应用HPLC测定生物制品中甲醛含量的研究

研究了直接用于液相色谱测定甲醛与2,4-二硝基苯肼的反应条件,使反应在菌疫苗类生物制品自身的pH范围内即弱酸性条件下,无需酸碱度调节,反应产物甲醛衍生物不需要有机溶剂萃取,避免损失,可直接用于液相色谱分析。在检出限及精确度方面更是优越于分光光度法。将样品用2,4-二硝基苯肼溶液衍生后,经C18化学键合硅胶为固定相,以乙睛∶水(60∶40,体积比)为流动相。紫外检测波长360 nm进行测定。方法的平均回收率为95.4%(n=5),两种生物制品6次独立测定的相对标准偏差分别为重组乙型肝炎疫苗(CHO)RSD=0.92%,吸附白喉破伤风联合疫苗RSD=3.92%。甲醛(0.6~3.0μg/mL)范围内,浓度与吸收面积值呈良好的线性关系。结果显示,该研究方法的色谱条件能准确定量、灵敏、准确、重复性好,优于药典的分光光度法,可用于实际检测分析。     

一种测定蛹虫草中虫草酸含量的酶标仪微量反应方法

研究和建立一种基于酶标仪-96孔板高通量测定虫草酸含量的检测方法,并对该方法进行性能评价。以酶标仪为检测仪器,在96孔板内按照设定反应条件微量加入样品和试剂进行显色反应,利用酶标仪测定吸光度值并计算虫草酸含量。通过检测精密度、重复性、回收率,并与分光光度计法进行比较,综合评价该方法的准确度、精确度。结果表明,测定数据具有较高的精密度(样品CM1的RSD值0.829%;样品CM2的RSD值1.772%)、重复性(标准样品B40的RSD值2.061%;样品CM2的RSD值1.599%)、回收率(平均回收率99.24%,RSD值3.666%),测定结果与分光光度法检测结果无显著差异(P>0.05)。结果表明,酶标仪微量法测定准确、重复性好,并可大大减少样品和试剂的用量,该方法方便、快捷、高效,可以替代分光光度法用于虫草酸含量的测定。     

肉苁蓉总黄酮含量的测定

目的：从肉苁蓉中提取并测定总黄酮含量,选择最佳提取工艺条件。方法：以芦丁为对照品,用分光光度法在最大吸收波长510nm对其含量进行测定。结果：测得样品中总黄酮含量C=9．33％,最佳提取工艺：乙醇浓度为70％、料液比1：40、回流时间2h、回流温度70℃。结论：选用芦丁为对照品应用于紫外分光光度法测定肉苁蓉总黄酮含量准确度较高,方法简单,是切实可行含量测定方法。     

紫外-可见分光光度法检测高纯度人C1酯酶抑制剂蛋白质含量的经验公式

目的建立高纯度人C1酯酶抑制剂(C1 esterase inhibitor,C1-INH)蛋白质含量(质量浓度)检测的紫外-可见光分光光度法(简称A_{280 nm}法)，得到双对数方程的经验公式，用于C1-INH精纯阶段含目标蛋白样品的蛋白质含量在线检测。方法以1批高效液相色谱(high performance liquid chromatography,HPLC)纯度98.49%的C1-INH原液作为对照品，用注射用水稀释获得不同质量浓度的稀释品，分别检测稀释品A_{280 nm}值，以稀释品的A_{280 nm}值与理论质量浓度进行不同方程的线性拟合，选取回收率在理论值100%±10%、R²>0.99的质量浓度区间建立最佳拟合方程。在此线性范围内，用原始值和消光值分别拟合得到不同的方程，对2种拟合方程的准确度、精密度和稳定性进行验证。通过原始值均值方程和不同基质液的检测均值来建立经验公式，用该经验公式计算不同批次纯化工艺中间样品的蛋白质质量浓度并与免疫比浊法和凯氏定氮法的检测结果进行比较，确定其准确性。用...     

