Enzyme-assisted extraction of cordycepin and adenosine from cultured Cordyceps militaris and purification by macroporous resin column chromatography

Enzyme-assisted extraction (EAE) coupled with macroporous resin column chromatography (MRCC) was successfully used for the extraction and purification of cordycepin and adenosine from cultured Cordyceps militaris. After optimized by the response surface methodology (RSM), the extraction yields of cordycepin and adenosine were 86.45% and 70.06%, respectively. Subsequently, under the optimal separation conditions on NKA-II resins (loading 1 BV of extraction solution with pH 8.0, and eluting with 4 BVs of 70% ethanol at a flow rate of 3 BV/h), cordycepin (purity of 32.5%), and adenosine (purity of 39.9%) were obtained with an overall recovery rate more than 90%.     

蛹虫草菌深层液体发酵耦合大孔树脂吸附高效生产虫草素

虫草素是一种核苷类抗生素,具有抗癌、抗肿瘤等活性,主要由蛹虫草菌液体发酵生产,然而低产量限制了其应用。考虑到高浓度虫草素积累对其生物合成过程产生强烈反馈抑制,提出了应用发酵分离耦合技术移除部分虫草素、弱化反馈抑制作用以提高虫草素产量的策略。通过静态吸附实验,筛选出虫草素吸附和解吸性能良好的大孔树脂NKA-Ⅱ,并优化了大孔树脂使用量（60 g·L^-1）、吸附温度（37℃）和解吸剂种类（100%乙醇）。在30 g·L^-1葡萄糖的分批发酵体系中,经过两次树脂吸附,发酵21 d后虫草素产量达到644.50 mg·L^-1,较对照组提高32.07%;随后,将树脂吸附分离策略应用于补料分批发酵体系,虫草素产量达到787.50 mg·L^-1,较对照组提高35.78%。这些结果表明,将虫草素液体发酵与大孔树脂吸附分离耦合,有效弱化了虫草素反馈抑制,显著提高虫草素产量,为虫草素高效生产分离提供了新思路。     

Response surface analysis of microwave‐assisted extraction of polysaccharides from cultured Cordyceps militaris

BACKGROUND: Microwave‐assisted extraction was employed to extract polysaccharides from cultured Cordyceps militaris. The optimal conditions for polysaccharide extraction were determined by response surface methodology. Box–Behnken design was applied to evaluate the effects of three independent variables (microwave power, extraction time and ratio of solution to solid) on the polysaccharide yield of C. militaris. RESULTS: Correlation analysis of the mathematical‐regression model indicated that a quadratic polynomial model could be employed to optimize the microwave‐assisted extraction of polysaccharide. Response surface plots showed that microwave power, extraction time and ratio of solution to solid exhibited independent and interactive effects on the extraction of polysaccharide. The optimal conditions to obtain the highest polysaccharide yield of C. militaris were a microwave power of 744.8 W, an extraction time of 4.2 min and a ratio of solution to solid of 31.1 mL g^?1. CONCLUSION: Under these optimal conditions, the experimental values agreed with the predicted values, using analysis of variance, indicating the goodness of fit of the model used and the success of response surface methodology for optimizing microwave‐assisted extraction of polysaccharide from C. militaris. Copyright © 2009 Society of Chemical Industry     

High‐performance liquid chromatography separation characteristics of molecular‐imprinted poly(methacrylic acid) microparticles prepared by suspension polymerization

Molecular‐imprinted poly(methacrylic acid) was synthesized with a template of retinoic acid to separate retinoid derivatives. The suspension polymerization technique was used to prepare round microparticles for high‐performance liquid chromatography (HPLC) packing column materials. The effects of the types and amounts of the dispersing agents and surfactants on the structure and size of the prepared molecular‐imprinted‐polymer particles were investigated. The separation of retinoic acid from its derivatives was more efficient when the perfluorocarbon dispersing agent was used instead of water, as the latter reduced the binding force between the objective molecules and monomers. HPLC separation features were also affected by the size and distribution of particles loaded in the column. A higher retention volume was obtained for smaller particles with a broader size distribution. © 2005 Wiley Periodicals, Inc. J Appl Polym Sci 96: 200–212, 2005     

