Microcolumn High-performance Liquid Chromatography Research Articles

STUDY OF THE COMPONENT COMPOSITION OF LEAVES OF SPECIES OF THE GENUS JUGLANS L. BY HPLC

Leaves of species of the genus Juglans L. of the Juglandaceae family are promising types of officinal medicinal plant materials, which preparations have an antimicrobial, restorative effect. In our opinion, the contribution to the antimicrobial activity, along with naphthoquinones, is also made by flavonoids contained in the leaves of various species of the genus Juglans L. Therefore, there is a need to determine the chemical composition of the leaves of species of the genus Juglans L. using modern methods of analysis. This article discusses the results of studying the component composition of the leaves of Juglans nigra L., Juglans regia L. and Juglans cinerea L. by microcolumn high performance liquid chromatography (HPLC) at an analytical wavelength of 360 nm. The conditions for chromatographic separation were established for the analysis of extracts from the leaves of species of the genus Juglans L. It was determined that in the leaves of Juglans nigra L. using HPLC it is possible to identify flavonoids - myricitrin, quercitrin, which are dominant and diagnostically significant for this type of raw material. In addition, the medicinal plant contains the aglycone of myricitrin – myricetin. A similar flavonoid profile was revealed in two types of medicinal plant raw materials – leaves of Juglans regia L. and Juglans cinerea L. Hyperoside, quercitrin, and juglanin were found in these species.

Tincture of <i>Juglans nigra</i> L. bark: peculiarities of standardization

BACKGROUND: Juglans nigra L. is a species of trees in the Juglandaceae family. This plant of the genus Juglans L. has not been sufficiently studied in comparison with other species, including Juglans regia L. It is a perspective medicinal plant raw material, its derivatives have an antimicrobial, general tonic effect. The known antimicrobial and general tonic activity of Juglans L preparations presented on the pharmaceutical market is probably due to substances of a flavonoid nature. These data indicate the relevance of studying the flavonoids of the Juglans nigra L. bark.
 AIM: The aim of this study is to develop methods for qualitative analysis and quantitative determination of biologically active compounds for tincture of Juglans nigra L. bark with the using of spectrophotometry, thin layer chromatography, microcolumn high performance liquid chromatography.
 MATERIALS AND METHODS: The object of the study was the tincture of Juglans nigra L. bark, samples of which were prepared in March-April 2020 on the territory of the Botanical Garden of Samara University, standard samples of myricitrin, myricetin. UV spectra were recorded with the use of Specord 40 spectrophotometer (Analytik Jena). Chromatographic analysis was carried out by thin-layer chromatography (qualitative analysis), reversed-phase high performance liquid chromatography on a Milikhrom-6 microcolumn liquid chromatograph (quantitative analysis).
 RESULTS: As a result of the chromatographic study, myricitrin were revealed, it appeared to be the dominant flavonoid substance. This compound has Rf value of about 0.4 in the solvent system of chloroform-ethanol-water (25:18:2). A method for the quantitative determination of the total flavonoids calculated on myricitrin in tincture of Juglans nigra L. bark was developed by means of differential spectrophotometry. It was determined that the content of the total flavonoids calculated on myricitrin in the tincture of Juglans nigra L. bark was 0.84 0.07%. A method for the quantitative determination of myricitrin in tincture of Juglans nigra L. bark by high performance liquid chromatography was developed. The content of the dominant flavonoid as myricitrin (myricetin-3-O--L-rhamnopyranoside) in the tincture of Juglans nigra L. bark was 0.42 0.06%.
 CONCLUSIONS: The methods of qualitative analysis and quantitative determination of flavonoids for tincture of Juglans nigra L. bark with the use of modern instrumental methods of analysis (spectrophotometry, thin layer chromatography, high performance liquid chromatography) has been developed. These results can be used to standardize the tincture of Juglans nigra L. bark.

