凝胶层析法纯化纤维素酶的研究

The gel filtration was carried out for purification of cellulase.The influences of chromatographic parameters on the resolution were studied to determine the optimal conditions for purification.The purified endoglucanase was obtained by gel filtration by Superdex 75 prep grade with an activity recovery of 92.8% and the purification factor 4.2.The sample volume should be below 6% of the column bed volume and the column bed height L≥12.0 cm.The optimum catalysis temperature and pH for the enzyme were 55℃and 4.5-.0 respectively.The cellulase was stable at pH ranging from 4.0 to 6.0 and temperature below 60℃.     

硫酸铵沉淀和层析法分离纯化纳豆激酶的研究

利用生产纳豆激酶的Bacillus subtilis进行深层发酵.采用发酵液离心除菌,20%～60%饱和度的硫酸铵沉淀,Superdex 75凝胶过滤层析和SP Sepharose Fast Flow离子交换层析对活性组分进行分离提纯.用纤维平版法测定了活力,并用十二烷基磺酸钠-聚丙烯酰胺凝胶电泳法(SDS-PAGE)对分离纯化的效果进行了检验.结果表明在SDS-PAGE中观察到单一条带,分子量27.7 kD,最终纯化倍数和酶活回收率分别为8.4和49%.     

阴离子交换层析法纯化聚唾液酸工艺

以大肠杆菌发酵所产聚唾液酸粗品(含蛋白质、核酸、无机盐和色素等杂质)为纯化对象,通过静态吸附和梯度洗脱实验选择了分离效果较好的阴离子交换介质Q-琼脂糖凝胶FF,对聚唾液酸的纯化工艺条件进行优化. 得到最佳洗脱条件为：洗脱液为pH 7.2的NaCl-0.02 mol/L磷酸钠缓冲液体系,流速0.8 mL/min,洗脱线性梯度方程为CNaCl=0.0017VEluent (CNaCl为NaCl浓度,VEluent为洗脱液体积),层析柱体积46 mL时最大进样量4.0 mL. 该条件下聚唾液酸回收率在86.0%以上,纯化后样品中的蛋白质含量从1.9%降低至0.04%,纯度在98%以上. 紫外吸收光谱和高效凝胶过滤色谱分析表明,聚唾液酸产品组分均一,重均分子量为303 kDa.     

悬浮法交联PVC树脂中凝胶含量的测定

试验研究了悬浮法交联PVC树脂中凝胶含量的测定条件,确定了空白试验的必要性以及最佳抽提温度和抽提时间等.通过优化测试条件,缩短了测定时间,提高了测定悬浮法交联PVC树脂中凝胶含量的可操作性及测试结果的重现性.     

马来酸噻吗洛尔眼用凝胶的含量测定

建立一种马来酸噻吗洛尔眼用凝胶中噻吗洛尔的含量测定方法。以C18柱为分析柱,以水（1‰三乙胺,用磷酸调节pH值至5．0）-甲醇（60：40）溶液为流动相,检测波长为295nm,柱温为室温,外标法测定,线性范围为0．05--2．0mg·mL^-1,线性方程为A=1．442×10^7c＋1．809×10^4,r=1．000,回收率为99．74％,RSD为0．44％。本法简单、快速、准确,可用于马来酸噻吗洛尔眼用凝胶的含量测定。     

聚氨酯凝胶点的测定

本文用RM-15流变仪研究了端羟基聚丁二烯预聚物和甲苯二异氰酸酯反应时,其粘度随反应时间的增长到产生凝胶化的过程,并通过数据处理和作图方法相结合来确定凝胶点,为研究高分子的凝胶化过程和凝胶点测定,提供了一种可行的方法。     

