Development Of Separation Methods Research Articles

Ultra high-performance liquid chromatography method development for separation of formoterol, budesonide, and related substances using an analytical quality by design approach

The application of the Quality by Design (QbD) principles in developing a new ultra high performance liquid chromatography method for the analysis of formoterol/budesonide and related substances using Fusion QbD® software is explored. The effect of various chromatographic parameters including, column stationary phase, pH, temperature, flow rate, and gradient time on separations were systematically investigated. Results show that optimal separations of these compounds in a standard solution can be achieved using a BEH C18 column (2.1 × 1.7 μm × 10 cm) applying a pH of 8.2, a temperature of 35 °C, a flow rate of 0.35 mL min−1 and a gradient time of 25 min. Furthermore, the results show that the main parameters affecting the performance of the method were the mobile phase pH, gradient time, and the temperature. For example, the most important factor for peak tailing was the pH of the mobile phase and the critical factors affecting resolution of the analytes were the gradient time and the temperature. As an application, the method was further used to analyze budesonide and formoterol in a sample obtained from a Symbicort® metered dose inhaler and it was found to provide similar separations to those obtained with the standard solution. These findings indicate that applying the QbD principles in analytical method development can be very advantageous not only in obtaining deep understanding of the effect of input parameters but also potential regulatory flexibility.

Fundamental distribution coefficient data and separations using eichrom extraction chromatographic resins

Distribution coefficient (Kd) data provides the analyst with a powerful set of tools for designing elemental chemical separations. While considerable Kd data exists in the literature for traditional anion and cation exchange resins, a comprehensive Kd study for the majority of the periodic table has not been published to date for several newer Eichrom extraction chromatographic resins. Kd values for the sorption of 41 different of elements on TEVA, TRU, UTEVA, and Diphonix resin were determined in this work. The power of Kd data in designing chemical separations is then demonstrated through development of separation methods for several challenging chemical separations including U/Mo/Te, Zr/Nb/Mo, and Cd isolation. Separation methodologies are finally applied to soil samples, where the ability for each separation to perform under complex matrix conditions is evaluated.

Ultra high performance liquid chromatography method development for separation of omeprazole and related substances on core‐shell columns using a Quality by Design approach

An updated and improved method for analysis of omeprazole/esomeprazole and related substances on core-shell columns was developed using Fusion LC Method Development™. The method was optimized with respect to column type, column temperature, mobile phase pH level, and gradient time. Four different core-shell columns were examined to develop a method suitable for both high performance- and ultra-high performance liquid chromatography using a Quality by Design approach. The final method offers two alternative columns: Poroshell EC C18 (3.0×100mm, 2.7µm) or Poroshell HPH (3.0×100mm, 2.7µm) with the same gradient elution condition and mobile phase composition. Total run time is 18min with 12min of gradient elution. Phosphate buffer (15mM, pH 7.8) is selected as the aqueous mobile phase and acetonitrile as the organic mobile phase. Column temperature is set at 40°C and ultraviolet detection at 302nm. Furthermore, by studying parameters in a systematic way, an understanding of the effect of the input parameters enhances the method robustness and should allow for regulatory flexibility in terms of post-approval changes. Compared to the current United States Pharmacopeia method, the updated method is faster, more efficient and performs well above acceptance criteria.

A new strategy for development of eco-friendly RP-HPLC method using Corona Charged Aerosol Detector and its application for simultaneous analysis of risperidone and its related impurities

Green analytical chemistry is primarily directed towards minimization of the amount of waste associated with either the sample preparation or analysis. Among different chromatographic methods, liquid chromatography is considered the least green, allowing for various possibilities for greening. Using green solvents such as ethanol or acetone in RP-HPLC, as an alternative to acetonitrile, is recently attracting an attention. Both ethanol and acetone are characterized with low toxicity, with certain drawbacks limiting their regular use in RP-HPLC. Ethanol has low eluotropic strength and causes high backpressures, while acetone shows high UV cut-off, making it unsuitable for UV/Vis detection. To overcome the existing problems, Corona Charged Aerosol Detector was employed for development of RP-HPLC methods for separation of risperidone and its structurally related impurities with either ethanol or acetone as organic modifier. The methods were optimized by experimental design methodology, while optimal conditions for separation were determined using Derringer's desirability function. Detailed assessment of 3D surface plots of Derringer's desirability function enabled selection of 0.6 mL min−1 flow rate and 20% (v/v) organic modifier content as optimal when using ethanol, while in case of acetone mobile phase flow rate was 0.8 mL min−1 and organic modifier content 17% (v/v). Methods were validated and their eco-friendly character was confirmed through Green Analytical Procedure Index (GAPI). Although both methods are ecologically acceptable, the main drawback is reflected in the fact that no recycling or another waste treatment method exist. In the end, acetone was prioritized over ethanol, due to lower health hazard and decreased amount of generated waste.