DYNAMICS OF SECRETION OF PROSTAGLANDINS E2 AND D2 WITH DIBOTHRIOCHEPHALLUS DENDRITICUS (CESTODA) PLEROCERCOIDS IN RESPONSE TO EXPOSURE OF BLOOD SERUM OF BAIKAL CISCO, THE HOST

Parasites regulate host immune response via secretion of soluble mediators which interact in a certain way with cells and molecules of the immune system. The aim of the study was to determine changes of prostaglandins E2 и D2 content in Dibothriochephallus dendriticus plerocercoids and also in the incubation media during incubation of tapeworms with addition of blood serum from the intermediate host – Baikal cisco. D. dendriticus plerocercoids were retrieved from the host body cavity, ashed in the physiological solution and placed in incubation media. Microcolumn high-performance liquid chromatography was used to detect prostaglandins. Concentration of prostaglandins in the plerocercoid organism increased insignificantly after incubation in the Hanks’ solution with addition of blood serum, when compared with incubation in the Hanks’ solution only. Concentration of prostaglandin E2 increased significantly in the incubation media after incubation of plerocercoids in the Hanks’ solution with addition of blood serum. Prostaglandin D2 was also identified in high concentrations after 12 h and 24 h of incubation, whereas prostaglandin D2 was not quite detected during incubation in Hanks’ solution only.

Q Sepharose micro-column chromatography: A simple screening method for identifying beta thalassemia traits and hemoglobin E carriers

ObjectivesFor beta thalassemia control program in pregnancy, mass screening of the carrier state by determination of the hemoglobin (Hb) A2 and Hb E proportions and mutation analysis is a preferred method for making prenatal diagnoses. Q Sepharose micro-column chromatography, developed for the determination of Hb A2 and Hb E for screening purposes, was compared with high performance liquid chromatography (HPLC) to ascertain its relative accuracy and reliability. Design and methodsResults using Q Sepharose micro-column chromatography in 350 blood specimens, including 50 samples genetically proven to be beta thalassemia heterozygotes, were compared to HPLC for validation. An additional study was conducted to test a clinical application on a large-scale survey for beta thalassemia in 1581 pregnant women and their spouses. ResultsThe mean (±SD) Hb A2 proportions in the normal and genetically proven beta thalassemia heterozygotes were 2.70±0.40% and 6.30±1.23%, respectively, as determined by Q-Sepharose micro-column chromatography, and 2.65±0.31% and 5.37±0.96%, respectively, as determined by HPLC. The mean Hb E proportions in the Hb E heterozygotes were 23.25±4.13% and 24.72±3.5% as determined by Q Sepharose micro-column chromatography and HPLC, respectively. In the large-scale survey for beta thalassemia, 23 at risk couples were detected. Seven affected fetuses were identified by prenatal diagnosis. ConclusionsQ Sepharose micro-column chromatography was found to be reliable, reproducible and well-suited for large-scale surveys. Additionally, by being reusable and convenient, this simple and economical chromatography method may be an alternative means to screen for beta thalassemia and Hb E carriers in the mass population.

Optimization of the conditions for the detection of psychoactive compounds of the cholinoblocker and antidepressant groups with the use of the microcolumn HPLC technique

The objective of the present study was to evaluate the possibility of using the BD-2003 database to separate the mixtures of various antidepressants by high-performance liquid chromatography (HPLC) based on the MiLiChrome A-02 complex with an EcoNova UV detector. The parameters of identification are described. The possibility of modification of the standard conditions for the analysis is demonstrated as exemplified by the identification of certain cholinoblockers. The proposed approach to the separation of psychoactive preparations provides a basis for the development of methods for the determination of these substances in biological tissues and fluids.

Surface Ionization Detector for High-Performance Liquid Chromatography

Here is reported the combination of microcolumn high-performance liquid chromatography with an on-line surface ionization detector (HPLC-SID). This device was developed using commercial gas chromatography instrumentation and an interface to vaporize the chromatographic effluent. Addition of air at a flow rate of at least 50 mL/min to the effluent enhanced sensitivity and reduced baseline noise. Tributylamine was detected by the device with a minimum detectable amount of 3.0 × 10−12 g/sec at a signal-to-noise ratio of three in a mobile phase composed of acetonitrile/water at a flow rate of 6 µL/min. The signal was linear with concentration for at least 3 orders of magnitude. Preliminary results for effluents of aromatic hydrocarbons are presented as an example of the capabilities of the HPLC-SID system.

