Two-dimensional liquid chromatography-mass spectrometry for lipidomics using off-line coupling of hydrophilic interaction liquid chromatography with 50 cm long reversed phase capillary columns.

Comprehensive characterization of the lipidome remains a challenge requiring development of new analytical approaches to expand lipid coverage in complex samples. In this work, offline two-dimensional liquid chromatography-mass spectrometry was investigated for lipidomics from human plasma. Hydrophilic interaction liquid chromatography was implemented in the first dimension to fractionate lipid classes. Nine fractions were collected and subjected to a second-dimension separation utilizing 50 cm capillary columns packed with 1.7 µm C18 particles operated on custom-built instrumentation at 35 kpsi. Online coupling with time-of-flight mass spectrometry allowed putative lipid identification from precursor-mass based library searching. The method had good orthogonality (fractional coverage of ∼40%), achieved a peak capacity of approximately 1900 in 600 min, and detected over 1000 lipids from a 5 µL injection of a human plasma extract while consuming less than 3 mL of solvent. The results demonstrate the expected gains in peak capacity when employing long columns and two-dimensional separations and illustrate practical approaches for improving lipidome coverage from complex biological samples.

Comparison of microcapillary column length and inner diameter investigated with gradient analysis of lipids by ultrahigh-pressure liquid chromatography-mass spectrometry.

Biological samples in lipidomic studies can consist of extremely complex mixtures due to the diverse range of species and isomerism. Herein, highly efficient, in-house packed microcapillary columns introduce the potential to better separate these complex mixtures. We compared the effects of changing column length (15, 30, and 60 cm) and inner diameter (75 and 100 µm) on lipid separation efficiency by reversed-phase gradient analysis using ultrahigh-pressure liquid chromatography coupled to mass spectrometry with operating pressures ranging from 450 to 2200 bar. Seven lipid standards composed of phosphatidylcholine and triacylglycerol species were analyzed at four different gradient rates to calculate conditional peak capacity. The longest column, 60 cm, at the shallowest gradient of 2% gave the highest peak capacity of 359 with a separation window of 2 h. The intermediate column length of 30 cm with 75 µm inner diameter provided a peak capacity of 287 with a separation window of 1 h. There was no significant difference in peak capacity between 75 and 100 µm inner diameter columns. This study showed that using highly efficient microcapillary columns increased peak capacity and resolution of lipids, and thus, this technique seems promising for enhancing lipid coverage and enabling better discovery of lipid biomarkers.

Ultrahigh-Performance capillary liquid chromatography-mass spectrometry at 35 kpsi for separation of lipids.

Improvements in sample preparation, separation, and mass spectrometry continue to expand the coverage in LC-MS based lipidomics. While longer columns packed with smaller particles in theory give higher separation performance compared to shorter columns, the implementation of this technology above commercial limits has been sparse due to difficulties in packing long columns and successfully operating instruments at ultrahigh pressures. In this work, a liquid chromatograph that operates up to 35 kpsi was investigated for the separation and identification of lipid species from human plasma. Capillary columns between 15-50 cm long were packed with 1.7 µm BEH C18 particles and evaluated for their ability to separate lipid isomers and complex lipid extracts from human plasma. Putative lipid class identifications were assigned using accurate mass and relative retention time data of the eluting peaks. Our findings indicate that longer columns packed and operated at 35 kpsi outperform shorter columns packed and run at lower pressures in terms of peak capacity and numbers of features identified. Packing columns with relatively high concentration slurries (200 mg/mL) while sonicating the column resulted in 6-34% increase in peak capacity for 50 cm columns compared to lower slurry concentrations and no sonication. For a given analysis time, 50 cm long columns operated at 35 kpsi provided a 20-95% increase in chromatographic peak capacity compared with 15 cm columns operated at 15 kpsi. Analysis times up to 4 h were evaluated, generating peak capacities up to 410 ±â€¯5 (n = 3, measured at 4σ) and identifying 480 ±â€¯85 lipids (n = 2). Importantly, the results also show a correlation between the peak capacity and the number of lipids identified from a human plasma extract. This correlation indicates that ionization suppression is a limiting factor in obtaining sufficient signal for identification by mass spectrometry. The result also shows that the higher resolution obtained by shallow gradients overcomes possible signal reduction due to broader, more dilute peaks in long gradients for improving detection of lipids in LC-MS. Lastly, longer columns operated at shallow gradients allowed for the best separation of both regional and geometrical isomers. These results demonstrate a system that enables the advantages of using longer columns packed and run at ultrahigh pressure for improving lipid separations and lipidome coverage.