BCA法检测组分百日咳疫苗中间品蛋白含量的可行性评价

目的评价BCA法检测组分百日咳疫苗中间品蛋白含量的可行性,并进行验证。方法以《中国药典》三部(2015版)Lowry法2的检测结果为对照,对干扰BCA法测定组分百日咳疫苗中间品蛋白质含量的物质进行分析,明确干扰物质的限度;对BCA法的专属性、线性范围、重复性、准确性进行验证,并与Lowry法2的检测结果进行比较。结果 30%硫酸铵对Lowry法2和BCA法测定蛋白质含量有明显干扰,其他缓冲体系均无影响;硫酸铵浓度≤25%时,对Lowry法2检测结果无干扰,低于5%的硫酸铵可通过1 mol/L的盐酸消除,不影响BCA法的测定;凯氏定氮法测定结果显示,组分百日咳疫苗中间品中硫酸铵浓度<5%。BCA法的专属性、线性范围、重复性、准确性均符合验证要求;Lowry法2与BCA法检测结果差异无统计学意义(P> 0.05)。结论 BCA法操作简便、快速、准确、高通量,可用于检测组分百日咳疫苗中间品的蛋白含量。     

利用控制图准确测定丁二烯橡胶的凝胶含量

对性能稳定质量优良的国产丁二烯橡胶(BR)9000样品,进行凝胶含量测定,根据测定结果各统计量绘制凝胶含量测定的X-R(或S)控制图。根据X图、R图和S图,可以综合判断出实验室丁二烯橡胶样品凝含量的测定结果处于良好的受控状态,实验室具有较高的系统和随机控制的能力,测定结果准确可靠。在测定丁二烯橡胶凝胶含量时,系统误差控制在±0.02%范围内,随机误差应小于0.05%,标准偏差应小于0.05。     

两种凝胶过滤介质纯化Sabin株脊髓灰质炎病毒效果的比较

目的比较两种不同的凝胶过滤介质对Sabin株脊髓灰质炎病毒的分离纯化效果。方法将Ⅰ、Ⅱ、Ⅲ型Sabin株脊髓灰质炎病毒收获液各3批经超滤浓缩后,分别采用Sepharose CL-6B和Sepharose 6FF填料进行凝胶过滤纯化,分段收集各纯化峰,检测D抗原及蛋白含量,计算比活性、蛋白去除率及抗原回收率,确定病毒峰收集范围。取抗原含量最高的病毒收获液,利用DEAE Sepharose FF离子交换填料进行纯化后,检测Vero细胞DNA残留量、Vero细胞蛋白残留量、牛血清白蛋白残留量、抗生素残留量。结果 Sepharose 6FF填料按6%柱体积上样进行凝胶纯化,纯化的Ⅰ、Ⅱ、Ⅲ型病毒收获液抗原回收率均值分别为78.69%、78.03%和78.37%,与Sepharose CL-6B填料按3%柱体积上样(77.29%、78.60%和77.36%)相比,差异无统计学意义(P0.05)。两种填料纯化的各型收获液再经离子交换纯化后,蛋白去除率、比活性、Vero细胞DNA残留量、Vero细胞蛋白残留量、牛血清白蛋白残留量、抗生素残留量检测结果均符合《中国药典》三部(2015版)相关要求,两种填料纯化的各型收获液比活性差异无统计学意义(P0.05)。结论使用Sepharose 6FF纯化的各型病毒纯化液抗原回收率和比活性与Sepharose CL-6B无明显差异,但可以减少纯化的上样次数,大幅提高纯化效率,可用Sepharose 6FF替换Sepharose CL-6B。     

