Open Tubular Columns Research Articles

Rapid preparation and evaluation of chiral open-tubular columns supported with bovine serum album and zeolite imidazolate framework-8 for mini-capillary electrochromatography

In this work, a class of novel and eco-friendly open-tubular (OT) chiral column was presented for the first time by one step preparation with zeolite imidazolate framework-8 (ZIF-8) and bovine serum album (BSA) based on electrostatic adsorption and adsorption affinity. This stationary phase materials combined the features of large surface areas and adsorption affinity of ZIF-8, and also the multiple chiral binding sites of BSA, which contributes to the π-interaction and hydrophobic interaction with the analytes. The separation performance of BSA@ZIF-8-OT chiral columns was evaluated with a miniaturized capillary electrochromatography and amperometric detection (mini-CEC-AD) system; in particular, nine groups of model molecules, including homologues, structural isomers, and chiral compounds, were baseline separated under the certain optimum conditions. The RSDs of run-to-run, day-to-day, column-to-column, and batch-to-batch reproducibility were less than 13.8 %. Furthermore, the prepared OT columns were successfully applied to fast analysis of ephedrine isomers in Chinese herb ephedra, and the LODs achieved 1.5-2.0 ng mL−1 (S/N=3) by an electrophoretic stacking technique of moving chemical reaction boundary. This mini-CEC-AD system with BSA@ZIF-8-OT chiral columns provides a promising potential in pharmaceutical analysis due to its fast, sensitive, enantioselective, and low-cost characteristics.

Evaluation of chiral separation based on bovine serum albumin-conjugated carbon nanotubes as stationary phase in capillary electrochromatography.

In this work, we utilized adsorbed BSA and multiwalled carbon nanoparticles (BSA/MWCNTs) as a stationary phase in open tubular (OT) capillary for separation of chiral drugs. (3-Aminopropyl)triethoxysilane was used to assist fabrication of BSA/MWCNTs-coated OT column by covalent bonding. Incorporation of MWCNTs nanomaterials into a polymer matrix could increase the phase ratio and take advantage of the easy preparation of an open tubular CEC column. SEM was carried out to characterize the BSA/MWCNTs OT columns. The electrochromatographic performance of the OT columns was evaluated by separation of ketoprofen, ibuprofen, uniconazole, and hesperidin. The effects of MWCNTs concentration, background solution pH and concentration, and applied voltage on separation were investigated. Chiral separations of ketoprofen, ibuprofen, uniconazole, and hesperidin were achieved using the BSA/MWCNTs-coated OT column with resolutions of 24.20, 12.81, 1.50, and 1.85, respectively. Their optimas were found in the 30mM phosphate buffers at pH 5.0, 6.5, 7.0, and 6.5, respectively. In addition, the columns demonstrated good repeatability and stability with the run-to-run, day-to-day, and batch-to-batch RSDs of migration times less than 3.5%.

Microgeometry-independent equation for measuring infinite dilution activity coefficients using gas-liquid chromatography with static-wall-coated open-tubular columns

Gas-liquid chromatography is an effective method to determine infinite dilution activity coefficients (γ∞). Wall-coated open-tubular (WCOT) column which offers more advantages over packed column should be a preferable column type; however, the small carrier gas flow rate and stationary phase amount in WCOT columns limit its application in the determination of γ∞. Mathematical strategy made some progress to avoid the quantification problem in the determination of γ∞ by static-wall-coated open-tubular (SWCOT) columns. However, the previously reported strategy was based on the assumption that SWCOT column was geometrically an ideal hollow cylinder, which indeed deviates from the reality. In this study, without that assumption, we derived a new microgeometry-independent equation by using the relationship between the hold-up volume (VM) and the volume of stationary phase (VL), and used it to measure the γ∞ of various organic solutes in two ionic liquids (ILs) 1‑butyl‑3-methylimidazolium dicyanamide and 1,3-dibutyronitrile-imidazolium bis((trifluoromethyl)sulfonyl)imide, both of which contain double cyano groups in the anion or cation. Phase loading study was adopted to eliminate the influence of interfacial adsorption to partition. The infinite dilution partial molar excess enthalpy, selectivity and capacity were directly calculated from the experimental γ∞ values, and the linear solvation energy relationship (LSER) model was used to characterize the specific properties of both ILs. This new established equation will promote the application of SWCOT columns in thermodynamic measurement and benefit the fast screening of novel solvents for chemical separation processes.

