively. Similarly, the mean through-pore diameters and size distributions of silica monoliths fabricated via phase separation by polymerization of tetramethoxysilane (TMOS) in the presence of poly(ethylene glycol) (PEG) verified that a greater number of through-pores with small diameters were prepared in columns with higher PEG content in the prepolymer mixture. The CFP system was also used to study the effects of column inner diameter and length on through-pore properties of polymeric monolithic columns. Typical monoliths based on butyl methacrylate (BMA) and poly(ethylene glycol) diacrylate (PEGDA) in capillary columns with different inner diameters (i.e., 50–250 μm) and lengths (i.e., 1.5–3.0 cm) were characterized. The results indicate that varying the inner diameter and/or the length of the column had little effect on the through-pore properties. Therefore, the through-pores are highly interconnected and their determination by CFP is independent of capillary length.
Co-reporter:Tai V. Truong, Aaron N. Nackos, John R. Williams, Douglas N. VanDerwerken, Jon A. Kimball, Jacolin A. Murray, Jason E. Hawkes, Donald J. Harvey, H. Dennis Tolley, Richard A. Robison, Calvin H. Bartholomew and Milton L. Lee
Analytical Methods 2010 vol. 2(Issue 6) pp:638-644
Publication Date(Web):12 Apr 2010
DOI:10.1039/B9AY00198K
A simple method to detect and differentiate Bacillus anthracis (BA), Bacillus thuringiensis (BT), Bacillus atrophaeus (BG), and Bacillus cereus (BC) endospores using biomarker compounds, including dipicolinic acid methyl ester (DPAME) and fatty acid methyl esters (FAMEs), has been developed. The method is based on thermochemolysis methylation (TCM) of the endospores and gas chromatography-mass spectrometry (GC-MS) of the reaction products. A suspension of the sample mixed with sulfuric acid (H2SO4) and tetramethylammonium hydroxide (TMAH) in methanol (MeOH) at room temperature is sampled using a coiled wire filament (CWF) device, which consists of a tiny platinum helical wire coil attached to a retractable plunger that moves the coil in and out of a syringe needle housing. Sampling is accomplished by dipping the CWF in an endospore sample suspension, evaporating the suspension liquid, and then introducing the CWF into the injection port. While DPAME can be used for the general detection of endospores, specific saturated and unsaturated C15, C16, and C17 fatty acid methyl esters provide additional information for differentiating various Bacillus species grown at different temperatures and in different media. DPAME could be detected in samples containing as few as 6000 endospores, and the GC-MS peak area percent reproducibility for FAMEs varied from 3 to 13% (RSD). Better than 97% correct predictability of Bacillus species identity was obtained from a blind experiment consisting of 145 samples.
Co-reporter:Yun Li, H. Dennis Tolley and Milton L. Lee
Analytical Chemistry 2009 Volume 81(Issue 11) pp:4406
Publication Date(Web):April 30, 2009
DOI:10.1021/ac900364d
Protein-resistant poly(ethylene glycol methyl ether acrylate-co-polyethylene glycol diacrylate) monoliths were prepared in 150 μm i.d. capillaries using novel binary porogenic solvents consisting of ethyl ether and poly(ethylene oxide)-poly(propylene oxide)-poly(ethylene oxide) (PEO-PPO-PEO) or PPO-PEO-PPO copolymer with molecular weights (MWs) from 2700 to 5800. The effects of the MWs and concentrations of these surfactants in the porogenic solvent mixture on the pore properties of the resultant monoliths were investigated. Several of the monoliths showed improvements in protein and peptide separations over an extended MW range compared to monoliths synthesized using non-surfactant porogens (i.e., low MW organic liquids). The pore size distributions were examined using inverse size-exclusion chromatography (ISEC) of a select series of proteins and peptides covering a wide MW range. It was found that the best monolith had relatively large fractions of micropores (<2 nm, 11.9%) and mesopores in the range from 2.8 to 15.7 nm (8.5%), which are important for size-exclusion separation of peptides and proteins, respectively. The new monoliths possessed high mechanical rigidity that enabled them to withstand pressures up to ∼4000 psi.
Co-reporter:Yuanyuan Li, H. Dennis Tolley and Milton L. Lee
Analytical Chemistry 2009 Volume 81(Issue 22) pp:9416
Publication Date(Web):October 19, 2009
DOI:10.1021/ac9020038
Rigid poly[hydroxyethyl acrylate-co-poly(ethylene glycol) diacrylate] monoliths were synthesized inside 75 μm i.d. capillaries by one-step UV-initiated copolymerization using methanol and ethyl ether as porogens. The optimized monolithic column was evaluated for hydrophobic interaction chromatography (HIC) of standard proteins. Six proteins were separated within 20 min with high resolution using a 20 min elution gradient, resulting in a peak capacity of 54. The effect of gradient rate and initial salt concentration on the retention of proteins were investigated. Mass recovery was found to be greater than 96%, indicating the biocompatibility of this monolith. The monolith was mechanically stable and showed nearly no swelling or shrinking in different polarity solvents. The preparation of this in situ polymerized acrylate monolithic column was highly reproducible. The run-to-run and column-to-column reproducibilities were less than 2.0% relative standard deviation (RSD) on the basis of the retention times of protein standards. The performance of this monolithic column for HIC was comparable or superior to the performance of columns packed with small particles.
Co-reporter:Xuefei Sun, Dan Li and Milton L. Lee
Analytical Chemistry 2009 Volume 81(Issue 15) pp:6278
Publication Date(Web):July 2, 2009
DOI:10.1021/ac9001832
Recently, we reported the synthesis, fabrication, and preliminary evaluation of poly(ethylene glycol) (PEG)-functionalized polymeric microchips that are inherently resistant to protein adsorption without surface modification in capillary electrophoresis (CE). In this study, we investigated the impact of cross-linker purity and addition of methyl methacrylate (MMA) as a comonomer on CE performance. Impure poly(ethylene glycol) diacrylate (PEGDA) induced electroosmotic flow (EOF) and increased the separation time, while the addition of MMA decreased the separation efficiency to approximately 25% of that obtained using microchips fabricated without MMA. Resultant improved microchips were evaluated for the separation of fluorescent dyes, amino acids, peptides, and proteins. A CE efficiency of 4.2 × 104 plates for aspartic acid in a 3.5 cm long microchannel was obtained. Chiral separation of 10 different d,l-amino acid pairs was obtained with addition of a chiral selector (i.e., β-cyclodextrin) in the running buffer. Selectivity (α) and resolution (Rs) for d,l-leucine were 1.16 and 1.64, respectively. Good reproducibility was an added advantage of these PEG-functionalized microchips.