几种中药DNA提取方法的比较研究

以丹参、绞股蓝、三七为材料,分别采取CTAB法和SDS法提取基因组DNA,并通过紫外分光光度法和琼脂糖凝胶电泳对所提取的DNA样品进行检测,将它们在DNA产量、质量等方面的优缺点进行总结。结果表明,CTAB法能从丹参、绞股蓝中提取高质量的DNA,而三七的DNA更适合用SDS法提取。     

5种常见植物DNA提取效率的比较

以小麦、玉米、甘蓝、花生、菠菜的幼嫩叶片为实验材料,采用高盐低pH值法、SDS法和CTAB法3种不同的DNA提取方法,提取其总DNA,用琼脂糖凝胶电泳检测和紫外分光光度法对所得DNA进行比较分析.结果表明:玉米和菠菜采用SDS法提取基因组DNA效果明显优于CTAB和高盐低pH法.CTAB法对高脂肪花生的DNA提取,所得浓度较高.而高盐低pH值法建议减少使用.     

濒危植物七子花DNA的提取及分析

用不同的方法抽提濒危植物七子花嫩叶DNA ,利用紫外分光光度法对其质量进行鉴定 ,以紫外分光光度法及琼脂糖凝胶电泳 -溴化乙锭染色法对其含量进行双重测定 ,显示适合七子花DNA抽提的最佳方法是改进的SDS法 ,该法适合RAPD分析。用此法对七子花植株不同器官的DNA进行抽提 ,其含量分别是 :嫩叶 >枝芽 >老叶 >嫩茎 >老茎。     

分光光度法测定地骨皮中牛磺酸含量

用分光光度法测定地骨皮中是否含有牛磺酸。在一定条件下,牛磺酸与乙酰丙酮和甲醛反应生成带色的配合物,建立了测定牛磺酸含量的分光光度法。结果表明,地骨皮中含有牛磺酸,已测定样品1中牛磺酸的质量分数为3.124 mg.g-1,样品2中牛磺酸的质量分数为6.203 mg.g-1,且样品2中的牛磺酸质量分数极显著高于样品1(p<0.01)。研究结果表明,地骨皮中含有牛磺酸,而且分光光度法成本低,干扰少,是测定地骨皮中牛磺酸质量分数的较好方法。     

问:高中生物实验“观察根对矿质元素离子的交换吸附现象”中的亚甲基蓝阳离子是否进入了根细胞内部?

答：亚甲基蓝,又称次甲基蓝、美蓝,是一种碱性染料,常用作活体染色,为重要的细胞核染剂,可用于细菌染色,神经组织活体染色,与曙红配合用于血液染色。当配制成0．01％的浓度时对根细胞基本无毒害作用,可用于验证根对矿质元素离子的交换吸附。亚甲基蓝在水中可离解成亚甲基蓝阳离子和氯离子,如果将根长时间泡在其蓝色溶液里,亚甲基蓝阳离子可有3个去向：一部分阳离子通过自由扩散进人细胞壁中的纤维素、半纤维素、果胶交织成的空隙里或者粘在根表皮外,这部分阳离子易被水洗掉,即所谓的浮色;在细胞壁中,果胶质的核基可电离而带负…     

β-榄香烯乳剂逆转多药耐药细胞株MCF-7/ADM对阿霉素耐药性研究

目的：测定中药β-榄香烯乳剂对MCF-7/ADM细胞的无毒剂量,并检测此无毒剂量是否有逆转MCF-7/ADM细胞对化疗药物阿霉素（ADM）的多药耐药（multidrug resistance,MDR）性。方法：采用四甲基偶氮唑蓝（MTT）法测定药物的细胞毒性及耐药细胞逆转倍数;荧光分光光度法测定细胞内药物浓度。结果：无毒剂量的β-榄香烯乳剂(6μg/ml）能显著降低化疗药物ADM对乳腺癌耐药细胞株MCF-7/ADM细胞的IC50,明显增加耐药细胞内药物浓度。结论：初步研究表明β-榄香烯乳剂具有逆转MCF-7/ADM细胞MDR的作用。     

Metal contaminants in commercial thiazine dyes.

The iron, potassium, sodium and zinc content of commercial samples of the thiazine dyes azure A (C.I. 52005), azure B (C.I. 52010), azure C (C.I. 52002), methylene blue (C.I. 52015), new methylene blue (C.I. 52030), polychrome methylene blue, thionine (C.I. 52000) and toluidine blue (C.I. 52040) have been determined by atomic absorption spectrophotometry. The metal concentration varied widely in the 38 samples examined--iron, potassium, sodium and zinc together comprised between 0.02% and 25.35% of individual samples.     