Comparison of Micellar Electrokinetic Chromatography and Microcolumn High Performance Liquid Chromatography for Resveratrol Analysis

Resveratrol, occurring in trans- and cis-isomeric forms, is a phytoalexin with pharmacological activities, for example, anti-cancer, anti-cardiovascular disease, and life-span increasing properties. Two analytical methods, namely, micellar electrokinetic chromatography (MEKC) and microcolumn high performance liquid chromatography (mu-HPLC), were developed and compared for resveratrol isomer separation. Both methods are suitable for resveratrol analysis owing to their performances of high speed, good reproducibility, and low consumption of solvents and samples.

Configuration, Column Construction, and Column Packing for a Capillary Liquid Chromatography System

INTRODUCTIONCapillary or microcolumn (<0.5-mm internal diameter, I.D.) high-performance liquid chromatography (HPLC) is an extremely powerful technique for separating small quantities of proteins and peptides. This protocol describes a procedure for adapting conventional HPLC systems to provide accurate low-flow rates (0.4-4 μl/min) and gradients required to operate slurry-packed capillary columns. A key component of this system is a commercial axial-beam longitudinal flow cell that can be fitted to several commercial UV detectors. Procedures are described for the fabrication of 0.32-mm I.D. polyimide-coated fused-silica columns, slurry-packed with reversed-phase chromatographic supports.

Ground, sieved, and C18 modified monolithic silica particles for packing material of microcolumn high-performance liquid chromatography

We here report a new type of stationary phase for microcolumns. C18 modified silica monolith particles were prepared by grinding and sieving the silica monolith followed by C18 modification and end-capping, and were used as packing material. Ground silica monolith particles were not spherical but irregular with some residual monolithic network structure. The separation efficiency of the stationary phase made of sieved monolith particles (5–10 μm) was better than that of the stationary phase made of unsieved particles. The microcolumn packed with the sieved C18 ground monolith particles (5–10 μm) showed quite good separation efficiency (height equivalent to theoretical plate, HETP, as low as 15 μm) and it was even superior to the microcolumn packed with a commercial spherical 5 μm C18 stationary phase. The column pressure drop of C18 monolith particles was about two-third of that of the commercial spherical C18 phase. The preparation method of C18 stationary phase with ground and sieved silica monolith particles presumably suggests advantages of simplicity and convenience in modification and washing procedures compared to bulk silica monolith. It also showed both improved separation efficiency and low back pressure.

Axial temperature gradient and mobile phase gradient in microcolumn high-performance liquid chromatography

The effect of axial temperature gradient (ATG) along a microcolumn on the separation performance at both isocratic and gradient elution mode was investigated. A thermostat system was designed to form an ATG along the packed column. Polycyclic aromatic hydrocarbons (PAHs) were separated on a 0.53 mm × 150 mm i.d. 5 μm C 18 microcolumn, with water and acetonitrile as mobile phase. The separation results obtained at mobile phase gradient (MPG) and ATG in microcolumn HPLC were compared with the results performed at ambient conditions. Extrapolated curves of peak width at half height ( w h ) versus ln k showed that w h is narrower at the same retention time when ATG was applied in addition to MPG. The column efficiency was enhanced 20–30% and the resolution was slightly reduced because of reduction of selectivity at elevated temperature at ATG condition. The RSD of retention time in ATG mode was less than 2.5%.

Packing Capillary Columns for RP-HPLC

INTRODUCTIONCapillary or microcolumn (<0.5-mm internal diameter [I.D.]) high-performance liquid chromatography (HPLC) is an extremely powerful technique for separating small quantities of proteins and peptides. This protocol describes a procedure for adapting conventional HPLC systems to provide accurate low-flow rates (0.4-4 μl/min) and gradients required to operate slurry-packed capillary columns. A key component of this system is a commercial axial-beam longitudinal flow cell that can be fitted to a number of commercial UV detectors. Procedures are described for the fabrication of 0.32-mm I.D. polyimide-coated fused-silica columns, slurry-packed with reversed-phase chromatographic supports.

