Mobile Solvent Research Articles

Study of system properties in reversed-phase liquid chromatography for binary and ternary solvent mobile phase compositions using the solvation parameter model

System maps for the individual system constant of the solvation parameter model and five binary solvent containing 20–70% (v/v) acetonitrile, acetone, methanol, 2-propanol, and tetrahydrofuran on a single octadecylsiloxane-bonded silica column (Luna C18) are used to provide insight into the variation of system properties with mobile phase composition and solvent type. Selectivity differences are dominated by variation in solute size and hydrogen-bond basicity with solvent-dependent variation in dipole-type and hydrogen-bond acid interactions. Interactions involving lone pair electrons are important only in the case of the alcohols and to a lesser extent tetrahydrofuran. Selectivity differences are also dependent on solvent strength. To expand the selectivity space four ternary solvent systems (acetonitrile-methanol-water, acetonitrile-2-propanol-water, methanol-tetrahydrofuran-water, and tetrahydrofuran-2-propanol-water) 1:1:2% (v/v) containing 50% (v/v) total organic solvent are compared with the binary solvent systems at the same organic solvent composition. There is no simple model that links the system properties of the ternary solvents to the binary solvent systems, but it is demonstrated that the ternary solvent systems expand the selectivity space available for reversed-phase separations. The solvation properties of the bulk organic solvents provide insufficient information to predict selectivity in RPLC because of the dominant contribution of water and effects related to the selective solvation of the stationary phase and possible changes in the solvent-dependent microstructure of the mobile phase.

HPLC BASED PROCEDURE DEVELOPMENT FOR MONITORING ACETIC ACID IN DACLATASVIR DRUG

A process for determination of acetic acid (AAD) in Daclatasvir (DAC) active medicinal ingredients applying HPLC technique was developed. The process involved separation AAD using Phenomenex made Synergi polar RP-80A column with gradient manner of elution, employing mobile solvent phase with 0.1% phosphoric acid (solvent A) and acetonitrile (solvent B), at flow velocity of 0.6 ml/min and UV sensor examination at 210 nm. AAD linearity range was 5.01289μg/ml to 30.07733μg/ml with sensitivity of 1.65425μg/ml (LOD) and 5.01289μg/ml (LOQ). Accuracy towards AAD recoveries were 111.3 to 113.0%, 86.7 to 94.4% and 90.9 to 95.7%, and precision towards AAD’s RSD was 1.197% and 3.367%. The projected HPLC process could be applied to analysis of AAD in DAC active medicinal ingredients with sharp selectivity, better sensitivity, and high point accuracy.

A simple generic method for analyzing water sensitive pinacol boronate compounds by hydrophilic interaction liquid chromatography

Pinacol boronates are widely used in the pharmaceutical and biomedical industries as primary building blocks for many chemical reactions, such as Suzuki coupling. However, pinacol boronate esters are often susceptible to hydrolysis and degrade into corresponding boronic acids. This causes a challenge for analyzing these boronates by reversed-phase HPLC (RPLC), where hydrolysis occurs on the column. On the other hand, boronic acids are polar compounds and difficult to be retained in RPLC. Presently, there is a lack of practical and versatile analytical methods that can accurately and simultaneously analyze a wide range of boronate esters and boronic acids. In this work, a simple generic method was developed using hydrophilic interaction liquid chromatography (HILIC) separation to overcome these challenges. More than 40 pinacol boronate compounds in a broad range of diverse structures were screened using ten different HILIC stationary phases. Among all the stationary phases studied, ZIC-cHILIC column demonstrated excellent selectivity and retention of pinacol boronate esters and boronic acids for all of the compounds studied. The high percentage of organic solvent mobile phase in HILIC mode effectively prevents the on-column hydrolysis and provides excellent solubility for boronate ester samples. The compatibility of HILIC mobile phase allows the peaks to be identified directly by mass spectrometry.