Zinc Chloride Methylene Blue. I. Biological Stain History, Physical Characteristics and Approximation of Azure B Content of Commercial Samples

Zinc chloride methylene blue appeared on the market almost contemporaneously with the zinc-free medicinal form. The former has rarely been reported as being used in blood stains. Recent suspension of manufacture of medicinal methylene blue by it. principal American producer has excited interest in the use of the zinc chloride form for the preparation of blood stains. According to Lillie (1944a,b) the azure B content of zinc chloride methylene blue may have varied from 5 to 30% in the samples studied. Taking the Merck Index (1968, 1976) figures for the spectroscopic absorption maximum (λmax) of 667.8 and 668 nm as standard, recent samples of zinc chloride methylene blue are calculated to contain 6-8% azure B. These figures are baaed on 1) the shift of λmax after exhaustive pH 9.5 chloroform extraction, 2) evaluation of the actual ratio of the observed TiCl₂ dye content to the theoretical for pure zinc chloride methylene blue, 3) comparison of spectroscopic and staining effects of graded hot dichromate oxidation products with those of highly purified azure B-methylene blue mixtures of known proportions.

As far as can be found, medicinal methylene blue is almost the exclusive source of cosin polychrome methylene blue blood stains. Lillie (1944c) included a short series comparing 5 zinc chloride methylene blues with a dozen medicinal methylene blue samples; all were oxidized with hot dichromate to produce successful Wright stains. No effort was made to remove the zinc Exhaustive pH 9.5 chloroform extraction of zinc chloride methylene blue (lot MCB 12-H-29) yielded a small amount of red dye which when extracted into 0.1 N HCI gave λmax = 650. The extraction moved the absorption peak of the zinc chloride methylene blue from 667 to 668 nm and the midpoint of the 90% maximum absorption band, 18 nm wide, from 666.5 to 667.5 nm.     

Visual detection of microencapsulated insecticides with selective staining and scanning electron microscopy

Selective staining with Sudan IV and methylene blue for light microscopy and scanning electron microscopy (SEM) were investigated to determine their potential for detecting and quantifying microencapsulated insecticides. Penncap-M (microencapsulated methyl parathion), Penncapthrin (microencapsulated permethrin), and Dyfonate (microencapsulated fonofos) were selectively stained with Sudan IV but not with methylene blue. Selective staining was not possible for Altosid SF-10 or SR-20 (microencapsulated methoprene) with either stain. Sudan IV enabled detection of some microencapsulated formulations in the digestive content of selected aquatic invertebrates and prepared contaminated pollen samples. Staining intensity with Sudan IV was greatest with acetone but capsular damage was high. A solvent ratio of 50:50 and 20:80 acetone/xylene minimized capsular collapse and maintained good staining intensity. The use of SEM for capsule identification and quantification depended upon the method of sample preparation: the slide smear method was superior to samples prepared by incision or microtomy. SEM was most suitable for investigation of formulations such as methoprene, for which selective staining was not possible. The chemical basis of staining with Sudan IV and potential application of both identification techniques are discussed.     

The purification of methylene blue and azure B by solvent extraction and crystallization.

Detailed schemes are described for the preparation of purified methylene blue and azure B from commercial samples of methylene blue. Purified methylene blue is obtained by extracting a solution of the commercial product in an aqueous buffer (pH 9.5) with carbon tetrachloride. Methylene blue remains in the aqueous layer but contaminating dyes pass into the carbon tetrachloride. Metal salt contaminants are removed when the dye is crystallized by the addition of hydrochloric acid at a final concentration of 0.25 N. Purified azure B is obtained by extracting a solution of commercial methylene blue in dilute aqueous sodium hydroxide (pH 11-11.5) with carbon tetrachloride. In this pH range, methylene blue is unstable and yields azure B. The latter passes into the carbon tetrachloride layer as it is formed. Metal salt contaminants remain in the aqueous layer. A concentrated solution oa azure B is obtained by extracting the carbon tetrachloride layer with 4.5 X 10(-4)N hydrobromic acid. The dye is then crystallized by increasing the hydrobromic acid concentration to 0.23 N. Thin-layer chromatography of the purified dyes shows that contamination with related thiazine dyes is absent or negligible. Ash analyses reveal that metal salt contamination is also negligible (sulphated ash less than 0.2%).     

