Sub-2 Μm Research Articles

High-Speed, High-Efficiency Achiral SFC on a 3 × 20-mm Column Packed with 1.8-µm Particles Facilitated by a Low-Dispersion Chromatograph

There are almost no reports using 20–30 mm long (or shorter) columns packed with sub-2 µm packings for SFC separation of achiral solutes in the literature, probably because current commercial SFCs have extra-column dispersion of > 80 µL2. A modified supercritical fluid chromatograph (SFC) with extra-column dispersion below 5 µL2 produced reduced plate heights as low as 2.2, using a 3 × 20-mm column packed with 1.8-µm RX-Sil, bare silica particles, with 7.5% methanol at 2.5 mL min−1. The calculated and observed peak fidelities were similar at ≈ 0.85 suggesting the chromatographs extra-column dispersion was slightly limiting the observed efficiency, which suggests the column might be even better packed. Increasing the flow rate to 5 mL min−1 with 15% methanol resulted in the resolution of 7 solutes, from 4 different solute families, in less than 8 s. A few of the peaks required a UV detector filter setting of 120 Hz (< 0.016 s. response time) to avoid extra-column peak broadening. The column was quite susceptible to the strong sample solvent effect. Even moderate sized injection volumes (of MeOH) and feed volumes (of IPA) degraded efficiency significantly.

Mode-locked dysprosium fiber laser: Picosecond pulse generation from 2.97 to 3.30 μm

Mode-locked fiber laser technology to date has been limited to sub-3 μm wavelengths despite significant application-driven demand for compact picosecond and femtosecond pulse sources at longer wavelengths. Erbium-doped and holmium-doped fluoride fiber lasers incorporating a saturable absorber are emerging as promising pulse sources for 2.7–2.9 μm, yet it remains a major challenge to extend this coverage. Here, we propose a new approach using a dysprosium-doped fiber with frequency shifted feedback (FSF). Using a simple linear cavity with an acousto-optic tunable filter, we generate ∼33 ps pulses with up to 2.7 nJ energy and 330 nm tunability from 2.97 to 3.30 μm (∼3000–3400 cm−1)—the first mode-locked fiber laser to cover this spectral region and the most broadly tunable pulsed fiber laser to date. Numerical simulations show excellent agreement with experiments and also offer new insights into the underlying dynamics of FSF pulse generation. This highlights the remarkable potential of both dysprosium as a gain material and FSF for versatile pulse generation, opening new opportunities for mid-IR laser development and practical applications outside the laboratory.

Measurement of the Band Broadening of UV Detectors used in Ultra-high Performance Liquid Chromatography using an On-tubing Fluorescence Detector

In modern ultra-high performance liquid chromatography set-ups, short columns (max. 10 cm) with a narrow ID (2.1 mm) packed with small, (sub-2 µm) fully or superficially porous particles are used. Since the volume and corresponding peak variance arising from these columns are very small, the dispersion contribution from the chromatographic system has a large effect on the overall separation performance. In gradient elution, the on-column focusing of the sample band at the start of the gradient results in the elimination of the pre-column band broadening. However, since gradient elution separations yield very narrow sample peaks at the outlet of the column, any post-column band broadening has a large effect on the obtained separation quality. In this contribution, the main factor of post-column band broadening is investigated, i.e., that from the UV detector, by comparing the peak width measured on capillary directly in front of the UV detector using an LIF detector and the peak widths obtained in the UV detector. These experiments show that there is a clear increase in peak variance with flow rate up to around 0.4–0.6 mL/min (depending on the investigated flow cell). It is found that modern low-dispersion flow cells generate a dispersion contribution around 0.7 µL2 at high flow rates, whereas standard flow cells can have a contribution up to 5.8 µL2. For the investigated nano-flow cell (80 nL), no significant dispersion was observed.

