Mobile Phase Of Acetonitrile-water Research Articles

Large-scale preparative isolation of bergenin standard substance from Saxifraga atrata using polyamide coupled with MCI GEL® CHP20P as stationary phases in medium pressure chromatography

In this study, polyamide and MCI GEL® CHP20P were employed as stationary phases in medium pressure chromatography (MPC) for the efficient preparative separation of bergenin from Saxifraga atrata. Ethanol–water, methanol-water, and acetonitrile-water mobile phases all showed good enrichment capacity for bergenin fraction when polyamide was used as a stationary phase. After 5 cycles of polyamide MPC using acetonitrile/water, 1.2 g of bergenin fraction was isolated from 180 g Saxifraga atrata herb. Further purification of this fraction was conducted using MCI GEL® CHP20P styrene-divinylbenzene beads. The bergenin fraction was separated into two fractions, and after three runs of MPC, 714.2 mg of bergenin with purity above 99% was obtained. The results demonstrate that the combination of polyamide and styrene-divinylbenzene MPC can be utilized for preparative isolation of compounds from natural products with high yield and purity.

Insights from molecular simulations about dead time markers in reversed-phase liquid chromatography

Among the most popular compounds to estimate the hold-up time in reversed-phase liquid chromatography (RPLC) are acetone and uracil, which are considered as too small and too polar, respectively, for retention by the hydrophobic stationary phase, although their observed elution behavior does not fully support this assumption. We investigate how acetone and uracil as solutes interact with the chromatographic interface through molecular dynamics simulations in an RPLC mesopore model of a silica-supported, endcapped, C18 phase equilibrated with a water (W)‒acetonitrile (ACN) mobile phase. The simulation results provide a molecular-level explanation for the observed elution behavior of acetone and uracil, but also question whether true dead time markers for RPLC exist. Both solutes have a density maximum in the interfacial region in addition to a low presence in the bonded-phase region, but these density peaks clearly differ from the adsorption and partitioning peaks of true analytes. Acetone partially behaves like a co-solvent of ACN and partially like the analyte acetophenone. Like ACN, acetone can be found in the first and second layer of solvent molecules at the silica surface; like acetophenone, acetone adsorbs to the bonded-phase chains by orienting its polar group to the bulk region to sustain hydrogen bonds with W molecules. Uracil behavior is governed by a need for extensive hydrogen-bond coordination by W molecules. Uracil adsorbs to the very edge of the bonded-phase chains, on the bulk-region side of the ACN density maximum in the interfacial region. Further penetration into the chains is prevented by the absence of W molecules, which are not found deeper in the bonded phase, except at the silica surface. Contrary to true analytes, accumulation of uracil and acetone in the interfacial region ceases at an equimolar presence of W and ACN in the mobile phase (at 70‒80% ACN volume fraction). Uracil achieves a closer approximation of the stationary-phase limit than acetone, but carries the risk of HILIC retention at high ACN fraction in the mobile phase.

Relationships of the Enantioselective Retention of Chiral Oxazolopyrroloquinolones on a Stationary Phase with Grafted Antibiotic Ristocetin A

A study is performed of mechanisms of the retention and separation of enantiomers of a series of chiral oxazolopyrroloquinolones on a stationary phase with grafted macrocyclic antibiotic ristocetin A under the conditions of high-performance liquid chromatography using water–acetonitrile mobile phases. It is found that the dependence of the retention factor on the concentration of CH3CN in the mobile phase has a U-shape. An explanation of this phenomenon is given. The thermodynamics of the oxazolopyrroloquinolone adsorption in different parts of this dependence is studied. It is assumed that the adsorption complex forms through inclusion of the oxazole moiety of the adsorbate molecule in the chiral cavity of the grafted selector. The effect the nature of the substituent on the chiral carbon atom in oxazolopyrroloquinolones on enantioseparation is discussed.