Co-reporter:Yun Li, Binghe Gu, H. Dennis Tolley, Milton L. Lee
Journal of Chromatography A 2009 Volume 1216(Issue 29) pp:5525-5532
Publication Date(Web):17 July 2009
DOI:10.1016/j.chroma.2009.05.037
Two novel polymeric monoliths for anion-exchange capillary liquid chromatography of proteins were prepared in a single step by a simple photoinitiated copolymerization of 2-(diethylamino)ethyl methacrylate and polyethylene glycol diacrylate (PEGDA), or copolymerization of 2-(acryloyloxy)ethyl trimethylammonium chloride and PEGDA, in the presence of selected porogens. The resulting monoliths contained functionalities of diethylaminoethyl (DEAE) as a weak anion-exchanger and quaternary amine as a strong anion-exchanger, respectively. An alternative weak anion-exchange monolith with DEAE functionalities was also synthesized by chemical modification after photoinitiated copolymerization of glycidyl methacrylate (GMA) and PEGDA. Important physical and chromatographic properties of the synthesized monoliths were characterized. The dynamic binding capacities of the three monoliths (24 mg/mL, 56 mg/mL and 32 mg/mL of column volume, respectively) were comparable or superior to values that have been reported for various other monoliths. Chromatographic performance was also similar to that provided by a modified poly(GMA-ethylene glycol dimethacrylate) monolith. Separation of standard proteins was achieved under gradient elution conditions using these monolithic columns. Peak capacities of 34, 58 and 36 proteins were obtained with analysis times of 20–30 min. This work represents a successful attempt to prepare functionalized monoliths via direct copolymerization of monomers with desired functionalities. Compared to earlier publications, additional surface modifications were avoided and the PEGDA crosslinker helped to improve the biocompatibility of the monolithic backbone.
Co-reporter:Xuefei Sun, Paul B. Farnsworth, H. Dennis Tolley, Karl F. Warnick, Adam T. Woolley, Milton L. Lee
Journal of Chromatography A 2009 Volume 1216(Issue 1) pp:159-164
Publication Date(Web):2 January 2009
DOI:10.1016/j.chroma.2008.11.031
Electric field gradient focusing (EFGF) is a technique used to simultaneously separate and concentrate biomacromolecules, such as proteins, based on the opposing forces of an electric field gradient and a hydrodynamic flow. Recently, we reported EFGF devices fabricated completely from copolymers functionalized with poly(ethylene glycol), which display excellent resistance to protein adsorption. However, the previous devices did not provide the predicted linear electric field gradient and stable current. To improve performance, Tris–HCl buffer that was previously doped in the hydrogel was replaced with a phosphate buffer containing a salt (i.e., potassium chloride, KCl) with high mobility ions. The new devices exhibited stable current, good reproducibility, and a linear electric field distribution in agreement with the shaped gradient region design due to improved ion transport in the hydrogel. The field gradient was calculated based on theory to be approximately 5.76 V/cm2 for R-phycoerythrin when the applied voltage was 500 V. The effect of EFGF separation channel dimensions was also investigated; a narrower focused band was achieved in a smaller diameter channel. The relationship between the bandwidth and channel diameter is consistent with theory. Three model proteins were resolved in an EFGF channel of this design. The improved device demonstrated 14,000-fold concentration of a protein sample (from 2 ng/mL to 27 μg/mL).
Co-reporter:Tai V. Truong, Aaron N. Nackos, Jacolin A. Murray, Jon A. Kimball, Jason E. Hawkes, Donald J. Harvey, H. Dennis Tolley, Richard A. Robison, Calvin H. Bartholomew, Milton L. Lee
Journal of Chromatography A 2009 Volume 1216(Issue 40) pp:6852-6857
Publication Date(Web):2 October 2009
DOI:10.1016/j.chroma.2009.08.022
A simple device for field sampling and concentration of analytes for subsequent introduction into an injection port for gas chromatographic (GC) analysis has been developed. It consists of a tiny, coiled platinum wire filament (CWF) that is attached to a retractable plunger wire, which fits inside a syringe needle housing. Sampling is accomplished by dipping the end of the CWF in a liquid sample, which is drawn into the wire coil by capillary action, and introducing it into the injection port either before or after allowing the solvent to evaporate. The CWF can be used with or without a nonvolatile chemical coating. A major advantage of this sampling device is that nonvolatile sample matrix components remain on the wire coil, reducing the required injection port and liner cleaning frequency and contamination of the head of the chromatographic column. The coil itself can be easily cleaned between analyses by rinsing and/or burning off residual material in a small flame. The sampling coil facilitates specifically designed chemical reactions in the injection port, such as thermochemolysis and methylation. Applications demonstrated in this work include: (1) direct introduction of samples with little or no pre-treatment, (2) simultaneous thermochemolysis and methylation of lipid-containing samples such as bacteria and bacterial endospores for analysis of biomarkers, and (3) solid phase micro-extraction (SPME) using temporary wire coatings. The CWF allowed for significant reduction in sample preparation time, in most cases to less than a few minutes. The peak shapes examined for polycyclic aromatic hydrocarbon analytes (PAHs) were significantly better (asymmetry factors <1.3) when using the CWF sampling technique compared to splitless and on-column injection techniques (asymmetry factors >1.3). Extraction efficiencies for SPME (especially for high boiling point components such as PAHs) improved by an average of 2.5 times when using the CWF compared to the performance of commercially available SPME fibers. Coiled wire filaments and GC injection port liners were used for more than 100 Bacillus endospore thermochemolysis methylation analyses without the need for cleaning or replacement.
