魔芋中神经酰胺类物质的HPLC-ELSD分析及其含量测定

建立高效液相色谱-蒸发光散射检测器分析神经酰胺的方法并进行了含量的测定.色谱柱:ZORBZX Eclipse XDB-C18(4.6mm×250mm,5μm),洗脱方法:梯度洗脱,柱温:35℃,流动相:甲醇/水,流速:1ml/min;检测器:蒸发光散射检测器,漂移管温度:40℃,氮气流速:1.5L/min.系统探讨了梯度洗脱的起始浓度、洗脱的时间和洗脱梯度的程序设置,最佳的梯度洗脱条件为5min内,甲醇浓度从60%线性增加为90%,从5min到25min,甲醇浓度线性增加为95%,在此条件下样品和标准品的分离色谱峰对称性较好.随后测定了各种样品中神经酰胺的含量,并进行了方法学验证,结果神经酰胺在0.2～2μg之间线性关系良好,最低检测限为0.01mg/ml,R2=0.9992;平均回收率为93.3%,RSD=1.65%(n=5).本法灵敏、方便、准确,重现性好,可用于魔芋神经酰胺类物质的分离及其含量的测定.     

牡丹皮化学成分系统化色谱分离方法研究

以牡丹皮为研究对象,探索中药复杂成分系统化色谱分离方法。通过对牡丹皮甲醇提取物进行系统溶剂萃取,TLC、HPLC分析化合物组成变化,确定牡丹皮化学成分色谱分离的前处理方法;以系统化TLC方法筛选确定了牡丹皮Fr1.3组分制备色谱分离条件;预测了台阶梯度洗脱条件下待分离物质的保留体积。结果显示:牡丹皮甲醇提取物经正己烷、乙酸乙酯依次萃取是有效的色谱分离前处理方法,减少了分离样品复杂性;建立的系统化TLC方法可以快速选定制备柱色谱的分离条件;台阶梯度洗脱分离情况下,分离目标化合物在初始洗脱溶剂下,其0.10Rf≤0.65时,多阶梯梯度预测方法准确预测了Fr1.3组分制备柱分离各化合物的保留体积;选定台阶梯度洗脱条件下,1/3理论上样量、理论上样量、最大上样量的三次制备柱色谱分离实践表明,Fr1.3组分中的6个化合物均得到TLC单点的纯化合物,其中3个化合物的HPLC纯度在95%以上。该方法也可用于其它中药成分的系统化分离。     

小鼠肠道菌群HPLC分析中色谱分离条件的优化

研究了在小鼠肠道菌群的高效离子交换色谱（HPLC）分析中,不同色谱分离条件对分离效果的影响,确立了最佳色谱条件：样品上样于Toyopearl TSKgel SuperQ-650c强阴离子交换树脂柱,以0.02mol/L哌嗪-HCl缓冲液（pH8.0）平衡,洗脱盐浓度为1.0mol/L NaCl,洗脱梯度为0.1-0.5mol/L NaCl线性梯度洗脱80min,再经0.5-0.75mol/L NaCl线性梯度洗脱25min,流速1mL/min,进样量为1mL。小鼠肠道菌群HPLC分析方法的建立,为深入研究小鼠肠道菌群的组成和动态变化奠定了基础。     

化工上的应用

<正>反相高压液相色谱梯度洗脱理论基础的发展,有助于最佳分离条件的选择。采用乙内酞苯硫脲氨基酸(PTH一氨基酸)做模拟系统,选择好梯度洗脱条件,在20分钟内,可以测定19种普通的PTH一氨基酸。虽然,要求平均溶剂强度和理论     

不同参数对蛋白免疫印迹法结果的影响

[目的]探讨曝光时间、洗脱时间等参数对蛋白免疫印迹法结果的影响。[方法]通过测量灰度值,比较不同曝光时间(6 s和21 s)、洗脱时间(30 min和8 h)以及图像处理软件(Image J和Imagequant TL)对蛋白免疫印迹法结果的影响。[结果]上样量比值为2∶1的蛋白曝光6 s的灰度值读数分别为14766±724和7171±577(n=3),均数比值为2. 1∶1。延长曝光至21 s后相同样品灰度值分别为14932±1198和11994±616(n=3),均数比值减小为1. 2∶1;洗脱30 min和8 h后的曝光结果相比,上样量为1. 64μg蛋白灰度值分别为23713±1895和22892±571(n=3),差异无统计学意义; Image J和Imagequant TL两种软件对倍比梯度上样的蛋白样品读值,比值分别为1∶0. 56∶0. 41∶0. 22和1∶0. 59∶0. 40∶0. 15;对两组读值进行直线回归分析,可得斜率分别为0. 13和0. 14,提示两组读值趋势一致。[结论]曝光时间对蛋白免疫印迹法结果有明显影响,高丰度蛋白需要低曝光时间,才能保持确切的倍比关系;洗脱时间超过30 min对蛋白免疫印迹法灰度值结果没有直接影响;不同图像处理软件对灰度值结果分析趋势一致。     