Application of micellar electrokinetic capillary chromatography to the discrimination of red lipstick samples

The main aim of this study was to apply capillary electrophoresis (CE) to the discrimination of red lipsticks – the first step towards developing the methodology for forensic purposes. The separation method was optimized using a mixture of eight dyes and micellar electrokinetic capillary chromatography (MEKC). The influence of the buffer pH and the concentration of SDS on the separation of dye mixtures was investigated. The best separation (highly efficient with the large resolution capacity in relatively short time) was accomplished using a buffer at pH 9 enriched with 80 mM SDS. The developed separation method was evaluated, determining the stability of sample (5 h) and buffer (10 h), the limit of detection of dyes (LOD < 1 μg·mL−1), the repeatability of migration times in one day (RSD < 1.2%), and over three days (RSD < 2.4%), as well as the reproducibility using different capillaries and CE instruments.In order to develop a versatile extraction method for compounds from red lipsticks, various types of solvents, temperatures and durations of ultrasound assisted extraction (UAE) were tested. The use of a specially designed and constructed device for repetitive sampling without contamination was suggested. The extraction method was characterized in terms of precision, obtaining very good results for the qualitative (RSD of migration time < 2.0%) and quantitative (RSD of peak height < 10.0%) methods.The proposed UAE/MEKC-DAD method was successfully applied to distinguish red lipstick samples with a very similar hue, achieving a high value of discrimination power (DP = 0.998). It was found that it has great potential in forensic investigations.

Applications of Chiral Supercritical Fluid Chromatography.

Nowadays polysaccharide chiral stationary phases are very popular and this originates for many reasons: (1) their wide applications window, (2) numerous different chemistry availability either in coated or in immobilized versions, and (3) large loading capability useful for preparative scale. Indeed chiral separations remain a hot topic (particularly in the pharmaceutical market) and in this field supercritical fluid chromatography is emerging rapidly. However, its use is more complex than high-performance liquid chromatography. The presented example illustrates analytical scale chiral separation method development and the effect of each operating parameter, i.e., flow rate, outlet pressure, and temperature variations on a chiral separation.

Separation of catechins from green tea (Camellia sinensis L.) by microwave assisted acetylation, evaluation of antioxidant potential of individual components and spectroscopic analysis

Separation of individual green tea catechins has the limitation due to their structural resemblance and unequal distribution. Hence, the objective of this study was to develop separation methods based on 1.Preparative Thin Layer Chromatography (TLC) using oxalic acid impregnation and 2. Microwave (MW) mediated acetylation followed by classical silica gel column chromatography separation. The yield of total catechins by different extraction techniques was, conventional (7.3%), liquid-liquid (7.8%), Soxhlet (8.3%) and microwave (8.8%). Acetylation of total catechins under various MW conditions explored with acetic anhydride, triethylamine and standardized at 15 min at 1g scale and subjected to separation. Subsequently, the individual catechin acetates deacetylated under MW conditions and characterized by TLC, High-Resolution Mass Spectrometry (HRMS), Fourier Transform Infrared Spectroscopy (FT-IR), 1H and 13C Nuclear Magnetic Resonance (NMR) spectroscopic studies. The total polyphenol content of the extract was 931.17 ± 0.67 g GAE kg−1. The antioxidant activity of deacetylated catechins was evaluated by Ferric-Reducing Antioxidant Potential (FRAP) in terms of Trolox equivalence (TE) and % inhibition of 1, 1-diphenyl-2-picryl-hydrazyl (DPPH). Accordingly, the TE and DPPH % inhibition was epigallocatechingallate (12.3 and 71.9%), epicatechingallate (11.5 and 55.5%), epigallocatechin (9.10 and 46.0%), epicatechin (7.40 and 35.1%) respectively.