Co-reporter:Xuefei Sun, Dan Li, Adam T. Woolley, Paul B. Farnsworth, H. Dennis Tolley, Karl F. Warnick, Milton L. Lee
Journal of Chromatography A 2009 Volume 1216(Issue 37) pp:6532-6538
Publication Date(Web):11 September 2009
DOI:10.1016/j.chroma.2009.07.050
Electric field gradient focusing (EFGF) uses an electric field gradient and a hydrodynamic counter flow to simultaneously separate and focus charged analytes in a channel. Previously, most EFGF devices were designed to form a linear field gradient in the channel. However, the peak capacity obtained using a linear gradient is not much better than what can be obtained using conventional CE. Dynamic improvement of peak capacity in EFGF can be achieved by using a nonlinear gradient. Numerical simulation results indicate that the peak capacity in a 4-cm long channel can be increased from 20 to 150 when changing from a linear to convex bilinear gradient. To demonstrate the increased capacity experimentally, an EFGF device with convex bilinear gradient was fabricated from poly(ethylene glycol) (PEG)-functionalized acrylic copolymers. The desired gradient profile was confirmed by measuring the focusing positions of a standard protein for different counter flow rates at constant voltage. Dynamically controlled elution of analytes was demonstrated using a monolith-filled bilinear EFGF channel. By increasing the flow rate, stacked proteins that were ordered but not resolved after focusing in the steep gradient segment were moved into the shallow gradient segment, where the analyte peak resolution increased significantly. In this way, the nonlinear field gradient was used to realize a dynamic increase in the peak capacity of the EFGF method.
Co-reporter:Xin Chen;H. Dennis Tolley
Journal of Separation Science 2009 Volume 32( Issue 15-16) pp:2565-2573
Publication Date(Web):
DOI:10.1002/jssc.200900255
Abstract
A strong cation-exchange (SCX) monolithic stationary phase was prepared in 75 μm id capillaries by direct in situ polymerization of sulfopropyl methacrylate and polyethylene glycol diacrylate in a ternary porogen system consisting of methanol, cyclohexanol, and water. The resulting monolith exhibited good dynamic binding capacity, fast kinetic adsorption of proteins, and high permeability. The monolith had a dynamic binding capacity of ˜52 mg/mL of column volume for lysozyme and cytochrome C. The monolith was evaluated for SCX capillary LC of synthetic peptides, natural peptides, and protein standards. Fast separation of proteins was achieved in less than 4 min. The average peak capacity for peptides was 28 using a relatively steep gradient when hydrophobic interactions were suppressed with 40% acetonitrile.
Co-reporter:Yun Li
Journal of Separation Science 2009 Volume 32( Issue 20) pp:3369-3378
Publication Date(Web):
DOI:10.1002/jssc.200900478
Abstract
The concept of biocompatibility with reference to chromatographic stationary phases for separation of biomolecules (including proteins and peptides) is introduced. Biocompatible is a characteristic that indicates resistance to nonspecific adsorption of biomolecules and preservation of their structures and biochemical functions. Two types of biocompatible polymeric monoliths [i. e., polyacrylamide- and poly(meth)acrylate-based monoliths] used for protein and peptide separations are reviewed in detail, with emphasis on size exclusion, ion exchange, and hydrophobic interaction chromatographic modes. Biocompatible monoliths for enzyme reactors are also included. The two main synthetic approaches to produce biocompatible monoliths are summarized, i. e., surface modification of a monolith that is not inherently biocompatible and direct copolymerization of hydrophilic monomers to form a biocompatible monolith directly. Integration of polyethylene glycol into the poly(meth)acrylate monolith network is becoming popular for reduction of non-specific protein interactions.
Co-reporter:Shu-Ling Lin, Yuanyuan Li, H. Dennis Tolley, Paul H. Humble, Milton L. Lee
Journal of Chromatography A 2006 Volume 1125(Issue 2) pp:254-262
Publication Date(Web):1 September 2006
DOI:10.1016/j.chroma.2006.05.041
Two electric field gradient focusing (EFGF) systems, one based on a hollow dialysis fiber and the other based on a shaped ionically conductive polymer were serially integrated to trap and concentrate selected proteins while simultaneously desalting and removing other unwanted proteins from the sample. A series of experiments were performed to test the EFGF systems individually and after integration. Online concentration of amyloglucosidase indicated a concentration limit of detection of approximatedly 20 ng mL−1 (200 pM) from a sample volume of 100 μL. Concentration of human α1-acid glycoprotein with simultaneous removal of human serum albumin was also demonstrated. Elimination of small buffer components while concentrating trypsin inhibitor, and selective concentration and separation of myoglobin from a mixture were performed using the integrated EFGF system.
Co-reporter:Yinhan Gong, Yanqiao Xiang, Bingfang Yue, Guoping Xue, Jerald S. Bradshaw, Hian Kee Lee, Milton L. Lee
Journal of Chromatography A 2003 Volume 1002(1–2) pp:63-70
Publication Date(Web):20 June 2003
DOI:10.1016/S0021-9673(03)00732-5
Two bonded chiral stationary phases (CSPs), 8-aminoquinoline-2-ylmethyl- and 8-aminoquinoline-7-ylmethyl-diaza-18-crown-6-capped [3-(2-O-β-cyclodextrin)-2-hydroxypropoxy]propylsilyl silica particles (non-porous, 1.5 μm), have been prepared and evaluated using capillary liquid chromatography at high pressures (≥8000 p.s.i.). High column efficiency (up to 400 000 plates m−1) was achieved for chiral separations. These CSPs with two recognition sites, i.e. substituted-diaza-18-crown-6 and β-cyclodextrin combined with high chromatographic efficiency provide good resolution of a variety of enantiomers and positional isomers in relatively short times under reversed-phase conditions. After inclusion of a Ni (II) ion from the mobile phase, the positively charged crown ether-capped β-cyclodextrin facilitates specific static, dipolar, and host–guest complexation interactions with solutes.
Co-reporter:Yanqiao Xiang, Bingwen Yan, Clayton V. McNeff, Peter W. Carr, Milton L. Lee
Journal of Chromatography A 2003 Volume 1002(1–2) pp:71-78
Publication Date(Web):20 June 2003
DOI:10.1016/S0021-9673(03)00733-7
In this study, 1-μm diameter polybutadiene-encapsulated non-porous zirconia particles were synthesized, slurry packed into 50-μm I.D. fused-silica capillary columns, and evaluated using ultrahigh pressure liquid chromatography. The dependencies of column efficiency and solute retention factor on pressure were investigated. Efficiencies as high as 280 000 plates per meter were obtained for the separation of anti-inflammatory drugs at a pressure of 1351 MPa. Comparing the reversed-phase behavior of the polybutadiene-encapsulated non-porous zirconia with octadecylsilane bonded non-porous silica, greater selectivity was found using the zirconia-based material for the applications reported in this study. The encapsulated non-porous zirconia particles demonstrated excellent thermal stability in the separation of polycyclic aromatic hydrocarbons at a temperature of 100 °C and a pressure of 1351 MPa.