Preparation of core-shell silica-carbon composite microspheres stationary phase and application in saccharide separation

In this study, core-shell mesoporous silica-carbon composite microspheres (Sil@MC) were prepared by one-step coating of the phenol formaldehyde polymer (PF) on SiO2 surface and by carbonizing the PF polymer under nitrogen atmosphere. The morphology observation of the Sil@MC stationary phase showed that it had good monodispersity. Surface area (302 m2/g), mean pore diameter (9.5 nm), and pore volume (0.63 cm3/g) of Sil@MC materials were also measured by pore structure analysis. The results showed that the Sil@MC was successfully immobilized on the silica particles via copolymerization and carbonization. As a stationary phase of HPLC, the Sil@MC column was filled by a slurry method. The Sil@MC materials formed after calcination of SiO2 coated with phenolic resin could be used for the separation of four polar sugar compounds (D-(+)-glucosamine hydrochloride, glucose, D-(+)-trehalose dihydrat and raffinose) with the mobile phase of acetonitrile-water (containing 0.1% (v/v) formic acid). However, the material formed by calcinating SiO2 without coating phenolic resin could not separate these polar sugar compounds by HPLC-MS. Finally, the representative oligosaccharide isomers of raffinose, melezitose and stachyose, nystose, and human milk oligosaccharide isomers, such as 3'-sialyllactose, 6'-sialyllactose and lacto-N-newtetraose, lacto-N-tetraose, were successfully separated by the Sil@MC column with good peak shapes. The results demonstrates that silica-carbon composites derived from phenolic resin have potential application in polar compounds chromatographic separation.

Selectivity evaluation of core-shell silica columns for reversed-phase liquid chromatography using the solvation parameter model

For the selectivity evaluation of core-shell silica, siloxane-bonded reversed-phase columns by the solvation parameter model a minimal set of thirty-five calibration compounds were identified for applications with mobile phases containing from 20-70 % (v/v) methanol- or acetonitrile-water. The Kennard-Stone uniform mapping algorithm was employed to select the calibration compounds from a larger database of compounds with known retention properties used previously for column selectivity evaluation. The number and identity of the calibration compounds was optimized by considering the statistical quality of the calibration models, the absolute value for the standard deviation of the individual system constants, and the closeness of the estimated system constants for the calibration models to the values for the full data set on seven core-shell columns with different morphology for mobile phases containing 50 % (v/v) methanol- or acetonitrile-water. The predictive capability of the calibration models was evaluated for an external test set for 50 % (v/v) methanol-water for the same columns with a typical root mean square error of prediction (log k) of 0.028 for the thirty-five calibration compounds (n = 35) models. For all columns and mobile phase compositions the calibration models with n = 35 had a range for the coefficient of determination from 0.999 to 0.970 with < 5 % smaller than 0.99 (84 models) and a standard error of the estimate from 0.011 to 0.057 with 75 % < 0.030. System constants determined at 10 % (v/v) increments of organic solvent were utilized for the construction of system maps and correlation diagrams on Kinetex columns with octadecylsiloxane-bonded (C18 and EVO C18), diisobutyloctadecylsiloxane-bonded (XB-C18) octylsiloxane-bonded (C8), biphenylsiloxane-bonded (Biphenyl), phenylhexylsiloxane-bonded (Phenyl-Hexyl) and pentafluorophenylpropylsiloxane-bonded (F5) silica stationary phases for 20-70 % (v/v) methanol- and acetonitrile-water mobile phases. System maps for the Kinetex Phenyl-Hexyl column and correlation diagrams for columns of similar selectivity (Kinetex C8 and Phenyl-Hexyl) and dissimilar selectivity (Kinetex EVO C18 and Biphenyl) is used to illustrate the general approach to selectivity evaluation.

Selection of calibration compounds for selectivity evaluation of siloxane-bonded silica columns for reversed-phase liquid chromatography by the solvation parameter model