New approaches to coupling TLC with TOF-SIMS

Summary A new hyphenated technique that enables coupling of thin-layer chromatography (TLC) with time-of-flight secondary-ion mass spectrometry (TOF-SIMS) has been used for identification of gibberellic acid, as model analyte. TOF-SIMS has outstanding capabilities for molecular surface analysis, trace metal determination on surfaces, and surface imaging. When TLC and TOF-SIMS are coupled on-line, the chromatographic thin layer must be modified to avoid TOF-SIMS background signal activity from the chromatographic material or solvents used. Two different types of TLC plate were used for coupling with TOF-SIMS. The first was an aluminum backed plate modified to form a universal porous TLC–TOF-SIMS coupling interface. The second was a commercially available plate with a monolithic silica layer. Low TOF-SIMS background signal intensity, surface homogeneity, and integral analyte transfer characterize this novel TLC–TOF-SIMS interface. TOF-SIMS images of the TLC surfaces with separated gibberellic acid were used for identification purposes. SIMS enables analyte detection with high mass resolution at a level of concentration not achieved by other methods. The system described can easily be coupled with dynamic microcolumn high-performance liquid chromatography (µHPLC).

Analysis of Vegetable Oils by Microcolumn High-Performance Liquid Chromatography

A procedure for determining triglycerides of vegetable oils by microcolumn reversed-phase HPLC with UV detection at 210 nm was developed. Acetonitrile and diethyl ether mixtures (10 : 4–6.5, by volume) were proposed as eluants. The procedure was applied to the identification of oils (linseed, corn, olive, sunflower, and pumpkin oils), to the detection of adulterated oils (using olive and sea-buckthorn oils as examples), and to the determination of the degree of the unsaturation of oils.

Study on Determination of Seven Transition Metal Ions in Water and Food by Microcolumn High-Performance Liquid Chromatography

A new method for the simultaneous determination of seven transition metal ions in water and food by microcolumn high-performance liquid chromatography has been developed. The lead, cadmium, mercury, nickel, cobalt, silver and tin ions were pre-column derivatized with tetra-(4-aminophenyl)-porphyrin (<TEX>$T_4$</TEX>-APP) to form the colored chelates which were then enriched by solid phase extraction with <TEX>$C_{18}$</TEX> cartridge. The enrichment factor of 50 was achieved by eluted the retained chelates from the cartridge with tetrahydrofuran (THF). The chelates were separated on a ZORBAX Stable Bound microcolumn (<TEX>$2.0{\times}50\;mm,\;1.8\;{\mu}m$</TEX>)with methanol-tetrahydrofuran (95 : 5, v/v, containing 0.05 mol/L pyrrolidine-acetic acid buffer salt, pH = 10.0) as mobile phase at a flow rate of 0.5 mL/min and detected with a photodiode array detector from 350-600 nm. The seven chelates were separated completely within 2.0 min. The detection limits of lead, cadmium, mercury, nickel, cobalt, silver and tin are 4 ng/L, 3 ng/L, 6 ng/L, 5 ng/L, 5 ng/L, 6 ng/L, 4 ng/L respectively in the original samples. This method was applied to the determination of the seven transition metal in water and food samples with good results.

Automation and optimization of multidimensional microcolumn size exclusion chromatography-liquid chromatography for the analysis of photocrosslinkers in Cyclotene 4000 series advanced electronic resins

Multidimensional chromatography using on-line, coupled, micro-column size exclusion chromatography and reversed-phase high performance liquid chromatography, has been successfully implemented for the quantitative determination of photoinitiator concentrations in photosensitive, interlayer dielectric resin formulations. The actual formulation consists of the prepolymer, aromatic solvent, antioxidant, and a variety of bis-azide photocrosslinkers. This paper describes the development of an automated system for Micro SEC-LC, as well as accuracy and precision data for the photoinitiator analysis. The technology is very effective for the analysis of small molecules (e.g. additives) in polymer products, and as such can have wide ranging applicability to other polymer and complex products. The use of microcolumns in the first dimension allows the introduction of the complete fraction containing the components of interest into a reversed-phase second dimension without deterioration of the separation when using a very strong mobile phase such as THF.