Turbulent Supercritical Fluid Chromatography in Open-Tubular Columns for High-Throughput Separations.

A method utilizing turbulent flow to perform ultrafast separations and screen chiral compounds in supercritical fluid chromatography (SFC) is described. Carbon dioxide at high flow rates (up to 4.0 mL/min) is delivered into gas chromatography (GC) open-tubular columns (OTC, 0.18 mm i.d., 20 m long, ∼0.2 μm stationary film thickness) to establish turbulent flow at Reynolds numbers (Re) as high as 9000. Postcolumn dispersion is eliminated by using a modified UV detector that takes measurements directly on column. Upon crossing the laminar-to-turbulent flow transition regime, a significant reduction in plate height is observed resulting in a nearly 3-fold increase in peak capacity from the laminar regime. This is explained by the massive reduction of the mass transfer resistance in the mobile phase due to a flatter flow profile and faster analyte dispersion across the open-tubular column (OTC) i.d.. Demonstrated in this work is a 9 s separation of four polycyclic aromatic hydrocarbons (PAHs) over a 2.2 s separation window using a poly(dimethylsiloxane-co-methylphenylsiloxane) coated OTC. Additionally, three chiral compounds and three chiral cyclodextrin-incorporated OTCs were evaluated at high temperatures (90-120 °C) and CO2 flow rates (3.3-3.7 mL/min) to demonstrate column stability and application of this method for rapid screening. Turbulent SFC provides a separation method for users desiring to achieve separation speeds above what is currently available with very high-pressure LC systems and do so without the resolution loss commonly observed at maximum allowable speed.

Kinetic performance factor – a proportional metric for comparing performance of differently structured liguid chromatography columns

Particle size (dp) of a column packing material is a subjective quantity, especially in open-tubular, monolithic, pillar-array and others non-particulate columns. It is possible to design a column internal structure so that the dimensionless plate height, h=H/dp, could be arbitrarily small without improving the column performance. Subsequently, h is unsuitable for comparing performance of differently structured columns. The problem is avoided if previously introduced effective diameter (deff) of a column – a measurable (objective) parameter – is used instead of dp. Previously introduced general definition of deff was suitable for LC with incompressible solvents and GC with compressible carrier gases. The latter feature complicates the concept of deff and its relation to other column parameters. Only LC with incompressible solvents is considered in this report. This substantially simplifies the concept of deff and objective performance metrics related to deff, including hc=H/deff and kinetic performance factor (q) proportional to (1/hc)1/2. Moreover, the Bristow-Knox separation impedance (E) – also an objective metric – can be expressed as E=32(H/deff)2. Metrics q, hc and E carry the same information, the ratio H/deff, only differently scaled and differently powered. However, q is the only metric proportional to external metrics like the peak capacity. Open-tubular columns (OTC) have the highest qmax among all other known structures. Having qmax close to one, OTC are convenient performance benchmark for other structures.Deficiencies of parameter h are demonstrated, properties of parameters deff and qmax are discussed and their numerical values found from published data for several column structures are compared.

β-Cyclodextrin-modified covalent organic framework as chiral stationary phase for the separation of amino acids and β-blockers by capillary electrochromatography.

