Exploring Biopharmaceutical Analysis with Compact Capillary Liquid Chromatography Instrumentation.

A recent trend in the design of liquid chromatography (LC) instrumentation is the move towards miniaturized and portable systems. These smaller platforms provide wider flexibility in operation, with the opportunity for conducting analysis directly at the point of sample collection rather than transporting the sample to a centralized laboratory facility. For the manufacturing of pharmaceutical and biopharmaceutical products, these platforms can be implemented for process monitoring and product characterization directly in manufacturing environments. This article describes a portable, miniaturized LC instrument coupled to a mass spectrometer (MS) for characterization of a biopharmaceutical monoclonal antibody (mAb).

Column selection considerations in compact capillary liquid chromatography.

Recent years have seen significant advances in compact, portable capillary LC instrumentation. This study explores the performances of several commercially available columns within the pressure and flow limits of both the columns and one of these compact LC instruments. The commercially available compact capillary LC system with UV-absorbance detector used in this study is typically operated using columns in the 0.15-0.3 mm internal diameter (i.d.) range. Efficiency measurements (i.e., theoretical plates, N) for six columns with i.d.s in this range and of varying lengths and pressure limits, packed with stationary phases of different particle diameters and morphologies, were made using a mixture of standard alkylphenones. Kinetic plot comparisons between columns that vary by one (or more) of these parameters are described, along with calculated kinetic performance and Knox-Saleem limits. These theoretical performance descriptions provide insight into optimal operating conditions when using capillary LC systems. Based on kinetic plot evaluation of available capillary columns in the 0.2-0.3 mm i.d. range with a conservative upper pressure limit of 330 bar packed with superficially porous particles, a 25 cm column could generate ∼47,000 plates in 7.85 min when operated at 2.4 µL/min. For comparison, more robust 0.3 mm i.d. columns (packed with fully porous particles) that can be operated at higher pressures than can be provided by the pumping system (conservative pump upper pressure limit of 570 bar), a ∼20 cm column could generate nearly 40,000 plates in 5.9 min if operated at 6 µL/min. Across all capillary LC columns measured, higher pressure limits and shorter columns can provide the best throughput when considering both speed and efficiency.

Online Monitoring of Small Volume Reactions Using Compact Liquid Chromatography Instrumentation.

A wide variety of analytical techniques have been employed for monitoring chemical reactions, with online instrumentation providing additional benefits compared to offline analysis. A challenge in the past for online monitoring has been placement of the monitoring instrumentation as close as possible to the reaction vessel to maximize sampling temporal resolution and preserve sample composition integrity. Furthermore, the ability to sample very small volumes from bench-scale reactions allows the use of small reaction vessels and conservation of expensive reagents. In this study, a compact capillary LC instrument was used for online monitoring of as small as 1 mL total volume of a chemical reaction mixture, with automated sampling of nL-scale volumes directly from the reaction vessel used for analysis. Analyses to demonstrate short term (~2 h) and long term (~ 50 h) reactions were conducted using tandem on-capillary ultraviolet absorbance followed by in-line MS detection or ultraviolet absorbance detection alone, respectively. For both short term and long term reactions (10 and 250 injections, respectively), sampling approaches using syringe pumps minimized the overall sample loss to ~0.2% of the total reaction volume.

Comparison of the Dynamic Thermal Gradient to Temperature-Programmed Conditions in Gas Chromatography Using a Stochastic Transport Model.

This paper compares dynamic (i.e., temporally changing) thermal gradient gas chromatography (GC) to temperature-programmed GC using a previously published stochastic transport model to simulate peak characteristics for the separation of C12-C40 hydrocarbons. All comparisons are made using chromatographic conditions that give approximately equal analyte retention times (tR). As shown previously, a static thermal gradient does not improve resolution (Rs) equally for all analytes, which highlights the need for a dynamic thermal gradient. An optimal dynamic thermal gradient should result in constant analyte velocities at any instant in time for those analytes that are actively being separated (i.e., analytes that have low retention factors). The average separation temperature for each analyte is used to determine the thermal gradient profile at different times in the temperature ramp. Because many of the analytes require a similar thermal gradient profile when actively being separated, the thermal gradient profile in this study was held fixed; however, the temperature of the entire thermal gradient was raised over time. From the simulations performed in this study, optimized dynamic thermal gradient conditions are shown to improve Rs by up to 13% over comparative temperature-programmed conditions, even with a perfect injection (i.e., zero injection bandwidth). In the dynamic thermal gradient simulations, all analytes showed improvements in Rs along with slightly shorter tR values compared to simulations for traditional temperature-programmed conditions.

