Evaluation of Analytical Countercurrent Chromatographs: High-Speed Countercurrent Chromatograph-4000 Vs. Analytical Toroidal Coil Centrifuge

Abstract

Performance of two countercurrent chromatographic models, high speed countercurrent chromatograph (HSCCC-4000) and analytical toroidal coil centrifuge (TCC), is evaluated in terms of theoretical plate number, resolution factor and separation time to assess their analytical capability. A series of experiments was conducted to investigate the effects of internal diameter and length of the coiled column, and flow rate of the mobile phase on the separation of indole auxins in two-phase solvent systems composed of n-hexane-ethyl acetate-methanol-water at different volume ratios. The three components of indole auxins were completely resolved in 16 min with a HSCCC system equipped with a multilayer coil of a 0.55 mm I.D. PTFE tube with theoretical plates ranging from 1290 to 829. Similar separation was achieved in 24 min with a TCC systems equipped with a 0.3 mm I.D. PTFE tube with theoretical plates ranging from 1811 to 969. It is concluded that both systems have comparable analytical capability at the present stage of development.     

Large-scale preparative countercurrent chromatography for separation of polar compounds

Gram quantity separations of polar compounds (tryptophyl-leucine and valyl-tyrosine) have successfully been accomplished with the use of a horizontal coil planet centrifuge. Two columns of different length fluorinated ethylene propylene tubing but of same internal diameter (0.55 cm) were coaxially coiled around a holder 7.5 cm or 15 cm in diameter and used to assess the preparative capabilities of the apparatus in terms of stationary phase retention and sample peak resolution. Optimal operating conditions derived from preliminary studies with the short column were applied to a column 7 times in length and volume. Volume capacities were 114 ml and 750 ml respectively. A hydrophilic solvent system of n-butanol, acetic acid and water (4:1:5) was used with both the aqueous and non-aqueous phases being used as the mobile phase. Preliminary studies revealed that the hydrodynamic distribution of the two phases was independent of the helical diameter while peak resolution was sensitive to both helical diameter and rpm setting.     

Gram Quantity Separation of DNP (Dinitrophenyl) Amino Acids with Multi-Layer Coil Countercurrent Chromatography (CCC)

Abstract

The efforts have been successfully made to extend the preparative capability of the high-speed CCC scheme with a multi-layer large capacity coiled column. The apparatus is a table top model of a horizontal flow-through coil planet centrifuge which produces a synchronous planetary motion of the column holder. The separation column was prepared from a single piece of 70 m long, 2.6 mm i.d., PTFE tubing coiled around the spool-shaped holder to form multiple layers of the coil with a total capacity of about 400 ml. The performance of the apparatus was assessed with a standard set of DNP amino acid samples and a two-phase solvent system composed of chloroform, acetic acid and 0.1N HCl (2:2:1). Preparative capability of the method was evaluated in terms of the retention level of the stationary phase and peak resolution for various sample size ranging from 0.05g to 2g. The effects of sample volume, sample concentration and the choice of the sample diluent on the separation were studied. The results indicated that both the retention level and the peak resolution tend to decrease with the increase of the sample volume applied at a given concentration. For separation of 1 gram quantity, best results were obtained by applying the sample dissolved in a small volume (10 ml) consisting of equal amounts of the two phases. Overall results indicate that the present scheme is capable of efficient separation for gram quantity of samples in a short period of time. The preparative capability may be further increased by the use of a larger-diameter and/or longer coil.     

Protein separation by nonsynchronous coil planet centrifuge with aqueous-aqueous polymer phase systems