反相C18固相萃取小柱用于蛋白质组分析中少量样品预处理

针对少量且复杂蛋白质组样品,开发一种耗时短、操作简便的分离方法。以人的脑海马组织蛋白样品为研究对象,采用反相C18固相萃取小柱,采用不同乙腈浓度对酶切后样品进行梯度洗脱,与反相高效液相色谱法对比分离效果。通过比较不同乙腈梯度洗脱方案所鉴定到的谱图数、非冗余多肽数、蛋白数和各洗脱组分间重复率分析,确定一种样品量少、简单易行、分离效果好的实验方案。虽然反相C18固相萃取小柱法鉴定蛋白总数占高效液相色谱法的85.5%,但其操作简单,耗时少。通过4种不同乙腈浓度方案比较,确定乙腈洗脱浓度优化为5%、15%、20%和90%时,分离30μg人的海马多肽混合物可以得到较优的分离效果。其蛋白鉴定数为反相液相色谱法的67.0%,且重复性良好。该结果证实反相C18固相萃取小柱分离效果比反相高效液相色谱法稍差,但此方法可以分离少量复杂蛋白质组样品。该方法分离样品充分,操作易行,耗时短,是进行蛋白质组学分析中少量样品的一个简便预处理方法。     

八种樟属肉桂组药用植物中肉桂酸、肉桂醛含量的测定

目的 建立反相高效液相色谱法测定8种樟属肉桂组药用植物中肉桂酸和肉桂醛的含量.方法 采用Liehrospher-C18(4.6mm × 150 mm,5μm)色谱柱,以流动相乙腈和水在0～10 min内由35:65至38:62线性梯度洗脱,在10～15 min内由38:62至50:50线性梯度洗脱,流速1 mL/min,检测波长290 nm,柱温30℃.结果 在8种药用植物中肉桂酸、肉桂醛的色谱峰与共存组分完全达到基线分离,线性范围分别为0.208～4.16 μg(r=0.999 6)和0.013 6～0.34μg(r=0.9999).结论 肉桂和清化桂可以作为肉桂药用,其它6种不能作为肉桂代用.     

四川GAP基地虎杖药材HPLC指纹图谱研究

为了建立虎杖GAP基地药材HPLC指纹图谱,采用梯度洗脱法,对虎杖野生与种植药材进行了HPLC代测定。流动相为乙腈-0．1％磷酸水溶液线性梯度洗脱,检测波长为230nm;记录时间：70min;采用中南大学出版的指纹图谱相似度比较软件进行比较。通过软件的比较,虎杖野生与种植药材的指纹图谱相似度均大于0．90。说明运用梯度洗脱能很好分离虎杖的各类成分,本文所建立的方法可作为虎杖药材质量标准制定的参考依据。     

蛇毒的研究和利用 Ⅱ.湖南产眼镜蛇毒(Naja naja atra)的柱层析分离及各组分的毒性和酶活力测定

应用羧甲基葡聚糖C-25和盐浓度直线梯度洗脱的方法,对湖南产眼镜蛇毒进行了柱层析分离,获得14个蛋白峰,并对柱层析的各组分进行了毒性和酶活性的测定。     

HPLC法同时测定人参皂苷Rb1、Rc、Rd、Rg3、CK和Rh2

目的:建立高效液相色谱法同时测定人参皂苷Rb1、Rc、Rd、Rg3、CK和Rh2的方法.方法:采用ODSC18(4.6 mm×150 mm)色谱柱,流动相乙腈-0.05%磷酸水,梯度洗脱,流速1 Ml/min,检测波长203 nm,柱温35 ℃.结果:人参皂苷Rb1、Rc、Rd、Rg3、CK和Rh2分离效果良好,线性关...     

双水相体系逆流色谱技术在蛋白质分离中的应用

双水相体系逆流色谱技术结合了逆流色谱的高效率、高制备量以及双水相体系适于蛋白质分离的特点,因此在蛋白质的分离方面具有独特的应用价值。本文综述了近年来基于正交轴逆流色谱仪器的双水相体系逆流色谱技术在多种蛋白质分离中的应用。并对一些新兴的蛋白质逆流色谱分离技术及新型逆流色谱柱分离系统进行了介绍。     

Effect of CCC on the gibberellin content of excised sunflower organs

Summary CCC was shown to be effective in retarding stem growth of sunflower; this effect was overcome by gibberellic-acid application. Using an agar-diffusion technique, the gibberellin (GA) content of sunflower apices treated with CCC was found to be significantly reduced as compared to controls. Similarly, the GA content of agar diffusates obtained from 2-day-old sunflower root tips treated with CCC was also significantly reduced as compared to controls.Root exudate or bleeding sap obtained from mature CCC treated sunflower plants contained no measurable GA-like substance, although it could not be argued that this was due to suppression of GA synthesis in the root systems.     