For the faster evaluation of selectivity in reversed-phase liquid chromatography of siloxane-bonded silica columns using the solvation parameter model a minimal set of calibration compounds is described suitable for mobile phase composition from 20-70% (v/v) methanol-, acetonitrile-, or tetrahydrofuran-water. The Kennard-Stone uniform mapping algorithm is used to select the calibration compounds from a larger database of compounds with known retention properties used earlier for column selectivity evaluation. Thirty-five compounds are shown to be necessary to minimize the standard deviation of the system constants and to minimize the difference between the system constants determined by conventional calibration and the values obtained for the reduced calibration compounds. The models for SunFire C18 with methanol-, acetonitrile- and tetrahydrofuran-water mobile phase compositions and XBridge Shield RP18, XBridge C8, XBridge Phenyl and Discovery HS F5 with methanol- and acetonitrile-water mobile phase compositions had an average coefficient of determination of 0.996 (standard deviation = 0.003, n = 11) and average standard error of the estimate 0.025 (standard deviation = 0.005, n = 11) for the reduced calibration compounds. Some octadecylsiloxane-bonded silica stationary phases with a high bonding density and methanol-water mobile phase compositions containing ≤ 30% (v/v) methanol exhibit extreme retention factors (log k > 2.5) for the low-polarity, two-ring aromatic compounds in the thirty-five compound calibration set. Alternative calibration compounds with more favorable retention properties are suggested as replacements in these cases. The predictive capability of the calibration models is validated using external test sets characterized by the average error, average absolute error and root mean square error of prediction. For the thirty-five calibration compounds sets the average absolute error 0.026 (standard deviation = 0.009, n = 11) and root mean square error of prediction 0.032 (standard deviation = 0.010, n = 11) confirm the suitability of the calibration models for column selectivity evaluation. System maps for XBridge Shield RP18 for 20-70% (v/v) methanol-water and Synergi Hydro-RP and 50% (v/v) methanol-water at temperatures from 25-65 °C together with a correlation diagram for XBridge Shield RP18 and SunFire C18 are presented as representative applications of the reduced calibration compounds for column selectivity evaluation.

In house validation for the direct determination of 5-hydroxymethyl-2-furfural (HMF) in “dulce de leche”

In this work an HPLC-DAD determination method for free and total HMF in “dulce de leche” was developed and validated. The separation was carried out using a mobile phase of acetonitrile-water (4.5:95.5, v/v) on Acclaim™120 C18 (4.6 × 150 mm, 5 μm), at 30 °C and a flow rate of 1 mL/min. The detection was done at 284 nm.The method showed good selectivity and linearity (determination coefficient over 0.999). Low LOD and LOQ values were obtained (19.51 μg/L and 65.05 μg/L respectively). Relative standard deviation (RSD) for repeatability and intermediate precision was <4.12 % and <5.98 % respectively. Recovery rates were between 89.04 % and 91.89 %. In addition, the HMF contents in commercial “dulce de leche” from Chile was reported, ranging from 2.33 to 10.31 mg/kg for free HMF and 314.61–464.98 mg/kg for total HMF.

Determination of zearalenone in cereals by high-performance liquid chromatography and liquid chromatography-electrospray tandem mass spectrometry

Zearalenone is a mycotoxin produced mainly by fungi belonging to the genus Fusarium in foods and feeds. A rapid and accurate method to quantify zearalenone in cereals was described. The determination of zearalenone was performed using immunoaffinity column for clean-up, high-performance liquid chromatography (HPLC)/fluorescence for quantification and liquid chromatography/electrospray tandem mass spectrometry (LC/ESI-MS-MS) for confirmation. Cereal samples were extracted with acetonitrile-water (90:10, v/v) prior to a Vicam ZearalaTest™ immunoaffinity column clean-up. Zearalenone was eluted from the column with methanol and quantified by HPLC with fluorescence detection (λex = 274 nm, λem = 440 nm). A mobile phase of acetonitrile-water (50:50, v/v) and a flow rate of 1.0 mL/min-1 were used. Average recoveries of zearalenone from cereals spiked at levels of 5, 20, 100 and 200 ppb ranged from 66.4% to 96.1%. The limit of quantitation was 5 ppb. The selective determination of zearalenone was achieved by LC/MS-MS with an electrospray (ESI) ionization interface. Using the negative ion mode, the parent ion of m/z 317 and the product ion of m/z 175 were selected. The quantification limit of 3 ppb was achieved. Seven mixed cereals samples, seven corn samples, five wheat samples, five rice samples and two oat samples were analyzed. The results showed that zearalenone was detected in four corn samples ranging from 7.9 to 9.0 ppb. Using the consumption data from "Nutrition and Health Survey in Taiwan 1993-1996", the zearalenone consumption for Taiwanese adults (male and female are 0.00297 and 0.00478 μg/kg b.w. respectively) was much lower than the tolerable daily intake of 0.5 μg/kg b.w. established by JECFA.