Counter-current chromatographic separation of proteins was performed using a rotary-seal-free nonsynchronous coil planet centrifuge (CPC) fabricated in our laboratory. This apparatus has a unique feature that allows a freely adjustable rotational rate of the coiled separation column at a given revolution speed. The separation was performed using a set of stable proteins including cytochrome c, myoglobin and lysozyme with two different types of aqueous-aqueous polymer phase systems, i.e., PEG (polyethylene glycol) 1000-dibasic potassium phosphate, and PEG 8000-dextran T500 in 5 mM potassium phosphate buffer. Using a set of multilayer coiled columns prepared from 0.8 mm I.D. PTFE tubing with different volumes (11, 24, 39 ml), the effect of the column capacity on the partition efficiency was investigated under a given set of experimental conditions. Among these experiments, the best separation of proteins was attained using the 39 ml capacity column with a 12.5% (w/w) PEG 1000-12.5% (w/w) dibasic potassium phosphate system at 10 rpm of coil rotation under 800 rpm. With lower phase mobile at 0.2 ml/min in the head-to-tail elution, the resolution between cytochrome c and myoglobin was 1.6 and that between myoglobin and lysozyme, 1.9. With upper phase mobile in the head-to-tail elution, the resolution between lysozyme and myoglobin peaks was 1.5. In these two separations, the stationary phase retention was 35.0 and 33.3%, respectively. Further studies were carried out using a pair of eccentric coil assemblies with 0.8 mm I.D. PTFE tubing at a total capacity of 20 ml. A comparable resolution was obtained using both lower and upper phases as a mobile phase in a head-to-tail elution. The results of our studies demonstrate that the nonsynchronous CPC is useful for protein separation with aqueous-aqueous polymer phase systems.     

对甲苯磺酸正丁铵毛细管色谱柱的制备及性能考察

用动态涂敷法制备了对甲苯磺酸正丁铵玻璃毛细管柱柱１（固定液浓度为７．５％,柱尺寸为３５ｍ×０．３５ｍｍｉ．ｄ．）和柱２（固定液浓度为９．２％,柱尺寸为３５ｍ×０．３０ｍｍｉ．ｄ．）,所用溶剂是丙酮和甲醇（２：１Ｖ／Ｖ）的混合溶剂,并对柱性能进行了考察。结果表明,这种色谱固定相具有较强的极性并对饱和烃、芳香烃、饱和酸、芳香酸、极性异构体等具有良好的分离效果。     

Advantages of a small-volume counter-current chromatography column

Counter-current chromatography (CCC) works with a support-free liquid stationary phase. This allows for preparative separations and purifications. However, there are serious technical constraints because of the need to keep a liquid stationary phase in a column. Centrifugal fields are used. A new commercial hydrodynamic 18 mL column made with a narrow-bore 0.8 mm Teflon tubing was evaluated by comparing it with older hydrodynamic CCC columns and a similar 19 mL column but made with 1.6 mm Teflon tubing. A small-volume CCC column allows for reliable and fast solute partition coefficient determination. When resolution is required, both high efficiency and liquid stationary phase retention are needed. Unfortunately, these two requirements bear technical contradictions. A column coiled with a narrow tubing bore will provide a high chromatographic efficiency while a column containing wider tubing bore will achieve higher stationary phase retention. In all cases, increasing the magnitude of the centrifugal field also increases the stationary phase retention. The solution is to build centrifuges able to produce high fields that will provide acceptable liquid phase retention with narrow-bore tubes. The new 18 mL 0.8 mm tubing bore column is able to rotate as fast as 2100 rpm generating a 240 × g field. The two older CCC columns cannot compete with the new one. However, the small 19 mL column with 1.6 mm bore tubing can be useful when fast results are desired without top resolution.     

Elution order in gas chromatography

The retention behavior of a heterogeneous group of solutes has been examined on seven different stationary phases under isothermal and temperature-programmed conditions. Both ΔH_v (enthalpy of solute vaporization from the stationary phase) and ΔS_v (entropy of solute vaporization from the stationary phase) values were determined for each solute – stationary phase combination under isothermal conditions. Both program rate and carrier gas velocity were shown to affect solute elution order. Unless these and other experimental factors discussed are controlled, column equivalency studies based on solute elution order have dubious value.     

Effect of high-temperature on high-performance liquid chromatography column stability and performance under temperature-programmed conditions