Axial heterogeneities in capillary ultrahigh pressure liquid chromatography columns: Chromatographic and bed morphological characterization.

We study axial heterogeneities in capillary ultrahigh pressure liquid chromatography (UHPLC) columns through kinetic performance and bed morphological analysis. Two columns are used in this work, a 75 µm i.d. × 100 cm column packed with 1.3 µm C18-silica particles and a 75 µm i.d. × 45 cm column packed with 1.9 µm C18-silica particles. The long column is chromatographically characterized and is afterwards sectioned into three segments, each analyzed individually. The column packed with the 1.9 µm particles is subjected to a bed morphological analysis using confocal laser scanning microscopy near the inlet, center, and outlet of the column. Chromatographic and morphological characterizations reveal highest separation efficiency and most homogeneous bed microstructure towards the column outlet. Kinetic performance data for inlet and central packing segments indicate enhanced contributions from wall effects to a transcolumn flow heterogeneity. Bed morphological data reveal systematic changes in geometrical and frictional wall effects along the bed: from inlet to outlet, bed morphologies increasingly reflect packing microstructures associated with concentrated slurries. Variations in separation efficiency and bed morphology can be related to the constant-pressure packing mode; the decrease in packing rate along the bed leaves fewer chances for particle rearrangement and bed consolidation from inlet to outlet. It explains the relatively uniform bed morphology towards the outlet and also the relatively loose wall region near the inlet. Bed microstructural features are discussed in a context of previous observations made in the characterization of capillary UHPLC columns.

Retention and effective diffusion of model metabolites on porous graphitic carbon.

The study of metabolites in biological samples is of high interest for a wide range of biological and pharmaceutical applications. Reversed phase liquid chromatography is a common technique used for the separation of metabolites, but it provides little retention for polar metabolites. An alternative to C18 bonded phases, porous graphitic carbon has the ability to provide significant retention for both non-polar and polar analytes. The goal of this work is to study the retention and effective diffusion properties of porous graphitic carbon, to see if it is suitable for the wide injection bands and long run times associated with long, packed capillary-scale separations. The retention of a set of standard metabolites was studied for both stationary phases over a wide range of mobile phase conditions. This data showed that porous graphitic carbon benefits from significantly increased retention (often >100 fold) under initial gradient conditions for these metabolites, suggesting much improved ability to focus a wide injection band at the column inlet. The effective diffusion properties of these columns were studied using peak-parking experiments with the standard metabolites under a wide range of retention conditions. Under the high retention conditions, which can be associated with retention after injection loading for gradient separations, Deff/Dm∼0.1 for both the C18-bonded and porous graphitic carbon columns. As C18 bonded particles are widely, and successfully utilized for long gradient separations without issue of increasing peak width from longitudinal diffusion, this suggests that porous graphitic carbon should be amenable for long runtime gradient separations as well.

High resolution separations of charge variants and disulfide isomers of monoclonal antibodies and antibody drug conjugates using ultra-high voltage capillary electrophoresis with high electric field strength.

Ultra-high voltage capillary electrophoresis with high electric field strength has been applied to the separation of the charge variants, drug conjugates, and disulfide isomers of monoclonal antibodies. Samples composed of many closely related species are difficult to resolve and quantify using traditional analytical instrumentation. High performance instrumentation can often save considerable time and effort otherwise spent on extensive method development. Ideally, the resolution obtained for a given CE buffer system scales with the square root of the applied voltage. Currently available commercial CE instrumentation is limited to an applied voltage of approximately 30kV and a maximum electric field strength of 1kV/cm due to design limitations. The instrumentation described here is capable of safely applying potentials of at least 120kV with electric field strengths over 2000V/cm, potentially doubling the resolution of the best conventional CE buffer/capillary systems while decreasing analysis time in some applications. Separations of these complex mixtures using this new instrumentation demonstrate the potential of ultra-high voltage CE to identify the presence of previously unresolved components and to reduce analysis time for complex mixtures of antibody variants and drug conjugates.

