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探索了大规模生产制备基因工程白细胞介素-2(rIL-2)的新工艺,采用等度和线性梯度相结合的方法,用大型反相高效液相制备色谱法分离提纯rIL-2,并用电泳仪等测量了提纯物的纯度,结果表明:用新方法制备的rIL-2纯度大于95%,冻干后成品比活大于10~(10)IU/g,且上样量大,在实际生产中应用价值很高。 相似文献
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探索了大规模生产制备基因工程白细胞介素-2(rIL-2)的新工艺,采用等度和线性梯度相结合的方法,用大型反相高效液相制备色谱法分离提纯rIL-2,并用电泳仪等测量了提纯物的纯度,结果表明:用新方法制备的rIL-2纯度大于95%,冻干后成品比活大于10~(10)IU/g,且上样量大,在实际生产中应用价值很高。 相似文献
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高效液相色谱法测定橡胶促进剂TBBS 总被引:3,自引:0,他引:3
采用反相高效液相色谱法(HPLC)测定了橡胶促进剂N-叔丁基-2-苯并噻唑次磺酰胺(TBBS)的纯度,标准偏差小于0.3。 相似文献
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反相高效液相色谱法测定3,5-二硝基邻甲苯酰胺含量翟素琴,吕青涛(山东建筑工程学院,济南250010)(山东中医学院,济南250014)3,5-二硝基邻甲苯酰胺(Zoalene)是一种饲料添加剂,可有效地预防和治疗鸡球虫病 ̄[1],是抗家畜球虫病的重... 相似文献
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非水反相高效液相色谱法同时检测人血清中VA,VD3,VE和β—胡萝卜素 总被引:2,自引:1,他引:2
介绍一种用非水反相高效液相色谱法及外标法同时检测人血清中的视黄醇(VA)、胆钙化醇(VD3)、α-生育酚)VE)和β-胡萝卜素含量的方法。此方法实际用于检测胃癌病例及对照血清标本188份,有操作简便,出限低的特点。 相似文献
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用反相高效液相色谱法分析测定Cd,Hg,Pb和Cu 总被引:5,自引:0,他引:5
用直接进样(C18柱)反相高效液相色谱法研究了Men+-Dz(二硫腙)体系的色谱行为,建立了同时测定Cd,Hg,Pb和Cu的分析方法。方法的线性范围为0.01~2.0mg/L,最低检出质量浓度为2.4~5.0μg/L,相对标准偏差为1.8%~9.7%,回收率为94%~103%(Hg除外)。直接进样反相高效液相色谱法比萃取进样正相液相色谱法更快速,更简便,更容易操作,已用于人发测定。 相似文献
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用高效液相色谱法优化苯与对氯氯苄烷基化反应催化剂刘红霞张书胜袁倬斌*(郑州大学分析测试中心郑州)(中国科学技术大学研究生院北京100039)关键词氯-二苯甲烷,制备,三氯化铝,硝基苯,硝基乙烷,高效液相色谱法1997-03-31收稿,1997-09-... 相似文献
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Although linear salt gradient elution ion-exchange chromatography (IEC) of proteins is commonly carried out with relatively short columns, it is still not clear how the column length affects the separation performance and the economics of the process. The separation performance can be adjusted by changing a combination of the column length, the gradient slope and the flow velocity. The same resolution can be obtained with a given column length with different combinations of the gradient slope and the flow velocity. This results in different separation time and elution volume at the same resolution. Based on our previous model, a method for determining the separation time and the elution volume relationship for the same resolution (iso-resolution curve) was developed. The effect of the column length and the mass transfer rate on the iso-resolution curve was examined. A long column and/or high mass transfer rate results in lesser elution volume. The resolution data with porous bead packed columns and monolithic columns were in good agreement with the calculated iso-resolution curves. Although the elution volume can be reduced with increasing column length, the pressure drop limits govern the optimum conditions. 相似文献
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Kato Y Nakamura K Kitamura T Tsuda T Hasegawa M Sasaki H 《Journal of chromatography. A》2004,1031(1-2):101-105
We explored chromatographic conditions to obtain high resolution in protein separations by ion-exchange chromatography (IEC) on a macroporous anion-exchange resin of 10 microm in particle diameter. We studied effects of flow-rate, gradient time (steepness of salt concentration gradient) and column length on resolution in wide ranges. It was found that very high resolutions are attainable at long gradient times with long columns. The resolution continuously became higher as the gradient time and the column length became longer except in some special cases. The dependence of resolution on gradient time was particularly great when the column was long and the gradient time for the change of 0-0.5 M NaCl was longer than 2 h. On the other hand, the effect of flow-rate on resolution was very small. Although the separations at long gradient times with long columns have not been popular in high-performance IEC and it takes several hours for one separation, such separations should be advantageous when very high resolutions are required like in proteomics research. 相似文献
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We applied the model described in our previous paper to the rapid scale-up in the ion exchange chromatography of proteins, in which linear flow velocity, column length and gradient slope were changed. We carried out linear gradient elution experiments, and obtained data for the peak salt concentration and peak width. From these data, the plate height (HETP) was calculated as a function of the mobile phase velocity and iso-resolution curve (the separation time and elution volume relationship for the same resolution) was calculated. The scale-up chromatography conditions were determined by the iso-resolution curve. The scale-up of the linear gradient elution from 5 to 100mL and 2.5L column sizes was performed both by the separation of beta-lactoglobulin A and beta-lactoglobulin B with anion-exchange chromatography and by the purification of a recombinant protein with cation-exchange chromatography. Resolution, recovery and purity were examined in order to verify the proposed method. 相似文献