Six commercially available analytical (4.1 or 4.6 mm i.d.) columns were evaluated under temperature-programmed high-temperature liquid chromatography (HTLC) conditions to access their stability and performance at extreme temperatures. Seven components consisting of acidic, basic and neutral compounds were analyzed under temperature-programmed conditions and solvent gradient conditions using three different mobile phase compositions (acidic, basic and neutral). Each column was checked with a two-component test mix at various stages of the evaluation to look for signs of stationary phase collapse. Three zirconia based stationary phases studied exhibited column bleed under temperature-programmed conditions. The other three columns, a polydentate silica column, a polystyrene-divinylbenzene (PS-DVB) polymeric column, and a graphitic carbon column performed well with no evidence of stationary phase degradation. The R.S.D. for the retention times and efficiencies were less than 10% for most conditions, and not more than 15% during the course of the evaluation for each column. The polydentate silica stationary phase was temperature programmed to 100 degrees C, the PS-DVB stationary phase was temperature programmed up to 150 degrees C, and the graphitic carbon column was used with temperature programming up to 200 degrees C. Comparable peak capacities and similar retention behaviors were observed under solvent gradient and temperature-programmed conditions. Temperature programming with dynamic mobile phase preheating can replace solvent gradient analysis without a loss of peak capacity when used with 4.1 or 4.6 mm columns.     

开管离子色谱柱的制备与表征

近年来,开管离子色谱(OTIC)的研究已取得重大进展,尤其是兼容OH^-淋洗液的OTIC柱的成功研制使OTIC梯度洗脱得以实现。该文介绍了各种OTIC柱的制备和表征方法,涵盖早期的二氧化硅开管柱和新近出现的有机聚合物基质开管柱,对静电乳胶附聚的OTIC柱的一系列表征方法进行了系统阐述,包括开管柱柱容量的实验测定和理论计算方法、固定相相同时填充柱与开管柱之间的联系、柱效改善方法以及开管柱均一性表征等。     

填充毛细管液相色谱-毛细管气相色谱在线联用分析八角茴香挥发油

首次将填充毛细管高效液相色谱-毛细管气相色谱在线联用技术(μ-HPLC-CGC)用于分离分析八角茴香果实的挥发油成分。液相色谱选用氰基分析柱(250 mm×0.32 mm i.d.),正己烷-乙腈-二氯甲烷(体积比为80∶8∶12)为流动相,对挥发油样品做族组分分离,得到的5个族组分被依次存放在多位储存接口内,然后不分流分别转入毛细管气相色谱仪做详细分析。气相色谱柱由10 m×0.53 mm i.d.保留间隔柱和30 m×0.53 mm i.d.×1.0 μm SE-54分析柱组成。采用了不分流柱内进样模     

Development and evaluation of gold-centered monolayer protected nanoparticle stationary phases for gas chromatography

The current status for the development of novel open-tubular gas chromatography (GC) stationary phases consisting of thin films of gold-centered monolayer protected nanoparticles (MPNs) is reported. Dodecanethiol MPNs, in which the monolayer is dodecanethiol linked to the gold nanoparticle, have shown great promise as a GC stationary phase with efficient columns having been produced in a variety of capillary i.d.'s with stationary phase film depths ranging from 10 to 60 nm, +/-2 nm at a given film depth. Stationary phase operational parameters are discussed including maximum operating temperature, sample capacity, and stationary phase lifetime and robustness. An overview of the general method employed for column production is also included. The sample capacity was determined for a 2.5 m, 250 microm i.d. column with a stationary phase film thickness of 40 nm, at 50 degrees C using anisole (k' = 1.86) as the probe analyte. The sample capacity was experimentally found to be 2.3 ng under these conditions, similar to values reported for thicker, polymer stationary phases. The efficiency of the dodecanethiol MPN stationary phase was determined with a 100 microm i.d. capillary and found to have a reduced plate height hmin value of 0.95 for octane (k' = 0.68). Areas of application illustrated and discussed utilizing the dodecanethiol MPN stationary phase include complementary separations such as two-dimensional GC (GC x GC), potential utilization within a model system for a micro-fabricated GC (microGC), as well as efficient single dimension high-speed separations. Initial development of polar stationary phases utilizing 4-chlorobenzenethiol MPNs and 4-(trifluoromethyl)benzenethiol MPNs is discussed. Included is a selectivity comparison of the retention behavior of the 4-chlorobenzenethiol MPN stationary phase and the dodecanethiol MPN stationary phase.     