A high-efficiency cross-flow micronebulizer for inductively coupled plasma mass spectrometry.

A pneumatically driven, high-efficiency cross-flow micronebulizer (HECFMN) is introduced for inductively coupled plasma (ICP) spectrometries. The HECFMN uses a smaller nozzle orifice for nebulizer gas (75 microm in diameter) and a replaceable and adjustable fused-silica capillary for sample uptake. The HECFMN is optimally operated over a wide range of sample uptake rate (5-120 microL/min) at a rf power of 1100 W and nebulizer gas flow rates of 0.8-1.0 L/min when a 50 microm i.d. by 150 microm o.d. capillary is used. The aerosol quality is qualitatively examined in a simple manner, and the transport efficiencies are determined by direct filter collection. Compared with conventional cross-flow nebulizers (CFNs), the HECFMN produces much smaller and more uniform droplets and thus provides much higher analyte transport efficiencies (generally 24-95%) at the sample uptake rates of 5-100 microL/min. Several analytical performance indexes are acquired using an Ar ICPMS system. The sensitivities and detection limits measured with the HECFMN at 50 microL/min sample uptake rate are comparable to or improved over those obtained with a conventional CFN consuming 1 mL/min sample, and the precisions with the HECFMN (typically 1.1-1.7% RSDs) are slightly better than those with the CFN (1.6-2.3% RSDs). The ratios of refractory oxide ion-to-singly charged ion (CeO+/Ce+) are typically in the range from 0.7 to 3.3% for the sample uptake rates of 5-100 microL/min. The free aspiration rate of the HECFMN is 8.9 microL/min for distilled deionized water at the nebulizer gas flow rate of 1.0 L/min without any effect of pressure. The features of the HECFMN suggest good potential for HECFMN use in interfacing ICPMS with capillary electrophoresis and microcolumn high-performance liquid chromatography.

Simultaneous measurement of corticosterone and 11-dehydrocorticosterone in the adrenals and plasma in acute stress

Adrenal and plasma levels of corticosterone and 11-dehydrocorticosterone were measured in intact and stressed (laparotomy) rats by microcolumn high performance liquid chromatography. In contrast to humans, in rats plasma level of basic glucocorticoid is essentially higher than the level of its reversible metabolite. Acute stress (laparotomy) led to an increase in the adrenal concentration of 11-dehydrocorticosterone and plasma concentration of corticosterone, which was regarded as creation of a reserve corticosterone pool.

Microcolumn High-Performance Liquid Chromatography of ortho-Substituted Hydrophobic Phenol Derivatives

The influence of the structure of phenol derivatives with hydrophobic orthosubstituents on their retention in microcolumn normal-phase high-performance liquid chromatography was considered. The dependence of the retention parameters of ortho-substituted phenols on the total polarity of the mobile phase was analyzed. The revealed trends were taken into account in the optimization of the chromatographic separation of the above compounds.

Optical Properties of Binary Mobile Phases as Constituent Objective Functions in Optimizing the Conditions for High-Performance Liquid Chromatography of Hydrophobic Phenol-Like Compounds

The dependence of the absorbance and the UV absorption limits of typical binary mobile phases for high-performance liquid chromatography (HPLC) on the volume fraction of modifiers was analyzed. The applicability of the found relationships to the multiparametric optimization of the mobile-phase composition with a generalized objective function using the Microsoft Excel 2000 spreadsheet program was considered by the example of microcolumn HPLC of hydrophobic phenol-like additives.

Determination of UV-absorbing anions in environmental samples by microcolumn high-performance liquid chromatography

The Chromatographic retention and separation of NO 2 - , Br-, NO 3 - , and I- anions on a column packed with Nucleosil 100-5 C18 dynamically modified with trimethyloctadecylammonium bromide (TMODAB) was studied using direct UV detection. A procedure was developed for the determination of the above anions in the concentration range 0.06-2500 Μg/L without preconcentration. Sample preparation involved only the elimination of organic impurities (if present) from the test sample. The procedure was used for the determination of NO 2 - and NO 3 - anions in Baikal Lake water and in an aerosol extract.