高效液相色谱-二极管阵列检测法快速测定化妆品中褪黑激素

建立了一种简单、快速测定化妆品中褪黑激素的分析方法。样品经甲醇提取后经反相C18柱(4.6×250 mm,5μm)分离,体积比65∶35的水-乙腈作为流动相,等度洗脱,流速为1.0 mL/min,二极管阵列检测器进行检测,检测波长为223 nm,外标法定量。在优化实验条件下,褪黑激素在0.5~100 mg/L范围内线性关系良好,在空白化妆品中添加0.5、5.0、50.0 mg/kg 3个浓度水平时,得到的回收率为91%~101%,相对标准偏差为2.1%~3.9%。方法的检出限(S/N=3)为1.0 mg/kg,定量限(S/N=10)为5.0 mg/kg。该方法简单、快速、准确,可满足化妆品中褪黑激素的检测要求。     

Synthesis of a chiral stationary phase with poly[styrene‐b‐cellulose 2,3‐bis(3,5‐dimethylphenylcarbamate)] by surface‐initiated atom transfer radical polymerization and its chiral resolution efficiency

A chiral stationary phase (CSP) with poly[styrene‐b‐cellulose 2,3‐bis(3,5‐dimethylphenylcarbamate)] was synthesized by the surface‐initiated atom transfer radical polymerization (SI‐ATRP) of cellulose 2,3‐bis(3,5‐dimethylphenylcarbamate)‐6‐acrylate after the SI‐ATRP of styrene on the surface of silicon dioxide supports in pyridine. The successful preparation of the CSP with poly[styrene‐b‐cellulose 2,3‐bis(3,5‐dimethylphenylcarbamate)] was confirmed via Fourier transform infrared spectroscopy, field emission scanning electron microscopy, X‐ray photoelectron spectroscopy, elemental analysis, and thermal analysis. The applicability for the chiral resolution of the CSP with poly[styrene‐b‐cellulose 2,3‐bis(3,5‐diphenylcarbamate)] was evaluated with high‐performance liquid chromatography with 10 racemates under various mobile phases of hexane/alcohol, hexane/tetrahydrofuran (THF), and hexane/chloroform. The results show that the CSP with poly[styrene‐b‐cellulose 2,3‐bis(3,5‐diphenylcarbamate)] could be used in THF and chloroform as eluents. The chiral resolutions of the commercial Chiracel OD, the CSP with cellulose 2,3‐bis(3,5‐dimethylphenylcarabmate), and the CSP with poly[styrene‐b‐cellulose 2,3‐bis(3,5‐dimethylphenylcarbamate)] prepared by SI‐ATRP were examined. © 2011 Wiley Periodicals, Inc. J Appl Polym Sci, 2011     

HPLC法测定建筑涂料及乳液中的苯甲醛

建立了建筑涂料及乳液中苯甲醛的液相色谱检测方法。样品经甲醇提取,用50%甲醇饱和氯化钙溶液(V/V)沉淀聚合物,离心后将上层清液过滤进样。采用C18色谱柱进行分离,甲醇-水为流动相洗脱,二极管阵列检测器进行目标物检测,外标法定量。结果表明:在0~10μg/mL范围内,质量浓度和峰面积呈现良好的线性关系(R=0.9998)。方法检出限0.44 mg/kg,定量限1.47 mg/kg,当添加水平在25 mg/kg、50 mg/kg、100 mg/kg时,加标回收率在87.60%~91.10%,相对标准偏差RSD(n=6)均小于1.5%,该方法可用于建筑涂料及乳液中的苯甲醛的快速检测。     

