Percentage Of Organic Modifier Research Articles

Optimization and validation of a fast ultrahigh‐pressure liquid chromatographic method for simultaneous determination of selected sulphonamides in water samples using a fully porous sub‐2 μm column at elevated temperature

High temperature in HPLC is considered a valuable tool helping to overcome the increase in the column backpressure when using small packing particles such as sub-2 μm, as it allows reduction in the mobile-phase viscosity. In this study, a fast analytical method based on HPLC-UV was developed using a sub-2 μm column at elevated temperature for the simultaneous determination of nine sulphonamides. Owing to the lower viscosity of the mobile phase, the separation could be achieved in 3 min at 60°C for all analytes. The effect of temperature, the organic modifier percentage and the flow rate on the retention time was studied. The method developed was used for the determination of selected sulphonamides in surface and wastewater samples. Sample preparation was carried out by solid-phase extraction on Oasis HLB cartridges. The method developed was validated based on the linearity, precision, accuracy, detection and quantification limits. The recovery ranged from 70.6 to 96 % with standard deviations not higher than 4.7%, except for sulphanilamide. Limits of detection ranged from 1 to 10 μg/L after optimization of all analytical steps. This method has the highest performance in terms of analytical speed compared with other published HPLC-UV methods for the determination of sulphonamides in water.

Enantioselective separation and determination of citalopram enantiomers in pharmaceutical dosage form and bulk drug using experimental design approach on Chiralcel® OC as a chiral stationary phase

Summary An enantioselective HPLC method was developed and validated for the separation and the estimation of citalopram (CIT) enantiomers in bulk drug and pharmaceutical preparations. The method was validated for its linearity (correlation coefficient = 0.9994 and 0.996 for S-(+)-enantiomer and R-(−)-enantiomer, respectively), accuracy, robustness, and intermediate precision. Experimental design was applied during intermediate precision (full factorial 23 design) and robustness testing (Box Benken as a factorial design), for robustness test three factors were considered: percentage of organic modifier, flow rate, and temperature. The separation was achieved on cellulose tris(phenylcarbamate) known as Chiralcel® OC (25 cm, 4.6 mm i.d.) derivatized cellulose (phenyl carbamate), with UV detection at 245 nm using n-hexane-isopropanoldiethylamine (85:15:0.2, υ/υ/υ) as mobile phase at flow rate 0.7 mL min−1. A decrease in the flow rate results in decreasing the selectivity factor (α), while varying the percenta...

Towards multimodal HPLC separations on humic acid-bonded aminopropyl silica: RPLC and HILIC behavior

The stationary phase characteristics of the material obtained through immobilization of humic acid (HA) to aminopropyl silica (APS) via amide-bond formation were investigated. The material was characterized in terms of elemental analysis, FTIR, thermogravimetric analyses, pH point of zero charge measurements, potentiometric titrations, and contact angle measurements. Amount of HA bonded to APS was determined from the elemental analysis results, and found as 170mgHA/gAPS. Stability of the material was studied in aqueous media at different pH values, and amount of HA released at pH=8 did not exceed 2% of the total immobilized HA. Stationary phase characteristics of the well-characterized material were investigated in an HPLC system by using some low-molecular weight polar compounds (i.e. some nucleosides and nucleobases) as test solutes. Effect of some experimental variables such as column conditioning, composition of mobile phase, and temperature on the chromatographic behavior of the studied compounds was studied. Role of ammonium solutions at different pH values on retentive properties of the species was also studied. Retention factors (k′) versus volume percentage of organic modifier exhibited a U-curve, which was evaluated as an indication for RPLC/HILIC mixed-mode behavior of the stationary phase. Orthogonality between RPLC and HILIC modes was analyzed through geometric approach, and found as 48.5%. Base-line separation for the studied groups of compounds was achieved under each studied mode, and some differentiations were observed in elution order of the compounds depending on the HPLC mode applied. Chromatograms recorded under RPLC and HILIC modes were compared with those recorded on APS under similar conditions, and thus the influence/importance of HA immobilization process was evaluated in detail. In light of the obtained results, immobilized HA is represented as a useful stationary phase for HPLC separations.