Comparison of Static Thermal Gradient to Isothermal Conditions in Gas Chromatography Using a Stochastic Transport Model.

This paper compares static (i.e., temporally unchanging) thermal gradient gas chromatography (GC) to isothermal GC using a stochastic transport model to simulate peak characteristics for the separation of C12-C14 hydrocarbons resulting from variations in injection bandwidth. All comparisons are made using chromatographic conditions that give approximately equal analyte retention times so that the resolution and number of theoretical plates can be clearly compared between simulations. Simulations show that resolution can be significantly improved using a linear thermal gradient along the entire column length. This is mainly achieved by partially compensating for loss in resolution from the increase in mobile phase velocity, which approximates an ideal, basic separation. The slope of the linear thermal gradient required to maximize resolution is a function of the retention parameters, which are specific to each analyte pair; a single static, thermal gradient will not affect all analytes equally. A static, non-linear thermal gradient that creates constant analyte velocities at all column locations provides the largest observed gains in resolution. From the simulations performed in this study, optimized linear thermal gradient conditions are shown to improve the resolution by as much as 8.8% over comparative isothermal conditions, even with a perfect injection (i.e., zero initial bandwidth).

Simulating Capillary Gas Chromatographic Separations including Thermal Gradient Conditions.

This article presents a method of simulating molecular transport in capillary gas chromatography (GC) applicable to isothermal, temperature-programmed, and thermal gradient conditions. The approach accounts for parameter differences that can occur across an analyte band including pressure, mobile phase velocity, temperature, and retention factor. The model was validated experimentally using a GC column comprised of microchannels in a stainless-steel plate capable of isothermal, temperature-programmed, and thermal gradient GC separations. The parameters governing retention and dispersion in the transport model were fitted with 12 experimental isothermal separations. The transport model was validated with experimental data for three analytes using four temperature-programmed and three thermal gradient GC separations. The simulated peaks (elution time and dispersion) give reasonable predictions of observed separations. The magnitudes of the maximum error between simulated peak elution time and experiment were 2.6 and 4.2% for temperature-programmed and thermal gradient GC, respectively. The magnitudes of the maximum error between the simulated peak width and experiment were 15.4 and 5.8% for temperature-programmed and thermal gradient GC, respectively. These relatively low errors give confidence that the model reflects the behavior of the transport processes and provides meaningful predictions for GC separations. This transport model allows for an evaluation of analyte separation characteristics of the analyte band at any position along the length of the GC column in addition to peak characteristics at the column exit. The transport model enables investigation of column conditions that influence separation behavior and opens exploration of optimal column design and heating conditions.

Portable capillary liquid chromatography for pharmaceutical and illicit drug analysis.

A newly developed portable capillary liquid chromatograph was investigated for the separation of various pharmaceutical and illicit drug compounds. The system consists of two high-pressure syringe pumps capable of delivering capillary-scale flow rates at pressures up to 10 000 psi. Capillary liquid chromatography columns packed with sub-2 µm particles are housed in cartridges that can be inserted into the system and easily connected through high-pressure fluidic contact points by simply applying a specific, predetermined torque rather than using standard fittings and less precise sealing protocols. Several over-the-counter analgesic drug separations are demonstrated, along with a simple online measurement of tablet dissolution. Twenty illicit drug compounds were also separated across six targeted drug panels. The results described in this study demonstrate the capability of this compact liquid chromatography instrument to address several important drug-related applications while simplifying system operation, and greatly reducing solvent usage and waste generation essential for onsite analysis.

Stainless-Steel Column for Robust, High-Temperature Microchip Gas Chromatography.

This paper reports the first results of a robust, high-performance, stainless-steel microchip gas-chromatography (GC) column that is capable of analyzing complex real-world mixtures as well as operating at very high temperatures. Using a serpentine design, a 10 m column with an approximately semicircular cross-section with a 52 µm hydraulic diameter ( Dh) was produced in a 17 × 6.3 × 0.1 cm rectangular steel chip. The channels were produced using a multilayer-chemical-etch and diffusion-bonding process, and metal nuts were brazed onto the inlet and outlet ports allowing for column interfacing with ferrules and fused silica capillary tubing. After deactivating the metal surface, channels were statically coated with a ≈0.1 µm layer of 5% phenyl-1% vinyl-methylpolysiloxane (SE-54) stationary phase and cross-linked with dicumyl peroxide. By using n-tridecane ( n-C13) as a test analyte with a retention factor ( k) of 5, a total of 44 500 plates (≈4500 plates per meter) was obtained isothermally at 120 °C. The column was thermally stable to at least 350 °C, and rapid temperature programming (35 °C/min) was demonstrated for the boiling-point range from n-C5 to n-C44 (ASTM D2887 simulated-distillation standard). The column was also tested for separation of two complex mixtures: gasoline headspace and kerosene. These initial experiments demonstrate that the planar stainless-steel column with proper interfacing can be a viable alternative platform for portable, robust microchip GC that is capable of high-temperature operation for low-volatility-compound analysis.

