High Plate Numbers Research Articles

Preparation and kinetic performance assessment of thick film 10–20 μm open tubular silica capillaries in normal phase high pressure liquid chromatography

It is well-known that the open tubular column design basically can offer very high plate numbers. Experimental realization has however not kept pace with theoretical considerations, lacking efficient methods for the deposition of a thick film porous layer within a microbore capillary. A previously published sol–gel synthesis method was extended from 20μm to 10μm inner diameter fused silica capillaries and the resulting columns were compared to a monolithic reference capillary in terms of kinetic performance at pressure maximum. Column permeability was investigated and pressure/flow-diagrams were obtained with a 400-fold permeability gain for the open tubes. Structural characterizations regarding layer thickness and surface porosity were carried out and displayed via scanning electron microscopy and nitrogen sorption analysis. Chromatographic results in normal phase mode at elevated mobile phase flow rate reveal the intrinsic performance potential of this column format when it comes to kinetic performance limitation plots, which were constructed for all columns prepared and compared to the monolithic silica reference capillary.

Performance evaluation of thick film open tubular silica capillary by reversed phase liquid chromatography

The unquestioned potential of open tubular column design for miniaturized liquid chromatography systems has been assessed recently. Instrument limitation and insufficient mass loadability of the columns, however, have prevented experimental breakthrough in this field, the latter requiring new methods for the deposition of thick films as the stationary phase. In this work, a previously proposed synthesis strategy has been applied for the preparation of a C8 modified 15μm inner diameter variant of 3m length, providing a silica porous layer with a thickness of approximately 500nm. The capillary column has been evaluated in terms of usability, permeability and chromatographic efficiency in reversed phase mode. Data was compared to both a monolithic and a particulate, commercially available C18 capillary column. High theoretical plate numbers have been generated in a test mixture separation composed of small molecules and the applicability of this new type of column was demonstrated by two reversed phase applications.

Advantages and limitations of a new cationic coating inducing a slow electroosmotic flow for CE-MS peptide analysis: a comparative study with commercial coatings

Capillary coatings are crucial for high-quality separation performance in capillary electrophoresis analysis of proteins or peptides as they prevent analyte adsorption at the capillary wall. These coating materials have to fulfill many requirements such as a good separation performance and ensuring a good repeatability. The number of commercially available coating materials is still limited, especially with regard to the charge density on the coating material and the induced electroosmotic flow (EOF) velocity. In this work, we compare the separation performance of the novel self-made cationic capillary coating OHNOON and two commercially available coating materials, the acrylamide based, neutral LN® and the cationic hexadimethrine bromide (Polybrene), using the same coating procedure for all three coating materials. The coatings are investigated regarding the separation efficiency, analyte resolution, coating stability, and migration time stability in tryptic peptide analysis. Good separation performance was confirmed for all three coating materials: all coatings provided high plate numbers of up to 400,000-500,000 and a repeatability of the EOF and the analyte migration times in the range of 1% relative standard deviation or below. Our results reveal a moderate EOF velocity for the novel OHNOON coating in comparison to the Polybrene coating. We present a detailed discussion of the impact of this reduced EOF velocity and the separation performance. The results presented here will help to define the necessary properties of coating materials to achieve the best compromise between speed of analysis and resolution for the respective application. We show that our novel OHNOON coating is especially valuable for the analysis of low mobility analytes and for samples with a broad range of analyte mobilities.

Synthesis and characterization of new generation open tubular silica capillaries for liquid chromatography

The chromatographic performance gain of open tubular (OT) silica capillaries over packed beds has been demonstrated theoretically. However, experimental progress in the preparative realization of these columns is still lacking behind as thick films are required for a sufficient mass loadability. Here, silica sol–gel chemistry in confined spaces was applied to the preparation of thick-film OT capillaries comprising a mesoporous layer with a thickness of approximately 500nm covalently attached to the capillary wall. Samples were synthesized varying both in length and inner diameter (ID) and the resulting layer morphology was investigated using scanning electron microscopy. The chromatographic performance of these first generation columns was evaluated by normal phase chromatography utilizing standard capillary-LC equipment. Separations of small molecules in a 15μm ID column provided high theoretical plate numbers (up to 170,000) and a good retention capacity within reasonable retention times (<1h), bearing the unavoidable trade-off between column efficiency and analysis time.