Co-reporter:Yanqiao Xiang, Daniel R Maynes, Milton L Lee
Journal of Chromatography A 2003 Volume 991(Issue 2) pp:189-196
Publication Date(Web):4 April 2003
DOI:10.1016/S0021-9673(03)00171-7
Ultrahigh pressure liquid chromatography (UHPLC) is an emerging technique which utilizes pressures higher than 10 000 p.s.i. to overcome the flow resistance imposed when using very small particles as packing materials in fused-silica capillary columns (1 p.s.i.=6894.76 Pa). This technique has demonstrated exceptionally high separation speeds and chromatographic efficiencies. However, safety is a concern when extremely high pressures are used. In this study, the safety aspects of capillary column rupture during operation were identified and carefully evaluated. First, liquid jets may be formed as a result of blow-out of the on-column frits or from rupture of the capillary at or near the column inlet. Second, incorrect installation of the capillary at the injector, failure of the ferrule used in the capillary connection, or rupture of the capillary can produce high speed projectiles of silica particles or column fragments. Experiments were carried out in the laboratory to produce liquid (water) jets and capillary projectiles using a UHPLC system, and the power density, an important parameter describing water jets in industrial practice, was calculated. Experimental results were in accordance with theoretical calculations. Both indicated that water jets and capillary projectiles under ultrahigh pressures might lead to skin penetration under limited conditions. The use of a plexiglass shroud to cover an initial length of the installed capillary column can eliminate any safety-related concerns about liquid jets or capillary projectiles.
Co-reporter:Qinggang Wang, Shu-Ling Lin, Karl F. Warnick, H.Dennis Tolley, Milton L. Lee
Journal of Chromatography A 2003 Volume 985(1–2) pp:455-462
Publication Date(Web):24 January 2003
DOI:10.1016/S0021-9673(02)01399-7
Electromobility focusing (EMF) is a relatively new protein separation technique that utilizes an electric field gradient and a hydrodynamic flow. Proteins are focused in order of electrophoretic mobility at points where their electrophoretic migration velocities balance the hydrodynamic flow velocity. Steady state bands are formed along the separation channel when equilibrium is reached. Further separation and detection can be easily achieved by changing the electric field profile. In this paper, we describe an EMF system with on-line UV absorption detection in which the electric field gradient was formed using a dialysis hollow fiber. Protein focusing and preconcentration were performed with this system. Voltage-controlled separation was demonstrated using bovine serum albumin and myoglobin as model proteins. The limitations of the current method are discussed, and possible solutions are proposed.
Co-reporter:Yanqiao Xiang, Bingwen Yan, Bingfang Yue, Clayton V McNeff, Peter W Carr, Milton L Lee
Journal of Chromatography A 2003 Volume 983(1–2) pp:83-89
Publication Date(Web):3 January 2003
DOI:10.1016/S0021-9673(02)01662-X
Capillary columns packed with small diameter particles typically lead to low permeability and long separation times in high-performance liquid chromatography. Ultrahigh pressures (>10 000 p.s.i.; 1 p.s.i. ≡6894.76 Pa) can be used to overcome the limitations that small particles impose. Ultrahigh-pressure liquid chromatography (UHPLC) has demonstrated great potential for high-speed and high-efficiency separations. Decreasing the viscosity of the mobile phase by elevating the temperature could additionally reduce the pressure drop and facilitate the use of longer columns or smaller particles to achieve even higher total plate numbers. For this reason, we investigated the use of elevated temperatures in UHPLC. Water-resistant, flexible heater tape covered with insulation was used to provide the desired heat to the column. Polybutadiene-coated 1 μm nonporous zirconia particles were used because of their chemical stability at elevated temperature. A column efficiency as high as 420 000 plates m−1 was obtained. The effects of temperature and pressure on the separation of parabens were investigated. Separation of five herbicides was completed in 60 s using 26 000 p.s.i. and 90 °C.
Co-reporter:Naijun Wu, J.Andreas Lippert, Milton L Lee
Journal of Chromatography A 2001 Volume 911(Issue 1) pp:1-12
Publication Date(Web):9 March 2001
DOI:10.1016/S0021-9673(00)01188-2
A novel pressure-balanced injection valve was evaluated for use with ultrahigh pressure liquid chromatography (UHPLC) at pressures up to 120 MPa (1200 bar). Fused-silica capillaries (30–33 cm×100 μm I.D.) packed with nonporous 1.5 μm isohexylsilane-modified (C6) silica particles were employed to study maximum pressure, injection reproducibility, injection time, and sample amount consumed for an injection. The new valve was more reproducible, convenient, and required much less sample than previously used injection systems. The effect of column diameter on efficiency and sensitivity was studied. The 100 μm I.D. columns demonstrated approximately 40% lower efficiency but 10-fold higher sensitivity than the 29 μm I.D. columns. Columns packed with nonporous C6 particles produced higher efficiencies than columns packed with a 1.5 μm porous octadecylsilane-modified (C18) material.
Co-reporter:Christopher R Bowerbank, Philip A Smith, Dean D Fetterolf, Milton L Lee
Journal of Chromatography A 2000 Volume 902(Issue 2) pp:413-419
Publication Date(Web):15 December 2000
DOI:10.1016/S0021-9673(00)00852-9
A separation technique known as solvating gas chromatography (SGC), which utilizes packed capillary columns and neat carbon dioxide as mobile phase, was used for the separation of nitroglycerine (NG) and other nitrogen-containing explosives including 2,6-dinitrotoluene (2,6-DNT), 2,4-dinitrotolulene (2,4-DNT), 2,4,6-trinitrotoluene (2,4,6-TNT), and pentaerythritol tetranitrate (PETN). SGC was coupled for the first time to a selective chemiluminescence thermal energy analyzer (TEA) detector for nitro-functional group specificity and sensitive detection of these compounds. TEA calibration curve for NG showed linearity in the sub-μg ml−1 range. Soil samples containing NG were used to test the validity of the technique. Detector response of SGC–TEA versus SGC–flame ionization detection for NG was also evaluated.
