Separon SGX Research Articles

Phosphorylated calix[4,8]arenes improve the RP HPLC separation of benzene derivatives

Aim. To study the effect of 5,11,17,23-tetrakis(diisopropoxyphosphonyl)-25,26,27,28-tetrapropoxycalix[4]arene and oсtаkis(diethoxyphosphoryloxy)-tert-butylcalix[8]аrene additives to the MeCN – H2O mobile phase (86:14) on the selectivity of the separation of aromatic compounds by the reversed-phase high performance liquid chromatography (RP HPLC) using a Separon SGX C18 support.Results and discussion. The process of complexation of phosphorylated calix[4,8]arenes with benzene derivatives in the mobile phase plays a key role in the RP HPLC separation of analytes. The stability constants of the inclusion complexes and the chromatographic separation coefficients of the analytes depend on the nature of the aromatic compounds and the cavity size of the calixarene macrocycle.Experimental part. The HPLC analysis was performed in acetonitrile – water (86:14) solution using a Separon SGX C18 column. The stability constants of the calixarene complexes were determined using the dependence of 1/k’ chromatographic parameters of benzene derivatives on the calixarene concentration in the mobile phase. Molecular modelling of the calixarene complexes was carried out using a Hyper Chem 8.0 program.Conclusions. The phosphorus-contained calixarenes due to their ability to form supramolecular complexes with aromatic molecules can be used as additives to the RP HPLC mobile phase and improve separation of benzene derivatives.Received: 14.05.2020Revised: 24.06.2020Accepted: 27.08.2020

QSRR models for potential local anaesthetic drugs using high performance liquid chromatography

Quantitative structure–retention relationships (QSRR) were proposed for Separon SGX C18 and Separon SGX Phenyl columns using physico-chemical molecular descriptors for the compounds, which are potential local anaesthetic drugs. Chemometrical methods were used for the QSRR studies of the HPLC retention factor k of 59 esters of alkoxyphenylcarbamic acid, which exhibit surface and/or infiltration anaesthetic activity. Four separation systems were used: phenyl column and acetonitrile/water mobile phase, phenyl column and methanol/water mobile phase, C18 column and acetonitrile/water mobile phase, and C18 column and methanol/water mobile phase. The values of log P and log S and 13C and 1H NMR chemical shifts were simulated and utilized in calculating the corresponding QSRR models and predicting the retention factors by artificial neural networks (ANN). In addition, principal component analysis and cluster analysis were used for a closer characterization of alkoxyphenylcarbamic acid esters. The proposed ANN models, based on optimally selected species descriptors, showed a high degree of correlation between k predicted and k measured. The intercepts and the slopes of the obtained dependences were close to the theoretically expected values of 0 and 1, respectively.

Influence of Calixarenes on Chromatographic Separation of Benzene or Uracil Derivatives

The addition of calix[4]arenes to MeCN/H2O or MeOH/MeCN/THF/H2O mobile phases improves LC separation of benzene or uracil derivatives on Separon SGX C18 or Separon SGX NH2 supports. Structure of the calixarenes and their host–guest supramolecular complexes with the analytes are discussed in context of the LC separation.

Determination of Quinic and Shikimic Acids in Products Derived from Bees and their Preparates by HPLC

An HPLC method was developed for determination of some organic acids in propolis, honey, and pastilles. The on‐line coupled Separon SGX C18 and Polymer IEX H‐form column with mobile phase composed of sulphuric acid 9 mmol/L and methanol (95:5) at a flow rate 0.8 mL/min and spectrophotometric detection at 215 nm were used for the determination of quinic acid and shikimic acid. Limit of detection of quinic acid was 10 µg/mL and shikimic acid 0.43 µg/mL. Limits of quantitation were 30 µg/mL for quinic acid and 1.26 µg/mL for shikimic acid. Shikimic acid concentrations from 4.2 to 309.0 µg/g and quinic acid concentrations from 0.2 to 6.2 mg/g were determined in all tested bees products. The differences in the acid concentrations were observed for propolis samples from East and West Slovakia.