Covalent organic frameworks (COFs) as a novel stationary phase have attracted much attention in the field of chromatography owing to their permanent nanoscale porosity, higher surface area, and exceptional stabilities. Here, a novel isocyanate-β-cyclodextrin-modified COF (MDI-β-CD-modified COF) was synthesized using isocyanate-β-cyclodextrin as the chiral selector and imine-based TpPa-1 COF as the matrix by a bottom-up strategy. The reaction condition and the structure of MDI-β-CD-modified COF were optimized and characterized by X-ray diffraction (XRD), Fourier-transform infrared (FT-IR) spectra, nitrogen adsorption/desorption (Brunauer-Emmett-Teller [BET]), and thermogravimetric analysis (TGA). And then the coated open-tubular column (OT column) was prepared using MDI-β-CD-modified COF as chiral stationary phase (CSP) by in situ growth approach, which exhibited excellent stability and repeatability. For seven consecutive runs, the intraday and interday relative standard deviations (RSDs) were in range from 0.35% to 2.21% for the migration time of histidine. The column-to-column reproducibility ranged from 2.39% to 3.08%. Meanwhile, the separation of eight compounds including four amino acids and four β-blockers by capillary electrochromatography sufficiently verified the favorable chiral resolution properties of the MDI-β-CD-modified COF-coated OT column. This strategy of fabricating MDI-β-CD-modified COF-coated OT column expanded the application of imine-based COFs in chromatographic analytical fields.

Optimum Cell Pathlength or Volume for Absorbance Detection in Liquid Chromatography: Transforming Longer Cell Results to Virtual Shorter Cells.

The cell volume permissible for a specified degree of loss of efficiency can be computed from response volume considerations. For an open tubular column, the permissible illuminated length can be computed for the unretained peak. Similar estimations can be made for the maximum permissible cell volume with a packed column for a given column efficiency and flow rate. The packed column case does require an assumption on the degree to which the cell behaves as a mixer. An altogether different question is if the data from a long cell, with its considerable advantage in S/N, can be used and the associated dispersion mathematically removed. Experimental data for a variable path length (0-60 mm) HPLC detection cell indicate that an exponential model fits the observed dispersion. Once fit parameters are determined, the same can be applied to a peak, not part of the original training set, obtained in a longer path cell and the effects of dispersion mathematically reversed without major loss of S/N. Results with shorter path cell dispersion characteristics are then obtained with much higher S/N. A comparison is made with Fourier transform-inverse Fourier transform based deconvolution that can be used to achieve the same ends.

Picoflow Liquid Chromatography-Mass Spectrometry for Ultrasensitive Bottom-Up Proteomics Using 2-μm-i.d. Open Tubular Columns.

In many areas of application, key objectives of chemical separation and analysis are to minimize the sample quantity while maximizing the chemical information obtained. Increasing measurement sensitivity is especially critical for proteomics research, especially when processing trace samples and where multiple measurements are desired. A rich collection of technologies has been developed, but the resulting sensitivity remains insufficient for achieving in-depth coverage of proteomic samples as small as single cells. Here, we combine picoliter-scale liquid chromatography (picoLC) with mass spectrometry (MS) to address this issue. The picoLC employs a 2-μm-i.d. open tubular column to reduce the sample input needed to greatly increase the sensitivity achieved using electrospray ionization (ESI) with MS. With this picoLC-MS system, we show that we can identify ∼1000 proteins reliably using only 75 pg of tryptic peptides, representing a 10-100-fold sensitivity improvement compared with the state-of-the-art liquid chromatography (LC) or capillary electrophoresis (CE)-MS methods. PicoLC-MS extends the limit of separation science and is expected to be a powerful tool for single cell proteomics.

Application of porous layer open tubular capillary columns with narrow inner diameter in nanoflow-high performance liquid chromatography for N-derivatized amino acid enantiomers

Porous layer open tubular capillary columns with a narrow inner diameter (NPLOT) have potential in life science, especially in single-cell analysis. In this work, quinidine-based chiral NPLOT columns were prepared by in-situ thermal-initiated polymerization. The porous layer of the organic polymer was prepared with a 6 μm i. d capillary by in-situ thermal-initiated polymerization, following which the thickness and morphology of the NPLOT column were observed for different durations (3 h, 6 h, and 9 h) of polymerization. The thicknesses of the porous layer were 103±51 nm and 210±51 nm when polymerization was conducted for 3 h and 6 h, respectively. Subsequently, the columns prepared under 3 h of polymerization were applied in nanoflow-high performance liquid chromatography for the enantioseparation of N-derivatized amino acid enantiomers. Baseline separation was achieved in 2 min. The sample volume consumed was of the picoliter level, which would provide a means of research for single-cell analysis.