Determination of steviol glycosides in commercial extracts of Stevia rebaudiana and sweeteners by ultra-high performance liquid chromatography Orbitrap mass spectrometry

Stevia rebaudiana extracts are used as sweeteners in several countries worldwide. Several extracts of diverse composition are available on the market, and their taste depends on the contents of the various steviol glycosides. This study presents an accurate method for the qualitative and quantitative determination of steviol glycosides in 40 Stevia extracts, 7 sweeteners and 3 Stevia-sweetened beverages by a UHPLC coupled to an Orbitrap mass spectrometer. The sub-2 μm amide column provided the separation of all the target analytes in a run time of 30 min with high resolution. The effect of different eluent compositions on the ionisation efficiency of the steviol glycosides was studied. The optimal ionisation conditions were achieved in negative mode using 0.05% formic acid. Under this condition, adducts were not found, [M-H]− were the main ions and the spontaneous loss of a glucose residue at C19 was reduced. The %RSD for intra- and inter-day precision for all eleven analytes varied from 2.1 to 4.2% and 3.0–5.1%, respectively. The recoveries from spiked Stevia extract samples were greater than 95% for all analytes. Rebaudioside A was the most abundant, ranging from 23 to 102%. Nine Stevia extracts and one drink were not compliant with the European Regulation. Isosteviol was under the LOD in all samples and steviol was found in four samples in quantities in the range 0.01–0.03%.

Direct analysis of chiral active pharmaceutical ingredients and their counterions by ultra high performance liquid chromatography with macrocyclic glycopeptide-based chiral stationary phases

In this work the simultaneous separation of chiral active pharmaceutical ingredients (API) in salt form from their counterions has been performed by using different high-efficiency macrocyclic glycopeptide-based chiral stationary phases (CSPs). Not only a new zwitterionic vancomycin-based CSP has been prepared (similarly to what was done for teicoplanin) but macrocyclic selectors have also been bonded to sub-2 μm fully porous silica particles through traditional ureidic linkage to obtain versions of CSPs suitable for ultra-high performance applications. The direct separation of chiral APIs and counterions is particularly attracting since it simplifies the workflow traditionally used with reduction of analysis time and costs. The wide selection of macrocyclic antibiotics CSPs now available has allowed to manage different cases that can happen in the simultaneous separation of APIs and their counterions (either cations or anions). Indeed, while inorganic cations are retained on traditional vancomycin- and teicoplanin-based CSPs, inorganic anions are almost unretained (due to Donnan’s effect). On the other hand, cations and anions can be both retained on the zwitterionic versions of these CSPs. Afterwards, zwitterionic CSPs allowed the separation of other compounds including N-derivative amino-acids, profens, polyols, sugar anomers, oligosaccharides and inorganic anions/cations opening new perspectives in the use of this family of CSPs.

Unmatched Kinetic Performance in Enantioselective Supercritical Fluid Chromatography by Combining Latest Generation Whelk-O1 Chiral Stationary Phases with a Low-Dispersion in-House Modified Equipment.

This proof-of-concept work investigates the ultimate kinetic limits reachable in chiral supercritical fluid chromatography (SFC) with modern columns and advanced technological solutions. A commercial equipment (Waters Acquity UPC2) has been in-house modified to minimize its overall extra-column variance through a series of technical adjustments including low-volume connecting tubings, reduced-volume flow cell, an in-house made external column oven, external low-dispersion injection system, and electronic temperature controller. Compared to the original (as-shipped) configuration, the extra-column variance on the low-dispersion equipment was reduced by more than 97%, from about 85 to slightly more than 2 μL2 (measured at 2.0 mL/min). This was mainly achieved thanks to the occurrence of fully developed turbulent regime with a proper selection of capillary i.d. at significantly smaller flow rates (1.5-4 mL/min; CO2/methanol 80/20, v/v; 35 °C; back pressure regulator (BPR), 105 bar) than in entry-1 configuration. Ultrahigh efficiency columns of different geometries in-house packed with latest generation sub-2 μm UHPC-FPP-Whelk-O1 Chiral Stationary Phase (CSP) have been employed under sub- and supercritical fluid conditions. By carefully modulating the length and the internal diameter of connecting tubings in the function of column geometry, state of the art efficiencies (estimated in roughly 300 000 theoretical plates/m with reduced HETP of roughly 1.85) have been obtained on 4.6 mm i.d. chiral columns. Remarkably, for 3.0 mm × 100 mm (i.d. × length) columns, the efficiency gain on the fully modified SFC system (compared to an instrumental configuration where only the standard injector was replaced by the low-dispersion one) was greater than 90% for compounds with a retention factor of 1 and as large as 25% for retention factors of 2.5.