Zinc Chloride Methylene Blue, Ii. Preparation of Giesma and Wright Type Low Azure B Blood Stains from Dichromate Oxidized Commercial Dye

TO determine the amount of K₂Cr₂O₇ required to produce optimal Giemsa type staining, six 1 g amounts (corrected for dye content) of zinc methylene blue were oxidized with graded quantities of K₂Cr₂O₇ to produce 4, 8, 12, 16, 20 and 24% conversion of methylene blue to azure B. These were heated with a blank control 15 minutes at 100 C in 60-65 ml 0.4 N HCI. cooled, and adjusted to 50 ml to give 20 mg original dye/ml. Aliquots were then diluted to 1% and stains were made by the “Wet Giemsa” technic (Lillie and Donaldson 1979) using 6 ml 1% polychrome methylene blue, 4 ml 1% cosin (corrected for dye content), 2 ml 0.1 M pH 6.3 phosphate buffer, 5 ml acetone, and 23 ml distilled water. The main is added last and methanol fixed blood films are stained immediately for 20-40 min.

For methylene blue supplied by MCB 12-H-29, optimal stains were obtained with preparations containing 20 and 24% conversion of methylene blue to azure B. With methylene blue supplied by Aldrich (080787), 16% conversion of methylene blue to azure B was optimal. Eosinates prepared from a low azure B/methylene blue preparation selected in this way give good stains when used as a Wright stain in 0.3% methanol solution. However, when the 600 mg eosinate solution in glycerol methanol is supplemented with 160 mg of the same azure B/methylene blue chloride the mixture fails to perform well. The HCI precipitation of the chloride apparently produces the zinc methylene blue chloride salt which is poorly soluble in alcohol. It appears necessary to have a zinc-free azure B/methylene blue chloride to supplement the probably zinc-free eosinate used in the Giemsa mixture.     

Analysis of methylene blue in human urine by capillary electrophoresis

A capillary electrophoresis method for the determination of the dye methylene blue (tetramethylthionine, MB) in human urine depending on liquid/liquid-extraction and diode array detection has been developed, validated, and applied to samples of healthy individuals, who had been dosed with methylene blue within clinical studies. After extraction with dichloromethane and sodium hexanesulfonate, sample extracts were measured on an extended light path capillary. The dye was detected simultaneously at 292 and 592 nm using methylene violet 3 RAX as internal standard. The limit of quantification was 1.0 microg/ml. The accuracy of the method varied between -15.2 and +0.8% and the precision ranged from 2.0 to 12.0%. The method was linear at least within 1.0 and 60 microg/ml. In contrast to earlier indirect determinations no leuco methylene blue (LMB) was directly detected in urine, whereas in aqueous test solutions containing surplus amounts of ascorbic acid leuco methylene blue was well separated from MB in a single run.     

Quantification of sodium dodecyl sulfate in microliter biochemical samples by gas chromatography

A novel gas chromatography (GC) method has been developed to accurately quantitate sodium dodecyl sulfate (SDS) in aqueous biochemical samples. This method is based on the quantitative conversion of SDS to 1-dodecanol in the GC injection port at elevated temperature, and the thermal degradation product 1-dodecanol was analyzed to determine SDS concentration. It was found that the addition of guanidinium chloride (GnHCl) to SDS samples (via direct dilution with GnHCl/MeOH solution) is necessary to ensure accurate quantitation. The presence of GnHCl enables quantitative conversion of SDS to 1-dodecanol, improves sensitivity, and virtually eliminates interference from proteins and other chemicals commonly present in biochemical samples. The method features direct analysis of diluted SDS samples, is free from interference, and is capable of quantifying less than 1 ng SDS in biochemical samples. It is also suitable for samples with limited volume, with as little as 1 microl sample being sufficient for quantitation.     