Performing flow injection chromatography using a narrow open tubular column

Flow injection chromatography (FIC) or sequential injection chromatography (SIC) is a low-pressure liquid chromatography technique that uses flow injection or sequential injection hardware. Due to the constraints of this hardware, the separation resolution is low; often no more than 3–5 components are resolved. We have recently demonstrated the excellent resolving power of narrow open tubular (OT) columns for various biomolecules, and only moderate elution pressures are needed to carry out these separations. In this paper, we incorporate a narrow OT column with FIC and construct an FIC system using a pressure chamber and two injection valves to implement gradient elution. The resultant system not only improves the resolution but also reduces the system cost. When we use the system to separate peptides from trypsin-digested cytochrome C, we can resolve dozens of peptides (with resolutions of 0.5 or greater) at a speed of 12 samples per hour. When we use this system to separate a mixture containing 3 amino acids, we can base-line resolve these compounds at a speed of 1800 sample per hour.

Recent developments of monolithic and open-tubular capillary electrochromatography (2017-2019).

Capillary electrochromatography, which combined the high selectivity of high-performance liquid chromatography and the high separation efficiency of capillary electrophoresis, is an attractive separation tool. In this review, the developments on monolithic and open tubular capillary electrochromatography during 2017 to August 2019 are summarized. Considering the development of novel stationary phases is the most active research field in capillary electrochromatography, monolithic capillary electrochromatography is classified according to the polymer-based and hybrid monolithic columns, while open-tubular capillary electrochromatography is categorized by cyclodextrin, silica, polymer, nanomaterials, microporous materials, and biomaterials-based open tubular columns.

Zeolite silica nanoparticles-supported open-tubular columns for isomer and chiral separation using capillary electrochromatography coupled with amperometric detection

Zeolite SiO2NPs OT columns with/without BSA were used for the separation of structural isomers, epimers and enantiomers by CEC-AD.

A novel open-tubular capillary electrochromatography using carboxymethyl-β-cyclodextrin functionalized gold nanoparticles as chiral stationary phase.

Enantioselective open tubular capillary electrochromatography with carboxymethyl-β-cyclodextrin conjugated gold nanoparticles as stationary phase was developed. This novel open tubular column was fabricated through layer-by-layer self-assembly of gold nanoparticles on a 3-mercaptopropyl-trimethoxysilane-modified fused-silica capillary and subsequent surface functionalization of the gold nanoparticles through self-assembly of 6-mercapto-β-cyclodextrin. The 6-mercapto-β-cyclodextrin was firstly synthesized and determined by extensive spectroscopic data. Scanning electron microscopy, energy dispersive X-ray analysis spectroscopy, and electroosmotic flow experiments were carried out to characterize the prepared open tubular column. Then, the separation effectiveness of the open tubular column was verified by two pairs of ɑ-tetralones derivatives enantiomers and two pairs of basic drug enantiomers (tramadol hydrochloride and zopiclone) as mode analytes. Factors that influence the enantioseparation were optimized, and under the optimized conditions, satisfactory separation results were obtained for the four enantiomers: compound A, compound B, tramadol hydrochloride, and zopiclone with resolutions of 3.79, 1.56, 1.03, 1.60, respectively. For the combination of gold nanoparticles and negatively charged carboxymethyl-β-cyclodextrin, the open tubular column exhibited wider separation range for neutral and basic drugs. Moreover, the repeatability and stability of the column were studied through the run-to-run and day-to-day investigations.

Ionic liquid-copolymerized monolith based porous layer open tubular column for CEC-MS analysis

Developing novel capillary electrochromatography (CEC) column with high separation efficiency and capacity is an essential work in CEC. In this work, porous layer open tubular (PLOT) column has been developed by co-polymerization of quaternary ammonium typed ionic liquid (IL), 1-allyl-methylimidazolium chloride (AlMeIm+Cl−) with styrene (IL-styrene) and ethylene dimethacrylate (EDMA) for CEC-MS analysis. The PLOT-CEC column possesses high separation efficiency and capacity while keeping the inherent advantages of open tubular (OT) column unaltered, which is suitable for CEC analysis. Due to the uniform branch-like structure with large specific surface area, the IL-styrene based PLOT-CEC column showed excellent separation efficiency towards multiple kinds of analytes with theoretical plates up to N = 322,277 N/m for phloroglucinol. And thanks to the high permeability and large EOF mobility generated by positively charged ionic liquid, the PLOT-CEC column showed good performance in CEC-MS analysis of parabens, amino acids and peptides with stable separation and ESI spray. Good repeatability with relative standard deviations for intra-day, inter-day runs and between tubes less than 4.94% was obtained.