Comparison of small size fully porous particles and superficially porous particles of chiral anion-exchange type stationary phases in ultra-high performance liquid chromatography: effect of particle and pore size on chromatographic efficiency and kinetic performance

Recent advancements in particle design are common in reversed-phase liquid chromatography (RPLC), but in chiral separations their use is still sporadic in commercially available chiral stationary phases (CSPs). Due to reported lower mass transfer resistance, they might be a promising opportunity to increase efficiency and reduce time of analysis since the relatively higher mass transfer resistance term of CSPs caused by slow adsorption-desorption kinetics is the most performance-limiting factor in enantioselective chromatography. This study was dedicated to the evaluation of new support materials for tert-butylcarbamoylquinine (tBuCQN) based CSP to provide highly efficient and fast enantioseparations. As the main focus of this study, the chiral selector tBuCQN was immobilized on sub-2 μm fully porous particles (FPPs) and 2.7 μm superficially porous particles (SPPs) and their column performance in enantioseparation was evaluated in comparison to 5 μm FPPs by van Deemter and Knox analyses as well as kinetic plots using racemic Fmoc-Phe. Both new particle types outperformed the 5 μm FPP benchmark in terms of speed and efficiency, with wider pore materials (160 or 200 Å) being advantageous (over 90 or 120 Å). Basically decisive for the performance gain was the 10-times smaller mass transfer resistance. Furthermore, 2.7 μm 160 Å SPPs outperformed their fully porous sub-2 μm 120 Å counterpart (HminR = 4.64 μm vs. HminR = 8.94 μm) due to various parameters affording reduced plate height h of 1.7. Caused by the inaccessible core, separations were about 2-times faster. Packing of 2.7 μm core-shell particles provided a very homogeneous column bed, and, owing to its higher permeability, the column backpressure was much lower. It enables packing of longer columns providing theoretically separation efficiencies of up to 106 plates per m (as indicated by kinetic plots) and versatile use without the necessity of UHPLC systems. Investigating the effect of particle size reduction (FPPs: 5 μm, 3 μm, 1.7 μm; SPPs: 2.7 μm, 2 μm) and wider pores (FPPs: 120 Å, 200 Å; SPPs: 90 Å, 160 Å), a significantly reduced mass transfer resistance was the driving force for performance gain. Individual contributions of peak dispersion were deconvoluted for 5 μm FPP CSP and confirmed that slow adsorption-desorption kinetics is the most significant contribution to peak broadening in this chromatographic system.

Applicability of supercritical fluid chromatography – mass spectrometry to metabolomics. I – Optimization of separation conditions for the simultaneous analysis of hydrophilic and lipophilic substances

The aim of this study was to evaluate the suitability of SFC-MS for the analysis of a wide range of compounds including lipophilic and highly hydrophilic substances (log P values comprised between −6 and 11), for its potential application toward human metabolomics. For this purpose, a generic unified chromatography gradient from 2 to 100% organic modifier in CO2 was systematically applied. In terms of chemistry, the best stationary phases for this application were found to be the Agilent Poroshell HILIC (bare silica) and Macherey-Nagel Nucleoshell HILIC (silica bonded with a zwitterionic ligand). To avoid system overpressure at very high organic modifier proportion, columns of 100 × 3 mm I.D. packed with sub-3 μm superficially porous particles were selected. In terms of organic modifier, a mixture of 95% MeOH and 5% water was selected, with 50 mM ammonium formate and 1 mM ammonium fluoride, to afford good solubility of analytes in the mobile phase, limited retention for the most hydrophilic metabolites and suitable peak shapes of ionizable species. A sample diluent containing 50%ACN/50% water was employed as injection solvent.These conditions were applied to a representative set of metabolites belonging to nucleosides, nucleotides, small organic acids, small bases, sulfated/sulfonated metabolites, poly-alcohols, lipid related substances, quaternary ammonium metabolites, phosphate-based substances, carbohydrates and amino acids. Among all these metabolites, 65% of the compounds were adequately analyzed with excellent peak shape, 23% provided distorted peak shapes, while only 12% were not detected (mostly metabolites having several phosphate or several carboxylic acid groups).

