Number Of Theoretical Plates Research Articles

Ultra high efficiency/low pressure supercritical fluid chromatography with superficially porous particles for triglyceride separation

This paper reports the development of the separation of vegetable oil triglycerides (TG) in supercritical chromatography (SFC), using superficially porous particles (SPPs). The SPP, having a small diameter (2–3μm), provide a higher theoretical plate number (N), which allows to improve separation of critical pairs of compounds. However, compared to fully porous particles of larger diameter (5μm), the pressure drop is also increased. Fortunately, supercritical fluids have a low viscosity, which allows coupling several columns to achieve high N values, while maintaining flow rate above 1ml/min, ensuring a ultra high efficiency (UHE) at low pressure (LP) (below 40MPa), with regards to the one reached with liquid and sub-two micron particles (around 100MPa). The use of two detector systems (UV and ELSD) connected in series to the UHE–LP-SFC system provides complementary responses, due to their specific detection principles. Working in a first part with three coupled Kinetex C18 columns (45cm total length), the effect of modifier nature and percentage were studied with two reference oils, argan and rapeseed, chosen for their different and well-known TG composition. The analytical method was developed from previous studies performed with fully porous particles (FPP). Optimized conditions with three Kinetex were as follows: 17°C, 12% of ACN/MeOH (90/10; v/v). With these conditions, and by using an increased length of Kinetex C18 column (60cm), another additional column was selected from ten different commercial SPP C18 bonded phases, by applying a Derringer function on varied parameters: theoretical plate number (TPN), separation index (SI) for critical pairs of peaks (the peaks of compounds difficult to separate due to subtle structural differences), the analysis duration, and the total peak number. This function normalizes the values of any parameters, between 0 and 1, from the worst value to the better, allowing to take account of various parameters in the final choice. Finally, by using four Kinetex C18 plus one Accucore C18 (75cm total column length), a high-performance separation of triglycerides was achieved, with reasonable analysis duration and isocratic conditions. These conditions can be applied to varied vegetable oils. Identification of the numerous separated peaks of rapeseed oil was achieved by using published data and chromatographic retention behaviour.

High-Resolution Hydrodynamic Chromatographic Separation of Large DNA Using Narrow, Bare Open Capillaries: A Rapid and Economical Alternative Technology to Pulsed-Field Gel Electrophoresis?

A high-resolution, rapid, and economical hydrodynamic chromatographic (HDC) method for large DNA separations in free solution was developed using narrow (5 μm diameter), bare open capillaries. Size-based separation was achieved in a chromatographic format with larger DNA molecules being eluting faster than smaller ones. Lambda DNA Mono Cut Mix was baseline-separated with the percentage resolutions generally less than 9.0% for all DNA fragments (1.5 to 48.5 kbp) tested in this work. High efficiencies were achieved for large DNA from this chromatographic technique, and the number of theoretical plates reached 3.6 × 10(5) plates for the longest (48.5 kbp) and 3.7 × 10(5) plates for the shortest (1.5 kbp) fragments. HDC parameters and performances were also discussed. The method was further applied for fractionating large DNA fragments from real-world samples (SacII digested Arabidopsis plant bacterial artificial chromosome (BAC) DNA and PmeI digested Rice BAC DNA) to demonstrate its feasibility for BAC DNA finger printing. Rapid separation of PmeI digested Rice BAC DNA covering from 0.44 to 119.041 kbp was achieved in less than 26 min. All DNA fragments of these samples were baseline separated in narrow bare open capillaries, while the smallest fragment (0.44 kbp) was missing in pulsed-field gel electrophoresis (PFGE) separation mode. It is demonstrated that narrow bare open capillary chromatography can realize a rapid separation for a wide size range of DNA mixtures that contain both small and large DNA fragments in a single run.