Analysis of cytokinin nucleotides by capillary zone electrophoresis with diode array and mass spectrometric detection in a recombinant enzyme in vitro reaction

A capillary zone electrophoresis (CZE) method for separation of adenosine and N6-isopentenyladenosine (cytokinin) nucleotides was developed, optimized and validated. Aqueous solutions of several amino acids were evaluated as the background electrolyte constituents. Separation of six nucleotides in less than 20min with high theoretical plate number (up to 400000 for isopentenyladenosine triphosphate) was achieved using a 100mM sarcosine/ammonia buffer at pH 10.0. The detection limits of the CZE-UV method are in the low micromolar range (0.69–1.27μmolL−1). Good repeatability of migration times (within 1.3%), peak areas (within 1.8%) and linearity (R2>0.999) was achieved over the concentration range 5–1000μmolL−1. The method was used to assay the activity of the recombinant Arabidopsis thaliana isopentenyltransferase 1 (AtIPT1). Baseline separation of isopentenylated nucleotides by CE–ESI-MS using a volatile buffer (30mM ammonium formate; pH 10.0) was accomplished. The identities of the reaction products – isopentenyladenosine di- and triphosphate were confirmed by HPLC-QqTOF-MS. Dephosphorylation of ATP was observed as a parallel reaction.

Monolithic Silica for Fast HPLC: Current Success and Promising Future

As an approach for fast HPLC, monolithic silica has proven to be highly effective. It is especially successful for routinely obtaining fast isocratic HPLC analyses of small drug molecules. The low cost of monolithic compared with other approaches, such as UPLC, makes it more convenient for everyday application. It is also the more developed and widely applied technique compared with superficially porous particles. It offers the possibility for gaining high plate numbers through column coupling, but not at the expense of run time if a proper flow program is subsequently applied. Good precision and batch reproducibility are now achieved with commercially available monolithic silica columns. The application of monolithic silica columns is already well developed in various fields. It invades the field of bio-analysis and proteomics. Hundreds of analytical methods have already been successfully transferred to or developed on monolithic silica columns. An updated strategy based on Snyder’s method for rapid method development using monolithic column has been provided. However, more is still to be expected from monolithic silica in term of chemistry variation, application, and instrument compatibility. The future of monolithic silica is promising when considering the high demand for fast chromatographic analysis.

Chirally functionalized anion‐exchange type silica monolith for enantiomer separation of 2‐aryloxypropionic acid herbicides by non‐aqueous capillary electrochromatography

A silica based monolithic capillary column derivatized with O-9-(tert-butylcarbamoyl)quinidine was prepared for CEC enantiomer separation of chiral 2-aryloxypropionic acid herbicides including inter alia dichlorprop, mecoprop and fenoprop. The silica monolith had a relatively low surface coverage with chiral cationic selector moieties. Due to the low selector density retention factors were low as well, yet still enabling enantiomer separations of the target solutes. Both electrophoretically and chromatographically dominated migration and separation modes, respectively, could be established depending on the employed conditions. In the former mode, enantiomers migrated in front of the EOF marker, and faster separations and higher plate numbers could be achieved. In the latter mode, stronger adsorption translated into a typical chromatographic separation in which the enantiomers eluted after the EOF marker whereby separation factors were slightly enhanced compared to the aforementioned separation mode. Reasonable baseline separations of enantiomers were accomplished for all analytes after optimization of relevant mobile phase parameters in the anion-exchange CEC system including sample loadability, and the separations were comparable to such obtained on an optimized high density quinidine-carbamate modified organic polymer monolith column. Overall, it is concluded that monoliths with a high surface density of chiral ion-exchange moieties are favorable because of their enhanced sample loadabilities and improved chromatographic performance with regard to separation factors, plate numbers and peak symmetries. The resultant accompanying longer analysis times may rather be reduced by adjusting effective column length than by reducing selector coverage of the monolith.