Chromatographic performance of monodisperse macroporous particles produced by modified seeded polymerization. II. The effect of the diluent/seed‐latex ratio

Poly(styrene‐co‐divinylbenzene)‐based monodisperse macroporous particles were obtained by a modified seeded polymerization technique. The monodisperse polystyrene particles, obtained by dispersion polymerization in sufficiently large sizes and with suitable average molecular weights, were directly used as the seed latex in the production of macroporous particles. Therefore, the number of swelling and polymerization stages in the multistage production was reduced. In the first stage, the seed particles were swollen with a diluent, dibutyl phthalate (DBP), and then with a monomer phase including styrene, divinylbenzene, and, as an initiator, benzoyl peroxide. The monodisperse macroporous particles were obtained by the repolymerization of the monomer mixture in the seed particles. The particles, having different porosity characteristics, were synthesized through variations in the dibutyl phthalate/seed‐latex (DBP/SL) ratio. Selected macroporous particle samples were slurry‐packed into stainless steel high‐performance liquid chromatography columns (300 mm long × 7.8‐mm i.d.). The separation of the protein mixture by these columns in the reversed‐phase chromatography (RPC) mode was investigated. Liquid chromatograms with high resolutions were obtained under an acetonitrile/water gradient over a wide range of flow rates (i.e., 0.5–3 mL/min), especially for the particles produced with a monomer/seed‐latex (M/SL) ratio of 3.0 mL/g. In the RPC experiments, the particles produced with an M/SL ratio of 3.0 mL/g and DBP/SL ratios of 1.0 and 1.5 mL/g exhibited better chromatographic performance than the other samples. The maximum theoretical plate number was 3500 for the particles produced with the M/SL ratio of 3.0 mL/g and DBP/SL ratio of 1.5 mL/g with albumin as the analyte. The size exclusion chromatography (SEC) calibration curves and the back‐pressure/flow‐rate relationships of the produced columns were also determined. The particles obtained with an M/SL ratio of 3.0 mL/g and a DBP/SL ratio of 1.5 mL/g exhibited the best performance in SEC applications. © 2004 Wiley Periodicals, Inc. J Appl Polym Sci 92: 3685–3696, 2004     

Quantification of Phorbol Esters in Jatropha curcas by HPLC‐UV and HPLC‐ToF‐MS with Standard Addition Method

Established analytic methods for the quantification of phorbol esters (PE), which are some toxic components in Jatropha curcas L., include HPLC with UV‐detection with the commercially available phorbol myristate acetate (PMA) as internal standard or HPLC coupled with MS detection with an external calibration, mostly also with PMA. The differences in the fatty acid side chains and connection to the base structure of PMA compared to PE leads to different UV absorption and MS ionization effects and cause problems for exact quantitative measurements. In this paper, a method is presented which combines both detection types and shows differences between both results. For this purpose, an extraction routine is performed on a PE‐containing seed oil to get a PE standard in high purity, which was used for a standard addition method on two real J. curcas oil samples, derived from Ghana and Mexico. Furthermore, a detection window of ±10 ppm for the high accurate ToF‐MS detection is set to eliminate isobaric interferences from co‐eluting material. Method evaluation of inter‐ and intra‐day variance as well as the recovery rate are performed and determined. With this method a limit of detection of 62 ng mL^?1 (UV) and 11 ng mL^?1 (MS) can be achieved. Practical Applications: Due to the good biological and technical properties of Jatropha curcas L., its seed oil seems perfect for the application as biodiesel feedstock. The toxicity on the other hand could cause problems when converting side products from the oil production to products of higher value. With the here described method an accurate and precise analysis procedure for the quantification of the toxic compounds namely, phorbol esters, could be applied for toxicity studies or routine checks in industry which is converting plant material from J. curcas, so that no toxic material is used for example as animal feed. In this paper, an exact and robust analysis method is described for the quantification of phorbol esters (PE) in Jatropha curcas L. seed oil. This method procedure includes the extraction of PE in methanol, chromatographic separation on a reverse phase C18 HPLC column and the quantification by standard addition method. For the standard addition method a highly pure PE standard is used, which is extracted and purified by semi preparative HPLC right before the measurements. The used detector for identification and quantification is UV set at 280 nm and ESI‐ToF‐MS with a ±10 ppm mass difference of the deprotonated and formate adduct pseudo molecular ion of PE.

