High Number Of Theoretical Plates Research Articles

Analysis of Salicylic Acid in Tobacco Leaves Using Capillary Zone Electrophoresis with UV Detection

AbstractA simple and inexpensive capillary zone electrophoresis (CZE) with UV detection method was used to determine salicylic acid (SA) and six structural analogs (benzoic acid, 3-hydroxybenzoic acid, 4-hydroxybenzoic acid, acetylsalicylic acid, methyl salicylate, and jasmonic acid). All of the compounds were successfully separated within a migration period of 6 min, with a high number of theoretical plates (>37,000, calculated using the width at the base of the SA peak), in 20.0 mmol L-1 Na2HPO4/H3BO3 buffer (pH 9.0) containing 2.0% methanol and 2.0% acetonitrile. The regression line for SA was linear over the range 0.5–200 µmol L-1. Both the intra- and inter-day precisions of the migration time (relative standard deviations, RSDs, 0.6% and 4.7%, respectively) and peak area (RSDs 0.9% and 7.3%, respectively) were acceptable. The proposed method was used to determine the SA concentrations in tobacco leaves ( Nicotiana tabacum L.) from the Xanthi-nc ( NN genotype) and the Nt-NahG mutant strains that had been irradiated with UV (254 nm) for 20 min and the ones that had not been irradiated. Rapid separation (<6 min), good reproducibilities (RSDs < 11.9%), and good recoveries (94.7–118%) were obtained in the tobacco leaf analyses.

New Trend in the LC Separation Analysis of Pharmaceuticals – High Performance Separation by Ultra High-performance Liquid Chromatography (UHPLC) with Core-shell Particle C18 Columns –

This article presents a mini-review of the recent results in the ultra high-performance liquid chromatography (UHPLC) separation of pharmaceuticals by our group. High performance UHPLC separation employing core-shell particle C18 columns was demonstrated. High performance (high theoretical plate number of approximately 20000/10 cm, low theoretical plate height of 5 μm) was obtained without any specific devices in the conventional HPLC apparatus, only through changing detector sampling times and the inner diameter of the connecting tube. High theoretical plate numbers with low column back pressure obtained by the core-shell particle columns enabled fast separation of the analytes. Methanol, which gives high column pressure drops in the reversed-phase mode HPLC compared with acetonitrile, can be used without any trouble. One analysis of the purity testing of diltiazem hydrochloride was performed within 100 s. One analysis in the photostability testing of mecobalamin (vitamin B12 analogue) was successful within 180 s.

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.

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.

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.

HPLC Separation of Amino Acids is Appropriate

From the Figure, several sorts of amino acids are not base-line separation and require at least Rs (resolution) more than 1.5 for baseline separation. Amino acid chromatographies done by Shimazdu, Hitachi or Nihon Bunko were almost identical. They require baseline separation; otherwise reproducible determination cannot be attained. For that purpose of base-line separation, gradient elution condition must be innovated. Most innovated point is the use of column with higher theoretical plate number. So, I suppose why they do not use capillary-column with higher theoretical plate number. The above presented chromatogram is representative chromatogram, so analytical validation is further required and gradient condition and elution condition must be innovated to attain base-line separation chromatogram.

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.

High resolution separation by pressure-driven liquid chromatography in meander extended nanochannels

The rapidly developing interest in nanofluidics, which is used to examine liquids on an order that ranges from an attoliter to a femtoliter, correlates with the recent interest in decreased sample amounts, such as in the field of single-cell analysis. In this paper, we have succeeded in the normal phase separation of a 101 fL sample with a high number of theoretical plates (103 plates), and a fast separation (4s), using a pressure-driven flow in extended nanochannels. A meander separation channel 1μm wide was introduced and showed no band broadening at the turns, unlike microchannels (101–102μm wide), which is an important aspect in order to lengthen the channel in a space-limited micro/nanofluidic chip. Our device enables the fast separation of an ultra-small volume of sample with a high number of theoretical plates, and will serve as a general platform for the separation of aL to fL volumes of samples.

