Sequential centrifugal layer chromatography (SCLC): A New technique for the isolation of natural compounds. Part 1: Description of the method and practical aspects

Sequential centrifugal layer chromatography (SCLC), a new preparative planar separation technique, combines the advantages of preparative centrifugal layer chromatography (CLC) and analytical, horizontal, anticircular, and sequential thin-layer chromatography (TLC). In this on-line preparative method, solvent delivery is locally and temporally variable, which means that the mobile phase can be brought onto the plate at any desired place. The SCLC apparatus operates in two modes: circular, with the help of centrifugal force in an inclined position, and anticircular, by means of capillary action in a horizontal position. These two operation modes can be combined and repeated as often as required, hence the separation space becomes practically unlimited. In this way the capacity and separation performance is significantly increased. In the present paper the apparatus and the method are described and some practical aspects are mentioned.     

A Simple and Economical Apparatus for Developing Thin-layer Chromatography Plates in the Anticircular Mode

Abstract

An improved method for developing thin layer chromatography plates in the anticircular mode is described. The apparatus consists of two glass dishes arranged concentrically. The outer dish has a diameter of 98 mm × 15 mm deep, and the inner one a diameter of 92 mm × 13 mm deep. The inner dish rests on four small pieces of glass (5 × 5 × 2 mm). A paper wick, (16 mm wide × 1.5 mm thick) sits between the two dishes and transfers the solvent from the outer dish to the thin layer plates. The plate sits on the paper wick with the adsorbent facing down. To ensure an even solvent flow, the dishes are situated on a platform which has adjustable legs, and an horizontal level. Up to 50 samples can be analyzed on one 10 × 10 cm plate in 5 minutes. Less than 10 ml of solvent are needed.     

High-throughput preparative process utilizing three complementary chromatographic purification technologies

A high-throughput process was developed in which wells in plates generated from parallel synthesis are automatically channeled to an appropriate purification technique using analytical data as a guide. Samples are directed to either of three fundamentally different preparative techniques: HPLC with UV-triggered fraction collection, supercritical fluid chromatography (SFC) with UV-triggered fraction collection, or HPLC with MS-triggered fraction collection. Automated analysis of the analytical data identifies the product compound mass and creates work lists based on chromatographic properties exhibited in the data so that each preparative instrument cherry picks the appropriate list of samples to purify when a preparative-scale plate is loaded.     

Identification of subnanomole amounts of PTH-amino acids by high performance thin-layer chromatography

Summary High performance thin-layer chromatography (HPTLC) of PTH-amino acids on 5×5 cm silica gel plates precoated with a fluorescence marker gives 10–20 fold increase in sensitivity compared to ordinary silica gel plates. Separation of PTH-Leu from PTH-Ile is easily achieved in contrast to chromatography on polyamide sheets. Only two solvent systems are required and as many as 12 samples can be chromatographed on each plate. However, if the sample is contaminated with N-phenylthiourea a third solvent system is necessary.     

Preliminary results for 2‐D separation with high‐performance thin‐layer chromatography and pressurized planar electrochromatography

Preliminary results of 2‐D separation of test dye mixture using high‐performance thin‐layer chromatography (HPTLC) and pressurized planar electrochromatography (PPEC) are demonstrated. The advantage of 2‐D HPTLC/PPEC separation is based on different separation selectivities obtained in both HPTLC and PPEC systems. HPTLC RP18 W plates of 5×20 cm from Merck were used in the investigations. In the first dimension, a HPTLC process was performed using 5 cm length of the plate and in the second dimension PPEC separation was obtained applying plate of 20 cm length. PPEC process followed prewetting the chromatographic plate with sample zones on it, which were partly separated after first dimensional (HPTLC) separation. In the experiments, the modified version of PPEC device for 20 cm long chromatographic plate and the reservoir for prewetting the adsorbent layer were applied.     

The Use of Polystyrene Gels in Preparative Recycle-HPLC for the Separation of Structurally Related Molecules

Abstract

This paper deals with the use of polystyrene gels in high performance liquid chromatography for preparative separations of small molecules with very similar structures. The preparative chromatograph, equipped with a recycle device and made in our laboratory, is described. The column set, consisted of three columns packed with low porosity polystyrene gel. The mobile phase was diisopropylether. Three particle sizes (10μ, 20μ, 50μ) were studied for improving the preparative performance. We checked the very high efficiency of our recycling system versus sample load. High sample loadings (up to 100 grams) can be injected when the separation does not require a large number of plates but typical sample loads are 10–20 grams for high performance separations. Some examples of separation of diastereoisomers, configurational isomers or related structure molecules are given. The advantages of our preparative system are discussed.     