Study of Chiral Ionic Liquid as Stationary Phases for GC

Due to the synthetic flexibility and special enantioselectivity, chiral ionic liquids (CILs) have heightened interest and an increasing number of CILs has been designed and utilized. In this work, CIL named l-1-butyl-3-(2-propionic-1-ether) imidazolium bromide ([BAlaIM]Br) derived from natural amino acids was synthesized, with chiral center at cation moiety. Chiral stationary phases for gas chromatography were then prepared by mixing the CIL with polymeric ionic liquid ([PSOMIM][NTf₂], homemade) at different ratios (4:1, 2:1, and 1:1). The column efficiency was measured to be about 3,200 plates m⁻¹ (8 m × 0.25 mm i.d.) when the content of [BAlaIM]Br was 50 % (mass percent) in the mixed stationary phase. All columns were coated via the static coating method using 0.30 % (w/v) of stationary phases dissolved in methanol. The results showed that the CIL contributed to the selectivity of stationary phase toward positional isomers dichlorobenzenes, methylnaphthalenes and pinenes, etc. Meanwhile, [BAlaIM]Br showed better selectivity for enantiomers such as carvones, citronellals, limonenes and camphors. The interactions between chiral selector and enantiomers were also discussed.

     

Development and evaluation of a spiral tube column for counter-current chromatography

An improved type-J counter-current chromatography (CCC) planet centrifuge with two spiral tube columns (volume 2×15 mL, β value 0.3-0.7, tubing 0.8 mm id) was developed and evaluated for its retention ability of four typical different solvent systems including heptane-methanol (1:1, v/v) (A), hexane-ethyl acetate-methanol-water (1:1:1:1, v/v) (B), n-butanol-acetic acid-water (4:1:5, v/v) (C), PEG1000-K(2)HPO(4)-water (12.5:12.5:75, w/w) (D) under eight different operation modes. The results indicated that the spiral tube column could significantly increase the retention of four typical solvent systems compared with a traditional multilayer coil column with similar parameters (volume 35 mL, β value 0.3-0.7, tubing 0.8 mm id). The retention of stationary phase (S(f)) for the less polar system (A) and moderately polar solvent system (B) can be increased by about 10%, and for the polar system (C) and aqueous two-phase system (ATPS) (D) by 30-40%. The preliminary applications of this spiral tube column to the separation of small molecular compounds such as moderately polar theaflavins, polar anthocyanins and dipeptides were successful. Acceptable resolution can be obtained between cytochrome c and myoglobin, lysozyme and myoglobin when it was applied on protein separation; however, it still needs to be improved with regard to its column efficiency.     

大环糖肽抗生素键合相高效液相色谱法拆分7种氨基带保护基的氨基酸对映体

采用高效液相色谱法在装有大环糖肽抗生素键合相的手性柱上拆分了7种氨基带有芴甲氧羰基(fluorenylmethoxycarbonyl,Fmoc)保护的氨基酸对映体。比较了Fmoc-缬氨酸和相应的不带保护基的缬氨酸对映体在不同流动相体系中的色谱保留行为;考察了甲醇-醋酸-三乙胺流动相体系中醋酸和三乙胺的浓度以及它们二者的浓度之比对N-Fmoc氨基酸对映体拆分效果的影响。实验结果表明分离温度及流动相流速的变化也会对分离结果产生影响。该法简便快速,已成功地用于这类氨基酸的光学纯度测定。     

Evaluation and comparison of n‐alkyl chain and polar ligand bonded stationary phases for protein separation in reversed‐phase liquid chromatography

Protein retention is very sensitive to the change of solvent composition in reversed‐phase liquid chromatography for so called “on–off” mechanism, leading to difficulty in mobile phase optimization. In this study, a novel 3‐chloropropyl trichlorosilane ligand bonded column was prepared for protein separation. The differences in retention characteristics between the 3‐chloropropyl trichlorosilane ligand bonded column and n‐alkyl chain modified (C₂, C₄, C₈) stationary phases were elucidated by the retention equation . Retention parameters (a and c) of nine standard proteins with different molecular weights were calculated by using homemade software. Results showed that retention times of nine proteins were similar on four columns, but the 3‐chloropropyl trichlorosilane ligand bonded column obtained the lowest retention parameter values of larger proteins. It meant that their retention behavior affected by acetonitrile concentration would be different due to lower |c| values. More specifically, protein elution windows were broader, and retentions were less sensitive to the change of acetonitrile concentration on the 3‐chloropropyl trichlorosilane ligand bonded column than that on other columns. Meanwhile, the 3‐chloropropyl trichlorosilane ligand bonded column displayed distinctive selectivity for some proteins. Our results indicated that stationary phase with polar ligand provided potential solutions to the “on–off” problem and optimization in protein separation.     