Разработка методов разделения и идентификации пространственных изомеров N,N’- диметилгликолурила

В настоящее время одним из развивающих разделов химии является химиягетероциклических соединений, в частности особый интерес представляют гликолурилыи их производные. Производные гликолурила имеют широкий спектр биологическойактивности. Они применяются в качестве психотропных лекарственных средств. Изомерыдиметилгликолурила можно получать синтезом из метилмочевины и глиоксаля. Каждыйизомер обладает своей специфичностью действия. Изомеры N,N’- диметилгликолурилапроявляют разные биологические свойства. На примере, 2,6-дизамещённые изомеры взначительно большей степени, чем 2,4-дизамещённые, проявляют психотропную активность. Влитературных источниках нет конкретных сведений по разделению и идентификации изомеровN,N’- производных диметилгликолурила, что делает их интересными для изучения. Поэтомубыли разработаны новые методы хроматографического разделения и идентификации N,N’-производных диметилгликолурила.

NP-StructurePredictor: Prediction of Unknown Natural Products in Plant Mixtures.

Identification of the individual chemical constituents of a mixture, especially solutions extracted from medicinal plants, is a time-consuming task. The identification results are often limited by challenges such as the development of separation methods and the availability of known reference standards. A novel structure elucidation system, NP-StructurePredictor, is presented and used to accelerate the process of identifying chemical structures in a mixture based on a branch and bound algorithm combined with a large collection of natural product databases. NP-StructurePredictor requires only targeted molecular weights calculated from a list of m/z values from liquid chromatography-mass spectrometry (LC-MS) experiments as input information to predict the chemical structures of individual components matching the weights in a mixture. NP-StructurePredictor also provides the predicted structures with statistically calculated probabilities so that the most likely chemical structures of the natural products and their analogs can be proposed accordingly. Four data sets consisting of different Chinese herbs with mixtures containing known compounds were selected for validation studies, and all their components were correctly identified and highly predicted using NP-StructurePredictor. NP-StructurePredictor demonstrated its applicability for predicting the chemical structures of novel compounds by returning highly accurate results from four different validation case studies.

Kinetic study of niobium and tantalum hexameric forms and their substituted ions by capillary electrophoresis in alkaline medium

In this work a capillary electrophoretic (CE) method is used for the kinetic study of the intermetallic substitutions in hexameric ions of two strategic metals, tantalum and niobium in an alkaline medium. Recently proposed processes for the production and analytical separation of tantalum and niobium that are faster, more economical and environmental friendly are based on the use of highly alkaline media. It was previously established that in these media, tantalum and niobium exist as hexameric species, HxTa6O19X−8 (Ta6) and HxNb6019x−8 (Nb6), which can be analysed with a CE method using an alkaline electrolyte and UV detection. However, when using the above method on an industrial sample a minor species that should correspond to the substituted Ta1Nb5 form was observed. The purpose of the present study is to probe, by means of CE, the kinetic of the formation of substituted niobate-tantalate ions, Ta6−xNbx (1 ≤ x ≤ 5), starting from mixtures of pure hexaniobate and hexatantalate ions. This study required the development of a new CE method allowing the separation of all the five substituted ions and their two non-substituted hexameric parent ions in less than seven minutes. In details, a previously developed separation method was transferred to a Beckman instrument and the separation improved by adjusting the total length, the applied voltage, the injection volume, the rinsing steps and the internal standard. The kinetic study shows that samples initially containing non-substituted hexameric forms of tantalum and niobium in a 1:1M ratio naturally form the five possible substituted species Ta6−xNbx (1 ≤ x ≤ 5) after only a few hours which may represent an issue for future Nb-Ta separation processes operated in alkaline media. The developed method was also transferred to an Agilent instrument and the kinetic study repeated. Results obtained with the Agilent instrument corroborate those obtained with the Beckman instrument. The proposed electrophoretic separation method lays the ground for new analytical techniques that could help assessing the presence of substituted species that can be deleterious for Nb-Ta purification processes.

A fully automated simultaneous single-stage separation of Sr, Pb, and Nd using DGA Resin for the isotopic analysis of marine sediments.