工业色谱法分离制备7-木糖基-10-去乙酰紫杉醇酶解产物10-去乙酰紫杉醇

基于工业色谱法分离制备抗癌药物紫杉醇的半合成前体10-去乙酰紫杉醇(10-DAP)。7-木糖基-10-去乙酰紫杉醇(10-DAXP)在我国特有红豆杉品系(中华红豆杉)枝叶中含量丰富,以其为原料可制备紫杉醇最理想的半合成前体——10-DAP。本研究以部分纯化后的7-木糖基-10-去乙酰紫杉烷为原料,通过β-木糖苷酶水解该粗提物中的主要成分10-DAXP及其两个微量类似物7-木糖基-10-去乙酰三尖杉宁碱(10-DAXC)和7-木糖基-10-去乙酰紫杉醇C(10-DAXP C),脱去其C-7位上的木糖基,水解产物采用大孔吸附树脂吸附,正相快速柱分离和反相制备色谱分离,可获得高纯度的目标物10-DAP,产物纯度为96%,整个工艺的收率大于75%。该方法适合以10-DAXP为原料大规模制备紫杉醇的半合成前体化合物10-DAP,为工业化生产紫杉醇开辟了一条新途径。     

Odour evaluation,fraction collection and preparative scale separations with glass capillary columns

A simple but effective GC approach to odour evaluation and fraction collection with capillary columns is described. An allglass device splits the column effluent, with one splitter arm going to an FID. For odour evaluation, the other splitter arm is led via teflon tubing to the nose. Off-line heart-cutting is carried out by collecting the split effluent on a glass capillary microtrap, the inner walls of which are coated with OV-101. This allows recovery of nanogram amounts of material. Multipass operation or repeated collection leads to preparative capillary GC for further spectroscopic investigation. These techniques do not involve sophisticated equipment or valves. Applications of this type of smell analysis, heart-cutting and enrichment in the analysis of Rhododendron fragrantissimum essential oil, black pepper essential oil and hop essential oil, illustrating the versatility of the systems, are presented.     

Preconcentration of samples in sandwich tanks for continuous thin-layer chromatography

It is demonstrated that horizontai sandwich tank with two cover plates can be used to preconcentrate sample solutions. Narrow sharply-defined starting zones are obtained just outside the smaller cover plate when the series of spots of the sample solution are applied on the thin layer and predeveloped with a volatile solvent. After evaporation of the solvent, the whole area of the TLC-plate is covered with two plates and developed with a suitable solvent.     

Basalt fibers functionalized with gold nanoparticles for in‐tube solid‐phase microextraction

Basalt fibers were functionalized with gold nanoparticles and characterized by scanning electron microscopy and energy‐dispersive X‐ray spectroscopy. An in‐tube solid‐phase microextraction device was developed by packing the functionalized basalt fibers in a polyether ether ketone tube. The device was connected into high performance liquid chromatography equipment with a diode array detector to build online enrichment and analysis system. Eight polycyclic aromatic hydrocarbons were used as model analytes, important factors including sampling rate, sampling volume, organic solvent content in sample, and desorption time were investigated. Linear range (0.01–20 μg/L), detection limits (0.003–0.015 μg/L), and enrichment factors (130–1628) were given by the online analysis method. Relative standard deviations (n = 5) of extraction repeatability on one tube and tube‐to‐tube repeatability were less than 5.2 and 14.7%, respectively. The analysis method was applied to detect polycyclic aromatic hydrocarbons in environmental water samples, and relative recoveries ranged from 87 to 128%.     