Microchip gas chromatography columns, interfacing and performance.

Almost four decades of investigations have opened up many avenues to explore the production and utilization of planar (i.e., microchip) gas chromatographic columns. However, there remain many practical constraints that limit their widespread commercialization and use. The main challenges arise from non-ideal column geometries, dead volume issues and inadequate interfacing technologies, which all affect both column performance and range of applications. This review reflects back over the years on the extensive developments in the field, with the goal to stimulate future creative approaches and increased efforts to accelerate microchip gas chromatography development toward reaching its full potential.

Extending the upper temperature range of gas chromatography with all-silicon microchip columns using a heater/clamp assembly.

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 5.9m long microcolumn with rectangular cross section of 158µm×80µm, which approximately matches a 100µm 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.

Dual-wavelength light-emitting diode-based ultraviolet absorption detector for nano-flow capillary liquid chromatography.

The design of a miniaturized LED-based UV-absorption detector was significantly improved for on-column nanoflow LC. The detector measures approximately 27mm×24mm×10mm and weighs only 30g. 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 255nm and 275nm LEDs with only 216nL volume between the detectors A 100µm slit was used for on-column detection with a 150µm 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 2000nL/min, less than 3% variation was observed.

Controlled crosslinking of trimethylolpropane trimethacrylate for preparation of organic monolithic columns for capillary liquid chromatography.

Organic monolithic columns based on single crosslinking of trimethylolpropane trimethacrylate (TRIM) monomer were prepared in a single step by living/controlled free-radical polymerization. Full optimization of the preparation, such as using different percentages of TRIM and different amounts of radical promoter as well as various porogen solvents were explored. The resulting monolithic columns were characterized by scanning electronic microscopy and nitrogen sorption for structure morphology studies and surface area measurements, respectively. Using capillary liquid chromatography, 150 µm i.d. columns were applied to separate a mixture of small hydrophobic molecules. The results indicated that column performance is highly sensitive to the type and the amount of porogen solvents used in the polymerization mixture composition. Good resolution factors and methylene selectivity were obtained, indicating the promising potential of this material for capillary liquid chromatography separations.

Concentrically packed high flow air sampler for parts-per-trillion volatile and semi-volatile organica compounds.

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 25min with this sampling system using gas chromatography-mass spectrometry for analysis. The concentric packed trap can sample at high flow rates (>10L/min) because it has a large sampling surface cross-section and short combined sorbent bed. Additionally, the large sorbent amount (>1g) provides large breakthrough volume (>100L) 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.

Compact Ultrahigh-Pressure Nanoflow Capillary Liquid Chromatograph.

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.

Retention behavior of isomeric polycyclic aromatic sulfur heterocycles in gas chromatography on stationary phases of different selectivity.

Retention indices for 48 polycyclic aromatic sulfur heterocycles (PASHs) were determined using gas chromatography with three different stationary phases: a 50% phenyl phase, a 50% liquid crystalline dimethylpolysiloxane (LC-DMPS) phase, and an ionic liquid (IL) phase. Correlations between the retention behavior on the three stationary phases and PASH geometry (L/B and T, i.e., length-to-breadth ratio and thickness, respectively) were investigated for the following four isomer sets: (1) 4 three-ring molecular mass (MM) 184Da PASHs, (2) 13 four-ring MM 234Da PASHs, (3) 10 five-ring MM 258Da PASHs, and (4) 20 five-ring MM 284Da PASHs. Correlation coefficients for retention on the 50% LC-DMPS vs L/B ranged from r=0.50 (MM 284Da) to r=0.77 (MM 234Da). Correlation coefficients for retention on the IL phase vs L/B ranged from r=0.31 (MM 234Da) to r=0.54 (MM 284Da). Correlation coefficients for retention on the 50% phenyl vs L/B ranged from r=0.14 (MM 258Da) to r=0.59 (MM 284Da). Several correlation trends are discussed in detail for the retention behavior of PASH on the three stationary phases.

Retention behavior of alkyl-substituted polycyclic aromatic sulfur heterocycle isomers in gas chromatography on stationary phases of different selectivity.