Optimization of HPLC chromatographic conditions for determination of Transkarbam 12 and its degradation products

This paper deals with searching of HPLC chromatographic conditions for determination and separation of Transkarbam 12 (T 12) and its two main degradation products (ω-aminocaproic acid and dodecylalcohol). T 12 is a new substance which belongs to the group of accelerators of transdermal penetration. Chromatographic separation was achieved using Separon SGX C18 analytical column (150 mm × 3 mm i.d.; 5 μm). Mobile phase contained acetonitrile and sodium acetate buffer pH 4.5 at the flow rate of 1 ml/min. Separation was carried out under the conditions of gradient elution. After the modification of the structure by derivatization reagent (3,5-dinitrobenzoyl chloride) detection at wavelength 230 nm was realized. The aim of this study was not only the optimization of the separation of derivatization reagent and derivatized T 12, Ak and D but also optimal derivatization processes for all three substances.

Solid Phase Extraction of Phenol and Chlorophenols on Octadecylsilica and Amberlite XAD 2 Sorbents in the Presence of Cationic Surfactant

Solid phase extraction of mixtures of phenol 3 monochlorophenols, 4 dichlorophenols, 3 methyl-4-chlorophenol, 4 trichlorophenols, 2 tetrachlorophenols and pentachlorophenol was studied on octadecylsilica (Separon SGX C18) and Amberlite XAD 2 in the presence of hexadecyltrimethylammonium chloride (CTMA) in alkaline medium, and their efficiency for the preconcentration of phenols was compared. The HPLC on narrow bore octadecylsilica column was successfully used for the determination of phenols in methanol eluate, when gradient elution was applied with (36:65) methanol/phosphate buffer of pH 3.5 and methanol mobile phases, and a diode array detector was used for their evaluation.

Fluorimetric determination of the levels of urinary neopterin and serum thiobarbituric acid reactive substances in the nonagenarians

Twelve self-sustaining nonagenarians, 10 women and two men, aged 94±3 years, and eight institutionalised nonagenarians, eight women, aged 91±1 year as well as 11 control subjects, seven women and four men, aged 84±5 years entered the study. Urinary neopterin, an indicator of systemic immune activation, and serum thiobarbituric acid reactive substances (TBARS), a marker of lipoperoxidation, were determined initially, and collection of the blood and urine samples was repeated at 3-month interval. Neopterin was measured in the urine specimens by reversed-phase high performance liquid chromatography. A C 18 reversed-phase column 3.3×150 mm, 5 μm-diameter packing Separon SGX was used. Potassium phosphate buffer (15 mmol l −1, pH 6.4) at flow rate of 0.8 ml min −1 was used as mobile phase. After centrifugation (5 min, 1300× g) and diluting 100 μl of urine specimens with 1.0 ml of mobile phase containing 2 g of disodium–EDTA per litre, a 20 μl sample was injected on a column. Neopterin was identified by its native fluorescence (353 nm excitation, 438 nm emission). Creatinine was determined by Jaffé kinetic reaction after dilution of sample 1:50 (v/v). The concentration of neopterin in urine was expressed as neopterin/creatinine ratio (μmol mol −1 creatinine). TBARS were determined spectrofluorometrically using LS-5 spectrofluorimeter (excitation wavelength 528 nm, emission wavelength 558 nm) after extraction with n-butanol treatment with thiobarbituric acid. The significance of differences between nonagenarians and control group was examined by ANOVA–Kruskal–Wallis tests, using statistical software NCSS 6.0.21 (Kaysville, UT, 1996). The decision on significance was based on P=0.05. Urinary neopterin was significantly higher in institutionalised compared to self-sustaining subjects and controls (625±565 vs. 203±63 μmol mol −1 creatinine, and 198±128 μmol mol −1 creatinine, respectively, P=0.006). The serum TBARS were higher in both groups of nonagenarians (3.23±1.16 μmol l −1 and 2.69±0.39 vs. 2.12±0.83 μmol l −1 for the self-sustaining, institutionalised and controls, respectively, P=0.023). We conclude that the fluorimetric determinations of urinary neopterin and serum TBARS can be useful for the monitoring health status in the elderly patients.