A novel, fast and reliable sample preparation procedure for the simultaneous separation of Sr, Pb, and Nd has been developed for subsequent isotope ratio analysis of sediment digests. The method applying a fully automated, low-pressure chromatographic system separates all three analytes in a single-stage extraction step using self-packed columns filled with DGA Resin. The fully automated set-up allows the unattended processing of three isotopic systems from one sediment digest every 2h, offering high sample throughput of up to 12 samples per day and reducing substantially laboratory manpower as compared to conventional manual methods. The developed separation method was validated using the marine sediment GBW-07313 as matrix-matched certified reference material and combines quantitative recoveries (>90% for Sr, >93% for Pb, and >91% for Nd) with low procedural blank levels following the sample separation (0.07μgL-1 Sr, 0.03μgL-1 Pb, and 0.57μgL-1 Nd). The average δ values for Sr, Pb, and Nd of the separated reference standards were within the certified ranges (δ (87Sr/86Sr)NIST SRM 987 of -0.05(28)‰, δ(208Pb/206Pb)NIST SRM 981 of -0.21(14)‰, and δ(143Nd/144Nd)JNdi-1 of 0.00(7)‰). The DGA Resin proved to be reusable for the separation of >10 sediment digests with no significant carry-over or memory effects, as well as no significant on-column fractionation of Sr, Pb, and Nd isotope ratios. Additional spike experiments of NIST SRM 987 with Pb, NIST SRM 981 with Sr, and JNdi-1 with Ce revealed no significant impact on the measured isotopic ratios, caused by potential small analyte peak overlaps during the separation of Sr and Pb, as well as Ce and Nd.

1-2-3 低品位炭・バイオマス廃棄物の溶剤改質法における溶剤と抽出物の分離方法の検討(Session 1-2 重質油、コプロセッシング)

1-2-3 低品位炭・バイオマス廃棄物の溶剤改質法における溶剤と抽出物の分離方法の検討(Session 1-2 重質油、コプロセッシング)

No.11 低品位炭・バイオマス廃棄物の溶剤改質法における溶剤と抽出物の分離方法および改質機構の検討(改質・処理・クリーン化(2)、熱分解・コークス(3))

No.11 低品位炭・バイオマス廃棄物の溶剤改質法における溶剤と抽出物の分離方法および改質機構の検討(改質・処理・クリーン化(2)、熱分解・コークス(3))

Unusual selective extraction of Pu 4+ by some novel diamide ligands in a room temperature ionic liquid

Abstract Extraction of Pu4+ was carried out from nitric acid feeds employing ionic liquid solutions of four novel diamide ligands containing an aromatic bridging group. The ligands with iso-butyl, n-butyl, n-octyl and 2-ethylhexyl groups were termed as LI, LII, LIII, and LIV, respectively and showed significantly higher extraction of the tetravalent plutonium ion as compared to the hexa-valent uranyl ion. Using 0.05 M ligands, the extraction of Pu4+ was in the range of 80–95% when 3 M HNO3 was used as the feed and followed the order: LIII > LII > LI > LIV while the UO22+ ion extraction was less than 4–5% and no particular trend could be established. The extraction of UO22+ could be partly due to the anionic nitrate complex of uranyl ion which was extracted in the absence of the ligands, while the ligand assisted extraction contributing for the remaining. Out of the other metal ions studied, Cs+ showed some extraction which was not ligand assisted while almost no extraction of metal ions such as Sr2+, Am3+ and Eu3+ was noticed. Unusually high selectivity was seen for Pu4+ ion extraction out of all the metal ions studied making the solvent systems very important for a plutonium specific separation method development. The extraction was found to be increasing with the aqueous phase nitric acid concentration conforming to a ‘solvation mechanism’ of extraction where the extracted species conforming to Pu(NO3)4·LIL for all the four diamide extractants.

Novel approach to determine ghrelin analogs by liquid chromatography with mass spectrometry using a monolithic column.

In our project, ghrelin analogs possessing enhanced stability and potential to significantly increase food intake were used. Three newly synthesized ghrelin analogs with fatty acid residues consisting of 8, 10, and 14 carbon atoms were studied. The main goal of this work was to develop a suitable analytical method for the determination of the stability of the novel ghrelin analogs in plasma. An appropriate liquid chromatography-mass spectrometry method was developed and optimized. The results obtained were compared with the data measured by using a commercial enzyme-linked immunosorbent assay kit, and a good correlation was found. A preparation strategy for plasma samples was optimized and consisted of simple dilution of the plasma samples followed by direct injection onto a very short monolithic column in combination with mass spectrometric detection. The developed analytical method was utilized for the determination of the stability of the prepared lipopeptides in plasma and for the quantification of the lipopeptides in a preliminary pharmacokinetic study. The feasibility of the developed separation method was clearly demonstrated. Accuracy and precision were within 80-120% and ±20% limits, respectively. Calibration curves were constructed in the range of 1-250 μg/mL.