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Verstraeten M Broeckhoven K Dittmann M Choikhet K Witt K Desmet G 《Journal of chromatography. A》2011,1218(8):1170-1184
We report on a first series of experiments comparing the selectivity and the kinetic performance of constant flow rate and constant pressure mode gradient elution separations. Both water-methanol and water-acetonitrile mobile phase mixtures have been considered, as well as different samples and gradient programs. Instrument pressures up to 1200 bar have been used. Neglecting some small possible deviations caused by viscous heating effects, the experiments could confirm the theoretical expectation that both operation modes should lead to identical separation selectivities provided the same mobile phase gradient program is run in reduced volumetric coordinates. Also in agreement with the theoretical expectations, the cP-mode led to a gain in analysis time amounting up to some 17% for linear gradients running from 5 to 95% of organic modifier at ultra-high pressures. Gains of over 25% were obtained for segmented gradients, at least when the flat portions of the gradient program were situated in regions where the gradient composition was the least viscous. Detailed plate height measurements showed that the single difference between the constant flow rate and the constant pressure mode is a (small) difference in efficiency caused by the difference in average flow rate, in turn leading to a different intrinsic band broadening. Separating a phenone sample with a 20-95% water-acetonitrile gradient, the cP-mode leads to gradient plate heights that are some 20-40% smaller than in the cF-mode in the B-term dominated regime, while they are some 5-10% larger in the C-term dominated regime. Considering a separation with sub 2-μm particles on a 350 mm long coupled column, switching to the constant pressure mode allowed to finish the run in 29 instead of in 35 min, while also a larger peak capacity is obtained (going from 334 in the cF-mode to 339 in the cP-mode) and the mutual selectivity between the different peaks is fully retained. 相似文献
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Capillary electrochromatography of proteins with polymer-based strong-cation-exchanger microspheres 总被引:1,自引:0,他引:1
Monodisperse poly(glycidyl methacrylate-divinylbenzene) microspheres were functionalized with propyl sulfonic acid moieties to obtain beads negatively charged in a wide pH range. They were packed into fused-silica capillary of 50 micro, I.D. in order to separate proteins by capillary electrochromatography (CEC). Baseline separation of four basic proteins as well as three cytochrome c variants with an average column efficiency of 60,000 theoretical plates was obtained under isocratic elution conditions. The high efficiency is attributed to the uniformity of the column packing and the hydrophilic surface coverage of the polymer beads derived from the functionalization process. The effect of pH and salt concentration on protein separations was investigated and the results showed that the CEC separation mechanism is the combination of chromatographic retention and electrophoretic migration. Moreover, the column packed with the strongly acidic poly(glycidyl methacrylate-divinylbenzene) beads was also suitable for protein separations by micro-HPLC with a salt gradient. The comparison between the two kinds of elution modes shows that the column described here exhibited higher peak efficiency with isocratic elution in CEC than with gradient elution in micro-HPLC. 相似文献
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In order to develop efficient separation methods for nucleotides according to their size and heterocyclic composition, the application of ion-exchange, reverse-phase, and normal-phase adsorption HPLC has been studied. The comparative investigation of retention power and selectivity of various packings (non-polar bonded-phase and amino silicas) in relation to nucleotide length and composition yields data which enable suitable packings to be selected and a method of preparing the new packing for a particular separation problem to be formulated. Thus a new anion exchanger with high selectivity and dynamic mass transfer has been prepared for fractionation of large oligonucleotides. The effect of the eluent pH and composition (organic modifier, salt) on retention, selectivity, and resolution in ion-exchange and reverse-phase HPLC has been studied. The optimum separation conditions comprise elution with oppositely directed gradients of the salt and the modifier, use of a precolumn packing that provides the best protection for the main column without loss of its efficiency, and the optimum gradient program for the desired retention of the component of interest. The relation between loading and sample concentration has been studied and the system for gradient elution improved. Our work shows that two-dimensional separation is the most