Design, Modeling, and Fabrication of MEMS-Based Multicapillary Gas Chromatographic Columns

This paper describes different approaches to achieve high-performance microfabricated silicon-glass separation columns for microgas chromatography systems. The capillary width effect on the separation performance has been studied by characterization of 250-, 125-, 50-, and 25-?m -wide single-capillary columns (SCCs) fabricated on a 10 × 8 mm2 die. The highest plate number (12 500/m), reported to date for MEMS-based silicon-glass columns, has been achieved by 25-?m-wide columns coated by a thin layer of polydimethylsiloxane stationary phase using static coating technique. To address the low sample capacity of these narrow columns, this paper presents the first generation of MEMS-based ?multicapillary? columns (MCCs) consisting of a bundle of narrow-width rectangular capillaries working in parallel. The theoretical model for the height-equivalent-to-a-theoretical-plate (HETP) of rectangular MCCs has been developed, which relates the HETP to the discrepancies of the widths and depths of the capillaries in the bundle. Two-, four-, and eight-capillary MCCs have been designed and fabricated to justify the separation ability of these columns. These MCCs capable of multicomponent gas separation provide a sample capacity as large as 200 ng compared to 5.5 ng for 25-?m-wide SCCs.

Effect of detergent on electromigration of proteins: CE of very low density lipoprotein receptor modules and viral proteins

The different electrophoretic behavior of the members of two groups of proteins with respect to the absence or presence of detergent additives in the BGE was explored. Recombinant soluble concatemers of repeat 3 of the very low density lipoprotein (VLDL)-receptor fused at their N-terminus to maltose-binding protein (MBP) exhibited different electrophoretic mobilities in borate buffer (pH 8.3) in the absence and in the presence of dodecyl-PEG ether (D-PEG). This enabled the separation of the receptor fragments from MBP after enzymatic cleavage. In the presence of SDS, the mobilities of all proteins approached the same values with increase in detergent concentrations. In contrast, viral capsid proteins of a human rhinovirus (HRV) exhibited different migration in the presence of the additive. For the receptor proteins, extreme apparent high plate numbers were observed when the SDS concentration in the sample and the separation buffer differed. This effect might be erroneously interpreted as a high efficiency. However, it is due to the conductivity boundaries caused by the sample and leads to a total loss of separation.

Determination of five anthraquinones in medicinal plants by capillary zone electrophoresis with β-cyclodextrin addition

A simple and rapid method for the simultaneous determination of five anthraquinone derivatives including aloe-emodin, emodin, chrysophanol, physcoin and rhein in Rheum species and Polygonum cuspidatum was established by capillary zone electrophoresis (CZE) using β-cyclodextrin (CD) as modifier and urea to enhance its solubility. The apparent binding constants of these derivatives with β-CD were evaluated. After an optimization study, the best conditions were selected using 35 mM phosphate buffer (pH 11.0) containing 20 mM β-CD and 2 M urea, applied voltage 20 kV and detection at 254 nm. Under such conditions, all of the five anthraquinones were baseline-separated within a short analysis time of 12 min with symmetrical peaks and high theoretical plate numbers (189 000–314 000). The RSD values of the migration times and peak areas were 0.6–1.1, 1.3–1.9% (intra-day) and 0.6–1.5, 1.3–2.8% (inter-day, for a 5-day period), respectively. The limits of detection for the analytes (S/N = 3) were 0.33–0.62 μg/ml. The recoveries were ranged from 93.37 to 107.69%. The proposed method was successfully applied to the determination of anthraquinones in ethanol extracts of two kinds of Rheum plants ( R. palmatum and R. hotaoense) and P. cuspidatum.