Wet effluent parallel plate diffusion denuder coupled capillary ion chromatograph for the determination of atmospheric trace gases

We describe an inexpensive, compact parallel plate diffusion denuder coupled capillary IC system for the determination of soluble ionogenic atmospheric trace gases. The active sampling area (0.6x10 cm) of the denuder is formed in a novel manner by thermally bonding silica gel particles to the surface of Plexiglas plates. The effluent liquid from the parallel plate diffusion denuder is collected and preconcentrated on a capillary preconcentrator column before analysis using a capillary ion chromatograph. Using SO(2) as the test gas, collection efficiency is essentially quantitative at air sampling rates up to 500 ml min(-1). The system provides a limit of detection (LOD) of 1.6 parts per trillion for SO(2) for a 10 min sampling period.     

Vortex counter-current chromatography

A novel counter-current chromatographic system is developed by mounting a vortex column on a type-I coil planet centrifuge. The column is fabricated from a high-density polyethylene disk (16 cm diameter and 5 cm thick) by making multiple holes of various diameters (3-12.5 mm) each arranged in a circle and connected with narrow transfer ducts. The performance of this vortex column is tested with three different two-phase solvent systems with a broad range in hydrophobicity. The results indicated that the smallest diameter column (3mm diameter, 120 units with 42.8 ml capacity) yielded the best separation with the height equivalent to a theoretical plate of 2 cm compared with 20 cm required by the conventional multilayer coil column of high-speed CCC. By avoiding the use of an Archimedean Screw Force, the system shows a low column pressure which would permit safe operation of a large preparative column without a risk of leakage of solvent and column damage.     

高速逆流色谱分离同分异构体

应用高速逆流色谱法对同分异构体的分离纯化进行研究。实验结果表明,以正己烷-乙酸乙酯-水(体积比为1∶10∶10)为两相溶剂系统,上相为固定相,下相为流动相,进行二次高速逆流色谱分离,可从茶多酚中分离出g级的儿茶素同分异构体——(-)-表没食子儿茶素没食子酸酯(EGCG)和(-)-没食子儿茶素没食子酸酯(GCG),其高效液相色谱纯度均在98%以上。选择四氯化碳-氯仿-甲醇-水(体积比为7∶3∶7∶3)为溶剂系统,下相为固定相,上相为流动相,经一次高速逆流色谱即可将药物中间体溴代苯胺同分异构体进行有效的分离。     

薄层色谱法检测甘薯呋喃萜类毒素

甘薯呋喃萜类毒素是甘薯受到损害后产生的强制性代谢产物,可用乙醚提取。乙醚提取物分别用５％Ｎａ２ＣＯ３和蒸馏水洗涤,无水硫酸钠干燥,蒸去乙醚。残渣（粗品）采用薄层色谱法进行检测,以纯品甘薯酮和甘薯酮醇作标样。结果表明：在２０℃条件下,在石油醚－醋酸乙酯（２１,Ｖ／Ｖ）和苯－甲醇（９１,Ｖ／Ｖ）两种溶剂系统中,对甘薯酮（Ｃ）、甘薯酮醇（Ａ）、化合物Ｂ和Ｄ的呋喃萜类毒素具有较好的分离效果,Ｒｆ值的重复性也较好。展开后的薄层板可以通过ＣＳ－９３０ＴＬＣ扫描仪在５２７ｎｍ下扫描定量。     

液相微萃取-高效液相色谱联用分析化妆品中痕量的雌激素

建立了液相微萃取-高效液相色谱联用(LPME-HPLC)测定爽肤水中痕量的雌三醇、雌二醇、炔雌醇和雌酮的分析方法,研究了萃取溶剂种类、接受相体积、搅拌速度、萃取时间等对萃取效率的影响。结果表明,该方法对4种雌激素的富集倍数可达到247~343倍,方法的线性范围为1~200 μg/L,检出限为0.4~1.0 μg/L,6次平行测定的相对标准偏差为3.6%~7.3%,爽肤水中的加标回收率为101.2%~114.9%。方法简单快速、灵敏度高、环境友好,满足痕量雌激素分析的要求。     