Co-reporter:Yufeng Shen, Xiaowen Shao, Kim O’Neill, Jerald S Bradshaw, Milton L Lee
Journal of Chromatography A 2000 Volume 866(Issue 1) pp:1-14
Publication Date(Web):7 January 2000
DOI:10.1016/S0021-9673(99)01043-2
Multimodal copolymer-encapsulated particles for liquid chromatography were prepared by bonding 1-octadecene and unsaturated carboxylic acids on silica particles (5 μm diameter, 300 Å pores) for liquid chromatography of proteins. These multimodal copolymer-encapsulated particles can provide both hydrophobic and hydrogen bonding interactions with polar compounds. The chromatographic performance of these multimodal copolymer-encapsulated particles for peptide and protein separations was evaluated under reversed-phase conditions. Compared with typical C8-bonded silica, polymer-encapsulated particles were more stable in acidic mobile phases and provided better recoveries, especially for large proteins (Mr>0.5·106). Totally hydrophobic polymer-encapsulated particles were found to produce broad peaks for proteins, and significant improvements were observed by introducing hydrophilic groups (–COOH) onto the polymer-encapsulated surface to form a multimodal phase. For the reversed-phase liquid chromatography of peptides and proteins, improved selectivity and increased solute retention were found using the multimodal polymer-encapsulated particles. More peaks were resolved for the separation of complex peptide mixtures such as protein digests using the multimodal polymer-encapsulated particles as compared to totally hydrophobic polymer-encapsulated particles.
Co-reporter:S.J.M Butala, J.C Medina, R.J Hulse, C.H Bartholomew, M.L Lee
Fuel 2000 Volume 79(Issue 13) pp:1657-1664
Publication Date(Web):October 2000
DOI:10.1016/S0016-2361(00)00025-9
Three Argonne Premium Coals (Upper-Freeport, Pittsburgh #8, and Lewiston-Stockton) were extracted with benzene in both Soxhlet and pressurized fluid extraction (PFE) systems. The extracts were compared on the basis of dry mass yield and hydrocarbon profiles as obtained by gas chromatography/mass spectrometry. The dry mass yields (wt%, daf) indicated no difference by either method for the Upper-Freeport coal, but statistically different yields were obtained for the Pittsburgh #8 and Lewiston-Stockton coals. In this study, PFE required ∼90 vol.% less solvent compared to Soxhlet extraction. The gas chromatograms obtained from the Soxhlet extracts contained solvent contaminant peaks as dominant peaks, which were not observed in chromatograms of the PFE extracts. No thermal degradation, as indicated by excess phenolic compound concentrations, was noted in any of the PFE extracts.
Co-reporter:Iulia M Lazar, Milton L Lee
Journal of the American Society for Mass Spectrometry 1999 Volume 10(Issue 3) pp:261-264
Publication Date(Web):March 1999
DOI:10.1016/S1044-0305(98)00151-2
The effect of the electrospray ionization (ESI) needle voltage on the electroosmotic flow (EOF) in capillary electrophoresis (CE)-mass spectrometry (MS) was investigated. The radial electric field that penetrates across the CE capillary wall imposed by the ESI needle voltage modifies the typical EOF. This effect was investigated for buffers commonly used in CE-MS. Variations as high as ±30% were observed.
Co-reporter:
Analytical Methods (2009-Present) 2014 - vol. 6(Issue 1) pp:NaN37-37
Publication Date(Web):2013/11/28
DOI:10.1039/C3AY41364K
Reports of novel micro/nanostructures designed to separate biomacromolecules and bioparticles are increasing in number, and these studies have greatly advanced our understanding of nanoscale fluidics and nanoparticle behavior in confined channels. This review is aimed at summarizing previous developments in micro/nanofabricated systems for nanoparticle separations. These are discussed in three groups based on architecture, namely, micro/nanopillar array structures, nanoplane gap structures and artificial nanoporous membranes.
Co-reporter:Aaron N. Nackos, Tai V. Truong, Trenton C. Pulsipher, Jon A. Kimball, H. Dennis Tolley, Richard A. Robison, Calvin H. Bartholomew and Milton L. Lee
Analytical Methods (2009-Present) 2011 - vol. 3(Issue 2) pp:
Publication Date(Web):
DOI:10.1039/C0AY00270D
Co-reporter:Tai V. Truong, Aaron N. Nackos, John R. Williams, Douglas N. VanDerwerken, Jon A. Kimball, Jacolin A. Murray, Jason E. Hawkes, Donald J. Harvey, H. Dennis Tolley, Richard A. Robison, Calvin H. Bartholomew and Milton L. Lee
Analytical Methods (2009-Present) 2010 - vol. 2(Issue 6) pp:NaN644-644
Publication Date(Web):2010/04/12
DOI:10.1039/B9AY00198K
A simple method to detect and differentiate Bacillus anthracis (BA), Bacillus thuringiensis (BT), Bacillus atrophaeus (BG), and Bacillus cereus (BC) endospores using biomarker compounds, including dipicolinic acid methyl ester (DPAME) and fatty acid methyl esters (FAMEs), has been developed. The method is based on thermochemolysis methylation (TCM) of the endospores and gas chromatography-mass spectrometry (GC-MS) of the reaction products. A suspension of the sample mixed with sulfuric acid (H2SO4) and tetramethylammonium hydroxide (TMAH) in methanol (MeOH) at room temperature is sampled using a coiled wire filament (CWF) device, which consists of a tiny platinum helical wire coil attached to a retractable plunger that moves the coil in and out of a syringe needle housing. Sampling is accomplished by dipping the CWF in an endospore sample suspension, evaporating the suspension liquid, and then introducing the CWF into the injection port. While DPAME can be used for the general detection of endospores, specific saturated and unsaturated C15, C16, and C17 fatty acid methyl esters provide additional information for differentiating various Bacillus species grown at different temperatures and in different media. DPAME could be detected in samples containing as few as 6000 endospores, and the GC-MS peak area percent reproducibility for FAMEs varied from 3 to 13% (RSD). Better than 97% correct predictability of Bacillus species identity was obtained from a blind experiment consisting of 145 samples.