Large-Volume Sample Sweeping with a High Theoretical Plate Number Using a Coupled-Capillary in Capillary Electrophoresis

A large-volume sample injection (> 5 microL) with an extremely high theoretical plate number (N > 10(7)) was achieved when the sweeping-MEKC mode and a coupled-capillary (100 - 50 microm i.d.) were simultaneously used in a capillary electrophoresis (CE) separation. A low-cost and compact violet-LED ( approximately 2 mW) was used as the fluorescence excitation source. As a result, the theoretical plate numbers of the detected peaks (two model compounds: naphthalene-2,3-dicarboxaldehyde derivatized-dopamine and -norepinephrine) were 1.0 x 10(7) and 7.4 x 10(6), respectively. The limits of detection (at S/N = 3) of these were determined to be 2.8 x 10(-10) M (92 ppt) and 2.3 x 10(-10) M (83 ppt), respectively.

Application of polymeric surfactants in micellar electrokinetic chromatography‐electrospray ionization mass spectrometry of benzodiazepines and benzoxazocine chiral drugs

Chiral micellar EKC (CMEKC) coupled to ESI-MS using polymeric surfactants as pseudostationary phases is investigated for simultaneous enantioseparation of two benzodiazepines, (+/-)-oxazepam ((+/-)-OXA) and (+/-)-lorazepam ((+/-)-LOR), and one benzoxazocine, (+/-)-nefopam ((+/-)-NEF). First, enantioselectivity and electrospray sensitivity of six chiral polymeric surfactants for all three chiral compounds are compared. Second, using poly(sodium N-undecenoyl-L-leucinate) as pseudostationary phase, the organic modifiers (methanol (MeOH), isopropanol, and ACN) are added into the running buffer to further improve chiral resolution (RS). Next, a CMEKC-ESI-MS method for the simultaneous enantioseparation of two benzodiazepines is further developed by using a dipeptide polymeric surfactant, poly(sodium N-undecenoxy carbonyl-L,L-leucyl-valinate) (poly-L,L-SUCLV). The CMEKC conditions including nebulizer pressure, capillary length, ammonium acetate concentration, pH, poly-L,L-SUCLV concentration, and capillary temperature were optimized to achieve maximum chiral RS and highest sensitivity of MS detection. The spray chamber parameters (drying gas temperature and drying gas flow rate) as well as sheath liquid conditions (MeOH content, pH, flow rate, and ionic strength) were found to significantly influence MS S/N of both (+/-)-OXA and (+/-)-LOR. Finally, a comparative study between simultaneous UV and MS detection showed high plate numbers, better chiral RS, and enhanced detectability with CMEKC-MS. However, speed of analysis was faster using CMEKC-UV.

Separation and on-line concentration of saponins from Panax notoginseng by micellar electrokinetic chromatography

A simple, reproducible and sensitive micellar electrokinetic chromatography (MEKC) method was developed for the separation and determination of 10 saponins from Panax notoginseng. Field-enhanced sample injection with reverse migrating micelles (FESI–RMM) was used for on-line concentration of the saponins. The effects of concentrations of sodium dodecyl sulfate (SDS) and organic modifier, the sample matrix, the injection time of water plug, the injection voltage and injection time of sample on the separation and stacking efficiency were investigated. The optimum buffer contained 10 mM H 3PO 4, 140 mM SDS, 20% acetonitrile and 15% 2-propanol and the pH of buffer was 2.4. The sample solution was diluted with 15 mM SDS and injected for 15 s with −8 kV after injection of 2 s water plug. Under the optimum conditions, the analytes were well separated with very high plate number (8.0 × 10 5–1.2 × 10 6 N/m); the limits of detection of the analytes were 1.7–6.3 μg/mL. The high sensitivity permitted the determination of two minor saponins Rh1 (1.01 mg/g) and Rg2 (0.62 mg/g) in P. notoginseng, which were not determined in the literature.