     

Perfume‐containing polyurea microcapsules with undetectable levels of free isocyanates

Core‐shell polyurea microcapsules with a 40% fragrance load were prepared by interfacial polymerization of guanidine and a technical polyisocyanate prepolymer containing mainly the biuret trimer derived from hexamethylene di‐isocyanate (HDI). Residual free polyisocyanates were still present at a level slightly above 100 mg NCO functional group per kg as determined by liquid chromatography hyphenated with tandem mass spectrometry of HDI and of its biuret trimer. This level was decreased by a factor of about 10 when the polymerization process was allowed to proceed for a longer time and by a factor of about 500 when guanidine or NaOH were added to the microcapsule suspension to act as scavengers. In these cases, polyisocyanate conversion was observed to proceed for about one month when the microcapsules were stored at room temperature before reaching a plateau at a level below 1 mg NCO/kg. Overall, ammonia was the most efficient polyisocyanate scavenger as no residual HDI biuret trimer and only less than 2 μg NCO/kg as HDI were detected at the end of the process, a level which had dropped below the limit of detection of 0.25 μg NCO/kg after about 40 days of aging at room temperature. © 2009 Wiley Periodicals, Inc. J Appl Polym Sci, 2009     

Use of gradient,critical, and two‐dimensional chromatography in the analysis of styrene‐ and methyl methacrylate‐grafted epoxidized natural rubber

The growing number of heterogeneous polymeric species that are being synthesized places increasing demands on existing analytical techniques. Although size‐exclusion chromatography (SEC) has established itself as a powerful analytical tool, it has its limits when complex polymers, e.g., graft copolymers, must be analyzed. In this case, complementary techniques such as gradient HPLC and liquid chromatography at critical conditions (LCCC) are more favorable. The present study describes the synthesis and analysis of methyl methacrylate‐ and styrene‐grafted epoxidized natural rubber by different chromatographic techniques. The grafting efficiency was evaluated by gradient HPLC under normal and reversed phase conditions. Methyl methacrylate‐grafted ENR50 was further analyzed by LCCC, where separation of the rubber and grafted rubber occurred according to chemical composition but was independent of the molar mass of the methyl methacrylate homopolymers. This was followed by the combination of LCCC and SEC, where separation was achieved in two dimensions. Relevant deductions were made of both the chemical composition distribution and the molar mass distribution of the functional groups of methyl methacrylate‐grafted ENR50. © 2003 Wiley Periodicals, Inc. J Appl Polym Sci 88: 2530–2538, 2003     

Characterization of Low‐Molecular‐Weight PLA using HPLC

HPLC is applied and assessed as an effective tool to investigate both the production of PLA by polycondensation and its corresponding degradation. A new HPLC calibration procedure, through which it is possible to fully characterize LMW PLA samples by determining the concentration of each individual oligomer, is developed. A comparison between HPLC, ¹H NMR spectroscopy and non‐aqueous solution titration is also reported in order to confirm the reliability of the proposed method. Finally, the proposed analytical technique is applied to monitor the development of a polycondensation reaction performed at 150 °C and 133.3 mbar for 12 h.