100 Micrometer bore open tubular capillary column modified with linear co-polymer chains for application in low pressure liquid chromatography

The performance of a 100 um i.d. open tubular column modified with linear co-polymer chains has been explored under low pressure operating conditions. A 512 mm fused-silica capillary was reacted with 3-chloropropyl isocyanate followed by sodium diethyldithiocarbamate. A copolymer layer was then immobilized on the inner surface via reversible addition-fragmentation transfer polymerization of styrene, methacrylic acid and N-phenylacrylamide. The thickness of the polymer layer was ca 1.5 µm. The column was evaluated for the separation of a mixture composed of three peptides (Trp-Gly, angiotensin I, bradykinin). The number of theoretical plates (N) as high as 72,100 plates/column (corresponding to 140,800/m, 7.10 µm plate height) was achieved. The column to column and day to day reproducibility of our columns was found better than 2.1%. For comparison purpose, similar chromatographic data for a selected analyte was also obtained with a commercial microcolumn of 0.5 mm id packed with a conventional C18 phase (5 µm), and 17 µm was the plate height obtained at the optimum flow rate. The van Deemter plots and kinetic plots were also obtained for comparison between our open tubular capillary column and the commercial packed column of a narrow id.

Gas chromatography system constant database for 52 wall-coated, open-tubular columns covering the temperature range 60–140 °C

The solvation parameter model is used to characterize the separation properties of 52 fused-silica, wall-coated, open-tubular columns at 5 evenly-spaced intervals over the temperature range 60 to 140 °C. Statistical and principal component factor analysis are used to identify stationary phases of similar separation characteristics considered near selectivity equivalent and stationary phases with specific differences in selectivity for method development. The column database includes examples of all common stationary phase chemistries and manufacturing processes (surface preparation, deactivation, surface bonding, and immobilization). The influence of monomer composition on the separation properties of poly(siloxane) and poly(ethylene glycol) stationary phases is fully characterized and used to demonstrate where gaps in the selectivity space exist in the column database. Interfacial adsorption was observed as an important retention mechanism for biscyanopropylsiloxane-containing stationary phases for n-alkanes but for other compounds and stationary phases gas-liquid partition appears to be the dominant retention mechanism. There is poor agreement between McReynolds phase constants and the system constants of the solvation parameter model for the characterization of stationary phase selectivity.

Ultra-high thermal stability perarylated ionic liquids as gas chromatographic stationary phases for the selective separation of polyaromatic hydrocarbons and polychlorinated biphenyls

Ionic liquids (ILs) are well-known in the field of separation science for their unique selectivity when used as stationary phases in gas chromatography (GC). While a significant amount of knowledge has been attained in correlating structural features of an IL to separation selectivity, developments in producing IL-based stationary phases suitable for high temperature GC studies have lagged behind. Column bleed is a result of the stationary phase undergoing volatilization/decomposition at high temperatures and is undesirable in separations coupled to GC/MS. It has been well-known that traditional classes of ILs with long alkyl side chain substituents are susceptible to Hofmann elimination at elevated temperatures. In this study, a new class of IL stationary phases containing perarylated cations exhibiting improved thermal stability are introduced. These ILs were used to prepare wall-coated open tubular columns with high column efficiency and produced very low bleed at temperatures up to 350 °C. Their unique chemical structures provide stronger π-π interactions compared to many commercially-available stationary phases. To exploit the unique interactions provided by these stationary phases, the separation of two classes of environmentally hazardous aromatic compounds, namely, polycyclic aromatic hydrocarbons (PAHs) and polychlorinated biphenyls (PCBs), was examined. Both classes of compounds contain structural isomers with high boiling points that are often challenging to separate. The perarylated sulfonium and phosphonium IL-based stationary phases exhibited excellent thermal stability as well as unique selectivity toward isomers of PAHs as well as toxic PCB analyte pairs.