Determination of butocarboxim residue in agricultural products by HPLC with post-column derivatization system

A method using high performance liquid chromatography (HPLC) for the determination of butocarboxim in agricultural products was developed. Butocarboxim was extracted from samples with acetone and the extract solution was concentrated. The residue was dissolved in sodium chloride solution and partitioned with n-hexane. The aqueous phase was collected followed by extracted with dichloromethane, which was then evaporated to a volume of 2 mL prior to passing through an aminopropyl cartridge for sample clean-up. Determination of butocarboxim residue in crops was carried out by HPLC equipped with a post-column derivatization system and a fluorescence detector. HPLC separation was performed on a Lichrospher 60 RP-Select B column eluted with a mobile phase of acetonitrile-water (25:75, v/v). Butocarboxim was hydrolyzed at 90°C under alkaline conditions and subsequently reacted with o-phthaldialdehyde (OPA)/2-mercaptoethanol reagent via a post-column reactor to generate a fluorophore, which was then detected with a fluorescence detector at Ex 340 nm and Em 455 nm. Average recoveries from radishes and bamboo sprouts, which were spiked with 0.1~0.3 and 0.2 ppm butocarboxim, respectively, were in the range of 81.9~82.6%. The detection limit was 0.05 ppm. No butocarboxim residue was detected in 10 commercial products including radishes, carrots, and bamboo sprouts.

Establishment of regenerative callus, cell suspension system, and molecular characterization of Taxus wallichiana Zucc. for the in vitro production of Taxol

Taxus wallichiana, an endangered Taxus species native to Nepal, is a source of Taxol (paclitaxel), an anticancer drug, which if produced via plant cell culture in vitro not only reduces the pressure on natural habitat but also allows continuous production throughout the seasons. Gamborg’s B5 medium was the culture medium to develop callus and suspension culture. Quantification of Taxol was carried out via high performance liquid chromatography (HPLC) using C18 column, water-acetonitrile mobile phase, and 1 ml/minute flowrate at 227 nm wavelength. Genetic variation among population was determined using the POPGEN32, version 1.31. Best callus response was in medium with 1 mg/l 2,4-D for both stem and needle explants. For cell suspension culture, callus from needle explants showed better response compared to stem. Fructose and sucrose proved to be the best carbon sources. Cell count increased as carbon concentration increased. 2,4-D at low concentration (5 mg/l) showed higher cell growth rate. Inoculum volume was directly proportional to cell count. Extra addition of macronutrients had inhibitory effect on cell count, but the effect was reversed for MgSO4. Using HPLC, for bark, the highest Taxol yield was 0.057 mg/g distilled water (DW), while for needle was 0.056 mg/g DW. For suspension cultured HPLC samples, Taxol yield ranged at 0.004%. Size range of amplified Deoxyribonucleic acids was between 200 and 2,000 bp. Thus, this investigation generated a concrete data for Taxus population of Nepal providing efficient protocols for establishment of callus and cell suspension culture with the aim of producing Taxol in in vitro conditions.

Hydrophilic Liquid Chromatography versus Reversed-Phase Liquid Chromatography in the Absence and the Presence of 1-Hexyl-3-methylimidazolium Chloride for the Analysis of Basic Compounds

In reversed-phase liquid chromatography (RPLC), positively charged basic compounds yield broad and asymmetric peaks, as a result of ionic interactions with free silanols that remain on conventional silica-based columns. Diverse solutions have been proposed to mask the silanophilic activity, which is translated to an improved peak shape. In this work, the chromatographic performance of hydrophilic interaction liquid chromatography (HILIC) was evaluated as an alternative to the addition of an ionic liquid (IL) to the aqueous-organic mobile phase used with RPLC columns, for the analysis of eight β-adrenoceptor antagonists. ILs change the behavior of RPLC stationary phases owing to adsorption on their surface. Meanwhile, in HILIC, a layer of adsorbed water is formed on the stationary phase surface. The association of cationic basic compounds with the adsorbed additive ions, hydrophilic partitioning on the HILIC columns, and other interactions, give rise to complex retention mechanisms. The chromatographic behavior was examined in terms of retention, elution strength, selectivity, peak shape and resolution, using acetonitrile-water mobile phases buffered at pH 3. Both chromatographic modes, RPLC with added IL and HILIC, proved to be a viable solution to the problem of poor peak shape for basic compounds.