Study of System Properties in Reversed-Phase Liquid Chromatography for Binary and Ternary Solvent Mobile Phase Compositions Using the Solvation Parameter Model

Study of System Properties in Reversed-Phase Liquid Chromatography for Binary and Ternary Solvent Mobile Phase Compositions Using the Solvation Parameter Model

Evaluation of acetamiprid and azoxystrobin residues and their hormonal disrupting effects on male rats using liquid chromatography-tandem mass spectrometry.

Endocrine-disrupting compounds as pesticides affect the hormonal balance, and this can result in several diseases. Therefore, the analysis of representative hormones with acetamiprid (AC) and azoxystrobin (AZ) was a good strategy for the investigation of the endocrine-disrupting activity of pesticides. Hence, a sensitive and rapid analytical method using liquid chromatography-tandem mass spectrometry (LC-MS/MS) was developed. The method was validated for the analysis of AC, AZ, estriol, estrone, progesterone, and testosterone in the serum, testis, and liver of rats. The correlation between the residues of pesticides and the disturbance of the endocrine system was evaluated. The different mass parameters, mobile phase types, analytical columns, injection volumes, and extraction solvents were compared to get the lowest limit of detection of the studied compounds. The detection limits of AC, AZ, estriol, estrone, progesterone, and testosterone were 0.05, 0.05, 1.0, 10, and 1.0 ng/ml, respectively. The method developed was applied to evaluate the changes in these hormones induced by the duration of exposure to AC and AZ in rat testis and serum. The hormones level in rat serum and testis had a significant decrease as they were oral gavage treated with different high concentrations of studied pesticides. Both pesticides were distributed in the body of rats by the multi-compartment model (liver, testis, and serum).

Determination of 40 dyes in oxidative hair dye products by high performance liquid chromatography

氧化型染发产品中的多种染发剂具有不同程度的致敏性及其他毒性,建立快速、准确检测多种染发剂的方法,为该类产品监管提供有效的技术手段,十分必要。该研究建立了氧化型染发类产品中40种染发剂的高效液相色谱测定方法。染发产品经含70%乙醇的亚硫酸氢钠水溶液涡旋、超声提取,并经亚硫酸氢钠水溶液稀释后,以0.02 mol/L乙酸铵水溶液(含4%乙腈)和乙腈为流动相,采用Waters Atlantis® T3 MV Kit色谱柱(250 mm×4.6 mm, 5 μm)分离,配合柱温变化进行梯度洗脱,二极管阵列检测器检测,检测波长为235 nm和280 nm,外标法定量。结果表明,40种染发剂在各自范围内线性关系良好,相关系数均大于0.999; 40种染发剂的检出限为5~168 μg/g,定量限为16~504 μg/g;各染发剂在3个添加水平下的平均回收率为81.4%~109.6%, RSD均小于5%;各染发剂标准溶液在24 h内稳定性良好,RSD为0.2%~2.2%。与现行标准检验方法相比,该方法较大程度地增加了单一液相色谱条件下可测定的染发剂,特别是准用染发剂种类(36种),提高了检测效率,并可保证检测结果的灵敏度与准确性,适用于氧化型染发产品中多种染发剂的检测分析。

A Validated Reversed-Phase HPLC Analytical Method for the Analysis of Fenofibrate in Bulk Drug and Tablet Dosage Formulation

Aims: A accurate, precise, and stability-indicating Reversed-Phase HPLC technique has been established for the estimation of fenofibrate in tablet formulation.
 Study Design: Experimental study.
 Place and Duration of Study: Department of Pharmaceutical Sciences, RTM Nagpur University, Nagpur-440033, Maharashtra, India between June 2019 and March 2020.
 Methodology: The chromatographic separation was attained on RP Princeton column (C18) (250 mm x 4.6 mm, 5 µ) with mobile solvent system as a mixture of water (pH 3.0 along o-phosphoric acid) and acetonitrile in the proportion (40:60) v/v, flow rate 1.0 ml per minute, at 240 nm. The retention time of fenofibrate was 3.905 minutes.
 Results: The method demonstrated linearity in the concentration range of 87-232 µg/ml with a coefficient of correlation (r2) of 0.9994. The % RSD was ˂2% and percentage recovery was found to be 99.13-100.74%. The assay of marketed tablet formulations was found to be 99.98%.
 Conclusion: The developed and validated technique as per ICH rules for specificity, accuracy, precision, linearity, and system suitability. Reverse Phase-HPLC technique was utilized to the market formulation.