     

Effect of absorbent in solid‐phase extraction on quantification of phospholipids in palm‐pressed fiber

Palm‐pressed fiber (PPF) is a by‐product of palm oil milling and it has been found to contain a high percentage of phospholipids (PL). The aim of this work was to evaluate the best solid‐phase extraction (SPE) method to purify PL from PPF. The purified PL were analyzed using high‐performance liquid chromatography (HPLC) with an evaporative light‐scattering detector (ELSD). The oil was extracted from PPF using the Folch method and classes of PL were purified using SPE. Different solvent phases and normal‐phase SPE cartridges [silica (Si), aminopropyl‐bonded silica (NH₂) and diol‐bonded silica (2OH)] at the same ratio of oil to sorbent mass were used to study the separation of PL from the extracted oil. The recovery of each standard PL was performed in a model oil system with the same amount of standard PL being added, and the repeatability of the SPE method was investigated. The isolation of PL by SPE diol cartridge and subsequent analysis by HPLC/ELSD have shown to be an accurate and reproducible analytical method for the determination of phosphatidylethanolamine, phosphatidylglycerol, phosphatidylcholine and lysophosphatidylcholine in PPF. This method provided a high yield and rapid separation of PL in PPF with less contamination from other lipid groups.     

固相萃取-高效液相色谱法测定化妆品中的丙烯酰胺

建立了保湿防晒系列化妆品中丙烯酰胺含量的高效液相色谱测定方法.采用丙酮超声提取样品中的待测物,提取液经浓缩吹干后用水溶解,经固相萃取小柱净化后用反相高效液相色谱法测定.丙烯酰胺在0.01 mg/L～5.0 mg/L与对应峰面积呈良好线性关系(R=0.999 5).在添加量为0.05 mg/kg,0.50 mg/kg和1.00 mg/kg时,平均回收率在81.3%～83.8%,变异系数在2.3%～3.0%,方法检出限为0.05 mg/kg.结果表明,该法操作简便、灵敏、准确,适用于化妆品中丙烯酰胺含量的测定.     

Kinetic of epoxy resin formation by high‐performance liquid chromatography

This article is focused on the following of the cure of an epoxy resin by high‐performance liquid chromatography (HPLC) and the comparison of the data obtained with those obtained by differential scanning calorimetry (DSC) and dynamic mechanical analysis (DMA) techniques usually employed for characterize curing processes. A reversed‐phase HPLC method with UV detection is developed to study the kinetic of the curing reaction of diglycidyl ether of bisphenol A (DGEBA) with 1,3‐cyclohexanebismethylamine (1,3‐BAC) at 60, 70, and 80°C, before and after gelation. The limits of quantification obtained permit the application of the proposed method until the last steps of the formation kinetic. HPLC and DSC analysis show a good correlation. The gel conversions obtained by HPLC and DMA agree well. © 2003 Wiley Periodicals, Inc. J Appl Polym Sci 89: 497–504, 2003     

Separation Behavior of Octadecadienoic Acid Isomers and Identification of cis‐ and trans‐Isomers Using Gas Chromatography

Using a strongly polar cyanopropyl capillary column we have investigated the gas chromatography (GC) separation behaviors of 24 octadecadienoic acid methyl ester (18:2ME) isomers compared against saturated methyl stearate (18:0ME) and arachidic acid methyl ester (20:0ME), and the dependency on the GC column temperature. The 24 isomers were obtained by performing cis‐to trans‐isomerization of six regioisomers: five of the 18:2ME isomers were prepared by the partial reduction of methyl α‐linolenate and methyl γ‐linolenate C18 trienoic acids with different double bond positions, whereas the sixth isomer, 18:2ME (c5, c9), was obtained from a raw constituent fatty acid methyl ester (FAME) sample extracted from Japanese yew seeds. There are no reference standards commercially available for 18:2ME isomers, and in elucidating the elution order of these isomers this study should help the future identification of cis‐ and trans‐type of 18:2ME. We also report the identification method of cis‐ and trans‐type of FAME using equivalent chain lengths and attempt the identification of cis‐ and trans‐type of 18:2ME isomers from partially hydrogenated canola oil.     