Open-tubular liquid chromatography under turbulent and secondary flow conditions

Summary Liquid chromatography in open tubes at flow rates around 0.1 cm · s^–1 and less can generate an enormous number of theoretical plates but suffers from long analysis times. The Golay and van Deemter equations are also obeyed with high accuracy.If straight tubes of circular cross section and very smooth inner surface are used, secondary flow effects can be neglected. Thus the plate height should depend only on the flow velocity and the number of theoretical plates should decrease with increase in linear flow, whereas the number of plates per unit time should be constant.Under turbulent conditions, however, the plate height seems to be more or less independent of linear flow over a wide range. Increasing the flow to several hundred cm · s^–1 causes, for example, 0.2 mm ID capillaries to show the reverse effect and a slight decrease in plate height is found. Alkali treated and silanized capillaries below 100 m i. d. deviate from the Golay prediction before turbulence occurs and a relative decrease in the plate height and thus higher efficiency is found possibly caused be secondary flow effects in the coiled tube or the roughness of the inner walls. These effects allow an increase in the efficiency of given open-tubular systems simply by increasing the solvent velocity drastically. Although plate heights are in the range mm to cm, efficiencies, in theoretical plates per second, similar to classic HPLC, are found. Therefore the quite interesting possibility of overcoming the well-known restrictions of classical HPLC to achieve high efficiency liquid chromatograms exists.Presented at the 17th International Symposium on Chromatography, September 25–30, 1988, Vienna, Austria.     

Sampling of water, soil and sediment to trace organic pollutants at a river-basin scale

Sampling is considered a crucial step in the analysis of organic compounds in the environment. This review describes field sampling techniques and provides detailed step-by-step procedures for collection and preservation of all major environmental matrices (water, sediment and soil) integrated as part of the river-basin water cycle. Attention is given to the prerequisites for obtaining reliable samples, and the practical issues of sample collection (planning, field sampling, sampling strategies and equipment and data quality assessment) are considered. Considering the heterogeneity of environmental matrices, special considerations for each matrix are given to solve typical problems and to find the most appropriate solutions to ensure the quality of the sample. The procedures described in the next sections are commonly used protocols that reflect true field conditions and current state-of-the-art techniques used in the sampling of organic compounds. The aim is to signify the importance of sampling to the overall analytical procedure. Finally, quality control issues to be considered in environmental sampling are given.     

Determination of neutral cannabinoids in hemp samples by overpressured-layer chromatography

Summary The overpressured-layer chromatographic separation of neutral cannabinaids (Δ⁹ -tetrahydro-cannabinol, cannabidiol, cannabinol, cannabigerol and cannabichromene) has been achieved on amino HPTLC plates with dichloromethane as mobile phase. By use of bidirectional development up to 30 samples can be analysed on a 10 cm×20 cm plate within 4 min. Evaluation was performed by slit-scanning densitometry at 200 nm. System-suitability data confirm the applicability of the method. Presented at Balaton Symposium 01 on High-Performance Separation Methods, Siófok, Hungary, September 2–4, 2001     

修正高斯模型在制备液相色谱收集区间优化中的应用

近年来制备液相色谱得到了越来越广泛的应用。制备液相色谱操作条件的优化对提高其效率及节约成本非常重要。对制备高效液相色谱而言,其样品收集区间的确定及优化是其操作条件优化的重要步骤。该文以修正高斯模型(EMG模型)为理论基础,编写了一个小软件,可有效地帮助用户确定合理的样品收集区间。此软件对制备液相色谱实际应用有积极的指导及参考意义。     

An automated capillary electrophoresis system for high-speed separation of DNA fragments based on a short capillary

A high-speed DNA fragment separation system was developed based on a short capillary and a slotted-vial array automated sample introduction system. The injection process of DNA sample in a short capillary was investigated systematically with three injection techniques including constant-field-strength, low-field-strength and translational spontaneous injections. Under the optimized conditions, picoliter-scale sample plugs (corresponding to ca. 20-μm plug length) were obtained, which ensure the high-speed and high-efficiency separation for DNA fragments with a short effective separation length. Other separation conditions including the sieving matrix concentration, separation field strength and effective separation length were also optimized. The present system was applied in the separation of ΦX174-Hae III digest DNA marker. With an effective separation length of 2.5 cm, the separation could be achieved in <100 s with plate heights ranging from 0.21 to 0.74 μm (corresponding to plate numbers from 4.86 × 10(6) to 1.36 × 10(6)/m). The repeatabilities for the migration time of the eleven fragments were between 0.4 and 1.1% RSD (n=8). By using the automated continuous injection method, the separation for four different DNA samples could be achieved within 250 s. The present system was further applied in the fast sizing of real DNA samples of PCR products.