Multiobjective Optimization Approach in Evaluation of Chromatographic Behaviour of Zolpidem Tartrate and Its Degradation Products

Multiresponse optimization methodology in combination with experimental design was employed as a powerful technique for simultaneous optimization of input variables significant for evaluation of chromatographic behaviour of zolpidem tartrate, zolpacid, oxozolpidem, zolpyridine and zolpaldehyde towards various responses. In the first stage of the investigation fractional factorial design was used to decrease the number of variables that should be studied in detail. Among examined variables, pH of the mobile phase, percentage of organic modifier and buffer concentration showed to be statistically important and were consequently optimized with central composite design and Derringer’s desirability function. Four responses were considered, the retention factors of zolpacid and zolpaldehyde (the first and last peak) and the resolutions between critical peaks. Optimal conditions included Luna C18(2) analytical column (250 mm x 4.6 mm, 5 μm particle size), mobile phase consisted of methanol–10 mM ammonium acetate (68.4:31.6, v/v, pH 5.4) and column temperature of 35 °C. The flow rate of the mobile phase was 1 mL min−1 and the detection was performed at 254 nm. At the end, the method was successfully validated in accordance with ICH guideline and subsequently applied to the analysis of commercially available zolpidem tartrate tablets.

Effect of Functionalized Stationary Phases on the Mechanism of Retention of Fungicides in RP-LC Elution

The objectives of this study were to investigate the molecular mechanisms of retention of selected fungicides and to classify stationary phases used for RP-HPLC. Chromatographic measurements were made using eight physicochemically diverse HPLC columns which were evaluated for analysis of nine biologically active compounds (strobilurins and triazoles). QSRRs were derived for the logarithms of the retention factors normalized to a hypothetical zero percent organic modifier in the mobile phase, log k W. Structural descriptors of the pesticides were used to describe their chromatographic retention behaviour under the given LC conditions. The next step was the correlation between retention data, log k W, and the structural descriptors of the analytes from molecular modelling.

Preparation and Characterization of Gold Nanoparticle-Modified Silica Monolith for Capillary Electrochromatography

Abstract A novel silica monolithic stationary phase functionalized with octadecanethiol gold nanoparticles (GNPs) was prepared for capillary electrochromatography (CEC). The bare silica monolith was prepared by a sol-gel process and then modified chemically with 3-mercaptopropyltrimethoxysilane, followed by immobilization of GNPs and modification by octadecanethiol. The relationship between plot of plate height and the linear velocity on octadecanethiol GNPs-modified silica monolith (Silica-GNPs-C18) was investigated, and a minimum plate height of about 7.8 μm for toluene was obtained. The electroosmotic flow characteristics of the Silica-GNPs-C18 and bare silica monolith (Silica) were studied by varying the percentage of organic modifier in buffer and buffer pH. To evaluate the column performance, hydrophobic alkylbenzenes as model compounds were tested on the prepared Silica, thiol-modified silica monolith (Silica-SH), and Silica-GNPs-C18. The electrochromatographic retention behavior of alkylbenzenes on Silica-GNPs-C18 column was also investigated. The experiment results showed that Silica-GNPs-C18 column exhibits typical reversed-phase electrochromatographic behavior toward neutral solutes. Reversed-phase CEC separations of four selected polar phenols were also demonstrated on Silica-GNPs-C18 column using mobile phase containing 15% methanol.

Determination of polyphenols in wines by liquid chromatography with UV spectrophotometric detection

This paper describes a new chromatographic method for the determination of polyphenolic compounds in wines. The method is based on the separation of analytes by reversed-phase mode in a C18 column (2.6 μm particle size) and UV absorption spectroscopy. The elution gradient is generated from 0.1% formic acid aqueous solution and acetonitrile as an organic modifier. Experimental conditions including pH, percentage of organic modifier and elution gradient profile have been thoroughly optimized using experimental design. A multi-objective function has been defined as a criterion for obtaining a satisfactory compromise among number of compounds separated, resolution and analysis time. Multi-detection at 280, 310 and 370 nm has been utilized in order to work under the most appropriate wavelengths for each compound. Figures of merit including linearity ranges, precisions, detection limits and recoveries have been established under selected experimental conditions using synthetic standards and commercial red wines. The method has been applied to analyze red wines from various Spanish regions.