Fe3+‐immobilized nanoparticle‐modified capillary for capillary electrophoretic separation of phosphoproteins and non‐phosphoproteins

A fused-silica capillary modified with Fe³⁺-immobilized magnetic nanoparticles (Fe³⁺-IMAN) has been investigated for the capillary electrophoretic (CE) separation of phosphoproteins and non-phosphoproteins. The Fe³⁺-IMAN capillary was achieved by covalently immobilising epoxy-based magnetic silica nanoparticles (160 nm) on the prederivatized 3-aminopropyl-trimethoxysilane (APTMS) fused-silica capillary (75 μm id), followed by disodium iminodiacetate and Fe³⁺. The buildup process was examined by measuring the streaming potentials of the bare capillary, APTMS capillary, epoxy-based nanoparticle capillary and Fe³⁺-IMAN capillary by varying the buffer pH. An inverted fluorescence microscope was used to determine the surface features of the Fe³⁺-IMAN capillary derivatized with morin. Further experimental results confirmed that Fe³⁺-IMAN bonded on the inner wall of the APTMS capillary could provide sufficient solute-bonded phase interactions to allow for the CE separation of phosphoproteins and non-phosphoproteins at concentration levels down to 50 μg/mL. The highest number of theoretical plates obtained was about 233,000/m, and the relative standard deviation (RSD) for migration times was <2.57% for eight consecutive runs, respectively. Additionally, the Fe³⁺-IMAN modifing method was also applied to the analyses of bovine milk proteins. With simplicity, high resolving power, and high repeatability, the proposed method has shown great potential for phosphoproteomics applications.

Cyano bonded silica monolith—Development of an in situ modification method for analytical scale columns

This study investigates the synthesis and chromatographic behaviour of an analytical size cyanopropyl “cyano” bonded silica monolith. Surface modification was undertaken by treating a neat silica monolith with chloro(3-cyanopropyl)dimethyl silane in dry heptane over a two day period. The resulting monolith showed stability over the duration of the testing program that involved flushing the column with more than 2000 column volumes of mobile phase. Efficiency measurements before and after sylation verified that the integrity of the silica monolith itself was not affected by the modification process, the highest number of theoretical plates ( N/m) using anisole was 81,650. A brief selectivity test was then undertaken to assess methylene selectivity and phenyl selectivity. Elemental analysis was used to determine the homogeneity of the carbon load throughout the monolithic bed, and was compared to two commercial C18 and one ‘self’ modified C18 silica monoliths. The development of the in situ modification is also discussed.

Fast preparation of polystyrene-based monolith using microwave irradiation for micro-column separation

The conventional analytical HPLC was successfully developed for micro-column separation by using a simple eluate splitting system, a self-preparation of monolithic column and an on-capillary column detector in our laboratory. A typical polystyrene-based monolith was quickly prepared inside the fused-silica capillary, which in situ polymerization was carried out in 10 min by microwave irradiation. The reactant solution consisted of styrene (ST) as a functional monomer, divinylbenzene (DVB) as a cross-linking agent, toluene and isooctane as porogenic solvents, and azobisisobutyronitrile (AIBN) as an initiator. The monolith was proved to form in the center of the capillary and adhered to the column inner wall by the scanning electron micrograph. Its chromatographic behaviors were evaluated in detail by varying the flow rate and percentage of mobile phases, and under the optimal condition, baseline separation of the model analytes including thiourea, benzene, toluene, ethylbenzene was obtained with a highest theoretical plate number near 11,290 N/m by the developed capillary HPLC. Furthermore, the stability and porosity of the prepared monolith were systematically investigated by a simple flow method.

Open-tubular gas chromatography using capillary coated with octadecylamine-capped gold nanoparticles

The octadecylamine-capped gold nanoparticles (ODA-Au-NPs) were prepared and directly used to coat the capillary wall. The hydrophobic coating acted as the stationary phase for open-tubular gas chromatography (OTGC). The ODA-Au-NPs can be adsorbed tightly onto the inner surface of fused silica capillary column via electrostatic interaction and enhanced interaction of van der Waals between gold nanoparticles and the capillary wall. Thus, the modification of the inner surface of capillary column by ODA-Au-NPs can be achieved simply by flushing the capillary with a solution of ODA-Au-NPs and the resulted ODA-Au-NPs coating is very stable. No perceptible degradation in the ODA-Au-NPs-based separation was observed after ∼1900 sample runs. This type of columns also provided excellent chromatographic performances: high number of theoretical plates, outstanding run-to-run and column-to-column reproducibility, and high selectivity for a wide range of test mixtures. An efficiency of 2474 theoretical plates per meter for chlorobenzene was obtained on an ODA-Au-NPs-modified 1.6 m × 100 μm i.d. fused silica capillary column.