High-efficiency liquid chromatography on conventional columns and instrumentation by using temperature as a variable: I. Experiments with 25 cm × 4.6 mm I.D., 5 μm ODS columns

High plate numbers were obtained in conventional LC by coupling columns and by using temperature to reduce the viscosity of the mobile phase. At 80 °C up to eight columns of 25 cm × 4.6 mm I.D. packed with 5 μm ODS particles could be coupled generating 180,000 effective plates while the pressure drop was only 350 bar. For routine work, a set of four columns is preferred. The analysis times on one column operated at 30 °C and 1 mL/min flow rate and on four columns at 80 °C and 2 mL/min flow rate are the same in isoeluotropic conditions while the resolution is doubled. Multicolumn systems were successfully applied in isocratic and gradient mode for the analysis of pharmaceutical and environmental samples.

Gold Nanoparticle-Modified Etched Capillaries for Open-Tubular Capillary Electrochromatography

The use of gold nanoparticles in conjunction with etched capillary-based open-tubular capillary electrochromatography (OTCEC) to improve the efficiency of separation and the selectivity between selected solutes is described. The fused-silica capillaries (50-microm i.d.) were etched with ammonium hydrogen difluoride, followed by prederivatization of the new surface with (3-mercaptopropyl)trimethoxysilane (MPTMS) for the immobilization of dodecanethiol gold nanoparticles, for OTCEC. The electrochromatography of a "reversed-phase" test mixture and of selected polycylic aromatic hydrocarbons was investigated, and efficient separations and high theoretical plate numbers per meter were obtained. The electroosmotic flow characteristics of the etched gold nanoparticle capillary, unetched gold nanoparticle capillary, bare capillary, and etched bare capillary were studied by varying the percentage of organic modifier in buffer, buffer pH, and separation voltage. Optical microscopy and scanning electron microscopy were used to examine the process of etching and modification and the surface features of the etched gold nanoparticle capillary. The results confirm that dodecanethiol gold nanoparticles bonded on the etched inner wall of the fused-silica capillary can provide sufficient solute-bonded phase interactions to obtain OTCEC separations with reproducible retention, as well as characteristic reversed-phase behavior, even with the inner diameter of the capillary of 50 microm.

Preparation and use of packed capillary columns in chromatographic and related techniques

In this paper general considerations related to the various approaches and parameters influencing the preparation of highly efficient and stable capillary columns for use in liquid chromatography and related techniques are presented and the column production process is discussed in some detail. The different packing methods available for delivering a packing material into a capillary column are discussed from a practical viewpoint. Packing with a gas (dry packing), packing with a liquid solvent or a mixture of solvents (slurry packing), packing with supercritical carbon dioxide, electrokinetic packing, and sol-gel packing technologies are introduced and discussed throughout the paper. Practical recommendations for obtaining highly efficient (high plate numbers) and stable capillary packed columns are also addressed and discussed.

Capillary electrochromatography with polymeric continuous beds synthesized via free radical polymerization in aqueous media using derivatized cyclodextrins as solubilizing agents

A novel synthetic route to amphiphilic acrylamide-based monolithic stationary phases for capillary electrochromatography (CEC) employing water-soluble cyclodextrins as solubilizing agents was explored. N, N′-Octamethylenebisacryamide and N, N′-dodecamethylenebisacryamide were synthesized and their solubilization in aqueous solution with derivatized and underivatized cyclodextrins of different cavity size was studied. Amphiphilic stationary phases were synthesized by free radical copolymerization of the bisacrylamide–cyclodextrin host–guest complexes with hydrophilic monomers and an additional hydrophilic cross-linker in aqueous solution. Complex formation in solution and removal of the complexed cyclodextrin from the polymer during synthesis was studied with 1 H- NMR and solid state 13 C- NMR spectroscopy and cyclodextrin-modified micellar electrokinetic chromatography. The impact of the incorporated alkylene groups in the acrylamide-based macroporous polymer on retention was studied with neutral solutes by CEC in the normal-phase elution mode and in the reversed-phase elution mode. Batch-to-batch reproducibility of the synthesis procedure and day-to-day repeatability of the separations achieved were investigated. With these capillaries, a sufficiently high electroosmotic flow velocity, a high reproducibility and repeatability of separation parameters and high plate numbers (up to 200 000 m −1) were obtained.