Direct HPLC enantioseparation of chemopreventive chiral isothiocyanates sulforaphane and iberin on immobilized amylose-based chiral stationary phases under normal-phase, polar organic and aqueous conditions

Sulforaphane and iberin are promising chemopreventive chiral phytochemicals. The chirality of these organic isothiocyanates is due to the presence of a stereogenic sulfur atom. Investigations of the effectiveness of single enantiomers as chemoprotective agents highlight the key role played by sulfur chirality on biological activity. The predominant native (R)-enantiomer is active whereas the (S)-counterpart is inactive or poorly active.Here, we provide an enantioselective method for the direct and complete resolution of both chiral sulfoxides by high-performance liquid chromatography on immobilized amylose-derived chiral stationary phases. A set of five different columns was investigated utilizing normal-phase, polar organic and aqueous conditions. The effect of the composition of mobile phase on enantioselectivity and retention was carefully evaluated.U-shape retention maps, which are indicative of a double and competitive hydrophilic interaction liquid chromatography and reversed-phase liquid chromatography retention mechanism, were established by recording the retention factors of the enantiomers of sulforaphane on the Chiralpak IA-3 and Chiralpak IG-3 chiral stationary phases varying progressively the water content in the water-acetonitrile mobile phases.

Development and Validation of a UPLC-DAD Method for Quantitative Analysis of Coumarin, trans-Cinnamic Acid, trans-Cinnamaldehyde, and Eugenol in Encapsulated Cinnamon Flavoring Powder.

Cinnamon is a popular spice used in food products. Its flavor varies by its chemical profile. Cinnamon flavoring powder is a unique form of material with essential oil encapsulated in wall material, which improves the stability and homogeneity but also increases the difficulties for analysis. A specific and rapid method is needed to analyze the main components for its quality and safety. An analytical method for the quantification of cinnamon flavoring powder was developed and validated. The characteristic components for analysis were selected as coumarin, trans-cinnamic acid, trans-cinnamaldehyde, and eugenol. This quantitation method with ultra-performance liquid chromatography coupled with diode array detector analysis was achieved by material extraction followed by chromatographic separation on C18 columns eluted with a gradient acetonitrile-water mobile phase. The detected wavelength was determined as 280 nm. Linear regression of calibration curves for each component was validated (R2 > 0.9995). The specificity, LOD and LOQ, precision, accuracy, and ruggedness of the developed method were also evaluated. Such an approach is applicable for the simultaneous determination of these four characteristic constituents in cinnamon flavoring powder used in manufacturing and quality control of nutritional products. This study describes the selection of four components for analysis, the efficient extraction of them from cinnamon flavoring powder, and the rapid quantitation of these four characteristic components in these materials.

Enantiomeric fraction evaluation of the four stereoisomers of difethialone in biological matrices of rat by two enantioselective liquid chromatography tandem mass spectrometry methods: Chiral stationary phase or derivatization

The need for the control of rodent populations with anticoagulant rodenticides remains actual, and enantioselective analytical methods are mandatory to understand ecotoxicity issues of those chiral pesticides. This study presents two enantioselective methods to achieve the residue levels and differentiated persistence of the four stereoisomers of difethialone (called in this work E1-trans, E2-cis, E3-cis and E4-trans), which is one of the most toxic second generation anticoagulant rodenticide. Their enantiomeric fraction evaluation in biological matrices of rats was determined by two LC-MS/MS methods. The first one (chiral-LC-MS/MS) combined a chiral column employed in reversed-phase mode (with acetonitrile-water mobile phase) to be compatible with mass spectrometry detection. The second one was also a LC-MS/MS method but with a reversed phase column after a derivatization step with (1S)-(−)-camphanic chloride. Extraction process combined Solid-Liquid extraction and sorbent cartridges. The methods were fully validated. The chiral column was chosen as a reference method for our laboratory because it was quicker and cheaper, and enantioresolution and sensitivity were better. This chiral-LC-MS/MS method was used to measure the enantiomeric fraction of the four stereoisomers of difethialone in rodent biological matrices (liver, plasma, blood and feces) of female rats treated with 3.5 mg/kg of difethialone. The results showed that metabolism is not the same for all the stereoisomers: cis-E3-difethialone was the most persistent, and E4-trans-difethialone was the most quickly eliminated.This chiral-LC-MS/MS method will be used to study the pharmacokinetics of the four stereoisomers of difethialone, and for ecotoxicological surveillance to evaluate the specific persistence of each stereoisomer of difethialone in case of secondary exposure of wildlife non-target species.