Benefits and Drawbacks of Fully Porous sub-2 µm and Core-Shell Particles. Comparative UHPLC Applications in Food, Environmental, Forensic, Biopharmaceutical and Natural Products Analyses

Benefits and Drawbacks of Fully Porous sub-2 µm and Core-Shell Particles. Comparative UHPLC Applications in Food, Environmental, Forensic, Biopharmaceutical and Natural Products Analyses

Visualizing and studying frictional heating effects in reversed-phase liquid chromatography using infrared thermal imaging

Column temperature control is a fundamental component of liquid chromatography experiments. However, it is typically monitored indirectly by tracking the temperature of an adjacent heating element that exchanges heat with the column in a controlled environment. The practice of not directly measuring the column temperature means that uncontrolled contributions of heat, such as frictional heating inside the column, can be overlooked. The present work describes the use of a high-resolution infrared thermal imaging camera to directly measure the column heat map during mobile phase flow. The approach was used to measure the longitudinal temperature gradient formed with three common mobile phases: water, methanol, and acetonitrile, in two 50 mm reversed-phase columns, a 1.7 μm particle-packed column and a polystyrene divinylbenzene monolith. In a close approximation to an adiabatic environment, the temperature gradients (ΔT) observed with the 1.7 μm particle column at a linear velocity of 5.8 mm/s were up to +16.6 and + 12.8 °C above an ambient temperature of 23 °C for water and acetonitrile, respectively. In the case of water, the measured temperature gradient values (ΔT) were within 1% difference of theoretically-calculated values and on average within 10% for acetonitrile. By contrast, the ΔT observed in the monolith was negligible. The elevated temperatures that are generated through friction in sub-2 μm particle columns may be particularly important to consider for the design of experiments that measure structural features of temperature-sensitive analytes, such as biomolecules. While frictional heating is one important application of the thermal imaging approach described, the technique can be used to provide a data-rich profile of heat exchange in numerous experimental configurations, chromatographic or otherwise.

A stochastic view on column efficiency

A stochastic model of transcolumn eddy dispersion along packed beds was derived. It was based on the calculation of the mean travel time of a single analyte molecule from one radial position to another. The exchange mechanism between two radial positions was governed by the transverse dispersion of the analyte across the column. The radial velocity distribution was obtained by flow simulations in a focused-ion-beam scanning electron microscopy (FIB-SEM) based 3D reconstruction from a 2.1 mm × 50 mm column packed with 2 μm BEH-C18 particles. Accordingly, the packed bed was divided into three coaxial and uniform zones: (1) a 1.4 particle diameter wide, ordered, and loose packing at the column wall (velocity uw), (2) an intermediate 130 μm wide, random, and dense packing (velocity ui), and (3) the bulk packing in the center of the column (velocity uc).First, the validity of this proposed stochastic model was tested by adjusting the predicted to the observed reduced van Deemter plots of a 2.1 mm × 50 mm column packed with 2 μm BEH-C18 fully porous particles (FPPs). An excellent agreement was found for ui = 0.93uc, a result fully consistent with the FIB-SEM observation (ui = 0.95uc). Next, the model was used to measure ui = 0.94uc for 2.1 mm × 100 mm column packed with 1.6 μm Cortecs-C18 superficially porous particles (SPPs). The relative velocity bias across columns packed with SPPs is then barely smaller than that observed in columns packed with FPPs (+6% versus + 7%). uw=1.8ui is measured for a 75 μm × 1 m capillary column packed with 2 μm BEH-C18 particles. Despite this large wall-to-center velocity bias (+80%), the presence of the thin and ordered wall packing layer has no negative impact on the kinetic performance of capillary columns. Finally, the stochastic model of long-range eddy dispersion explains why analytical (2.1–4.6 mm i.d.) and capillary (<400 μm i.d.) columns can all be packed efficiently (1 <hmin< 2) with particles of size larger than 2 μm. In contrast, the model predicts that 0.4–1.2 mm i.d. columns and 2.1 mm i.d. columns cannot be packed well (hmin>3) with sub-2 μm particles and with 1 μm particles, respectively.