The influence of fluid dynamics on the selectivity of fast gas–liquid reactions in methanol

One important strategy for process intensification is the enhancement of yield and selectivity in fast gas–liquid reactions, like oxidations, hydrogenations or halogenations. However, the interplay between fluid dynamics and competitive chemical reactions has not yet been understood to an extent that allows tailoring the flow and concentration fields for intensified reactions. To understand the interplay, the fluid dynamic conditions surrounding Taylor bubbles rising in an organic solvent are studied and compared to data of aqueous systems from the literature. The local flow fields are measured using Particle Image Velocimetry (PIV) and compared to spectroscopically derived selectivity data of a competitive consecutive gas–liquid reaction. The general rising behavior of Taylor bubbles in methanol is confirmed to be similar to those of bubbles in aqueous systems. However, as surface active agents do not affect the interface mobility in organic solvents, the local flow structures in the bubble wake differ significantly from those of bubbles rising in water, impacting the mixing behavior. Finally, the flow fields are compared to the concentration fields of the main and side products. Thereby, a decisive influence of the fluid dynamics on yield and selectivity becomes apparent, unveiling the potential for process intensification.

Assessment of the greenness of micellar HPLC method for rapid separation and simultaneous estimation of chlorpheniramine maleate in presence of some co-administered drugs in three pharmaceutical dosage forms using single run

Abstract Sustainable chemistry established one of kind standards to maintain protection of environment through using safer mobile phase composition and/or lower solvent consumption. A fast green micellar HPLC method was developed and applied for the first time aiming at simultaneous determination of chlorpheniramine maleate, one of the most widely used antihistamine in combination with levochlopersatine fenodizoate or dextromethorphan hydrobromide or dexamethasone, in their pure forms, laboratory prepared mixtures and pharmaceutical dosage forms used in alleviating the symptoms of cough resulting from common colds and allergy. The separation was achieved on Kinetex C18 column (100 mm × 4.6 mm i.d., 2.6-μm particle size) using micellar aqueous mobile phase consisting of (30 mM sodium dodecyl sulfate and 50 mM sodium dihydrogen phosphate, pH 5) and ethanol (85:15) with UV detection at 230 nm. The four drugs were successfully separated using isocratic elution in a single run not exceeding 7 min. According to ICH guidelines, the method was confirmed to be linear, accurate and precise over the concentration ranges of 5–60 μg mL−1 for chlorpheniramine maleate, 10–100 μg mL−1 for levocloperastine fenodizoate and dextromethorphan hydrobromide and 5–30 μg mL−1 for dexamethasone. In addition, the greenness of the developed method was assessed using two different tools indicating their least hazardous effect on the environment.

DEVELOPMENT OF HPLC STABILITY DEMONSTRATING METHODOLOGY FOR QUANTIFYING AZELNIDIPINE AND TELMISARTAN IN TABLETS AND BULK TYPES: VALIDATION FOLLOWING ICH DIRECTIVES