Development of a conductivity-based photothermal absorbance detection microchip using polyelectrolytic gel electrodes.

The development and application of polyelectrolytic gel electrodes (PGEs) for a microfluidic photothermal absorbance detection system is described. The PGEs are used to measure changes in conductivity based on heat generation by analytes absorbing light and changing the solution viscosity. The PGEs are suitable for direct contact conductivity measurements since they do not degrade with exposure to high electric fields. Both a 2-electrode system with DC voltages and a 3-electrode system with AC voltages were investigated. Experimental factors including excitation voltage, excitation frequency, laser modulation frequency, laser power, and path length were tested. The limits of detection for the 3-electrode and 2-electrode systems are 500nM and 0.55nM for DABSYL-tagged glucosamine, respectively. In addition, an electrokinetic separation of a potassium, DABSYL-tagged glucosamine, Rhodamine 6G, and Rhodamine B mixture was demonstrated.

Bed morphological features associated with an optimal slurry concentration for reproducible preparation of efficient capillary ultrahigh pressure liquid chromatography columns.

Column wall effects and the formation of larger voids in the bed during column packing are factors limiting the achievement of highly efficient columns. Systematic variation of packing conditions, combined with three-dimensional bed reconstruction and detailed morphological analysis of column beds, provide valuable insights into the packing process. Here, we study a set of sixteen 75µm i.d. fused-silica capillary columns packed with 1.9µm, C18-modified, bridged-ethyl hybrid silica particles slurried in acetone to concentrations ranging from 5 to 200mg/mL. Bed reconstructions for three of these columns (representing low, optimal, and high slurry concentrations), based on confocal laser scanning microscopy, reveal morphological features associated with the implemented slurry concentration, that lead to differences in column efficiency. At a low slurry concentration, the bed microstructure includes systematic radial heterogeneities such as particle size-segregation and local deviations from bulk packing density near the wall. These effects are suppressed (or at least reduced) with higher slurry concentrations. Concomitantly, larger voids (relative to the mean particle diameter) begin to form in the packing and increase in size and number with the slurry concentration. The most efficient columns are packed at slurry concentrations that balance these counteracting effects. Videos are taken at low and high slurry concentration to elucidate the bed formation process. At low slurry concentrations, particles arrive and settle individually, allowing for rearrangements. At high slurry concentrations, they arrive and pack as large patches (reflecting particle aggregation in the slurry). These processes are discussed with respect to column packing, chromatographic performance, and bed microstructure to help reinforce general trends previously described. Conclusions based on this comprehensive analysis guide us towards further improvement of the packing process.

Performance comparison of three trypsin columns used in liquid chromatography.

Trypsin is the most widely used enzyme in proteomic research due to its high specificity. Although the in-solution digestion is predominantly used, it has several drawbacks, such as long digestion times, autolysis, and intolerance to high temperatures or organic solvents. To overcome these shortcomings trypsin was covalently immobilized on solid support and tested for its proteolytic activity. Trypsin was immobilized on bridge-ethyl hybrid silica sorbent with 300Å pores, packed in 2.1×30mm column and compared with Perfinity and Poroszyme trypsin columns. Catalytic efficiency of enzymatic reactors was tested using Nα-Benzoyl-l-arginine 4-nitroanilide hydrochloride as a substrate. The impact of buffer pH, mobile phase flow rate, and temperature on enzymatic activity was investigated. Digestion speed generally increased with the temperature from 20 to 37°C. Digestion speed also increased with pH from 7.0 to 9.0; the activity of prototype enzyme reactor was highest at pH 9.0, when it activity exceeded both commercial reactors. Preliminary data for fast protein digestion are presented.

Profiling the Photochemical-Induced Degradation of Rat Growth Hormone with Extreme Ultra-pressure Chromatography-Mass Spectrometry Utilizing Meter-Long Microcapillary Columns Packed with Sub-2-µm Particles.