Nanochitosan crosslinked with polyacrylamide as the chiral stationary phase for open‐tubular capillary electrochromatography

Nanoparticles exhibiting favorable surface-to-volume ratios create efficient stationary phases for electrochromatography. New nanomaterials derived from chitosan (CS) were immobilized onto modified capillaries for use as the chiral stationary phase (CSP) in open-tubular electrochromatography. This immobilization was achieved through the copolymerization of glycidyl methacrylate-modified nano-CS with methacrylamide (MAA) and bis-acrylamide crosslinkers (forming the MAA-CS capillary) rather than the attachment of nano-CS to the copolymer of glycidyl methacrylate, MAA, and bis-acrylamide (forming the MAA+CS capillary). The completed MAA-CS capillary and its precursors were examined by SEM and ATR-IR measurements. Before separating chiral samples, the MAA-CS capillary was characterized by electroosmotic flow measurements at varying pH values, concentrations, and volume percentages of organic modifiers in the running buffers. Tryptophan enantiomers were well separated by the MAA-CS capillary, whereas no enantioselectivity was observed in the MAA+CS capillary. With the addition of 80% MeOH into the phosphate buffer, the chiral separation of (±)-catechin was accomplished in a normal-phase mode. However, the new CSP has its limitations, as only two groups of α-tocopherol stereoisomers were separated.

Column-switching linked to large sample volumes to preconcentrate β-blockers at trace levels in environmental water

This paper describes about an automated on-line enrichment method for the simultaneous determination of seven β-blockers in river water using a short liquid chromatography column for preconcentration coupled with LC-DAD. The method performs the preconcentration of 30mL of river water samples (5% organic modifier) using a 50mm×4.6mm C18 column for enrichment and a 150mm×4.6mm C18 column for separation, allowing the determination of β-blockers at trace levels in river water. The analytical procedure was developed by optimizing the breakthrough parameters (flow rate, time of preconcentration and percentage of organic modifier added to the sample) in order to achieve the maximum sensitivity, and by optimizing the mobile phase (composition and flow rate) to get adequate separation of the components in a reasonable analysis time. Under the optimized conditions, the method was validated with respect to linearity, precision, limits of detection, limits of quantification and accuracy. Detection and quantitation limits ranged between 0.1 and 3.1 and between 1.0 and 5.0ngmL−1, respectively, whereas the RSD on inter-day precision was below 8%.To cope with the matrix effect in the determination of these drugs in river water samples, the standard addition methodology was successfully applied. Recoveries ranging from 81 to 115% proved the accuracy of the methodology proposed in this work.

Optimisation and validation of a fast HPLC method for the quantification of sulindac and its related impurities

The European Pharmacopoeia describes a liquid chromatography (LC) method for the quantification of sulindac, using a quaternary mobile phase including chloroform and with a rather long run time. In the present study, a new method using a short sub-2μm column, which can be used on a classical HPLC system, was developed. The new LC conditions (without chloroform) were optimised by means of a new methodology based on design of experiments in order to obtain an optimal separation. Four factors were studied: the duration of the initial isocratic step, the percentage of organic modifier at the beginning of the gradient, the percentage of organic modifier at the end of the gradient and the gradient time. The optimal condition allows the separation of sulindac and of its 3 related impurities in 6min instead of 18min. Finally, the method was successfully validated using an accuracy profile approach in order to demonstrate its ability to accurately quantify these compounds.

Prediction of the effects of methanol and competing ion concentration on retention in the ion chromatographic separation of anionic and cationic pharmaceutically related compounds