Objective: Azelnidipine (AZEL) and Telmisartan (TELM) combination is referred to the sufferers of hypertension. No analytical process has yet been mentioned for the TELM and AZEL combination analysis. We, therefore, have designed for its first time stability demonstrating methodology based on HPLC for analysing TELM and AZEL in the tablets and bulk. Methods: The assay of TELM and AZEL was get done on a 250 mm length C18 column (Supelco, 4.6 mm inner diameter, 5.0 μm particle size), and utilized 0.1M Na2SO4 (pH 3.6) and acetonitrile (55% volume: 45% volume) as the mobile solvents phase, at a stream rate 1.0 ml/min. HPLC recognition of TELM and AZEL was taken by a photodiode array sensor set at 258 nm. For validation of the stability demonstrating methodology proposed in terms of sensitivity, precision, specificity, linearity, device adequacy, robustness and accuracy, ICH directives were followed. Results: Calibration curves of TELM and AZEL were generated in the array of 20-60 µg/ml and 4-12 µg/ml with recovery percentage ranges of 99.62%-101.05% and 97.76%-100.17%, and detection limits of 0.020 µg/ml and 0.009 µg/ml, respectively. TELM and AZEL stability was inspected in the existence of acid, base, light, heat, and oxidation and it was realised to be more stable under oxidation degradation testing conditions employed when compared to acid, alkaline, photo, and heat degradation testing conditions applied. Conclusion: The observations demonstrated that the described HPLC stability demonstrating methodology was suitable for quantitating TELM and AZEL combination in tablets and bulk.

ISOLATION AND CHARACTERIZATION OF A SECONDARY METABOLITE FROM THE AERIAL PARTS OF Launaea (Lactuca) taraxacifolia

Launaea (Lactuca) taraxacifolia also known as wild lettuce is a medicinally important plant of the Asteraceae family. The plant was selected for this study due to its widespread use in traditional medicine and on the basis that the plant is almost going into extinct in Nigeria. The extraction was carried out by subjecting the air dried pulverized plant materials into trichloromethane solvent for a period of some days, followed by filtration and concentration. Separation and purification of the various constituents of the crude were done using thin layer and column chromatography. The mobile phases and solvent blends employed were: normal hexane, trichloromethane, ethyl ethanoate and methanol. The extraction, purification, concentration and chromatographic separations of the extracts were carried out at Post-graduates, chemistry laboratory, Michael Okpara University of Agriculture, Umudike, Nigeria. The NMR spectra analysis was investigated at Monash University Malaysia while the FTIR and GC-MS were carried out at Nanotechnology and Research Institute Malaysia. The NMR analysis was done using a Bruker VSP 500MHZ model machine. The GC-MS was performed using an Agilent 7000 model while the FTIR analysis was carried out on an Agilent 4500 series FTIR system. The structural elucidation of the proposed compound was deduced using GC-MS, FTIR, NMR (13C and 1H) and 2D COSY NMR spectra. This chemical investigation resulted in the isolation of a novel compound SB3 (Octadecahydro-10-(1,2,3,4-tetrahydronaphtalen-3-yl)-4,4-dimethoxy-6b,12b,14b-trimethyl Picen-3-(4H,6bH,14bH)-one. Compound SB3 was isolated using 50% hexane and 50% trichloromethane at an Rf value of 0.692. The MS spectrum of compound SB3 gave a molecular ion peak at m/z 545.81, which corresponded to a molecular formula of C37H53O3.

Dynamics of Opposing Polymer Brushes: A Computer Simulation Study.

Opposing polymer brush systems were synthesized and investigated by molecular modeling. Chains were restricted to a face-centered cubic lattice with the excluded volume interactions only. The system was confined between two parallel impenetrable walls, with the same number of chains grafted to each surface. The dynamic properties of such systems were studied by Monte Carlo simulations based on the dynamic lattice liquid model and using a highly efficient parallel machine ARUZ, which enabled the study of large systems and long timescales. The influence of the surface density and mean polymer length on the system dynamic was discussed. The self-diffusion coefficient of the solvent depended strongly on the degree of polymerization and on the polymer concentration. It was also shown that it is possible to capture changes in solvent mobility that can be attributed to the regions of different polymer densities.