In recent years protein therapeutics have seen increasing use in the therapeutic arena. As with traditional small molecule drug substances, one is obligated to ensure purity and stability of the various dosage forms. With these higher molecular weight therapeutics a common approach for analytical characterization is enzymatic digestion followed by gradient elution liquid chromatography with mass spectrometry detection to create a peptide map (bottom-up protein analysis). Due to the difficult to separate mixtures frequently encountered, there is the need for advanced chromatographic systems featuring increased resolution and/or peak capacity that can be operated in the gradient elution format. Presently we describe an extreme ultra-pressure liquid chromatography (XUPLC) system that has been implemented as an in-house add-on to a commercial ultra-pressure chromatography system. This add-on allows operation at the 38 Kspi range, accommodates the use of capillary columns in excess of one meter packed with sub-2 µm particles and can be operated in the gradient elution format. To evaluate the utility of this system, rat growth hormone was used as a model protein and was exposed to light (λ 254 nm) to create a stress environment. When enzymatic digests of control and stressed protein were analyzed with the XUPLC system using MS detection, greater than 92% peptide coverage was achieved, including the identification some peptides where pre-oxidation of Met residues had occurred, as well as chemistry specifically related to the photolysis of protein disulfide linkages. When the same samples were analyzed by commercial UPLC and compared to the XUPLC results, the utility of the increased peak capacity available with the XUPLC was apparent as previously co-eluting peaks were now well resolved. In particular one specific degradation route was identified where a pair of isobaric cis/trans diastereomerically related peptides were well resolved by XUPLC while they were unresolved by UPLC. Clearly the use of this system operating at the higher pressure regime with long capillary columns is and will be useful in continued investigations of protein stability, especially in cases where only subtle differences in the amino acid residues have occurred during degradation.

Development of a 45kpsi ultrahigh pressure liquid chromatography instrument for gradient separations of peptides using long microcapillary columns and sub-2µm particles.

Commercial chromatographic instrumentation for bottom-up proteomics is often inadequate to resolve the number of peptides in many samples. This has inspired a number of complex approaches to increase peak capacity, including various multidimensional approaches, and reliance on advancements in mass spectrometry. One-dimensional reversed phase separations are limited by the pressure capabilities of commercial instruments and prevent the realization of greater separation power in terms of speed and resolution inherent to smaller sorbents and ultrahigh pressure liquid chromatography. Many applications with complex samples could benefit from the increased separation performance of long capillary columns packed with sub-2µm sorbents. Here, we introduce a system that operates at a constant pressure and is capable of separations at pressures up to 45kpsi. The system consists of a commercially available capillary liquid chromatography instrument, for sample management and gradient creation, and is modified with a storage loop and isolated pneumatic amplifier pump for elevated separation pressure. The system's performance is assessed with a complex peptide mixture and a range of microcapillary columns packed with sub-2µm C18 particles.

Implementation of high slurry concentration and sonication to pack high-efficiency, meter-long capillary ultrahigh pressure liquid chromatography columns.

Slurry packing capillary columns for ultrahigh pressure liquid chromatography is complicated by many interdependent experimental variables. Previous results have suggested that combination of high slurry concentration and sonication during packing would create homogeneous bed microstructures and yield highly efficient capillary columns. Herein, the effect of sonication while packing very high slurry concentrations is presented. A series of six, 1m×75µm internal diameter columns were packed with 200mg/mL slurries of 2.02µm bridged-ethyl hybrid silica particles. Three of the columns underwent sonication during packing and yielded highly efficient separations with reduced plate heights as low as 1.05.

Extraction, Enrichment, Solubilization, and Digestion Techniques for Membrane Proteomics.

The importance of membrane proteins in biological systems is indisputable; however, their amphipathic nature makes them difficult to analyze. In this study, the most popular techniques for extraction, enrichment, solubilization, and digestion are compared, resulting in an overall improved workflow for the insoluble portion of Saccharomyces cerevisiae cell lysate. Yeast cells were successfully lysed using a French press pressure cell at 20 000 psi, and resulting proteins were fractionated prior to digestion to reduce sample complexity. The proteins were best solubilized with the addition of ionic detergent sodium deoxycholate (1%) and through the application of high-frequency sonication prior to a tryptic digestion at 37 °C. Overall, the improved membrane proteomic workflow resulted in a 26% increase in membrane protein identifications for baker's yeast. In addition, more membrane protein identifications were unique to the improved protocol. When comparing membrane proteins that were identified in the improved protocol and the standard operating procedure (176 proteins), 93% of these proteins were present in greater abundance (higher intensity) when using the improved method.