高效液相色谱-二极管阵列检测器法测定西瓜中氯吡脲的残留

采用高效液相色谱-二极管阵列检测器法(HPLC-DAD)测定西瓜中残留的膨大剂(即:氯吡脲).西瓜样品溶于乙酸乙酯进行提取,提取物经固相萃取柱(ENVI-18)富集、净化后,在265 nm处,流动相为甲醇-水(65∶35,V∶V)条件下进行检测.结果表明,质量浓度在2.5～80.0 mg/kg范围内呈良好的线性关系,相...     

Carboxymethylation of cellulosic material (average degree of polymerization 2600) isolated from cotton (Gossypium) linters with respect to degree of substitution and rheological behavior

Carboxymethylation of cellulosic material (average degree of polymerization 2600) containing 76.5% cellulose and 23.6% xylose isolated from cotton (Gossypium) linters was studied under varying reaction conditions with respect to maximum degree of substitution (DS). The influence of reaction conditions on the apparent viscosity of the prepared derivatives was also studied. The conditions optimized were sodium hydroxide concentration 3.75M, monochloroacetic acid concentration 2.05 mol/AGU, carboxymethylation time 3.5 h, and temperature 55°C, to yield carboxymethyl cellulose of DS 0.77. Rheological studies of 1 and 2% solutions of the optimized product revealed their non‐Newtonian pseudoplastic behavior. Repeating units of the optimized product were also determined by HPLC after complete depolymerization of the polymer. © 2005 Wiley Periodicals, Inc. J Appl Polym Sci 96: 1477–1482, 2005     

吸附树脂极性的高效液相色谱法测定及表征

建立了测定及表征吸附树脂极性的高效液相色谱法。DA-201极性树脂、AB-8弱极性树脂和D-101非极性树脂分别填充高效液相色谱柱,以去离子水为流动相,葡聚糖为示踪剂,流速1.0 mL/min,柱温30℃,测定树脂的极性。结果表明,正丁醇、乙醇均可作为单一探针分子,流出顺序可定性表征吸附树脂极性:DA-201极性树脂AB-8弱极性树脂D-101非极性树脂;采用乙醇/甲醇作为探针分子组,通过相对极性法计算,得到3种吸附树脂的定量极性值:DA-201极性树脂极性值100,AB-8弱极性树脂极性值18,D-101非极性树脂极性值0,相对标准偏差(RSD)均小于1.50%。方法稳定可靠,可以有效表达吸附树脂的极性。     

Delivery of bupivacaine included in poly(acrylamide‐co‐monomethyl itaconate) hydrogels as a function of the pH swelling medium

Copolymeric hydrogels of poly(acrylamide‐co‐monomethyl itaconate) (A/MMI) crosslinked with N,N′‐methylenbisacrylamide (NBA) were synthesized as devices for the controlled release of bupivacaine (Bp). Two compositions of the copolymer, 60A/40MMI and 75A/25MMI, were studied. A local anesthetic was included in the feed mixture of polymerization (2–8 mg Bp/tablet) and by immersion of the copolymeric tablets in an aqueous solution of the drug. A very large amount of Bp (36–38 mg Bp/tablet) was included in the gels by sorption due to interactions between the drug and the side groups of the hydrogels. Swelling and drug release were in accordance with the second Fick's law at the first stages of the processes. The swelling behavior of these copolymers depended on the pH of the medium. The equilibrium swelling degree (W_∞) was larger at pH 7.5 (W_∞ ≈ 90 wt %) than at pH 1.5 (W_∞ ≈ 52–64 wt %) due to the ionization of the side groups of the copolymer. Release of the drug also depended on the pH of the swelling medium; at pH 7.5, about 60% of the included drug was released, and at pH 1.5, about 80% was released. Bp release was controlled by the comonomer composition of the gels, their drug‐load, and the pH of the swelling medium. © 2002 Wiley Periodicals, Inc. J Appl Polym Sci 86: 327–334, 2002