A Study on the Stability of TiO2 Nanoparticles as an Electron Transport Layer in Quantum Dot Light-Emitting Diodes

We report highly efficient and robust quantum dot light-emitting diodes (QLEDs) with Li-doped TiO2 nanoparticles (NPs) as an electron transport layer (ETL). As core materials, ZnO-based inorganic NPs can enhance the performance of QLEDs due to their suitable energy level and solution processability. However, their fast electron mobility and instability in organic solvents are two main obstacles to practical display applications. The colloidal stability of TiO2 NPs in ethanol was confirmed after three day-storage, while ZnO NPs showed severe agglomeration. Inverted structure QLEDs using 3% Li-doped TiO2 NP were successfully fabricated and their optical/electrical properties were investigated. With 3% Li-doped TiO2 NPs, the charge balance in the emitting layer of the QLEDs was improved, which resulted in a maximum luminance of 159,840 cd/m<sup>2</sup> and external quantum efficiency (EQE) of 9.12%. These results were comparable to the performance of QLEDs with commonly used ZnO NPs. Moreover, the QLEDs with the Li-doped TiO2 NPs showed more stable characteristics than those with ZnO NPs after 7 days in ambient conditions. The EQE of the QLEDs with Li-doped TiO2 NPs was reduced by only 4.9%. These results indicate that Li-doped TiO2 NPs show great promise for use as a solution based inorganic ETL for QLEDs.

Anthropogenic emission inventory of multiple air pollutants and their spatiotemporal variations in 2017 for the Shandong Province, China

Shandong is the most populous and highly industrialized province in eastern China, and the resultant poor air quality is a cause for widespread concern. This study combines bottom–up and top–down approaches to develop a high-resolution anthropogenic emission inventory of air pollutants for 2017. The inventory was developed based on updated emission factors and detailed activity data. The emissions of sulfur dioxide (SO2), nitrogen oxides (NOx), particulate matter with aerodynamic diameters smaller than 2.5 and 10 μm (PM2.5 and PM10, respectively), carbon monoxide (CO), volatile organic compounds (VOCs), and ammonia (NH3) were estimated to be 1387.8, 2488.6, 5281.7, 3193.0, 9250.7, 2254.7, and 1210.6 kt, respectively. Power plants were the largest contributors of SO2 and NOx emissions accounting for 43.7% and 41.9% of the total emissions, respectively. CO emissions mainly originated from industrial processes (40.1%), mobile sources (24.8%), and fossil fuel burning (21.2%). The major sources of PM10 and PM2.5 emissions were industrial processes and fugitive dust, contributing 83.0% and 86.9% of their total emissions, respectively. Industrial processes (60.0%) contributed the largest VOC emissions, followed by mobile sources (16.8%) and solvent use (14.5%). Livestock and N-fertilizers were major emitters of NH3, accounting for 69.9% and 21.2% of the total emissions, respectively. Emissions were spatially allocated to grid cells with a resolution of 0.05 ° × 0.05 ° based on spatial surrogates, using Geographic Information System (GIS). Heavy pollutant emissions were mainly concentrated in the central and eastern areas of Shandong, while high NH3–emissions occurred in the western region. Most pollutant emissions from industrial sectors occurred in June and July, while low emissions were recorded between January and February. Range uncertainties in emission inventory were quantified using Monte Carlo simulations. Our inventory provides effective information to understand local pollutant emission characteristics, perform air quality simulations, and formulate pollution control measures.

Efficient Flexible Fitting Refinement with Automatic Error Fixing for De Novo Structure Modeling from Cryo-EM Density Maps