Retention indices for 10 sets of alkyl-substituted polycyclic aromatic sulfur heterocycles (PASHs) isomers (total of 80 PASHs) were determined using gas chromatography with three different stationary phases: a 50% phenyl phase, a 50% liquid crystalline dimethylpolysiloxane (LC-DMPS) phase, and an ionic liquid (IL) phase. Correlations between the retention behavior on the three stationary phases and PASH geometry [length-to-breadth (L/B) and thickness (T)] were investigated for the following PASHs: 4 methyl-substituted dibenzothiophenes (DBTs), 3 ethyl-substituted DBTs, 15 dimethyl-substituted DBTs, 8 trimethyl-substituted DBTs, 15 methyl-substituted naphthothiophenes, 30 methyl-substituted benzonaphthothiophenes, and 5 methyl-substituted tetrapheno[1,12-bcd]thiophene. Correlation coefficients for retention on the 50% phenyl phase vs L/B ranged from r=-0.28 (MeBbN23Ts) to r=0.92 (EtDBTs). Correlation coefficients for retention on the IL phase vs L/B ranged from r=0.13 (MeN12Ts) to r=0.83 (EtDBTs). Correlation coefficients for retention on the 50% LC-DMPS phase vs L/B ranged from r=0.22 (MeDBTs) to r=0.84 (TriMeDBTs).

Improvement in Liquid Chromatographic Performance of Organic Polymer Monolithic Capillary Columns with Controlled Free-Radical Polymerization.

Capillary columns containing butyl or lauryl methacrylate monoliths were prepared using two different free-radical polymerization methods: conventional free-radical polymerization and controlled/living free-radical polymerization, both initiated thermally, and these methods were compared for the first time. Both monolith morphology and chromatographic efficiency were compared for the synthesized stationary phases using scanning electronic microscopy (SEM) and capillary liquid chromatography, respectively. Columns prepared using controlled method gave better chromatographic performance for both monomers tested. The lauryl-based monolith showed 7-fold improvement in chromatographic efficiency with a plate count of 42,000 plates/m (corrected for dead volume) for a non-retained compound. Columns fabricated using controlled polymerization appeared more homogenous radially with fused small globular morphologies, evaluated by SEM, and lower column permeability. The columns were compared with respect to resolving power of a series of alkylbenzenes under isocratic and gradient elution conditions.

Retention behavior of alkyl-substituted polycyclic aromatic sulfur heterocycles in reversed-phase liquid chromatography.

Retention indices for 79 alkyl-substituted polycyclic aromatic sulfur heterocycles (PASHs) were determined by using reversed-phase liquid chromatography (LC) on a monomeric and polymeric octadecylsilane (C18) stationary phase. Molecular shape parameters [length, breadth, thickness (T), and length-to-breadth ratio (L/B)] were calculated for all the compounds studied. Based on separations of isomeric methylated polycyclic aromatic hydrocarbons on polymeric C18 phases, alkyl-substituted PASHs are expected to elute based on increasing L/B ratios. However, the correlation coefficients had a wide range of values from r=0.43 to r=0.93. Several structural features besides L/B ratios were identified to play an important role in the separation mechanism of PASHs on polymeric C18 phases. First, the location of the sulfur atom in a bay-like-region results in alkylated-PASHs being more retentive than non-bay-like-region alkylated-PASHs, and they elute later than expected based on L/B value. Second, the placement of the alkyl group in the k region of the structure resulted in a later elution than predicted by L/B. Third, highly nonplanar methyl-PASHs (i.e., 1-Me and 11-MeBbN12T) elute prior to the parent PASH (BbN12T).

Retention behavior of isomeric polycyclic aromatic sulfur heterocycles in reversed-phase liquid chromatography.

Retention indices for 70 polycyclic aromatic sulfur heterocycles (PASHs) were determined using reversed-phase liquid chromatography (LC) on a monomeric and a polymeric C18 stationary phase. Molecular shape parameters [length, breadth, thickness (T), and length-to-breadth ratio (L/B)] were calculated for all the compounds studied. Correlations between the retention on the polymeric C18 phase and PASH geometry (L/B and T) were investigated for six specific PASH isomer groups with molecular mass (MM) 184Da, 234Da, 258Da, 284Da, 334Da, and 384Da. Similar to previous studies for polycyclic aromatic hydrocarbons (PAHs), PASH elution order on the polymeric C18 phase was generally found to follow increasing L/B values. Correlation coefficients for retention vs L/B ranged from r=0.45 (MM 184Da) to r=0.89 (MM 284Da). In the case of smaller PASHs (MM≤258Da), the location of the sulfur atom in the bay-region of the structure resulted in later than expected elution of these isomers based on L/B. In the case of the larger PASHs (MM≥284Da), nonplanarity had a significant influence on earlier than predicted elution based on L/B values.