Rapid and direct analysis of statins in human plasma by column-switching liquid chromatography with restricted-access material

This study presents the development of a column-switching liquid chromatographic method with direct injection of human plasma for simultaneous determination of four statins (lovastatin, pravastatin, rosuvastatin and simvastatin), the main class of drugs used in the treatment of hyperlipidemia. By using a C18 (30 mm × 4.6 mm, 15 μm) a lab made bovine serum albumin restricted access material (RAM) column was prepared and compared with a commercial alquil-diol silica RAM column (C18, 25 mm × 4.0 mm, 25 μm) in terms of their protein exclusion capacity and micromolecules retention. Foreflush and backflush modes were compared for both RAM columns to the number of theoretical plates, asymmetry, resolution and chromatographic run time. The developed method was validated in the range from 125 to 876 ng mL(-1) for lovastatin, rosuvastatin and simvastatin, and from 500 to 2000 ng mL(-1) for pravastatin, presenting selectivity, precision and accuracy intra and inter-run. Total analysis time (sample preparation and chromatographic separation) was only 16 min when the backflush mode was employed in the column-switching system.

Catalyst assisted synthesis of initiator attached silica monolith particles via isocyanate-hydroxyl reaction for production of polystyrene bound chromatographic stationary phase of excellent separation efficiency

Dibutyltin dichloride (DBTDC) was used as a catalyst to chemically bind 4-chloromehtylphenylisocynate (4-CPI) to porous monolithic silica particles via isocyanate-hydroxyl reaction, and the reaction product was reacted with sodium diethyldithiocarbamate (SDDC) to yield initiator attached silica monolith particles. Reversible addition-fragmentation transfer (RAFT) polymerization was taken place on them to result in polystyrene attached silica particles that showed excellent separation efficiency when packed in a chromatographic column (1.0mm×300mm). The numbers of theoretical plates (N) of 56,500 is better than those of any commercially available HPLC or UHPLC column yet.

Chiral 3D Open‐Framework Material Ni(D‐cam)(H2O)2 Used as GC Stationary Phase

Metal-organic frameworks (MOFs) have been explored for analytical applications because of their outstanding properties such as high surface areas, flexibility and specific structure features, especially for chromatography application in recent years. In this work, a chiral MOF Ni(D-cam)(H2O)2 with unusual integration of molecular chirality, absolute helicity, and 3-D intrinsic chiral net was chosen as stationary phase to prepare Ni(D-cam)(H2O)2-coated open tubular columns for high-resolution gas chromatographic (GC) separation. Two fused-silica open tubular columns with different inner diameters and lengths, including column A (30 m × 250 µm i.d.) and column B (2 m × 75 µm i.d.), were prepared via a dynamic coating method. The chromatographic properties of the two columns were investigated using n-dodecane as the analyte at 120 °C. The number of theoretical plates (plates/m) of the two metal-organic framework (MOF) columns was 1300 and 2750, respectively. The racemates, isomer and linear alkanes mixture were used as analytes for evaluating the separation properties of Ni(D-cam)(H2O)2-coated open tubular columns. The results showed that the columns offered good separations of isomer and linear alkanes mixture, especially racemates.

Estimation of tea catechin levels using micellar electrokinetic chromatography: A quantitative approach

A simple, inexpensive micellar electrokinetic chromatography (MEKC) method with UV detection was used to determine seven catechins and one xanthine (caffeine) in tea. All the compounds were successfully separated (15kV) within a 15-min migration period with a high number of theoretical plates (>8.0×104) in a running buffer (pH 7) containing 10mmoll−1 sodium tetraborate, 4mmoll−1 sodium phosphate, and 25mmoll−1 SDS. The regression lines of all standard catechins were linear within the range of 0.03–4μgml−1. Green tea infused at 95°C for 10min showed higher levels of catechins (especially epigallocatechin galate, epicatechin gallate, and epicatechin) than tea infused at 80°C. In addition, major differences were observed in the levels of catechins in the first and second infusions (both brewed at 95°C for 10min). Finally, green tea leaves were infused separately with tap water, deionised water, spring water, reverse osmosis water, and distilled water at 95°C, and the catechin content of the infusions was investigated by the proposed method. In the infusion brewed with tap water, catechins appeared to be epimerisation from the epistructure to the nonepistructure. This epimerisation may take place more readily in tap water than in distilled water owing to the complexity of the ions present in tap water.