The mixed-mode separation of a selection of anionic and cationic pharmaceutically related compounds is studied using ion-exchange columns and eluents consisting of ionic salts (potassium hydroxide or methanesulfonic acid) and an organic modifier (methanol). All separations were performed using commercially available ion-exchange columns and an ion chromatography instrument modified to allow introduction of methanol into the eluent without introducing compatibility problems with the eluent generation system. Isocratic retention prediction was undertaken over the two-dimensional space defined by the concentration of the competing ion and the percentage of organic modifier in the eluent. Various empirical models describing the observed relationships between analyte retention and both the competing ion concentration and the percentage of methanol were evaluated, with the resultant model being capable of describing the separation, including peak width, over the entire experimental space based on six initial experiments. Average errors in retention time and peak width were less than 6% and 27%, respectively, for runs taken from both inside and outside of the experimental space. Separations performed under methanol gradient conditions (while holding the competing ion concentration constant) were also modelled. The observed effect on retention of varying the methanol composition differed between analytes with several analytes exhibiting increased retention with increased percentage methanol in the eluent. An empirical model was derived based on integration of the observed t R vs. %methanol plot for each analyte. A combination of the isocratic and gradient models allowed for the prediction of retention time using multi-step methanol gradient profiles with average errors in predicted retention times being less than 4% over 30 different 2- and 3-step gradient profiles for anions and less than 6% over 14 different 2- and 3-step gradient profiles for cations. A modified peak compression model was used to estimate peak widths under these conditions. This provided adequate width prediction with the average error between observed and predicted peak widths being less than 15% for 40 1-, 2- and 3-step gradients for anions and less than 13% over 14 1-, 2- and 3-step gradients for cations.

A capillary electrophoresis method for the determination of selected biogenic amines and amino acids in mammalian decomposition fluid

A simple capillary zone electrophoresis method for the determination of selected biogenic amines (tyramine and tryptamine) and amino acids (tryptophan, phenylalanine and tyrosine) in mammalian decomposition fluids is presented. Separations were carried out in a fused silica capillary (75microm i.d., total length 65cm, effective length 56cm) with detection by ultraviolet absorbance spectrophotometry at 200nm. In order to improve resolution and total analysis time, the method was subjected to optimisation utilising a chemometric approach. A screening design was carried out followed by a central composite design (CCD), using peak resolution and total analysis time as response factors. The influences of four experimental variables (pH, background electrolyte concentration, percentage of organic modifier (methanol) and applied voltage) were investigated. Optimum separation conditions were determined to be; a background electrolyte of boric acid (70mM) adjusted to pH 9.5 with 0.1M sodium hydroxide with 32% methanol (v/v). Applied voltage was 30kV, with the resulting current being less than 26microA. Under these conditions the analytes were separated within 12min. Tryptamine, tyramine, tryptophan, tyrosine and phenylalanine were identified by migration time and spiking in porcine decomposition fluids.

Development of an environmental friendly method for the analysis of organochlorine pesticides in sediments

This work proposes a pressurized hot water extraction (PHWE) followed by solid phase microextraction (SPME) and determination by gas chromatography–mass spectrometry method for the analysis of organochlorine pesticides in sediment samples. For SPME, extraction temperature and time, desorption temperature and desorption time, and effect of an organic modifier were studied. For PHWE, parameters such as organic modifier, percentage of organic modifier, temperature, and static extraction time were studied. When these parameters were selected, the figures of merit were calculated in order to assess the performance of the proposed method. Quantitative recoveries (80–115%) and satisfactory precisions were obtained. The detection and quantification limits were between 0.11 and 16 μg kg −1 and between 0.24 and 22 μg kg −1, respectively with good linearity between LOQs and 500 μg kg −1 for most of the studied pesticides. The method was validated by the analysis of a reference marine sediment material (SRM 1944). The obtained results are in excellent agreement with the certificate material. Another sediment reference material (SRM 1941b), with a very low concentration of pesticides was also analyzed with good results. In addition, this method was successfully applied to the analysis of three marine sediment samples. The developed method seems to be a nearly full automated, environmental friendly, sensitive, simple and less-time consuming method. Moreover this method, look for the implementation of the principles of green analytical chemistry and has demonstrated to be suitable for the analysis of organochlorine pesticides at trace levels in environmental matrices as sediment samples.