Isolation and characterization of Escherichia coli chromosomal mutants affecting plasmid copy number.

We have isolated chromosomal mutants of an Escherchia coli K-12 strain that maintain higher levels of an F' plasmid. The mutants are designated as plasmid copy number (pcn) mutants. They were detected by selecting for increased lactose fermentation in bacteria deleted for the lac operon but harboring an F'lacI,P pro+ plasmid. When examined for the amount of F' plasmid deoxyribonucleic acid (DNA) by the dye-CsCl isopycnic technique, the mutants show two to seven times as much covalently closed, circular (CCC) DNA as does the parental strain. The increased plasmid level in one mutant strain (pcn-24) was confirmed by DNA-DNA hybridization; however, this latter technique indicated about a twofold lower increase when compared with the increase measured for pcn-24 by the dye-CsCl technique. In mutant pcn-24 the increased amount of F' DNA reflects a proportional increase in monomeric-size plasmid molecules because oligomeric forms are not found. Also, in mutant pcn-24 the extra CCC plasmid copies do not seem to be randomly distributed throughout the cell's cytoplasm but appear complexed in situ with their host's folded chromosome. In all pcn mutants examined to date, the classical sex factor F is maintained at normal levels, whereas the viral plasmid Pl CM is maintained at two to three times the normal level. In all 17 pcn mutants isolated, the pcn mutation maps on the chromosome and not on the plasmid. Finally, the absolute amount of CCC F' DNA detectable in lysates of the six different pcn mutants examined decreased 50 to 90% upon incubation of the lysate at 37 C. In contrast, no loss of CCC DNA occurs when lysates of the parental F' strain are incubated at 37 C.     

Counter-current chromatography for the separation of terpenoids: a comprehensive review with respect to the solvent systems employed

Natural products extracts are commonly highly complex mixtures of active compounds and consequently their purification becomes a particularly challenging task. The development of a purification protocol to extract a single active component from the many hundreds that are often present in the mixture is something that can take months or even years to achieve, thus it is important for the natural product chemist to have, at their disposal, a broad range of diverse purification techniques. Counter-current chromatography (CCC) is one such separation technique utilising two immiscible phases, one as the stationary phase (retained in a spinning coil by centrifugal forces) and the second as the mobile phase. The method benefits from a number of advantages when compared with the more traditional liquid–solid separation methods, such as no irreversible adsorption, total recovery of the injected sample, minimal tailing of peaks, low risk of sample denaturation, the ability to accept particulates, and a low solvent consumption. The selection of an appropriate two-phase solvent system is critical to the running of CCC since this is both the mobile and the stationary phase of the system. However, this is also by far the most time consuming aspect of the technique and the one that most inhibits its general take-up. In recent years, numerous natural product purifications have been published using CCC from almost every country across the globe. Many of these papers are devoted to terpenoids—one of the most diverse groups. Naturally occurring terpenoids provide opportunities to discover new drugs but many of them are available at very low levels in nature and a huge number of them still remain unexplored. The collective knowledge on performing successful CCC separations of terpenoids has been gathered and reviewed by the authors, in order to create a comprehensive document that will be of great assistance in performing future purifications.     

Clinical implications of different calculation algorithms in breast radiotherapy: A comparison between pencil beam and collapsed cone convolution

BackgroundThis investigation focused on the clinical implications of the use of the Collapsed Cone Convolution algorithm (CCC) in breast radiotherapy and investigated the dosimetric differences as respect to Pencil Beam Convolution algorithm (PBC).Material and methods15 breast treatment plans produced using the PBC algorithm were re-calculated using the CCC algorithm with the same MUs. In a second step, plans were re-optimized using CCC algorithm with modification of wedges and beam weightings to achieve optimal coverage (CCCr plans). For each patient, dosimetric comparison was performed using the standard tangential technique (SWT) and a forward-planned IMRT technique (f-IMRT).ResultsThe CCC algorithm showed significant increased dose inhomogeneity. Mean and minimum PTV doses decreased by 1.4% and 2.8% (both techniques). Mean V95% decreased to 83.7% and 90.3%, respectively for the SWT and f-IMRT. V95% was correlated to the ratio of PTV and lung volumes into the treatment field. The re-optimized CCCr plans achieved similar target coverage, but high-dose volume was significantly larger (V107%: 7.6% vs 2.3% (SWT), 7.1% vs 2.1% (f-IMRT). There was a significantly increase in the ipsilateral lung volume receiving low doses (V5 Gy: 31.3% vs 26.2% in SWT, 27.0% vs 23.0% in f-IMRT). MUs needed for PTV coverage in CCCr plans were higher by 3%.ConclusionsThe PBC algorithm overestimated PTV coverage in terms of all important dosimetric metrics. If previous clinical experience are based on the use of PBC model, especially needed is discussion between medical physicists and radiation oncologists to fully understand the dosimetric changes.     