Kinetic efficiency of polar monolithic capillary columns in high-pressure gas chromatography

Poppe plots were used for analysis of kinetic efficiency of monolithic sorbents synthesized in quartz capillaries for utilization in high-pressure gas chromatography. Values of theoretical plate time and maximum number of theoretical plates occurred to depend significantly on synthetic parameters such as relative amount of monomer in the initial polymerization mixture, temperature and polymerization time. Poppe plots let one to find synthesis conditions suitable either for high-speed separations or for maximal efficiency. It is shown that construction of kinetic Poppe curves using potential Van Deemter data demands compressibility of mobile phase to be taken into consideration in the case of gas chromatography. Model mixture of light hydrocarbons C1 to C4 was then used for investigation of influence of carrier gas nature on kinetic efficiency of polymeric monolithic columns. Minimal values of theoretical plate times were found for CO2 and N2O carrier gases.

Studies on Citronellal Extract Column Reflux Ratio and Theoretical Plate Number Design

In this paper, the batch rectification operation is adopted to keep distillate constituting constant high vacuum, in the operation process, the operation of variable reflux ratio is carries out. Through studying the relational system of reflux ratio, column top product composition, rectification time and theoretical plate number, when the concentration of citronellal extract column product is 99%, the appropriate reflux ratio is 0.8-15, and the graphic method is used to design13 pieces of theoretical plate number of citronellal extract column, providing powerful data support for the process design of citronellal extract column.

Polyvinylsilazane layer coating and its application in poly(dimethylsiloxane) microchip electrophoresis

In this paper, reactive polyvinylsilazane (PVSZ) is employed as silicate source for poly(dimethylsiloxane) (PDMS) channel surface modification. The PVSZ submicron layer is simply constructed by flushing 1wt.% PVSZ-anhydrous DMF solution into PDMS channel for 1min. After UV and thermal curing, the reactive coating is subjected to hydrolysis reaction in an alkali solution for 1h to convert the consolidated polymer surface to silicate phase. The surface is characterized by SEM, AFM and contact angle measurement. The PVSZ silicate channel is of optical transparence and solvent resistance, and provides a long-term stable electroosmotic flow (EOF) with 4.3±0.1×10−4cm2V−1s−1 at pH9.00, and a reliable capillary electrophoresis (CE) performance. Theoretical plate numbers of five FITC-labeled amino acids were all over 14,000 (2.8μm height), and EOF repeatability is much superior to that of PDMS channel.

Core shell stationary phases for a novel separation of triglycerides in plant oils by high performance liquid chromatography with electrospray-quadrupole-time of flight mass spectrometer

A new method for the analysis of triglycerides (TAGs) in vegetable oils was developed using a Poroshell 120 EC-C18 column (3.0mm×50mm, 2.7μm) with a high resolution ESI-Q-ToF tandem mass spectrometer as detection system. We used an Agilent Poroshell column, which is characterized by a recently developed stationary phase based on non-porous core particles. The results highlighted the advantages of this column in terms of the dramatic improvement in the number of theoretical plates and in low column backpressure. The developed method enabled us to analyze complex mixtures of more than 40 TAGs within less than 25min and with a low backpressure (lower than 100bar), and represents the first application of a core-shell stationary phase in reverse phase HPLC using an ESI-Q-ToF as detection system. The method was optimized on standards of TAGs, validated and applied to several plant oils. By a quantitative point of view, the method showed a very good linearity (r2>0.999) in the range 0.1–2.4μg/g; high intra- and inter-day precision both in terms of retention times (RSD%<0.04%) and peak areas (RSD%<0.3%). Limits of detection and quantitation were lower than 0.03μg/g and 0.10μg/g, respectively.