Larger voids in mechanically stable, loose packings of 1.3µm frictional, cohesive particles: Their reconstruction, statistical analysis, and impact on separation efficiency.

Lateral transcolumn heterogeneities and the presence of larger voids in a packing (comparable to the particle size) can limit the preparation of efficient chromatographic columns. Optimizing and understanding the packing process provides keys to better packing structures and column performance. Here, we investigate the slurry-packing process for a set of capillary columns packed with C18-modified, 1.3µm bridged-ethyl hybrid porous silica particles. The slurry concentration used for packing 75µm i.d. fused-silica capillaries was increased gradually from 5 to 50mg/mL. An intermediate concentration (20mg/mL) resulted in the best separation efficiency. Three capillaries from the set representing low, intermediate, and high slurry concentrations were further used for three-dimensional bed reconstruction by confocal laser scanning microscopy and morphological analysis of the bed structure. Previous studies suggest increased slurry concentrations will result in higher column efficiency due to the suppression of transcolumn bed heterogeneities, but only up to a critical concentration. Too concentrated slurries favour the formation of larger packing voids (reaching the size of the average particle diameter). Especially large voids, which can accommodate particles from>90% of the particle size distribution, are responsible for a decrease in column efficiency at high slurry concentrations. Our work illuminates the increasing difficulty of achieving high bed densities with small, frictional, cohesive particles. As particle size decreases interparticle forces become increasingly important and hinder the ease of particle sliding during column packing. While an optimal slurry concentration is identified with respect to bed morphology and separation efficiency under conditions in this work, our results suggest adjustments of this concentration are required with regard to particle size, surface roughness, column dimensions, slurry liquid, and external effects utilized during the packing process (pressure protocol, ultrasound, electric fields).

Highly efficient capillary columns packed with superficially porous particles via sequential column packing.

Highly efficient capillary columns packed with superficially porous particles were created for use in ultrahigh pressure liquid chromatography. Superficially porous particles around 1.5µm in diameter were packed into fused silica capillary columns with 30, 50, and 75µm internal diameters. To create the columns, several capillary columns were serially packed from the same slurry, with packing progress plots being generated to follow the packing of each column. Characterization of these columns using hydroquinone yielded calculated minimum reduced plate heights as low as 1.24 for the most efficient 30µm internal diameter column, corresponding to over 500,000plates/m. At least one highly efficient column (minimum reduced plate height less than 2) was created for all three of the investigated column inner diameters, with the smallest diameter columns having the highest efficiency. This study proves that highly efficient capillary columns can be created using superficially porous particles and shows the efficiency potential of these particles.

Wide injection zone compression in gradient reversed-phase liquid chromatography.

Chromatographic zone broadening is a common issue in microfluidic chromatography, where the sample volume introduced on column often exceeds the column void volume. To better understand the propagation of wide chromatographic zones on a separation device, a series of MS Excel spreadsheets were developed to simulate the process. To computationally simplify these simulations, we investigated the effects of injection related zone broadening and its gradient related zone compression by tracking only the movements of zone boundaries on column. The effects of sample volume, sample solvent, gradient slope, and column length on zone broadening were evaluated and compared to experiments performed on 0.32mm I.D. microfluidic columns. The repetitive injection method (RIM) was implemented to generate experimental chromatograms where large sample volume scenarios can be emulated by injecting two discrete small injection plugs spaced in time. A good match between predicted and experimental RIM chromatograms was observed. We discuss the performance of selected retention models on the accuracy of predictions and use the developed spreadsheets for illustration of gradient zone focusing for both small molecules and peptides.

Repetitive injection method: a tool for investigation of injection zone formation and its compression in microfluidic liquid chromatography.