High speed hydrophilic interaction liquid chromatographic method for simultaneous determination of selected pharmaceuticals in wastewater using a cyano-bonded silica column

ABSTRACTIn this work, a fast analytical method based on hydrophilic interaction liquid chromatographic-Ultraviolet detection (HILIC-UV) using a short narrow bore cyano-bonded silica column packed with fully porous sub-2 µm particles has been developed for simultaneous determination of eight pharmaceuticals in wastewater. The method involved pre-concentration and clean-up by solid phase extraction using Oasis HLB extraction cartridges. The analytes were separated using a mobile phase consisted of acetonitrile and 5 mM ammonium acetate buffer (95:5 v/v) with a flow rate of 0.6 mL/min. The chromatographic separation was optimized in order to achieve short analysis time and good resolution for all analytes in a single run. Each analyte was detected at its maximum wavelength for higher sensitivity. All analytes could be separated in 5.7 min with resolution ≥2.7. The optimized method was validated based on linearity, precision, detection and quantification limits, selectivity and accuracy. The detection limits of the studied pharmaceuticals ranged from 0.6 to 3 µg/L, while limits of quantification were in the range from 2 to 10 µg/L with UV detection. The developed method is fast, reliable, cost-effective and could be used for the analysis of the studied analytes in other matrices such as food, pharmaceutical preparations and biological fluids.

Enantioselective ultra high performance liquid and supercritical fluid chromatography: The race to the shortest chromatogram.

The ever-increasing need for enantiomerically pure chiral compounds has greatly expanded the number of enantioselective separation methods available for the precise and accurate measurements of the enantiomeric purity. The introduction of chiral stationary phases for liquid chromatography in the last decades has revolutionized the routine methods to determine enantiomeric purity of chiral drugs, agrochemicals, fragrances, and in general of organic and organometallic compounds. In recent years, additional efforts have been placed on faster, enantioselective analytical methods capable to fulfill the high throughput requirements of modern screening procedures. Efforts in this field, capitalizing on improved chromatographic particle technology and dedicated instrumentation, have led to highly efficient separations that are routinely completed on the seconds time scale. An overview of the recent achievements in the field of ultra-high-resolution chromatography on column packed with chiral stationary phases, both based on sub-2μm fully porous and sub-3μm superficially porous particles, will be given, with an emphasis on very recent studies on ultrafast chiral separations.

High efficiency and fast separation of active proteins by HIC chromatographic pie with sub-2 μm polymer packings

This paper reports the development of hydrophobic interaction chromatography (HIC) by synthesizing sub-2 μm polymer packings which was packed into a chromatographic pie for fast separation of native proteins at low pressures demonstrating high efficiency. Using styrene as monomer and ethylene dimethacrylate (EDMA)as swelling agent, the polystyrene seeds with an average particle size of 0.8 μm and monodisperse polymeric microspheres with a particle size of 1.5–5.0 μm were synthesized through dispersion polymerization and one-step swelling method, respectively. In order to separate active proteins, the microspheres were modified to hydrophobic chromatographic packings through covalent bonding with benzene methanol. Compared with the traditional column chromatography, the sub-2 μm polymer packings in chromatographic pie exhibited higher column efficiency for protein separation at lower column pressures, even at higher flow rates. The van Deemter curve showed that the flow rate had insignificant effect on column efficiency of chromatographic pie. Seven example proteins were clearly separated within 3 min at a flow rate of 10 mL/min. The applicability of this method was further demonstrated by the separation of human serum samples. The results indicated that this chromatographic mode can be potentially applied for the fast separation of complex active proteins, such as protein drugs from natural products.