MICRO-SCALE METHOD FOR SEPARATION AND QUANTIFICATION OF ATENOLOL AND HYDROCHLOROTHIAZIDE BY SEQUENTIAL INJECTION CHROMATOGRAPHY

A microscale stopped-flow separation manifold, which is termed sequential injection chromatograph (SIC), was exploited for separating and quantifying atenolol and hydrochlorothiazide. A short C18 separation column (4.6 × 25 mm) with a monolithic structure was used to offer a rapid and sufficient separation. Microscale fiber optic spectrometric devices were coupled with the SIC manifold for UV detection at 220 nm. The proposed SIC method was optimized, validated, and applied to both formulations single and binary mixture. The mobile phase composition (MPC) was optimized using the 23 full-factorial design approach while other conditions were optimized using the univariate approach. The optimum MPC was 40 µmol/L phosphate:methanol:acetonitrile (85:7.5:7.5, v/v/v) at pH 3.0. Other optimum conditions were 40 µL/s for flow rate and 40 µL for sample volume. Satisfactory separation with a resolution of 1.6 and numbers of theoretical plates of more than 630 was achieved. The interesting features of the proposed SIC method are the microliter-scale of the consumed volume of mobile phase (3.50 mL) and high sample frequency (23 samples/hr).

Dress-up chiral columns for the enantioseparation of amino acids based on fluorous separation

In this paper, we report a new type of chiral high-performance liquid chromatography (HPLC) column--a so-called dress-up chiral column--featuring a chiral stationary phase adsorbed reversibly in a commercial fluorous HPLC column through fluorous interactions. We synthesized perfluroalkylated proline derivatives as chiral stationary phase compounds and then adsorbed them reversibly in the fluorous HPLC column through the pumping of their solutions. By using this dress-up chiral column and fluorophobic elution of an aqueous copper(II) sulfate/MeOH mixture, we could enantioseparate seven racemic amino acids within 40 min. When we washed the dress-up chiral column with fluorophilic tetrahydrofuran or MeOH, the adsorbed chiral stationary phase compounds desorbed from the column, completely destroying its enantioseparation ability. The relative standard deviation of the retention times, the number of theoretical plates, and the resolution for each of four preparations of the dress-up columns were all less than or equal to 9.53% in 20-times repeated analysis, and were all less than or equal to 18.7% in four different preparations, respectively.

High performance liquid chromatography of selected alkaloids in ion-exchange systems

A HPLC procedure on strong cation exchange column (SCX) has been developed for the analysis of selected alkaloids from different chemical groups. The retention, separation selectivity, symmetry of peaks and system efficiency were examined in different eluent systems containing different types or concentrations of buffers at various pH and the addition of organic modifiers: methanol (MeOH), acetonitrile (CH3CN), tetrahydrofuran (THF) or dioxane (Dx). The retention factors as the function of the concentration of buffers, the mobile phase pH and the percentage of modifier in the eluents were investigated. More symmetrical peaks and the highest theoretical plate number were obtained in eluents containing acetonitrile or tetrahydrofuran. In most cases, the increase of buffer concentration caused the decrease of alkaloids’ retention, the improvement of peaks’ symmetry and the increase of theoretical plate number. The improved peak symmetry and the efficiency of system for most investigated alkaloids were observed in the systems containing buffers at strongly acidic pH. The obtained results also reveal a large influence of salt cation used for buffer preparation. The results obtained on SCX column were compared with those obtained on a C18 column. The most efficient and selective systems were used for the separation of alkaloid standard mixture.