LC-UV METHOD DEVELOPMENT AND VALIDATION FOR THE NON STEROIDAL ANTI-INFLAMMATORY AGENT TENOXICAM

A rapid and sensitive reversed phase high performance liquid chromatographic (HPLC) method was developed and validated for the analysis of tenoxicam in raw material and its pharmaceutical formulation. The analysis was carried out on a reversed phase C18 column, using mixtures of buffer/acetonitrile (40:60, v/v) with flow rate was of 1 mL min−1. The method was validated statistically for its linearity (correlation coefficient = 0.9983), accuracy, robustness, and intermediate precision. An experimental design was used during validation to evaluate method robustness and for the determination of intermediate precision. To test robustness, four factors were considered, mainly, percentage of organic modifier in the mobile phase, pH, flow rate, and different wavelengths. An increase of the flow rate results in a decrease of the drug concentration found, while the percentage of organic modifier, pH, and wavelength have no significant effect on the response. For intermediate precision the factors examined were multiple analysts, multiple instruments, and multiple days. The RSD value (0.49%, n = 24) indicated a good precision of the analytical method. Due to its simplicity, accuracy, sensitivity, and precision the method may be used for routine quality control analysis.

EXPERIMENTAL DESIGN STRATEGIES IN LC METHOD DEVELOPMENT AND IN ROBUSTNESS TESTING FOR REVERSIBLE CHOLINESTERASE INHIBITOR RIVASTIGMINE IN PHARMACEUTICAL FORMULATION

An isocratic, reversed phase liquid chromatographic (RPLC) method was developed for the quantitative determination of rivastigmine hydrogen tartrate, in bulk drugs and in pharmaceutical formulation. The chromatographic separation was achieved on A Vertex Hypersil reversed phase C18 column (25 cm × 4.6 mm i.d., particle size 5 μm) using acetonitrile and phosphate buffer. The procedure was validated by linearity (correlation coefficient = 0.9983), accuracy, robustness, and intermediate precision. An experimental design was used during validation to calculate method robustness and intermediate precision. For the robustness test, three factors were considered: percentage v/v of acetonitrile, flow rate, and temperature; an increase in flow rate results in decrease of the concentration of the drug from the expected value, while the percentage of organic modifier and temperature have no effect on the response. For intermediate precision measurement, the considered variables were analyst, equipment, and days. The RSD value (1.10%, n = 24) indicate a good precision.

Chemometrically Assisted Development and Validation of LC for Simultaneous Determination of Carbamazepine and Its Impurities Iminostilbene and Iminodibenzyl in Solid Dosage Form

A chemometrical approach was applied to develop a reversed-phase liquid chromatographic method for simultaneous determination of carbamazepine and its impurities iminostilbene and iminodibenzyl in solid dosage form. According to contemporary literature, no method was developed for simultaneous determination of carbamazepine and these impurities by chemometrical approach. The fractional factorial design was used for selection of variables significantly influencing the chromatographic separation of the investigated substances. The investigated variables were: temperature of the column, the percentage of organic modifier, the acetate buffer concentration and pH of water phase. The first three variables were proved to be significant and were optimized by face centered, central composite design. Investigation was performed using C18 XBridge Shield analytical column (50 mm × 4.6 mm i.d., particle size 3.5 µm). The optimal conditions for the separation were established with the mobile phase composition of methanol–10 mM acetate buffer (pH adjusted to 2.21 with glacial acetic acid) (50:50, v/v) at a flow rate of 1.5 mL min−1, 25 °C column temperature and detection at 260 nm. Total analysis time was shortened to about 8 min. Finally, the method was successfully validated and subsequently applied to the analysis of commercially available carbamazepine tablets.

Prediction of retention in reversed-phase liquid chromatography by means of the polarity parameter model

The polarity parameter model previously developed: log k = (log k) 0 + p( P m N − P s N ) has been successfully applied to study several chromatographic systems involving new generation RPLC columns (Luna C18, Resolve C18, XTerra MSC18, and XTerra RP18). In this model the retention of the solutes (log k) is related to a solute parameter ( p), a mobile phase parameter ( P m N ) and two chromatographic system parameters [ P s N and (log k) 0]. The studied systems have been characterized with different acetonitrile–water and methanol–water mobile phases, using a set of 12 neutral solutes of different chemical nature. The polarity parameter model allows prediction of retention of any solute in any mobile phase composition just using the retention data obtained in one percentage of organic modifier and the polarity parameters established in the characterization of the chromatographic systems. This model also allows the solute polarity data transference between RPLC characterized systems, so it is possible to predict the retention in various RPLC systems working experimentally with just one of them. Moreover, the global solvation parameter model has also been applied to the same chromatographic systems using a wide set of solutes in order to compare its predictive ability with the one of the polarity parameter model. The results clearly show that both models predict retention with very similar accuracy but the polarity parameter model requires much less preliminary experimental measurements to achieve equivalent results than the global solvation approach.