On the relationship between radial structure heterogeneities and efficiency of chromatographic columns

The general dispersion theory of Aris is applied to predict the virtual asymptotic dispersion behavior of packed columns. The derived model is also used to estimate the actual pre-asymptotic dispersion behavior of modern 2.1 mm × 50 mm columns packed with sub-2 μm fully porous particles (FPPs) during the transient dispersion regime. The model accounts for the actual radial distribution of the flow velocity across the column diameter. From the wall to the center of the column, focused-ion-beam scanning electron microscopy (FIB-SEM) experiments were recently performed to reveal the existence of a thin (0.15dp wide, dp is the average particle diameter) hydrodynamic boundary layer (THBL), a thin (3dp wide) and loose orderly packed layer (TLOPL), a 60dp wide and dense randomly packed layer (WDRPL), and a large (≃460dp) randomly packed bulk central region [1].The theoretical calculations of the actual pre-asymptotic reduced van Deemter curves (2.1 mm × 50 mm column, sub-2 μm BEH-C18 FPPs, n-hexanophenone analyte, acetonitrile/water eluent, 80/20, v/v, flow rate from 0.05 to 0.35 mL/min) confirm that the impact of the sole THBL on column dispersion can be neglected. In contrast, the contribution of the TLOPL to the reduced plate height (RPH) is about 0.2 h unit at optimum reduced velocity. Most remarkably, the negative impact of the TLOPL on column performance may be fully compensated by the presence of the adjacent WDRPL if the depth of the velocity well were to be 5% of the bulk velocity. In actual 2.1 mm × 50 mm columns packed with sub-2 μm FPPs, this velocity depth is as large as 25% of the bulk velocity causing a significant RPH deviation of 0.7 h unit from the RPH of the bulk packing free from wall effects. Maximum column performance is expected for a reduction of WDRPL density. This suggests optimizing the packing process by finding the proper balance between the stress gradient across the WDRPL (responsible for the deep velocity well) and the friction forces between the packed particles (responsible for the rearrangement of the particles during bed consolidation). Past and recently reported RPH data support the theoretical insights: the stress gradient/particle friction balance in the WDRPL is better realized when packing superficially porous particles (SPPs) rather than FPPs in 2.1–4.6 mm i.d. columns (the RPH deviation is reduced to 0.4 h unit) or sub-2 μm particles in 100 cm × 75 μm i.d. capillaries combining high slurry concentrations and sonication (the RPH deviation is reduced to only 0.15 h unit).

Separation of isomers of new psychoactive substances and isotope-labeled amphetamines using UHPSFC-MS/MS and UHPLC-MS/MS

ABSTRACTThe use of sub-2 µm particles columns is finding its use in ultrahigh-pressure supercritical fluid chromatography (UHPSFC), opening up for fast analysis and high-resolution separations. The development of new and more robust systems also makes the technique more interesting for bioanalytical analysis, where the need for reproducible and fast analysis with little downtime is great. One area where the use of UHPSFC could become a useful tool is in the separation of structural isomers of new psychoactive substances (NPS). 2-, 3-, and 4- structural isomers of fluoroamphetamine, fluoromethamphetamine, and methylmethcathinone, isomeric pairs of the synthetic cannabinoids UR-144/UR-144 degradant, XLR-11/XLR-11 degradant, JWH-015/JWH-073, and JWH-019/JWH-122, as well as amphetamine and several stable isotope-labeled amphetamine internal standards were separated with UHPSFC-MS/MS and compared with ultrahigh-pressure liquid chromatography (UHPLC) MS/MS. NPS isomers that were difficult to separate with reversed-phase UHPLC-MS/MS were separated by UHPSFC; in most cases with an orthogonal retention order to UHPLC. In contrast to the behavior seen when using reversed-phase UHPLC, the deuterated amphetamines eluted later than amphetamine with UHPSFC. 13C6-labeled amphetamine coeluted with amphetamine for all conditions, making this the best choice of an internal standard.