Ambient laser ablation sampling for capillary electrophoresis mass spectrometry

Ambient laser ablation with mass spectrometric detection is a powerful method for direct analysis of biological samples in their native environment. Capillary electrophoresis (CE) can separate complex mixtures of biological molecules prior to mass spectrometry (MS) analysis and an ambient sampling interface for CE/MS will allow the detection of minor components. An infrared (IR) laser ablated and transferred sample materials under ambient conditions for direct loading onto the CE separation column. Samples were deposited on a transparent target and ablated in transmission geometry using a pulsed mid-IR laser. The ablated materials were captured in the exposed sampling solvent and then loaded into a capillary by electrokinetic injection for separation and analysis by electrospray ionization (ESI)-MS. The system was tested using mixtures of peptide and protein standards. It is estimated that tens of fmol of material was transferred from the ablation target for injection into the CE system and the theoretical plate number was between 1000 and 3000. A novel interface for ambient sampling to CE/MS was developed. The interface is generally applicable and has potential utility for mass spectrometry imaging as well as the loading of microfluidic devices from untreated ambient samples.

Determination of Thiamine in Pharmaceutical Preparations by Reverse Phase Liquid Chromatography Without Use of Organic Solvent

A novel green aqueous mobile phase modified with room temperature ionic liquids (RTILs) was employed in the absence of volatile organic solvents or ion-pairing reagents to analyze thiamine, a very polar compound, by reverse phase high performance liquid chromatography (RP-HPLC). Due to its strongly hydrophilic nature, thiamine was eluted near the column dead time (t0) using a mobile phase without adding RTILs or ion-pairing reagents, even if a 100% aqueous mobile phase, which has weak elution power under reverse phase conditions, was used. Thus, 1-ethyl-3-methyl-imidazolium hexafluorophosphate ([EMIM][PF6]), which has the strongest chaotropic effect, was selected as a mobile phase additive to improve retention and avoid baseline disturbances at t0. Various mobile phase parameters such as cation moiety, chaotropic anion moiety, pH and concentration of RTILs were optimized to determine thiamine at the proper retention time. Method validation was performed to assess linearity, intra- and inter-day accuracy and precision, recovery and repeatability; all results were found to be satisfactory. The developed method was also compared to the current official United States Pharmacopoeia (USP) and Korean Pharmacopoeia (KP) methods using an organic mobile phase containing an ionpairing reagent by means of evaluating various chromatographic parameters such as the capacity factor, theoretical plate number, peak asymmetry and tailing factor. The results indicated that the proposed method exhibited better efficiency of thiamine analysis than the official methods, and it was successfully applied to quantify thiamine in pharmaceutical preparations.

Evaluating active flow technology HPLC columns as a platform for multiplexed detection

Abstract Active flow technology is a new high performance liquid chromatography column format that has been shown to improve separation performance in terms of the sensitivity, resolution and number of theoretical plates. In this study active flow technology has been shown as a platform for multiplexed detection, with sample analysis being undertaken from each of four separated flow streams located at the column outlet: One central port and three peripheral ports. The flow homogeneity across each of these ports was studied using theoretical plates and sensitivity as the performance indicators. The experimental findings showed that the theoretical plates were lower for solutes that eluted from the peripheral ports compared to the performance observed from a conventional column. However, the sensitivity in UV detection that was obtained from any of the three peripheral ports was approximately the same as that from a conventional column. The study therefore concluded that detection from the peripheral ports was feasible, dependent on the attributes of the detector itself.

Cation exchange/hydrophobic interaction monolithic chromatography of small molecules and proteins by nano liquid chromatography†

In this study, vinyl phenyl boronic acid modified lauryl methacrylate-based monolithic column was successfully prepared for cation exchange/hydrophobic interaction monolithic chromatography of small molecules and proteins in nano LC. The polymeric mixture consisted of lauryl methacrylate, vinyl phenyl boronic acid as cation exchanger, ethylene dimethacrylate as cross-linker, polyethylene glycol and methanol as binary porogenic solvent, and azobisisobutyronitrile as initiator. The resulting monolith showed good permeability and mechanical stability. Different ratios of monomer and porogens were used for optimizing the properties of the column. The monolithic column performance with respect to hydrophobic and cation exchange interactions was assessed by the separation a series of alkyl benzenes and anilines, respectively. cis-Diol-containing compounds such as phenols were also utilized to evaluate the retention behaviors of the vinyl phenyl boronic acid modified monolithic column. The monolithic column showed cation exchange interactions in the separation of aniline compounds. Theoretical plate number up to 52,000 plates/m was successfully achieved. The prepared monolith was further applied to the proteins with different acetonitrile content.