The plant growth retardant CCC as inhibitor of gibberellin biosynthesis inFusarium moniliforme

Summary Gibberellin (GA) production inFusarium moniliforme (Gibberella fujikuroi) is suppresed by adding the plant growth retardant CCC [(2-chloroethyl)trimethylammonium chloride] to the culture medium. A concentration of 0.1 mg/l of CCC causes 50% inhibition whereas 10 mg/l and higher concentrations fully suppress GA production. Dry weight of the mycelium is not, or only slightly reduced in the presence of CCC.Thin-layer chromatography of acidic fractions of CCC-free cultures reveals fluorescent spots at 4 differentR _f values. No fluorescent spots can be detected on chromatograms of acidic fractions obtained from CCC cultures, thus demonstrating that production of all GA's is inhibited by CCC.If CCC is added to the medium 2 or 3 days after inoculation, further GA production is blocked, but the level of GA present at the time of CCC application is maintained. CCC does not enhance inactivation of GA₃ in sterile culture medium, nor in the presence of the fungus. It is therefore concluded that CCC inhibits the biosynthesis of GA in the fungus.Transfer of thoroughly washed mycelium from medium with CCC to fresh medium does not result in GA production because sufficient CCC is carried over in the mycelium to block GA biosynthesis completely.     

The Effect of Gibberellin A3 and (2-Chloroethyl)-trimethylammoniuin chloride on Assimilate Distribution in Gladiolus in Relation to Corm Growth1

The influence of GA and CCC on growth and assimilate translocationto the various plant organs in gladiolus was studied by labellinga single leaf with ¹⁴CO₂ and following the distribution of theassimilates. GA promotes inflorescence growth by directing assimilatemovement towards the inflorescence at the expense of the corm.CCC has a similar but smaller effect. GA and CCC both promoteassimilate translocation from the labelled leaf during the periodof inflorescence growth. When this period is over and the cormbecomes the main sink, translocation from the labelled leafis promoted by CCC but inhibited by GA. The effect of CCC maybe only partly explained on the basis of an increase in GA turnoverin gladiolus.     

Modeling the performance of pilot-scale countercurrent chromatography: scale-up predictions and experimental verification of erythromycin separation

Biosynthesis of polyketide antibiotics, such as erythromycin A (EA), can result in the formation of analogues of the main compound that are chemically and structurally extremely similar. The large-scale purification of these antibiotics by conventional high-performance liquid chromatography (HPLC) can be prohibitively expensive due to the large volume of both solvent and adsorbent required. This study examines the feasibility of using a novel pilot-scale countercurrent chromatography (CCC) machine as an alternative to HPLC. CCC is a low-pressure (typically <4000 kN m(-2)) liquid-liquid chromatographic technique that allows the separation of solutes on the basis of their partitioning between two immiscible liquid phases. The effects of mobile phase flow rate, column rotational speed, and sample injection volume on the attainable yield and purity of EA were investigated. Our results show that, at a mobile phase flow rate of 40 mL min(-1), a rotational speed of 1200 rpm, and an injection volume of 100 mL (10 g total erythromycin), EA could be satisfactorily fractionated with a purity of approximately 92% (w/w) and a recovery yield of approximately 100% (w/w). The total solute throughput was estimated to be 0.41 kg day(-1). More importantly, we demonstrated simple and predictive linear scale-up of the CCC separation based on data obtained from a single laboratory-scale CCC chromatogram, and verified this experimentally. The retention time and peak width of the target compound at the pilot scale could be predicted to within 4% for operation at a range of mobile-phase flow rates and injection volumes. This predictable nature of CCC separations, unlike HPLC methods, can greatly reduce process development times and enable a complete process-scale operating scenario to be planned.     

The Effect of CCC and Amo-1618 on Growth, Catalase, Peroxidase and Indoleacetic Acid Oxidase Activity of Young Barley Seedlings

Barley seedlings were grown in darkness on filter paper saturated with phosphate buffer or CCC and Amo-1618 buffered solutions. The effects of CCC and Amo-1618 on growth, catalase, peroxidase and lAA-oxidase were studied on coleoptile and primary leaves. Both growth-retarding chemicals cause an inhibition of growth, Amo-1618 being more effective than CCC. They have no effect on catalase activity. Increasing concentrations of CCC and Amo-1618 progressively stimulate peroxidase and IAA-oxidase activities (Amo-1618 more than CCC). The enzymatic activity in short-treated plants is higher than in the corresponding control plants of the same height. It is proposed that CCC and Amo-1618 exert their effect on the growth of barley by acting on auxin catabolism.