Development and validation of a method for phenobarbital in serum: anticonvulsant pharmacotherapy monitoring

The therapeutic drug monitoring (TDM) is an important strategy for the effectiveness and safety of long-term pharmacotherapy, such as the use of phenobarbital as an anticonvulsant drug in epilepsy. In this sense, HLPC has been presented as a technique for the measurement of phenobarbital in serum. However, the ideal conditions for carrying out the method must be established for each laboratory reality. An analytical method using HPLC was developed and validated in order to identify and quantify Phenobarbital in blood. The chromatographic conditions were C-18 column (Shimpack XR-ODS 50L x 3.0), acetonitrile-water mobile phase (30:70, v v-1), 0.2 mL min-1 flow and reading wavelength of 210 nm. Linearity was established in the range of 2.5 to 80 μg mL-1, the linear correlation coefficient was 0.9981. The average of the coefficient of variation of the precision was 5.30%. The relative standard error of the accuracy was -2.17% and of the recovery coefficient was 97.83%. In all eleven patients, phenobarbital concentrations were below the therapeutic range. The tested method was selective, linear, precise, accurate and showed good recovery.

Stability-indicating HPLC-DAD method for the simultaneous determination of fluoroquinolone in combination with a non-steroidal anti-inflammatory drug in pharmaceutical formulation

We developed and validated a stability-indicating assay method for the simultaneous determination of enrofloxacin and piroxicam in combination and in the presence of degradation products. Reversephase high-performance liquid chromatography analyses were carried out on a Vertisep C18 column and acetonitrile-water (48:52 v/v, pH 3.0) mobile phase with a 1.00 mL min−1 flow rate. The efficient chromatographic separation of these drugs and their forced degradation products was achieved in less than 5 min with a peak purity match factor higher than 950. The method used showed linearity in the concentration ranges of 0.25 to 16.0 μg mL−1 for enrofloxacin (r = 0.9997) and 0.125 to 8.0 μg mL−1 for piroxicam (r = 0.9999) as well as precision (relative standard deviation lower than 2%), accuracy (mean recovery 100 ± 2%), and robustness, according to ICH (International Conference on Harmonization) and AOAC (Association of Official Analytical Chemists) guidelines. This method can simultaneously determine the combination of these drugs in a veterinary formulation and separate the drug peaks from their forced degradation products. Additionally, its optimized chromatographic conditions can contribute to the quality control of this formulation in pharmaceutical manufacturing plants and minimize waste from the organic solvent.

An HPLC Method for detection of Anti-inflammatory Drugs in Bone and Cartilage health supplements

Many bone-building and cartilage repair supplements are available in the market and, many people especially the elderly people use these supplements. Manufacturers claim various therapeutic properties for these types of supplements. Therefore, it is likely that these products are included with chemical and anti-inflammatory drugs that are not listed in labels and brochures. There is not reported using HPLC method to check for chemical and anti-inflammatory drugs in bone and cartilage supplements, by the competent authorities. In this research, a valid, simple, affordable and selective method launched to quantify and identify some corticosteroids in bone and cartilage supplements. This method involves the extraction of the case from supplements using ethanol and injection directly to high-performance liquid chromatography (HPLC). The effect of different variables such as mobile phase, column type, pH and other variables was investigated. Optimum conditions including water-acetonitrile mobile phase with a ratio of 55: 45, ODS column, column temperature of 25°C, injection volume of 20 microliters, detector wavelength of 254 nm and the mobile phase flow rate 1ml/min were obtained. With this HPLC analysis method, cartilage repair products and bone-building supplements can be simply analyzed and the proportion of infected or free corticosteroids can also be identified.