reliable and informative method for preparation of homogeneous oligonucleotides. The hydrophobic-pair ion-exchange mechanism is proposed for ion-pair chromatography. Protected and partially deblocked oligonucleotides, chemically synthesized for genetic engineering studies, have been separated with high selectivity by adsorption (normal-phase) HPLC which is efficient for gradient elution with isohydric eluents. The analysis of a monomeric composition of nano-(pico-) molar amounts of oligonucleotides has been developed; the procedure involves microcolumn digestion of the oligonucleotides with immobilized enzymes followed by microcolumn separation of the nucleoside-mononucleotide mixture. Also, a new slurry method for packing stable HPLC columns with a tightly consolidated, nonshrinkable bed of particles has been developed. 相似文献
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Kato Y Nakamura K Kitamura T Tsuda T Hasegawa M Sasaki H 《Journal of chromatography. A》2003,1009(1-2):141-145
We explored chromatographic conditions to obtain high resolution in protein separations by ion-exchange chromatography (IEC) on a nonporous anion-exchange resin of 2.5 microm in particle diameter. We studied the effects of gradient time (steepness of salt concentration gradient), flow-rate and column length on resolution in much wider ranges than had been studied before. It was found that two distinct conditions exist that provide high resolution. The first is a condition which has widely been employed in current high-performance IEC, namely, a combination of short gradient time, high flow-rate and comparatively short column. Separation times are usually 5-30 min, and even more rapid (1-2 min) separations are possible. The second is the condition which has rarely been employed in high-performance IEC. It is a combination of long gradient time, low flow-rate and long column. Although it takes several hours for one separation, very high resolution is attainable. 相似文献
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An online two-dimensional (2D) strong cation-exchange (SCX)/reversed-phase (RP) nano-scale liquid chromatography/mass spectrometry (nanoLC/MS) method was developed for improved separation and hydrophobic peptide recovery. Sharper and more symmetric RP peaks were observed with the use of a "band re-focusing method", in which an analytical RP column with more hydrophobicity than the RP trap column was used in the system. To recover hydrophobic peptides still unreleased from the SCX column after a conventional salt step gradient due to hydrophobic interaction, a RP step gradient from 10% to 30% acetonitrile (ACN) was applied to the SCX column in the presence of a high salt concentration following the salt gradient. There were 301 unique hydrophobic E. coli peptides identified from the RP fractions. These peptides, which were 19% of all E. coli peptides identified from a 2D run, would not have been identified without the application of the RP gradient to the SCX column. 相似文献
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Jandera P 《Journal of chromatography. A》2002,965(1-2):239-261
Gradient elution is widely used for separation of complex samples in reversed-phase HPLC systems, but is less frequently applied in normal-phase HPLC, where it has a notoriously bad reputation for poor reproducibility and unpredictable retention. This behaviour is caused by preferential adsorption of polar solvents used in mixed mobile phases, which may cause significant deviations of the actual gradient profile from the pre-set program. Another important source of irreproducible retention behaviour is gradual deactivation of the adsorbent by adsorption of even traces of water during normal-phase gradient elution. To avoid this phenomenon, carefully dried solvents should be used. Finally, column temperature should be carefully controlled during normal-phase gradient elution if reproducible results are to be obtained. Working with dry solvents at a controlled constant temperature and using a sophisticated gradient-elution chromatograph, reproducibility of the retention data in normal-phase gradient elution better than 2% may be achieved even over several months of column use. The retention data in gradient elution can be calculated accurately if appropriate corrections are adopted for the gradient dwell volume and for the preferential adsorption of the polar solvents using experimental adsorption isotherms. The average error of prediction for the corrected calculated gradient retention data was lower than 2% for a silica gel column and lower than 3% for a bonded nitrile column, which may be suitable for the optimization of separation. Further, a simple approach is suggested for rapid estimation of changes in the retention induced by a change in the gradient profile in normal-phase HPLC. For such a rough estimation, it is not necessary to know the parameters of the dependence of the solute retention factors on the composition of the mobile phase. 相似文献