ON THE NATURE OF THE DYE PENETRATING THE VACUOLE OF VALONIA FROM SOLUTIONS OF METHYLENE BLUE

When uninjured cells of Valonia are placed in methylene blue dissolved in sea water it is found, after 1 to 3 hours, that at pH 5.5 practically no dye penetrates, while at pH 9.5 more enters the vacuole. As the cells become injured more dye enters at pH 5.5, as well as at pH 9.5. No dye in reduced form is found in the sap of uninjured cells exposed from 1 to 3 hours to methylene blue in sea water at both pH values. When uninjured cells are placed in azure B solution, the rate of penetration of dye into the vacuole is found to increase with the rise in the pH value of the external dye solution. The partition coefficient of the dye between chloroform and sea water is higher at pH 9.5 than at pH 5.5 with both methylene blue and azure B. The color of the dye in chloroform absorbed from methylene blue or from azure B in sea water at pH 5.5 is blue, while it is reddish purple when absorbed from methylene blue and azure B at pH 9.5. Dry salt of methylene blue and azure B dissolved in chloroform appears blue. It is shown that chiefly azure B in form of free base is absorbed by chloroform from methylene blue or azure B dissolved in sea water at pH 9.5, but possibly a mixture of methylene blue and azure B in form of salt is absorbed from methylene blue at pH 5.5, and azure B in form of salt is absorbed from azure B in sea water at pH 5.5. Spectrophotometric analysis of the dye shows the following facts. 1. The dye which is absorbed by the cell wall from methylene blue solution is found to be chiefly methylene blue. 2. The dye which has penetrated from methylene blue solution into the vacuole of uninjured cells is found to be azure B or trimethyl thionine, a small amount of which may be present in a solution of methylene blue especially at a high pH value. 3. The dye which has penetrated from methylene blue solution into the vacuole of injured cells is either methylene blue or a mixture of methylene blue and azure B. 4. The dye which is absorbed by chloroform from methylene blue dissolved in sea water is also found to be azure B, when the pH value of the sea water is at 9.5, but it consists of azure B and to a less extent of methylene blue when the pH value is at 5.5. 5. Methylene blue employed for these experiments, when dissolved in sea water, in sap of Valonia, or in artificial sap, gives absorption maxima characteristic of methylene blue. Azure B found in the sap collected from the vacuole cannot be due to the transformation of methylene blue into this dye after methylene blue has penetrated into the vacuole from the external solution because no such transformation detectable by this method is found to take place within 3 hours after dissolving methylene blue in the sap of Valonia. These experiments indicate that the penetration of dye into the vacuole from methylene blue solution represents a diffusion of azure B in the form of free base. This result agrees with the theory that a basic dye penetrates the vacuole of living cells chiefly in the form of free base and only very slightly in the form of salt. But as soon as the cells are injured the methylene blue (in form of salt) enters the vacuole. It is suggested that these experiments do not show that methylene blue does not enter the protoplasm, but they point out the danger of basing any theoretical conclusion as to permeability on oxidation-reduction potential of living cells from experiments made or the penetration of dye from methylene blue solution into the vacuole, without determining the nature of the dye inside and outside the cell.     

Analysis of rhamnolipid biosurfactants by methylene blue complexation

Rhamnolipids, produced by Pseudomonas aeruginosa, represent an important group of biosurfactants having various industrial, environmental, and medical applications. Current methods for rhamnolipid quantification involve the use of strong hazardous acids/chemicals, indirect measurement of the concentration of sugar moiety, or require the availability of expensive equipment (HPLC-MS). A safer, easier method that measures the whole rhamnolipid molecules would significantly enhance strain selection, metabolic engineering, and process development for economical rhamnolipid production. A semi-quantitative method was reported earlier to differentiate between the rhamnolipid-producing and non-producing strains using agar plates containing methylene blue and cetyl trimethylammonium bromide (CTAB). In this study, a rapid and simple method for rhamnolipid analysis was developed by systematically investigating the complexation of rhamnolipids and methylene blue, with and without the presence of CTAB. The method relies on measuring the absorbance (at 638 nm) of the rhamnolipid−methylene blue complex that partitions into the chloroform phase. With P. aeruginosa fermentation samples, the applicability of this method was verified by comparison of the analysis results with those obtained from the commonly used anthrone reaction technique.