Sample introduction in microfluidic liquid chromatography often generates wide zones rather than peaks, especially when a large sample volume (relative to column volume) is injected. Formation of wide injection zones can be further amplified when the sample is dissolved in a strong eluent. In some cases sample breakthrough may occur, especially when the injection is performed into short trapping columns. To investigate the band formation and subsequent zone focusing under gradient elution in situations such as these, we developed the Repetitive Injection Method (RIM), based on the temporally resolved introduction of two discrete peaks to a column, mimicking both the leading and trailing edges of a larger, singly injected sample zone. Using titanium microfluidic 0.32 mm I.D. columns, the results of RIM experiments were practically identical to injection of a correspondingly larger single zone volume. It was also experimentally shown that zone width (spacing between two injected peaks) decreases during gradient elution. We utilized RIM experiments to investigate wide sample zones created by strong sample solvent, and subsequent gradient zone focusing for a series of compounds. This experimental work was compared with computationally simulated chromatograms. The success of sample focusing during injection and gradient elution depends not only on an analyte's absolute retention, but also on how rapidly the analyte's retention changes during the mobile phase gradient.

1.1 µm superficially porous particles for liquid chromatography: part II: column packing and chromatographic performance.

The predicted advantages of superficially porous particles over totally porous particles are decreased eddy dispersion, longitudinal diffusion, and resistance to mass transfer contributions to the theoretical plate height. While sub-2 micron superficially porous particles are commercially available, further improvements in performance are predicted by decreasing the particle diameter and decreasing the porous layer thickness. 1.1 µm superficially porous particles with 187Å pores have been synthesized using a layer-by-layer method tuned for production of smaller diameter particles. Following synthesis, these particles were packed into 30 µm i.d. capillary columns and their chromatographic performance evaluated using electrochemical detection. Based on the initial studies, the column efficiency did not meet theory, but was similar to the commercially available products tested. It is believed that the column packing process plays a critical role in the sub-par column performance. To determine if column efficiency could be predicted by solvent-particle interactions, in-solution optical microscopy and sedimentation velocity of particles in various slurry solvents were investigated and compared to column performance. Aggregating slurry solvents, such as methanol were found to produce columns with increased efficiency. The hmin for a column packed with an acetone slurry and a methanol slurry at 3mg/mL were found to be 6.3 and 3.5, respectively. Increasing the slurry concentration to 25mg/mL further improved the efficiency, producing a column with an hmin of 2.6. These efficiency results were accurately predicted by in-solution optical microscopy.

Evaluation of preparative hydrodynamic chromatography of silica stationary phase supports.

Reducing the particle size distribution (PSD) of sub-2 µm chromatographic packing materials can improve the performance of capillary UHPLC columns, but several size refinement methods are only partially effective in this size range. To this end, a preparative scale hydrodynamic chromatography (HDC) method was developed to size-refine C18 functionalized sub-2 µm particles, but suffered from poor reproducibility and particle aggregation issues. Presented here are improvements based on the use of an ammonium hydroxide as the mobile phase. This mobile phase makes the method reproducible, decreases column conditioning requirements, and focuses on the preparation of bare silica material which allows for a wider variety of stationary phase bondings. Additionally, particle recovery for both non-porous silica size standards and bridged-ethyl hybrid (BEH) particles are detailed to highlight the advantages of this method. The data presented demonstrates the capability of this method to reduce the relative standard deviation (RSD) of the PSD of BEH particles by 33% in under 2 h with sufficient yield to pack several capillary columns.

Observation of enhanced heat dissipation in columns packed with superficially porous particles.

At high flow rates and pressures, columns packed with sub-2 µm particles suffer from efficiency losses due to frictional heating. The thermal environment of the column (insulated or isothermal) can decrease or magnify these losses. While a number of studies have been conducted demonstrating the improved performance (partially due to the benefits of enhanced thermal conductivity) of columns packed with superficially porous particles, none have made a comparison between sub-2 µm fully and superficially porous particles in an isothermal environment where radial thermal gradients are maximized and thermal broadening is amplified. Here we show that when such columns are characterized in a recirculating water jacket (providing an isothermal environment), efficiency loss and changes in retention and mobile phase temperature are reduced for sub-2 µm superficially porous particles compared to sub-2 µm fully porous particles.