Structural modeling of proteins from cryo-electron microscopy (cryo-EM) density maps is one of the challenging issues in structural biology. De novo modeling combined with flexible fitting refinement (FFR) has been widely used to build a structure of new proteins. In de novo prediction, artificial conformations containing local structural errors such as chirality errors, cis peptide bonds, and ring penetrations are frequently generated and cannot be easily removed in the subsequent FFR. Moreover, refinement can be significantly suppressed due to the low mobility of atoms inside the protein. To overcome these problems, we propose an efficient scheme for FFR, in which the local structural errors are fixed first, followed by FFR using an iterative simulated annealing (SA) molecular dynamics protocol with the united atom (UA) model in an implicit solvent model; we call this scheme “SAUA-FFR”. The best model is selected from multiple flexible fitting runs with various biasing force constants to reduce overfitting. We apply our scheme to the decoys obtained from MAINMAST and demonstrate an improvement of the best model of eight selected proteins in terms of the root-mean-square deviation, MolProbity score, and RWplus score compared to the original scheme of MAINMAST. Fixing the local structural errors can enhance the formation of secondary structures, and the UA model enables progressive refinement compared to the all-atom model owing to its high mobility in the implicit solvent. The SAUA-FFR scheme realizes efficient and accurate protein structure modeling from medium-resolution maps with less overfitting.

Analytical quality by design methodology for botanical raw material analysis: a case study of flavonoids in Genkwa Flos

The present study introduces a systematic approach using analytical quality by design (AQbD) methodology for the development of a qualified liquid chromatographic analytical method, which is a challenge in herbal medicinal products due to the intrinsic complex components of botanical sources. The ultra-high-performance liquid chromatography-photodiode array-mass spectrometry (UHPLC-PDA-MS) technique for 11 flavonoids in Genkwa Flos was utilized through the entire analytical processes, from the risk assessment study to the factor screening test, and finally in method optimization employing central composite design (CCD). In this approach, column temperature and mobile solvent slope were found to be critical method parameters (CMPs) and each of the eleven flavonoid peaks’ resolution values were used as critical method attributes (CMAs) through data mining conversion formulas. An optimum chromatographic method in the design space was calculated by mathematical and response surface methodology (RSM). The established chromatographic condition is as follows: acetonitrile and 0.1% formic acid gradient elution (0–13 min, 10–45%; 13–13.5 min, 45–100%; 13.5–14 min, 100–10%; 14–15 min, 10% acetonitrile), column temperature 28℃, detection wavelength 335 nm, and flow rate 0.35 mL/min using C18 (50 × 2.1 mm, 1.7 μm) column. A validation study was also performed successfully for apigenin 7-O-glucuronide, apigenin, and genkwanin. A few important validation results were as follows: linearity over 0.999 coefficient of correlation, detection limit of 2.87–22.41, quantitation limit of 8.70–67.92, relative standard deviation of precision less than 0.22%, and accuracy between 100.13 and 102.49% for apigenin, genkwanin, and apigenin 7-O-glucuronide. In conclusion, the present design-based approach provide a systematic platform that can be effectively applied to ensure pharmaceutically qualified analytical data from complex natural products based botanical drug.

Simultaneous determination of 22 sunscreen agents in cosmetics by ultra-high-performance liquid chromatography using diode array detector

建立了一种超高效液相色谱-二极管阵列检测法同时测定不同种类化妆品中22种防晒剂的方法。样品首先加入少量四氢呋喃,涡旋、分散、混匀(蜡质化妆品可于50 ℃超声,以便分散完全),然后加入0.1%(v/v)甲酸乙醇溶液振摇、超声提取,稀释、过滤后经Poroshell 120 EC-C18(100 mm×4.6 mm, 2.7 μm)分离,乙腈-异丙醇-50 mmol/L乙酸铵水溶液(含0.05%(v/v)甲酸)梯度洗脱,二极管阵列检测器检测,检测波长311 nm,外标法定量。结果表明,22种防晒剂在各自的范围内线性关系良好,相关系数(r)均在0.998以上。选取两种不同基质类型的样品,在低、中、高3个不同添加水平下的平均回收率为85.2%~112.3%,相对标准偏差(RSD)在0.5%~6.5%之间,方法的检出限和定量限分别为0.7~64 mg/kg和2.4~215 mg/kg。在2、10、50 mg/L3个水平的混合标准溶液的稳定性试验中,甲酚曲唑三硅氧烷组分12 h内稳定,其余组分36 h内稳定。采用该方法测定市售16份化妆品中的防晒剂,其中5份样品检测结果与厂家提供的配方值匹配良好。与《化妆品安全技术规范》(2015年版)收载方法相比,该方法在流动相组成和提取溶剂选择方面,摒弃了四氢呋喃和高氯酸等腐蚀性溶剂的使用,改善了弱极性组分的提取效率,同时增加了检测组分二苯酮-2,被测物稳定性更好,实验操作更便利高效,分离效果更好,结果更准确,兼顾绿色环保,可用于化妆品防晒剂的日常检测。