Polystyrene immobilized ionenes as novel stationary phase for ion chromatography

Several aliphatic ionenes (2-6-, 6-6-, 10-6-ionene) have been prepared as ion exchangers for the development of novel high-performance stationary phases for anion chromatography (IC). A macroporous polystyrene/divinylbenzene (PS/DVB) resin with adjusted cation exchange capacity was used as support. Therefore the immobilization of ionenes to polystyrene carriers with remaining positive surface charge became possible for the first time. Strong ion-exchange interactions, resulting in high retention times, between the stationary phase and inorganic as well as organic anionic analytes have been observed. The influence of different ionenes on the retention behaviour during the ion chromatographic separation was investigated. Additionally, partly aromatic and polar ionene backbones were prepared and their retention behaviour as anion exchanger was investigated. The highest number of theoretical plates obtained was about 90.000 per meter. The signal asymmetries were generally lower than obtained for surface functionalized anion exchangers.

Joule heating effects on separation efficiency in capillary zone electrophoresis with an initial voltage ramp

An analytical model is developed to quantify the Joule heating effects on the separation efficiency in CZE with an initial voltage ramp. This model considers the temporal variations of nonuniform temperature and flow fields in the course of voltage ramping. The temperature dependence of electrical conductivity, dynamic viscosity, and mass density of the fluid is also taken into account. We demonstrate that the application of an initial voltage ramp delays the development of pressure-driven flows induced passively by the axial temperature gradients. The thermal dispersion is thus significantly reduced, resulting in a higher theoretical plate number in CZE. Such improvement in the separation efficiency is apparent in noncoated capillaries at high electric fields with an appropriate voltage ramp-up time. These predictions are consistent with previous observations in both aqueous and nonaqueous CZE that took place in uncoated capillaries. In coated capillaries where the EOF is suppressed, however, our model predicts a lower plate number in the presence of an initial voltage ramp.

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.

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.

Peralkylated-β-cyclodextrin used as gas chromatographic stationary phase prepared by sol–gel technology for capillary column

For the first time, three peralkylated-β-cyclodextrins (β-CD), permethylated-β-CD, perethylated-β-CD and perpentylated-β-CD, were coated onto the fused-silica capillary by sol–gel method with simplicity and rapidity. Multiple steps in conventional column preparation technology were avoided. Also, these new columns demonstrated many inherent advantages, the main being the outstanding thermal stability (up to 300°C), high number of theoretical plates, excellent column-to-column and run-to-run reproducibility, and pronounced selectivity for positional isomers and enantiomers. Using n-tridecane as a test reagent (k=4.18), an efficiency value of 3520 theoretical plates/m was obtained on a sol–gel perpentylated-β-CD capillary column (15m×0.25mm I.D.). On the basis of the results, we proposed that the peralkylated-β-CD, which has no terminal hydroxyl group, is encapsulated in the sol–gel network and the whole matrix is chemically bonded to the surface of the fused-silica tubing.

Separation of stilbene isomers from red wine by overpressured-layer chromatography

A simple method is described for separation and determination of the isomers of trans-resveratrol and related compounds from different types of red wine by overpressured layer chromatography (OPLC). Comparison of OPLC with TLC clearly showed the advantages of the forced-flow technique (higher theoretical plate number, good resolution, etc.) over conventional planar layer liquid chromatography. It was established that the glycosides of resveratrol isomers were always present in higher concentrations than free stilbene isomers in red wine samples. This was especially true for the Pinot Noir wine. Exploitation of the advantages of OPLC provides further possibilities of analysis and isolation of stilbene isomers from grapes and wine.