Co-reporter:
Analytical Methods (2009-Present) 2013 - vol. 5(Issue 22) pp:
Publication Date(Web):
DOI:10.1039/C3AY41393D
A simple approach for preparing standard mixtures of volatile and semi-volatile organic compounds is reported. When placed in a closed container, standard mixture components partition between a polymeric material such as poly(dimethylsiloxane) (PDMS) and headspace to provide constant vapor concentrations. The granular form of heat-conditioned PDMS provides rapid equilibration with the headspace vapor and serves as a standard reservoir. Solid phase micro extraction (SPME) or gas-tight syringe can be used to deliver sample from the headspace to the analytical instrument. Quantitative calibration can be achieved with either active temperature control or by using a previously constructed look-up table. The effects of PDMS form and temperature on equilibrium distribution, initial equilibrium time, and re-equilibrium time after sampling were investigated. With respect to long term use and stability, analytes introduced onto 2.0 g of PDMS in a 7.4 mL vial were sampled more than 114 times during a test period of 43 days, giving chromatographic peak area %RSD values below 4.5% for all compounds. This device was designed to be solventless, quantitative, reproducible, environmentally friendly, and robust for routine evaluation and calibration of gas chromatography-mass spectrometry (GC-MS) systems.
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![Naphtho[8,1,2-bcd]perylene](http://img.cochemist.com/ccimg/200/188-89-6.png)
![Naphtho[8,1,2-bcd]perylene](http://img.cochemist.com/ccimg/200/188-89-6_b.png)
![Benzo[pqr]dinaphtho[8,1,2-bcd:2',1',8'-lmn]perylene](/data/chemimg/927500/188-11-4.png)
![Benzo[pqr]dinaphtho[8,1,2-bcd:2',1',8'-lmn]perylene](/data/chemimg/927500/188-11-4_b.png)
![6-Isoquinolinol,1-[(3,4-dimethoxyphenyl)methyl]-1,2,3,4-tetrahydro-7-methoxy-2-methyl- (9CI)](http://img.cochemist.com/ccimg/100/85-65-4.png)
![6-Isoquinolinol,1-[(3,4-dimethoxyphenyl)methyl]-1,2,3,4-tetrahydro-7-methoxy-2-methyl- (9CI)](http://img.cochemist.com/ccimg/100/85-65-4_b.png)
![6,7-Dihydrodipyrido[1,2-a:2',1'-c]pyrazine-5,8-diium bromide](http://img.cochemist.com/ccimg/100/85-00-7.png)
![6,7-Dihydrodipyrido[1,2-a:2',1'-c]pyrazine-5,8-diium bromide](http://img.cochemist.com/ccimg/100/85-00-7_b.png)
![3-(10,11-Dihydro-5H-dibenzo[b,f]azepin-5-yl)-N-methylpropan-1-amine](http://img.cochemist.com/ccimg/100/50-47-5.png)
![3-(10,11-Dihydro-5H-dibenzo[b,f]azepin-5-yl)-N-methylpropan-1-amine](http://img.cochemist.com/ccimg/100/50-47-5_b.png)
![3'-Hydroxy-[1,1'-biphenyl]-3-carbonitrile](http://img.cochemist.com/ccimg/154900/154848-43-8.png)
![3'-Hydroxy-[1,1'-biphenyl]-3-carbonitrile](http://img.cochemist.com/ccimg/154900/154848-43-8_b.png)
![[1,1'-Biphenyl]-2-carbonitrile, 3'-hydroxy-](http://img.cochemist.com/ccimg/154900/154848-41-6.png)
![[1,1'-Biphenyl]-2-carbonitrile, 3'-hydroxy-](http://img.cochemist.com/ccimg/154900/154848-41-6_b.png)
![3'-Methoxy-[1,1'-biphenyl]-4-carbonitrile](http://img.cochemist.com/ccimg/154900/154848-39-2.png)
![3'-Methoxy-[1,1'-biphenyl]-4-carbonitrile](http://img.cochemist.com/ccimg/154900/154848-39-2_b.png)
![3'-Methoxy-[1,1'-biphenyl]-3-carbonitrile](http://img.cochemist.com/ccimg/154900/154848-38-1.png)
![3'-Methoxy-[1,1'-biphenyl]-3-carbonitrile](http://img.cochemist.com/ccimg/154900/154848-38-1_b.png)
![3'-Methoxy-[1,1'-biphenyl]-2-carbonitrile](http://img.cochemist.com/ccimg/154900/154848-36-9.png)
![3'-Methoxy-[1,1'-biphenyl]-2-carbonitrile](http://img.cochemist.com/ccimg/154900/154848-36-9_b.png)
![[1,1'-Biphenyl]-2-carbonitrile, 2'-methoxy-](http://img.cochemist.com/ccimg/150800/150766-96-4.png)
![[1,1'-Biphenyl]-2-carbonitrile, 2'-methoxy-](http://img.cochemist.com/ccimg/150800/150766-96-4_b.png)
![[1,1'-BIPHENYL]-2-CARBONITRILE, 4'-HYDROXY-](http://img.cochemist.com/ccimg/137900/137863-68-4.png)
![[1,1'-BIPHENYL]-2-CARBONITRILE, 4'-HYDROXY-](http://img.cochemist.com/ccimg/137900/137863-68-4_b.png)
![3'-Hydroxy-[1,1'-biphenyl]-4-carbonitrile](http://img.cochemist.com/ccimg/127800/127703-35-9.png)
![3'-Hydroxy-[1,1'-biphenyl]-4-carbonitrile](http://img.cochemist.com/ccimg/127800/127703-35-9_b.png)