Photochemical stability of nimesulide

HPLC and TLC methods for monitoring of the photochemical stability of nimesulide are presented. Solution of nimesulide sodium salt was exposed to the light of wavelengths 254 nm. The presence of degradation products (2-phenoxy4-nitroaniline and methanesulfonic acid) was observed. In the exposed sample, 2-phenoxy4-nitroaniline was detected by HPLC analysis and sulfonic acid was detected by TLC analysis. An isocratic HPLC chromatographic condition was described for determination of nimesulide in a presence of its degradation product. The sample was analysed on Separon SGX, C 18, 250×4.6 i.d. 7 μm analytical column. The mobile phase was consisted of a mixture of acetonitrile and ammonium phosphate (pH 7.9; 0.02 M) (35:65 v/v). UV detector was performed at 245 nm. Propylparaben was employed as an internal standard. Standard area response was linear respect to concentration of nimesulide over range 150–500 μg/ml. As a validation of the method, the accuracy and between-day precision were done. The detection limit of 2-phenoxy4-nitroaniline was 0.12 μg/ml. The solvent system for TLC analysis was consisted of ethylacetate and cyclohexane (45:55), the samples were plotted on silica gel UV-254 nm. UV lamp (254 nm) and the chemical detection were used.

Symmetrical and inherently chiral water-soluble calix[4]arenes bearing dihydroxyphosphoryl groups

A series of symmetrical and inherently chiral water-soluble calix[4]arenes bearing one, two or four proton-ionisable dihydroxyphosphoryl groups at the narrow rim of the macrocycle has been synthesised by consecutive treatment of appropriate diethoxyphosphoryl derivatives with bromotrimethylsilane and methanol. The calix[4]arene phosphoric acids synthesised possess pKa-values 2.85–3.10 (CH3OH–H2O 70 : 30) and form salts with L-(−)-α-phenylethylamine or (1S,2R)-(+)-ephedrine in methanol solution. The salts of the inherently chiral calixarene phosphoric acids with chiral amines are easily separated into diastereomeric forms by the RP HPLC method on Separon SGX C18 or Partisil 5 ODS 3 achiral columns.

High-performance liquid chromatographic analysis and separation of N-feruloylserotonin isomers

The N-feruloylserotonin containing fraction was isolated from seeds of Leuzea carthamoides (Willd.) DC by solvent extraction followed by column chromatography on silica gel or on Sephadex LH-20. Nuclear magnetic resonance spectroscopic analysis of the isolated fraction showed the presence of four structurally related compounds. These compounds were identified as four isomers of N-feruloylserotonin: N-( Z)-feruloylserotonin, N-( Z)-isoferuloylserotonin, N-( E)-feruloylserotonin and N-( E)-isoferuloylserotonin. They were analyzed by HPLC on Separon SGX C 18, Separon SGX and Separon SGX phenyl, using various mobile phases. Separon SGX phenyl phase was found the most efficient for a rapid analysis and for the final separation of the N-feruloylserotonin isomers.

Determination of the Stability Constants of Inclusion Complexes of p-H-37-(2-carboxy-methyloxy)-calix-[6]-arene and p-sulphonato-37-(2-carboxy-methyloxy)-calix-[6]-arene with 15 Amino acids by RP-HPLC

Reversed-phase high performance liquid chromatography (Separon SGX CN, UV detection at 254~nm and water as mobile phase) was applied to thestudy of the host–guest complexation ofp-H-37-(2-carboxy-methyloxy)-calix-[6]-arene (1) and p-sulphonato-37-(2-carboxy-methyloxy)-calix-[6]-arene (2)with 15 amino acids in the mobile phase. It was established that the formation ofthe inclusion complexes results in changes in the retention times and capacity factorsof amino acids. Stability constants of the complexes were determined. The variations instability constant values may be explained in terms of the different interactions, whichmay occur between amino acids and 1 and 2.