Enantiomeric separation of phenylsuccinic acid by cyclodextrin-modified reversed phase high-performance liquid chromatography

The chiral separation of phenylsuccinic acid (PSA) was studied by reversed phase high-performance liquid chromatography (RP-HPLC) with cyclodextrins (CDs) as chiral mobile phase additives. The effects of types of CDs, concentration of hydroxypropyl-β-cyclodextrin (HP-β-CD), percentage of organic modifier, pH value and column temperature on enantioselective separation were investigated. The quantification property of the developed RP-HPLC method was examined. The chiral recognition mechanism of PSA was also discussed. The results show that a baseline separation of PSA enantiomers is achieved on a Lichrospher C18 column (4.6 mm (inner diameter)×250 mm, 5 μm) with HP-β-CD as chiral mobile phase additive. The capacity factors of R-PSA and S-PSA are 3.94 and 4.80, respectively. The separation factor and resolution are respectively 1.22 and 8.03. The mobile phase is a mixture of acetonitrile and deionized water (20:80, volume ratio) containing 10 mmol/L HP-β-CD and 0.05% trifluoroacetic acid (pH 2.5, adjusted with triethylamine) with a flow rate of 1.0 mL/min. The ultraviolet (UV) detector is set at 254 nm. The likely roles are inclusion interaction, induction and hydrogen bonding between HP-β-CD and PSA enantiomers.

HILIC separation and quantitation of water‐soluble vitamins using diol column

Hydrophilic interaction liquid-chromatography (HILIC) in conjunction with diode array detection has been applied for the separation of selected-water-soluble vitamins using an end-capped HILIC-diol column. Vitamins with significant biological importance, such as thiamine (B(1)), riboflavin (B(2)), nicotinic acid (B(3)), nicotinamide (B(3)), pyridoxine (B(6)), folic acid (B(9)), cyanocobalamin (B(12)) and ascorbic acid (vitamin C) were simultaneously separated. Chromatographic conditions including type and percentage of organic modifier in the mobile phase, pH, type and concentration of buffer salt and flow rate were investigated. ACN was shown to offer superior separation for the compounds tested as compared to methanol, isopropanol and THF. Isocratic separation and analysis were achieved for six vitamins (B(1), B(2), nicotinic acid/nicotinamide, B(6) and C) at ACN-H(2)O 90:10, containing ammonium acetate 10 mM, triethylamine 20 mM, pH 5.0, using a flow rate of 0.8 mL/min, while a gradient was necessary to resolve a mixture of all eight water-soluble vitamins. The HILIC method was validated and successfully applied to the analysis of a pharmaceutical formulation and an energy drink negating the need for time consuming clean-up steps.

Multiple Step Gradient Analysis in Stationary Phase Optimised Selectivity LC for the Analysis of Complex Mixtures

Stationary phase optimised selectivity liquid chromatography (SOSLC) is an approach to tune a given LC separation by combining different stationary phases in a multi- segment column set-up. The presently available SOSLC optimisation procedure and algorithm are, however, only applicable to isocratic conditions. This is a severe limitation for the analysis of mixtures composed of components covering a broad hydrophobicity range. A strategy is described to circumvent this limitation. The components of a mixture are divided into different groups according to hydrophobicity as elucidated by a gradient analysis on a C18 reversed-phase column. Each group separation is then individually optimised with a specific isocratic mobile phase composition using the original SOSLC strategy. The mobile phase composition thereby only differs in the percentage of organic modifier between the various groups. Finally, a combination of stationary phases that guarantees sufficient selectivity for all the groups is selected and the separation is performed by a multiple step gradient, whereby each level consists of the mobile phase composition applied for the SOSLC optimisation of the individual groups. The multi step gradient approach is demonstrated through the analysis of a mixture of 27 steroids covering a wide range of hydrophobicity.