Analysis on fat-soluble components of sinapis semina from different habitats by GC–MS

A simple and rapid gas chromatography/mass spectrometry (GC/MS) analysis method was developed for the determination of fat-soluble parts of sinapis semina. Four major compounds were chosen as marker compounds to evaluate the method. Various extraction techniques were evaluated and the greatest efficiency was observed with sonication extraction using diethyl ether. The method was valuated as follows: acceptable apparatus suitability was obtained by testing the resolutions, tailing factors and theoretical plate number of the marker compounds; the precision and reproducibility, expressed as relative standard deviation (RSD), fell within the prescribed limits. Eight samples of sinapis semina collected from markets in Xi'an were monitored by using the method. The fingerprints of those samples were analyzed by hierarchical cluster analysis (HCA) similarity analysis. The result indicated that the combination of fingerprint and HCA could be used to analyze sinapis semina from different habitats.

Simulation of Extractive Distillation for Recycling Tetrahydrofuran from Pharmaceutical Wastewater with Chem CAD Software

The functions and application of ChemCAD simulation software was introduced. Mathematical model of extraction distillation process was established. The extractive distillation process for preparation of tetrahydrofuran (THF) was simulated by SCDS rectification model in ChemCAD software, methanol-THF-solution and ethanedol- lithium chloride solution as extractant. Influence of extraction agent on vapor-liquid equilibrium curve of methanol- THF system and effects of theoretical plate number, feed and extractant input positions, extraction agent ratio (m(chlorinated lithium): v(ethanediol)), extractant ratio (m (extractant): m(feed)) and reflux ratio on tower top mass fraction of THF were investigated. The results of simulation were compared with experimental data. Under optimum extraction distillation conditions: theoretical plate 30, extractant plate 6, feed plate 18, extraction agent ratio 0.24g/mL, extractant ratio 2.7 and reflux ratio 5.0, mass fraction of THF on tower top can reach 97.2%. The distribution characteristic parameters of the column were simulated. The results of simulation and experimental data agree satisfactorily.

Separation of acetone–tetrahydrofuran azeotropic mixture by continuous extractive distillation

AbstractBackgroundAcetone and tetrahydrofuran are commonly used as solvents in the chemical industry. The separation of acetone–tetrahydrofuran mixtures is often faced in the pharmaceutical and special chemical industries. As acetone and tetrahydrofuran can form a minimum azeotrope, they cannot be separated by conventional distillation. But acetone and tetrahydrofuran are important organic raw materials and solvents, so the mixture should be separated for reuse.ResultsThe process of continuous extractive distillation was used to separate the mixture of acetone (62% mass fraction) and tetrahydrofuran (38% mass fraction) using butyl ether as solvent. The characteristics of the continuous extractive distillation were simulated via ASPEN and experiments also showed the feasibility of separating the acetone–tetrahydrofuran mixture. Effects of the reflux mass ratio (R), mixture feed stage (FS), the solvent feed stage (SFS) on the extractive distillation column and the volume ratio of solvent to mixture (S/F) on the distillate mass fraction of acetone and bottom product mass fraction of acetone were investigated. The results of the extractive distillation simulation were verified by experiment data. With the following operation conditions for the extractive distillation column: number of theoretical plates 53; mixture feed at 24th plate; solvent feed at 7th plate, solvent to mixture ratio 3 and reflux mass ratio 3, the mass fraction of acetone in the distillate can reach 99%.ConclusionsThe process of continuous extractive distillation using butyl ether as solvent can separate the acetone–tetrahydrofuran mixture. The solvent to mixture ratio and reflux mass ratio are important factors that affect the mass fraction of the product. © 2013 Society of Chemical Industry