Determination of Triandrin and Salicin in Salix viminalis L. by Reversed-Phase High-Performance Liquid Chromatography

The optimal separation of phenolic components of the bark of basket willow Salix viminalis L. was attained using reversed-phase high-performance liquid chromatography with isocratic elution. Triandrin (1-O-β-D-glucopyranoside of p-coumaryl alcohol) and salicin (1-O-β-D-glucopyranoside of salicyl alcohol) were identified. Regularities in the retention of triandrin and salicin in the Separon SGX C18–binary mobile phase (acetonitrile–water) system were considered. A procedure was developed for the quantitative determination of triandrin in raw basket-willow bark and its extract.

Analysis of Flavonoids in Silybum marianum Fruit by HPLC

Flavonoids of spotted milkweed (Silybum marianumL. Gaerth.) were optimally separated using reverse-phase HPLC with isocratic elution by CH 3 CN-H 2 O over Separon SGX C 18 . The flavolignans silybin, silydianin, silychristin, and the flavonoid taxifolin were identified in the chromatograms of extracts from this plant

A comparative study of the determination of the stability constants of inclusion complexes of p-sulfonatocalix[4]arene with amino acids by RP-HPLC and 1H NMR

Reversed-phase high-performance liquid chromatography (Separon SGX C 18, UV detection at 254 nm and acetonitrile–water–trifluoroacetic acid (70∶30∶0.5, v/v), and methanol–water–trifluoroacetic acid (3∶97∶0.5, v/v) as mobile phases) was applied to the study of the host–guest complexation of p-sulfonatocalix[4]arene (SC[4]A) with amino acids as guests in the mobile phase. It was established that the formation of the inclusion complexes results in changes in the retention times of the amino acids. Stability constants of the complexes were determined. The variations in stability constants may be explained in terms of the various interactions (ion-pairing, hydrophobic, aromatic–aromatic and electrostatic interactions) that may occur between a given amino acid and SC[4]A. For the amino acids aspartic acid and proline the association constants were also derived from 1H NMR experiments.

COMBINATION OF SOLID PHASE EXTRACTION WITH SUPERCRITICAL FLUID EXTRACTION FOR THE DETERMINATION OF CONTAMINANTS IN WATER

The applicability of supercritical fluid extraction was studied for the determination of mineral oil contaminants in the presence of non-ionic surfactants in water after a prior preconcentration on a solid sorbent. The species representative of the two types of contaminants were retained first on a solid sorbent Separon SGX C18. Under optimized conditions supercritical fluid extraction (SFE) with carbon dioxide was used in the first step to extract selectively mineral oil, to be determined by FTIR in a carbon tetrachloride solution. In the second step, SFE with carbon dioxide modified with 5 % (v/v) of methanol yielded a selective and quantitative separation of a non-ionic surfactant into methanol with UV spectrometric finish. A new design of column for retention of species from SFE with the use of a solid sorbent was also developed and tested.

Electrorheological behaviour of suspensions of various surface-modified porous silica particles

Electrorheological (ER) properties of suspensions in silicone oil of three silane-modified porous silicas Separon SGX and porous non-modified Separon SGX and compact cristobalite particles Sibelco (cristobalite flour M 3000) were studied. The experiments showed that the formation of chains of polarized particles in the electric field, which increases viscosity of the system, is strongly affected by the hydrophilic character of the particle modifier. Thus, while in suspensions of non-treated silica particles or particles treated with hydrophilic 3-aminopropyl(triethoxy)silane or 2-cyanoethyl(triethoxy)silane a strong enhancement of the apparent viscosity at low shear rates upon application of an electric field set in, in suspension of the particles modified with hydrophobic octadecyl(trichloro)silane, the ER effect was completely suppressed. According to the bulk polarization theory, polarizability of suspension particles in the electric field is controlled by particle dipole coefficient related to particle permittivity. This seems to be true for silica particles, where the dependence of viscosity of ER structure on particle dipole coefficient continuously increases. The change in the ER behaviour of silica and cristobalite on drying considerably differed. If the samples were heated at 100°C, the ER effect depended on a residual amount of water firmly adhering to the particles. An additional heating at 300°C, causing a complete removal of physically adsorbed water, leads to a dramatic decrease in ER viscosity of suspension of compact cristobalite particles while for silica, only a much smaller decrease in this quantity appeared, probably due to higher polarizable silanol groups present in the perfectly dry porous material.