High-performance countercurrent chromatography for lutein production from a chlorophyll-deficient strain of the microalgae Parachlorella kessleri HY1

Green microalgae are a recognized lutein source; however, processing for lutein production requires additional operations such as extraction and saponification, mainly due to the high green pigment and lipid content in the biomass. In this study lutein was isolated from a chlorophyll-deficient Parachlorella kessleri HY1 strain using high-performance countercurrent chromatography (HPCCC). The lower phase of the biphasic solvent system composed of n-heptane–ethanol–water, 5:4:1.5, v/v/v was used both as biomass extraction solvent and HPCCC mobile phase conferring a high selectivity to the lutein production process. For the HPCCC isolation, a multiple injection method was developed, and ten consecutive sample injections (300 mg per each) were performed. To favor the economics of the process, the HPCCC mobile and stationary phases were separately formulated based on nuclear magnetic resonance (NMR) analyses. This strategy enabled to avoid obtaining immiscible liquid phases from their parent biphasic solvent system, which led to the reduction of the separation process duration and solvent consumption. Overall, 3 g of P. kessleri HY1 strain extract was processed by HPCCC yielding 150 mg of lutein (95% purity, 97% recovery). The results presented here form an efficient and economical basis for the large-scale production of microalgae-sourced lutein.

Enhancing the stability of Geobacillus zalihae T1 lipase in organic solvents and insights into the structural stability of its variants

Critical to the applications of proteins in non-aqueous enzymatic processes is their structural dynamics in relation to solvent polarity. A pool of mutants derived from Geobacillus zalihae T1 lipase was screened in organic solvents (methanol, ethanol, propanol, butanol and pentanol) resulting in the selection of six mutants at initial screening (A83D/K251E, R21C, G35D/S195 N, K84R/R103C/M121I/T272 M and R106H/G327S). Site-directed mutagenesis further yielded quadruple mutants A83D/M121I/K251E/G327S and A83D/M121I/S195 N/T272 M, both of which had improved activity after incubation in methanol. The km and kcat values of these mutants vary marginally with the wild-type enzyme in the methanol/substrate mixture. Thermally induced unfolding of mutants was accompanied with some loss of secondary structure content. The root mean square deviations (RMSD) and B-factors revealed that changes in the structural organization are intertwined with an interplay of the protein backbone with organic solvents. Spatially exposed charged residues showed correlations between the solvation dynamics of the methanol solvent and the hydrophobicity of the residues. The short distances of the radial distribution function provided the required distances for hydrogen bond formation and hydrophobic interactions. These dynamic changes demonstrate newly formed structural interactions could be targeted and incorporated experimentally on the basis of solvent mobility and mutant residues.

A review on chromatography and advancement in paper chromatography technique

Chromatography is a Physical separation technique in which mixture is dissolved in a mobile phase and carried through a structure holding another material called stationary phase. The separation of components depends on differential partitioning between mobile and stationary phase. Literature survey reveals that paper chromatography has been used from ancient times, especially in countries like United State for separation of organic and inorganic components. Paper chromatography is a type of analytical tool which is used for separation of colored componentsusing paper fibers (stationary phase) and solvent (mobile phase) in the atmosphere. Further, investigation should be made to make new advancement in the field of chromatography separation involving identification of types of paper used and gel permeation process.