血浆特定蛋白检测在IgG层析法中试纯化工艺中的应用

目的通过血浆特定蛋白检测,对层析法纯化IgG的中试工艺进行质量控制。方法采用免疫散射比浊法及BNP特定蛋白分析仪及配套试剂,全面检测生产用和中试纯化工艺用原料血浆的组成,建立血浆中白蛋白(albumin,ALB)、IgG、IgA、IgM、补体3(compliment 3,C3)、C4、转铁蛋白(transferin,TRF)、α1-抗胰蛋白酶(α1-antitrypsin,AAT)、α2巨球蛋白(alpha2-macroglobin,A2M)、触珠蛋白(haptoglobin,HPT)、α1酸性糖蛋白(alpha1-acid glycoprotein,AAG)、铜蓝蛋白(ceruloplasmin,CER)、纤维蛋白原(fibrinogen,FIB)、抗凝血酶Ⅲ(antithrombinⅢ,ATⅢ)、IgG1、IgG2、IgG3、IgG4 18项特定蛋白分布的数据库;对3批中试纯化工艺各步骤中ALB、IgG、IgA、IgM、FIB、AAT的去除情况及IgG回收率进行监测,验证工艺的稳定性;将3批中试纯化工艺制备的原液及成品中IgG含量的检测结果与凯氏定氮法检测结果进行比较,验证免疫散射比浊法的准确度及不同人员重复检测的中间精密度;对中试纯化工艺及低温乙醇法制备的IgG原液的杂质含量及亚类进行比较分析。结果中试纯化工艺用原料混浆中仅TRF、A2M、ATⅢ含量与生产用原料混浆差异有统计学意义(P<0.05);中试纯化工艺用原料血浆经A2P亲和层析去除了大部分的ALB、AAT,辛酸盐沉淀去除了剩余的ALB、FIB和IgM,DEAE离子交换纯化去除了IgA和ALB,IgG总回收率高于70%,表明该工艺稳定性好,提高了目标蛋白的得率。中试纯化工艺制备的IgG原液及成品的检测CV值均<10%,回收率在90%¹10%之间,表明该方法准确度及精密度良好;与低温乙醇法制备的IgG原液相比,中试纯化工艺制备的原液杂质含量更低,IgG的亚类分布与所用的原料血浆相似。结论血浆特定蛋白检测在血浆蛋白纯化工艺的监控和优化中具有应用价值。     

环孢菌素A柱层析硅胶再生工艺

环孢菌素A工业化生产涉及真菌发酵、萃取、浓缩、结晶、硅胶柱层析、脱色、二次结晶等步骤,目前硅胶柱层析步骤的硅胶只能一次使用,现实生产过程产生了大量的废硅胶,环保处理压力大、企业成本高。据此本文开展了环孢菌素A柱层析硅胶再生工艺研究,针对硅胶上吸附的杂质性质,研究了硅胶再生洗脱溶剂系统,通过响应面法优化再生条件,结果表明：选用含有0.26%聚山梨醇的乙醇反向冲洗4个柱体积,再使用双氧水浓度为0.15%的去离子水正向冲洗2.1个柱体积,冲洗流速为5mL/min;使用压缩空气将再生后的硅胶压出,烘干至水分含量为6%。按照本方法得到的再生硅胶按原工艺使用时,环孢菌素A回收率完全达标,并且可以三次重复使用达标。本研究建立了一套切实可行的环孢素纯化硅胶再生工艺,可有效节约工厂生产成本和减轻环境压力。     

人工伴侣与凝胶过滤色谱耦合作用促进高浓度溶菌酶复性研究

研究了人工伴侣系统,即十六烷基三甲基溴化铵(CTAB)和β 环糊精(β CD)促进高浓度变性 还原溶菌酶的氧化复性。确定CTAB与β CD的浓度比为1∶4,CTAB与变性溶菌酶的结合时间在10—50min内复性收率较高。同时发现,利用人工伴侣和凝胶过滤色谱的耦合作用可有效提高溶菌酶的复性收率,在连续复性操作条件下可使进料质量浓度为1mg/mL的变性酶获得87%的总复性收率。     

气相色谱法测定三氯甲烷中微量磷酸三丁酯的条件实验

介绍气相色谱内标法测定三氯甲烷中微量磷酸三丁酯含量的条件实验。结果表明,气相色谱仪的柱温200℃、进样口温度300℃、检测器温度300℃、分流比20∶1、进样量1μL时,信号基线稳定、分峰效果好,相关系数为0.999 97,方法准确、可靠。     

Block copolymer molecular weight determination via gel permeation chromatography: Choosing a combining rule