Microcolumn high-performance liquid chromatographic assay for doxycycline in isolated alveolar macrophages

A procedure for the isolation of doxycycline from the alveolar macrophages is described. Due to the minimal amount of the sample, it was necessary to employ microcolumn HPLC. A great advantage of the methodology developed in the present paper consists in the direct injection of an aqueous supernatant over disintegrated macrophages without the use of liquid–liquid or solid–liquid extraction. Another advantage of the method is the fact that the injection of an aqueous solution on a microcolumn with a lipophilic stationary phase makes the analyzed substances concentrate, which increases the sensitivity of assay. Separation was performed on a Separon SGX C 18 microcolumn, 150×1 mm, detection at 254 nm by UV, 1 μl flow cell. Rabbits were treated intravenously with Vibramycin inj. sic. in a dose of 13 mg/kg body weight doxycycline. A procedure for washing macrophages from rabbit lungs was developed. Dry matter of macrophages ranged from 5.9 to 11.4 mg. In the samples of three rabbits the levels of doxycycline ranged from 110 to 270 ng per 1 mg of macrophages dry matter.

Determination of propafenone and its main metabolite 5-hydroxypropafenone in human serum with direct injection into a column-switching chromatographic system

Column-switching chromatographic systems using conventional reversed-phase Separon SGX C 18 and restricted access media LiChrospher ADS RP-18 precolumns were applied for the determination of propafenone and its main metabolite 5-hydroxypropafenone in human serum samples. The LiChrospher ADS RP-18 precolumn has been found to be more suitable for the sample clean-up. Serum samples were directly injected into the chromatographic system. Proteins and other endogenous compounds were removed by washing with 10% 2-propanol in water and the analytes separated on the Gromsil ODS AB analytical column. The chromatograms were detected at 246 nm. The method validation confirms the suitability of the column-switching system for the quantitation of propafenone and its metabolite. The presented assay shows good linearity with high correlation coefficients (0.992–0.999), high recoveries (96.6±6.1–103.5±5.8) and excellent values of the repeatabilities (1.23–4.5%). The limits of quantitation are 25–40 ng/ml for the injection volume of 50 μl. The complete analysis including the precolumn reconditioning and the sample clean-up requires 26 min, the sample throughput is approximately four samples in an hour.

Study of the Complexation of Octakis(diethoxyphosphoryloxy)-tetramethylcalix[4]resorcinarene with Benzene Derivatives by the RP HPLC Method

Stability constants of the host–guest complexes of octakis(diethoxyphosphoryloxy) tetramethylcalix[4]resorcinarene (RA) with different benzene derivatives (alkyl benzenes, halogenated benzenes, substituted aldehydes, phenols and benzoic acids) were determined by the reversed phase high-performance liquid chromatography (RP HPLC) method (Separon SGX C 18, UV detector at 254 nm and acetonitrile-water, 86 : 14, v/v, as mobile phase) from the relationship between the aromatic guest capacity factors and the RA host concentration in the mobile phase. The constants are within the range 17–596 M−1, dependent on the size, nature, position and quantity of the substituents in the benzene ring of the guest molecules.

Effect of Octakis(diethoxyphosphoryloxy)-tert-butyl-calix[8]arene in Mobile Phase on the Reversed-Phase Retention Behavior of Aromatic Compounds: Host-Guest Complex Formation and Stability Constants Determination

Under reversed-phase high-performance liquid chromatographic conditions (Separon SGX C 18 stationary phase, ultraviolet detection at 254 nm, and acetonitrile—water [86:14, v/v] as the mobile phase), addition of octakis(diethoxyphosphoryloxy)-tert-butylcalix[8]arene in concentrations of 1×10 -4 to 4 ×10 4M to the mobile phase leads to decreasing sorption of aromatic solutes on the sorbent's surface due to the formation of host-guest inclusion complexes between the calixarenes and the aromatic molecules. Stability constants of the complexes (608-2795M-1) are determined from the relationship between the solute capacity factors and the calixarene concentration in the mobile phase.