Fast ultra-high-performance liquid chromatography with diode array and mass spectrometry method for determination of tadalafil drug substance and its impurities.

Tadalafil is used for the treatment of erectile dysfunction. Its related patents expired in 2016, and so related generic drug production is predicted to be increased. This work is focused on developing a fast ultra-high-performance liquid chromatography with diode array detection and/or mass spectrometry detection for the separation and determination of tadalafil and its impurities in pharmaceutical samples. A modern reversed-phase stationary phase with sub-2 μm particle size, Zorbax StableBond Rapid Resolution High Definition with octylsilane chemically bonded phase to totally porous silica particles, was used for the solving this problem. Column temperature was set at 40 ± 0.1°C. A mobile phase consisting of acetonitrile and aqueous solution of 0.1% (v/v) trifluoroacetic acid for diode array detection detection and 0.05% (v/v) formic acid, both running at a flow rate of 0.62 mL/min, were used to achieve the required separation of all components within a 5 min run. The limit of detection was 3.5 μg/L and the limit of quantification was 10.0 μg/L for the method for both UV and MS detectors. Accurate mass spectra of tadalafil's related impurities are shown for advanced confirmation. The method is directly transferable to routine analysis of tadalafil in pharmaceutical and control laboratories.

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.

Preparation of sub-2 μm large-pore monodispersed mesoporous silica spheres using mixed templates and application in HPLC

Sub-2 μm large-pore monodispersed mesoporous silica spheres (MMSS) were synthesized by using mixed non-ionic surfactants of dodecylamine (DDA) and dodecylamine polyoxyethylene ether (AC1210) as templates through an alkali-catalyzed silica sol–gel technique combined with the template method. Methanol-H2O volume ratio on morphology was researched in detail. In addition, by using hydrothermal treatment, the average pore size of MMSS could be enlarged to 11 nm from 4.4 nm. The mesoporous silica particles were characterized by scanning electron microscopy (SEM), transmission electron microscopy (TEM), laser particle size analyzer, X-ray diffraction, N2 physisorption technique. The synthesized sub-2 μm monodispersed mesoporous silica microspheres were successfully functionalized by dimethyloctadecylchlorosilane and used as a packing material in high performance liquid chromatography (HPLC).

Fast separation of flavonoids by supercritical fluid chromatography using a column packed with a sub-2μm particle stationary phase.

The purpose of this study was to compare the effects of different chromatographic columns for the separation of seven flavonoids. Four different stationary phases are available, including bridged ethyl hybrid, BEH and the same hybrid phase modified with 2-ethylpyridine, CSH fluorophenyl, and HSS C18 SB. The analytes included calycosin, genistein, medicarpin, calycosin-7-O-β-d-glucoside, formononetin, formononetin-7-O-β-d-glucoside, and liquiritigenin. The CSH fluorophenyl column was determined to be the most suitable and provided the fastest separation within 17min using gradient elution with carbon dioxide as the mobile phase and methanol as the co-solvent. Good peak shapes were obtained, and the values of the peak asymmetry were close to 1.0 for all of the flavonoids. The resolution was more than 1.41 for all of the separated peaks. Baseline separation on the optimal columns was achieved by changing the co-solvent type and adjusting the temperature and pressure. Quantitative performance was evaluated under optimized conditions, and method validation was accomplished. The validation parameters, such as linearity, sensitivity, precision, and accuracy, were satisfactory. Good repeatability of both peak area (relative standard deviation <1.02%) and retention time (relative standard deviation <0.88%) was observed. The optimized chromatographic methods were successfully used for the determination of seven flavonoids in Radix astragali. The sensitivity was sufficient for the analysis of real samples.