New challenge in the lipophilicity determination and separation of biologically active 16,17-secoesterone derivatives by HPLC – Use of pentafluorophenyl-propyl column

The retention behavior, lipophilicity of 16,17-secoestrone derivatives and efficiency in the separation of halogenated derivatives by HPLC with pentafluorophenyl-propyl stationary phase (F5) were studied. Methanol-water (MeOH-W) and acetonitrile-water (ACN-W) were used as mobile phases in proportions according to nomograph of iso-eluotropic binary solvent mixtures. Since mobile phase ACN-W was more effective eluent, the retention behavior of 16,17-secoesterone derivatives has further been evaluated, with water content increasing in increments of 5%. The retention constants log k for mobile phases MeOH-W and ACN-W, have been compared by applying Student’s paired t-test and Fisher’s F-test to determine the applicability of the nomograph to this column. Linear correlations were obtained between parameters log k0 and log P, as well as φ0 and log P, for both MeOH-W and ACN-W eluents. Compared with log k0, the hydrophobicity index φ0 proved to be better parameter for lipophylicity estimation of these compounds. According to the separation efficacy of individual components, as well as mixtures of halogenated secoestone compounds, chromatographic columns can be placed in the following order: C18> F5> C8. This result is of huge importance for the field of synthesis, purification and characterization of novel biologically and pharmacologically active compounds and steroids among them.

Evaluation of additives on reversed-phase chromatography of monoclonal antibodies using a 1000 Å stationary phase

A wide pore (1000 Å) diphenyl stationary phase was evaluated for the analysis of monoclonal antibodies (mAbs), comparing a conventional mobile phase of acetonitrile-water containing overall 0.1% trifluoracetic acid (TFA) with a similar mobile phase incorporating in addition 5% butanol. Alternatively, TFA was replaced by ammonium formate (AF) buffer (pH 3.0) and by methane sulfonic acid. Addition of 5% butanol to the mobile phase reduces the minimum temperature at which suitable UV analysis of the mAbs can be obtained from about 70 °C with TFA alone to about 60 °C thus potentially improving column lifetime and reducing the possibility of sample degradation. AF buffers produce satisfactory UV sensitivity at 70 °C and have the advantage of reducing signal suppression in mass spectrometry (MS). Some peak tailing was noted in comparison with TFA separations. Methane sulfonic acid at the same molar concentration as TFA produced the best chromatographic peaks, maintaining reasonable UV sensitivity down to 50 °C, also giving acceptable results even at only 3 mM concentration of the additive. The good results with this additive were attributed to its stronger acidity and consequent suppression of the ionisation of column silanols. Surprisingly, peak response (as measured by the size of the peaks) was rather poorly correlated with the peak capacity of the gradient analysis. A possible explanation is self-deactivation of active column sites by a portion of the sample.

Separation of enantiomers of chiral sulfoxides in high-performance liquid chromatography with cellulose-based chiral selectors using acetonitrile and acetonitrile-water mixtures as mobile phases

The separation of 14 chiral sulfoxides was systematically studied on 12 cellulose-based chiral columns in acetonitrile and acetonitrile-water mobile phases. Out of all monosubstituted methylphenylcarbamates of cellulose the one having a methyl moiety in position 3 showed more universal chiral resolving ability compared to 2- and 4-substituted derivatives. Out of disubstituted phenylcarbamates of cellulose the ones with methyl substituents showed higher enantiomer resolving ability compared to chloro-substituted ones and substitution in positions 3 of the phenyl moiety was clearly advantageous. From disubstituted derivatives those possessing a combination of methyl- and chloro-substituents were advantageous compared to the ones having dimethyl- or dichloro-substituents. Chiral recognition ability of most chiral selectors towards studied sulfoxides was higher in pure acetonitrile compared to previously studied methanol. The effect of water addition to the mobile phase on analyte retention and enantioseparation was also quite different from that observed with methanol. In particular, with aqueous methanol by increasing the water content in the mobile phase retention increased in most cases and the separation factor improved. In contrast, with aqueous acetonitrile retention and separation factors decreased up to a certain water content in the mobile phase and then started to recover again for most of the studied analytes.