![4'-Methoxy-[1,1'-biphenyl]-2-carbonitrile](http://img.cochemist.com/ccimg/125700/125610-78-8.png)
![4'-Methoxy-[1,1'-biphenyl]-2-carbonitrile](http://img.cochemist.com/ccimg/125700/125610-78-8_b.png)
![2'-Methoxy-[1,1'-biphenyl]-4-carbonitrile](http://img.cochemist.com/ccimg/113600/113547-30-1.png)
![2'-Methoxy-[1,1'-biphenyl]-4-carbonitrile](http://img.cochemist.com/ccimg/113600/113547-30-1_b.png)
![[1,1'-Biphenyl]-4-carboxylic acid, 4'-(2-propenyloxy)-](http://img.cochemist.com/ccimg/93100/93001-09-3.png)
![[1,1'-Biphenyl]-4-carboxylic acid, 4'-(2-propenyloxy)-](http://img.cochemist.com/ccimg/93100/93001-09-3_b.png)
![2-methylphenanthro[4,5-bcd]thiophene](http://img.cochemist.com/ccimg/88200/88114-00-5.png)
![2-methylphenanthro[4,5-bcd]thiophene](http://img.cochemist.com/ccimg/88200/88114-00-5_b.png)
![Phosphonic acid, (benzo[b]thien-3-ylmethyl)-, diethyl ester](http://img.cochemist.com/ccimg/80100/80090-41-1.png)
![Phosphonic acid, (benzo[b]thien-3-ylmethyl)-, diethyl ester](http://img.cochemist.com/ccimg/80100/80090-41-1_b.png)
![11-METHYLNAPHTHO[2,3-B][1]BENZOTHIOLE](http://img.cochemist.com/ccimg/80000/79966-33-9.png)
![11-METHYLNAPHTHO[2,3-B][1]BENZOTHIOLE](http://img.cochemist.com/ccimg/80000/79966-33-9_b.png)
![Phenaleno[1,9-bc]thiophene](http://img.cochemist.com/ccimg/80000/79965-99-4.png)
![Phenaleno[1,9-bc]thiophene](http://img.cochemist.com/ccimg/80000/79965-99-4_b.png)
![PHENANTHRO[4,5-BCD]THIOPHENE, 1-METHYL-](http://img.cochemist.com/ccimg/79400/79313-24-9.png)
![PHENANTHRO[4,5-BCD]THIOPHENE, 1-METHYL-](http://img.cochemist.com/ccimg/79400/79313-24-9_b.png)
![PHENANTHRO[1,2-B][1]BENZOTHIOLE](http://img.cochemist.com/ccimg/56000/55969-62-5.png)
![PHENANTHRO[1,2-B][1]BENZOTHIOLE](http://img.cochemist.com/ccimg/56000/55969-62-5_b.png)
![[4,8-BIS(OCTYLOXY)THIENO[2,3-F][1]BENZOTHIENE-2,6-DIYL]BIS(TRIMET<WBR />HYLSTANNANE)](http://img.cochemist.com/ccimg/7400/7384-07-8.png)
![[4,8-BIS(OCTYLOXY)THIENO[2,3-F][1]BENZOTHIENE-2,6-DIYL]BIS(TRIMET<WBR />HYLSTANNANE)](http://img.cochemist.com/ccimg/7400/7384-07-8_b.png)
![[1,1'-Biphenyl]-3-carbonitrile, 6-hydroxy-](http://img.cochemist.com/ccimg/6800/6781-76-6.png)
![[1,1'-Biphenyl]-3-carbonitrile, 6-hydroxy-](http://img.cochemist.com/ccimg/6800/6781-76-6_b.png)
![Benzenemethanol,4-hydroxy-3-methoxy-a-[(methylamino)methyl]-](http://img.cochemist.com/ccimg/5100/5001-33-2.png)
![Benzenemethanol,4-hydroxy-3-methoxy-a-[(methylamino)methyl]-](http://img.cochemist.com/ccimg/5100/5001-33-2_b.png)
![6-methylbenzo[b]naphtho[2,1-d]thiophene](http://img.cochemist.com/ccimg/4600/4567-51-5.png)
![6-methylbenzo[b]naphtho[2,1-d]thiophene](http://img.cochemist.com/ccimg/4600/4567-51-5_b.png)
![[1,1'-Biphenyl]ol](http://img.cochemist.com/ccimg/1400/1322-20-9.png)
![[1,1'-Biphenyl]ol](http://img.cochemist.com/ccimg/1400/1322-20-9_b.png)
![Card-20(22)-enolide,3-[(6-deoxy-a-L-mannopyranosyl)oxy]-1,5,11,14,19-pentahydroxy-,(1b,3b,5b,11a)-](http://img.cochemist.com/ccimg/700/630-60-4.png)
![Card-20(22)-enolide,3-[(6-deoxy-a-L-mannopyranosyl)oxy]-1,5,11,14,19-pentahydroxy-,(1b,3b,5b,11a)-](http://img.cochemist.com/ccimg/700/630-60-4_b.png)
![[1]Benzothieno[3,2-b]pyridine](http://img.cochemist.com/ccimg/400/318-69-4.png)
![[1]Benzothieno[3,2-b]pyridine](http://img.cochemist.com/ccimg/400/318-69-4_b.png)
![5,10-Dioxatricyclo[7.1.0.04,6]decane](http://img.cochemist.com/ccimg/300/286-75-9.png)
![5,10-Dioxatricyclo[7.1.0.04,6]decane](http://img.cochemist.com/ccimg/300/286-75-9_b.png)
![[1]Benzothieno[2,3-b]pyridine](http://img.cochemist.com/ccimg/300/244-93-9.png)
![[1]Benzothieno[2,3-b]pyridine](http://img.cochemist.com/ccimg/300/244-93-9_b.png)
![[1]Benzothieno[2,3-c]pyridine](http://img.cochemist.com/ccimg/300/244-90-6.png)
![[1]Benzothieno[2,3-c]pyridine](http://img.cochemist.com/ccimg/300/244-90-6_b.png)
![5H-Benzo[b]carbazole](http://img.cochemist.com/ccimg/300/243-28-7.png)
![5H-Benzo[b]carbazole](http://img.cochemist.com/ccimg/300/243-28-7_b.png)
![Phenanthro[9,10-b]thiophene](http://img.cochemist.com/ccimg/300/236-01-1.png)
![Phenanthro[9,10-b]thiophene](http://img.cochemist.com/ccimg/300/236-01-1_b.png)
![Naphtho[2,1-b]thiophene](http://img.cochemist.com/ccimg/300/233-02-3.png)