Resolving Co-Elution Problems of Components in Complex Mixtures by Multiple Heart-Cutting 2D-LC

Multiple heart-cutting (MHC) 2D-LC was applied to resolve challenging peak co-elutions in a pesticidal natural product-derived extract, which exhibited a complex impurity profile. While the 1D separation using sub-2 μm particle column technology provided good separation efficiency, additional selectivity was required to separate overlapping peaks in several chromatographic regions. MHC 2D-LC using C18 columns in both dimensions, but different pH in the mobile phase, provided full separation of the components of interest. This article describes how the MHC 2D-LC technology has been applied for optimized analysis of target components as well as being a rapid tool for peak purity evaluation. It also discusses strategic benefits of the technology regarding method development for separation of very complex samples.

Development of separation methods for the chiral resolution of hexahelicenes

In this short communication we report optimized procedures for the chiral separation of non-charged [6]helicene (1) and cationic derivative 1-butyl-3-(2-methyl[6]helicenyl)-imidazolium bromide (2) using high-performance liquid chromatography (HPLC) and supercritical fluid chromatography (SFC) methods. The possibility of using capillary electrophoresis (CE) was also tested. The satisfactory results were obtained with SFC, where the highly selective resolution of four enantiopure 1 and 2 helicenes was achieved in a single run within 5min. The semi-preparative procedure for the isolation of P and M enantiomers of compound 2, including circular dichroism data, is reported here for the first time. The results could be used in further separations and analytical applications targeting carbohelicenes vs. positively charged helicene derivatives.

Chromatographic Studies of Protein-Based Chiral Separations.

The development of separation methods for the analysis and resolution of chiral drugs and solutes has been an area of ongoing interest in pharmaceutical research. The use of proteins as chiral binding agents in high-performance liquid chromatography (HPLC) has been an approach that has received particular attention in such work. This report provides an overview of proteins that have been used as binding agents to create chiral stationary phases (CSPs) and in the use of chromatographic methods to study these materials and protein-based chiral separations. The supports and methods that have been employed to prepare protein-based CSPs will also be discussed and compared. Specific types of CSPs that are considered include those that employ serum transport proteins (e.g., human serum albumin, bovine serum albumin, and alpha1-acid glycoprotein), enzymes (e.g., penicillin G acylase, cellobiohydrolases, and α-chymotrypsin) or other types of proteins (e.g., ovomucoid, antibodies, and avidin or streptavidin). The properties and applications for each type of protein and CSP will also be discussed in terms of their use in chromatography and chiral separations.

Development of methods for separation of binarized fragments of etching pits of semiconductor wafer

The article is devoted to the search of successful methods for separation of fragments belonging to the supposed etching pits of dislocation loops. The developed methods are revealed binarized fragments of etching pits among of the many other elements of surface image of a semiconductor wafer. The filtration method of binarized fragments of etching pits of wafer dislocation uses a roundness index of the specified range, received on the base of reference line width of dislocation loop at a ratio of 1:4. This optional feature allows separating fragments similar to lines of loops of etching pits on the basis of their size and shape. The method of removing the micro-defects loops reduces the number of fragments by eliminating of loops without signs of loops of etching pits. It is based on the use of the XOR subtraction operation between the binarized image of dislocation areas and the image with accentuated loops of the fragments. The criteria for allocation of the main significant loop fragments allow form the selection rules for the further processing of binarized image. The criteria for allocation of the main significant loop fragments, method of binarized fragments filtering, method of removing the loops of micro-defects of the semiconductor wafer are the part of a package of measures to carry out tasks on production management organization and creation of technical diagnostic system of output production quality.

TLC-Direct Bioautography as a High Throughput Method for Detection of Antimicrobials in Plants

The richness of bioactive compounds in plant materials encourages continuous development of separation methods and bioassays for their isolation and identification. Thin-layer chromatography-direct bioautography links separation on the adsorbent layer with biological tests performed directly on it. Therefore, the method is very convenient for searching plant constituents with biological activity, such as antibiotics. Test bacteria grow directly on a plate surface excluding places where antibacterials are located. They can be detected with reagents converted by living bacteria. TLC-DB is a high throughput method enabling analyses of many samples in parallel and the comparison of their activity. Both screening and semi-quantitative analysis is possible. The targeted compounds can be identified using spectroscopic methods, mostly mass spectrometry, that can be performed directly on a TLC plate. This paper discusses all above mentioned aspects of TLC-DB, illustrating them with literature, schemes and our own results.