Capillary electrophoresis in the determination of anionic catecholamine metabolites from patients’ urine

A micellar electrokinetic capillary chromatographic method for determining anionic catecholamine metabolites from patient urine samples was established. The optimum electrolyte solution (pH 10.6) was made of tetraborate and sodium dodecyl sulphate into water. Furthermore, studies were focused to optimize solid-phase extraction clean-up steps to concentrate patient urine samples for identification of catecholamines with UV detection. The water-micelle distribution coefficients (octanol–water partition coefficient, log P ow) for the analytes were determined by conductometric titration. High plate numbers (120 000–200 000/40 cm detection window) and small diffusion coefficients (2.00–3.50×10 −6 cm −2 s −1) resulted in high resolution and symmetry of the analyte zones. The standard deviations of the migration times and the peak heights were less than 3 and 7%, respectively. The octanol–water coefficients increased in the order of decreased p K a1 value of the analytes, why separation between structurally related vanillinic mandelic acids could be obtained. Limits of detection and quantification were around 0.05 and 0.1 μg/ml, respectively, except for dopamine. The concentrations of the catecholamine metabolites in the studied patient urines varied from 0.186 to 76.4 μg/ml. The results showed evidences of serious diseases among the patients.

Experiences with monolithic LC phases in quantitative bioanalysis

The applicability of monolithic liquid chromatographic (LC) phases in the field of quantitative bioanalysis has been evaluated. Two existing methods with fluorescence detection (the determination of bexarotene in plasma and the determination of dextromethorphan plus metabolites in urine) were successfully transferred from a conventional reversed-phase column to a 10 cm×4.6 mm i.d. monolith. By simply increasing the mobile phase flow-rate, run times were about 3-fold reduced, while the chromatographic resolution of the analytes remained unaffected. In both cases, a very good correlation was found between the results of clinical samples obtained with the original method and those obtained with the adapted method. Two methods with tandem mass spectrometric detection were set up. For one of these methods (nifedipine in plasma), the separation of the analyte from interfering matrix components did not need a high plate number; the resolution found on a 10-cm monolith at 6 ml/min and that on a 3-cm conventional column at 2 ml/min were comparable and achieved in the same period of time. As the validation results on both column types were similar and considering the limited compatibility of mass spectrometric detection with high solvent flow rates, the conventional column was concluded to be the best choice for this application. For the determination of estradiol in plasma, however, there was so much interfering material that needed to be separated from the analyte, that the best results were obtained with three 10-cm monolithic columns coupled in series and because of the possibility to apply a relatively high flow-rate, a reasonable run time was still achieved.

Enantioseparations in nonaqueous capillary liquid chromatography and capillary electrochromatography using cellulose tris(3,5-dimethylphenylcarbamate) as chiral stationary phase.

The potential of the widely used chiral stationary phase for high-performance liquid chromatography (HPLC) enantioseparations, cellulose tris(3,5-dimethylphenylcarbamate) (CDMPC, sold under the trade name Chiralcel OD) was evaluated under the conditions of nonaqueous capillary electrochromatography (CEC). The effect of the particle size of the silica gel, the loading of CDMPC on the silica gel and nature of the organic solvent, as well as electrolyte salts on the separation characteristics were investigated. This study illustrates the applicability of CDMPC for obtaining highly efficient enantioseparations under the conditions of nonaqueous CEC. Comparative study of enantioseparations in capillary liquid chromatography (CLC) and CEC indicated the significant advantages of CEC such as higher plate number at the similar linear flow rates of the mobile phase as well as better tolerance of higher linear flow rates.