![Naphtho[2,1-b]thiophene](http://img.cochemist.com/ccimg/300/233-02-3_b.png)
![Anthra[2,1-b]thiophene(8CI,9CI)](http://img.cochemist.com/ccimg/300/227-56-5.png)
![Anthra[2,1-b]thiophene(8CI,9CI)](http://img.cochemist.com/ccimg/300/227-56-5_b.png)
![Benz[a]acridine](http://img.cochemist.com/ccimg/300/225-11-6.png)
![Benz[a]acridine](http://img.cochemist.com/ccimg/300/225-11-6_b.png)
![Phenanthro[2,3-b]thiophene(7CI,8CI,9CI)](http://img.cochemist.com/ccimg/300/224-07-7.png)
![Phenanthro[2,3-b]thiophene(7CI,8CI,9CI)](http://img.cochemist.com/ccimg/300/224-07-7_b.png)
![phenanthro[2,1-b]thiophene](http://img.cochemist.com/ccimg/300/219-25-0.png)
![phenanthro[2,1-b]thiophene](http://img.cochemist.com/ccimg/300/219-25-0_b.png)
![Acenaphtho[1,2-b]pyridine(6CI,7CI,8CI,9CI)](http://img.cochemist.com/ccimg/300/206-49-5.png)
![Acenaphtho[1,2-b]pyridine(6CI,7CI,8CI,9CI)](http://img.cochemist.com/ccimg/300/206-49-5_b.png)
![pyreno[2,1-b]thiophenato(4-)](http://img.cochemist.com/ccimg/200/189-81-1.png)
![pyreno[2,1-b]thiophenato(4-)](http://img.cochemist.com/ccimg/200/189-81-1_b.png)
![[1,1'-Biphenyl]-4-carbonitrile, 4'-(2-propenyloxy)-](http://img.cochemist.com/ccimg/112000/111928-38-2.png)
![[1,1'-Biphenyl]-4-carbonitrile, 4'-(2-propenyloxy)-](http://img.cochemist.com/ccimg/112000/111928-38-2_b.png)
![dibenzo[b,d]thiophen-4-amine](http://img.cochemist.com/ccimg/72500/72433-66-0.png)
![dibenzo[b,d]thiophen-4-amine](http://img.cochemist.com/ccimg/72500/72433-66-0_b.png)
![dibenzo[b,d]thiophen-1-amine](http://img.cochemist.com/ccimg/29500/29451-76-1.png)
![dibenzo[b,d]thiophen-1-amine](http://img.cochemist.com/ccimg/29500/29451-76-1_b.png)
![Phenanthro[3,2-b]thiophene(7CI,8CI,9CI)](http://img.cochemist.com/ccimg/300/224-10-2.png)
![Phenanthro[3,2-b]thiophene(7CI,8CI,9CI)](http://img.cochemist.com/ccimg/300/224-10-2_b.png)
![4H-Benzo[def]carbazole](http://img.cochemist.com/ccimg/300/203-65-6.png)
![4H-Benzo[def]carbazole](http://img.cochemist.com/ccimg/300/203-65-6_b.png)
![Benzo[a]aceanthrylene](http://img.cochemist.com/ccimg/300/203-33-8.png)
![Benzo[a]aceanthrylene](http://img.cochemist.com/ccimg/300/203-33-8_b.png)
![dibenzo[b,d]thiophene-1-ol](http://img.cochemist.com/ccimg/69800/69747-83-7.png)
![dibenzo[b,d]thiophene-1-ol](http://img.cochemist.com/ccimg/69800/69747-83-7_b.png)
![Triphenyleno[1,12-bcd]thiophene](http://img.cochemist.com/ccimg/68600/68558-73-6.png)
![Triphenyleno[1,12-bcd]thiophene](http://img.cochemist.com/ccimg/68600/68558-73-6_b.png)
![Cyclopenta[cd]pyrene](http://img.cochemist.com/ccimg/27300/27208-37-3.png)
![Cyclopenta[cd]pyrene](http://img.cochemist.com/ccimg/27300/27208-37-3_b.png)
![11H-Benzo[a]carbazole](http://img.cochemist.com/ccimg/300/239-01-0.png)
![11H-Benzo[a]carbazole](http://img.cochemist.com/ccimg/300/239-01-0_b.png)
![Dibenzo[def,mno]chrysene](http://img.cochemist.com/ccimg/200/191-26-4.png)
![Dibenzo[def,mno]chrysene](http://img.cochemist.com/ccimg/200/191-26-4_b.png)
![Benzo[a]fluorene](http://img.cochemist.com/ccimg/30800/30777-18-5.png)
![Benzo[a]fluorene](http://img.cochemist.com/ccimg/30800/30777-18-5_b.png)
![Naphtho[2,3-b]thiophene](http://img.cochemist.com/ccimg/300/268-77-9.png)
![Naphtho[2,3-b]thiophene](http://img.cochemist.com/ccimg/300/268-77-9_b.png)
![4H-Cyclopenta[def]phenanthrene](http://img.cochemist.com/ccimg/300/203-64-5.png)
![4H-Cyclopenta[def]phenanthrene](http://img.cochemist.com/ccimg/300/203-64-5_b.png)
![3-Methyldibenzo[b,d]thiophene](http://img.cochemist.com/ccimg/16600/16587-52-3.png)
![3-Methyldibenzo[b,d]thiophene](http://img.cochemist.com/ccimg/16600/16587-52-3_b.png)
![Benzo[ghi]fluoranthene](http://img.cochemist.com/ccimg/300/203-12-3.png)
![Benzo[ghi]fluoranthene](http://img.cochemist.com/ccimg/300/203-12-3_b.png)
![Benzo[b]naphtho[2,3-d]thiophene](http://img.cochemist.com/ccimg/300/243-46-9.png)
![Benzo[b]naphtho[2,3-d]thiophene](http://img.cochemist.com/ccimg/300/243-46-9_b.png)
![Benzo[f]quinoline](http://img.cochemist.com/ccimg/100/85-02-9.png)
![Benzo[f]quinoline](http://img.cochemist.com/ccimg/100/85-02-9_b.png)
![Benzo[b]naphtho[2,1-d]thiophene](http://img.cochemist.com/ccimg/300/239-35-0.png)
![Benzo[b]naphtho[2,1-d]thiophene](http://img.cochemist.com/ccimg/300/239-35-0_b.png)