Experimentally Validating Open Tubular Liquid Chromatography for a Peak Capacity of 2000 in 3 h.

The advancements in life science research mandate effective tools capable of analyzing large numbers of samples with low quantities and high complexities. As an essential analytical tool for this research, liquid chromatography (LC) encounters an ever-increasing demand for enhanced resolving power, accelerated analysis speed, and reduced limit of detection. Although theoretical studies have indicated that open tubular (OT) columns can produce superior resolving power under comparable elution pressures and analysis times, ultrahigh-resolution and ultrahigh-speed open tubular liquid chromatography (OTLC) separations have never been reported. Here we present experimental results to demonstrate the predicted potential of this technique. We use a 2 μm i.d. × 75 cm long OT column coated with trimethoxy(octadecyl)silane for separating pepsin/trypsin digested E. coli lysates and routinely produce exceptionally high peak capacities (e.g., 1900-2000 in 3-5 h). We reduce the column length to 2.7 cm and exhibit the capability of OTLC for ultrafast separations. Under an elution pressure of 227.5 bar, we complete the separation of six amino acids in ∼800 ms and resolve these compounds within ∼400 ms. In addition, we show that OTLC has low attomole limits of detection (LOD) and each separation requires samples of only a few picoliters. Importantly, no ultrahigh elution pressures are required. With the ultrahigh resolution, ultrahigh speed, low LOD, and low sample volume requirement, OTLC can potentially be a powerful tool for biotech research, especially single cell analysis.

Ultrafast Gradient Separation with Narrow Open Tubular Liquid Chromatography.

Separation speed and resolution are two important figures of merit in chromatography. Often, one gains the speed at the cost of the resolution, and vice versa. Scientists have employed short-packed columns for ultrafast separations but encountered challenges such as limited mobile phase velocity, extra-column effect caused band broadening, and column packing difficulty. We have recently demonstrated ultrahigh resolutions of narrow open tubular liquid chromatography (NOTLC); this allows us to trade some of the resolution for speed. In this work, we explored NOTLC for ultrafast LC separations. We used a 2.7 cm (effective length) narrow open tubular (NOT) column and showed a baseline separation of 6 amino acids in less than 700 ms. Ways to further increase the speed were discussed. Using short narrow open tubular (NOT) columns to perform ultrafast separation we overcame the challenges from using short packed columns. To demonstrate the feasibility of using this ultrafast separation technique for practical applications, we separated complex protein digests; peptides were nicely resolved in ∼1 min.

Polydopamine-assisted immobilization of a zinc(II)-derived metal-organic cage as a stationary phase for open-tubular capillary electrochromatography.

A capillary column was modified with a soluble zinc(II)-derived metal-organic cage (MOC) [Zn2L] as the stationary phase to obtain a new coating layer for use in open-tubular capillary electrochromatography. The inner surface of the capillary was first coated with a layer of polydopamine. Then, a solution of the MOC in dichloromethane was introduced into the capillary upon which it is bonded both covalently and non-covalently. The resulting coating layer on the capillary was characterized by scanning electron microscopy and Fourier transform infrared spectroscopy. The results demonstrated the successful formation of the [Zn2L] modified open-tubular column. The column showed good separation performance towards neutral compounds (such as methylbenzene, ethylbenzene, n-propylbenzene and n-butylbenzene), acidic drugs (such as ibuprofen, ketoprofen, flurbiprofen and diclofenac sodium), food additives (such as parabens, vanillin and related phenolic compounds) and small biomolecules (such as nucleosides and nucleotide bases) by π-interaction and hydrophobic interaction. It also exhibited good precision, the relative standard deviations of the retention time for intra-day, inter-day runs and column-to-column being <1.6%, 2.8%, and 4.0%, respectively. Graphical abstract Schematic presentation of the open-tubular column modified with zinc(II)-derived metal-organic cage by polydopamine-assisted strategy onto the inner wall of capillary for electrochromatographic separations.