We present a method for accurately determining the true molecular weights of narrow‐distribution block copolymers, using only a basic gel permeation chromatograph (GPC) equipped with a refractive index detector and calibrated with polystyrene standards. Our approach is based on the well‐known observation that GPC calibration curves for different homopolymers in good solvents are essentially parallel, allowing the curves for different polymers to be described by simple hydrodynamic equivalence ratios r_B versus polystyrene. We present values of r_B, in both toluene and tetrahydrofuran, for various polydiene and hydrogenated polydiene homopolymers commonly incorporated into commercial styrenic block copolymers. These values of r_B must be combined to yield the hydrodynamic equivalence ratio of the block copolymer, from which the block copolymer's true molecular weight can be determined. Three combining rules proposed in the literature are tested against a series of symmetric polystyrene–polybutadiene diblock copolymers of varying molecular weight. A simple linear combining rule accurately represents the results. © 2001 John Wiley & Sons, Inc. J Appl Polym Sci 82: 2056–2069, 2001     

A study of micelles in a base lubricating oil by gel permeation chromatography

Micellar solutions of two polystyrene-poly(ethylene/propylene) block copolymers in a base HVI lubricating oil were studied by gel permeation chromatography (g.p.c.). The Styragel columns employed in the work were calibrated using polyisobutylene fractions. At 50°C each copolymer solution gave a single sharp peak which was well within the resolution of the columns, indicating that the micelles were able to enter the pores of the Styragel and elute through the columns as stable particles. Analysis of the chromatograms showed that the micelles had narrow size distributions. Light scattering provided a method of determining the weight-average molecular weights and apparent mean-square radii of gyration of the micelles. Molecular weights determined from g.p.c. using the universal calibration procedure were in fair agreement with the light scattering values. Unfortunately g.p.c. experiments attempted over the range 75°–150°C were unsuccessful because the Styragel strongly absorbed the copolymer at these higher temperatures.     

凝胶色谱法测试PVC树脂平均分子质量及分子质量分布

叙述了凝胶色谱法的测试原理,介绍了用凝胶色谱测试PVC树脂平均分子质量及分子质量分布的方法。     

Kinetics of petroleum pitch polymerization by gel permeation chromatography

The thermal polymerization kinetics of petroleum pitch molecules with molecular weights (MW) between 400–2000 were studied utilizing gel permeation chromatography (GPC). Two distinctly different reaction mechanisms of the petroleum pitch were observed from the GPC curves during transformation of the pitch to mesophase and semi-coke. When mesophase is forming in the pitch, molecules in the MW range of 400–700 were the most reactive, while molecules with MW greater than 1200 were essentially unreactivc. This lack of reactivity of the larger molecules ensures that the mesophase remains highly fluid during its formation. A change in reaction mechanism occurs after the mesophase has solidified as a result of the thermal polymerization reactions. At this stage, there is an abrupt decrease in the reactivity of molecules with MW less than 1100 and the reactivity of all measured molecules is the same. This significant kinetic change is due to a physical phenomenon in which solid state reactions occur in the infusible (vitrified) polymerized pitch. The significantly reduced reaction rates occurring in petroleum pitch after vitrification are similar to the results observed for other thermosetting materials.     

Can porosity properties of particles really be determined by inverse gel permeation chromatography?

Inverse gel permeation chromatography can be fruitfully used for the texture determination of porous mineral beads. However for swelling macroporous gels the same procedure may lead to flagrant errors, which are pointed out on some examples. Thermodynamic considerations explain such results and some ways to take into account perturbing effects are suggested.     

薄膜专用PP树脂加工性能的凝胶渗透色谱评价

用凝胶渗透色谱测定了不同批号的薄膜专用聚丙烯(PP)树脂T38F的相对分子质量及其分布,评价了产品的相对分子质量及其分布对加工性能的影响.测定结果的重复性和再现性良好.根据分析结果调整相关工艺条件后生产的PP T38F能够满足用户的加工要求.实验结果表明:薄膜专用PP树脂的重均分子量(Mw)控制在(25.0～29.0)...