Capillary liquid chromatography as a tool for separation of hydrophobic basic drugs. Relation between tests for column characterization and real analysis

Two capillary columns for reversed phase (RP) capillary liquid chromatography (CLC), viz. Nucleosil 100‐5 C18 and LiChrosorb RP‐select B, were characterized by the Walters test, i.e. the chromatographic test proposed for RP stationary phases. Hydrophobicity indices were determined not only in acetonitrile/water mobile phase, as proposed in the test, but they were also measured in buffered systems. This approach was used to quantify the influence of mobile phase composition on the modification of the surface of the stationary phases. In the next step, small basic compounds differing in their hydrophobicity and basicity were selected and their retention on the stationary phases in mobile phases of the same composition as used for column testing was examined. Furthermore, the retention of newly synthesized drugs, chemotherapeutics derived from thioacridine and pyridoquinoline, differing in their structures, basicity, and hydrophobicity, was also studied. The composition of the mobile phases had to be shifted to higher contents of organic modifiers – acetonitrile or methanol – in order to elute these hydrophobic compounds from the columns. The question we wanted to answer was: How is the method for testing of reversed phases related to retention, separation efficiency, and peak symmetry of various analytes?     

A comparative study of hydrophobicity of octadecyl and alkylamide bonded phases based on methylene selectivity

The conformational dynamics of home‐made monomeric and polymeric (octadecyl – C18 and alkylamide – AP) stationary phases have been investigated by analyzing the dependence of retention on the reciprocal temperature for different organic compounds. Theoretical considerations were based on the results of methylene selectivity determination. Two selected PAHs – naphthacene and benzo[a]anthracene, whose structures differ in the position of the ring – were chosen as solutes to study the thermodynamics of retention behavior. Standard enthalpies (ΔH ⁰) were obtained from the slope of the plots of the capacity factor logarithm (ln k) against the reciprocal temperature (1/T), i. e. van't Hoff plots, in a temperature range from 285 to 315 K. The conformation of the stationary phase (monomeric and polymeric) structure and the length of bonded ligands with their specific functional (N‐acylamide) groups had a significant effect on retention.     

单硫代和双硫代甘油醚在直链淀粉-三（3,5-二甲基苯基氨基甲酸酯）固定相上的手性拆分

 合成了直链淀粉-三(3,5-二甲基苯基氨基甲酸酯）手性固定相。用该固定相对6种单硫 代 甘油醚和4 种双硫代甘油醚进行了手性拆分。单硫代甘油醚对映异构体得到了较好的分离, 而双硫代甘油醚完全不能拆分。提出了样品与固定相的作用模式。     

万古霉素键合手性固定相高效液相色谱法直接分离泰妥拉唑对映体

利用反相高效液相色谱法在大环抗生素类手性固定相万古霉素键合手性固定相(Chirobiotic V)上直接分离了泰妥拉唑对映体。考察了缓冲溶液的种类、浓度和pH值,有机改性剂的种类和浓度,柱长和柱温等对手性分离的影响。优化后的色谱条件为:Chirobiotic V色谱柱(150 mm×4.6 mm,5 μm),流动相为0.02 mol/L 醋酸铵缓冲液(pH 6.0)-四氢呋喃(体积比为93∶7),流速为0.5 mL/min,柱温为20 ℃,检测波长为306 nm。在此条件下泰妥拉唑对映体达到了基线分离,分离度达1.68;对映体保留时间的相对标准偏差分别为0.48%和0.49%(n=6),峰面积的相对标准偏差分别为0.45%和0.55%(n=6)。所建立的手性分离方法具有简便快速及重复性好等优点。     

Enantiomeric separation of 2‐arylpropionic acid nonsteroidal anti‐inflammatory drugs by HPLC with hydroxypropyl‐β‐cyclodextrin as chiral mobile phase additive

The enantio‐separations of eight 2‐arylpropionic acid nonsteroidal anti‐inflammatory drugs (2‐APA NSAIDs) were established using reversed‐phase high‐performance liquid chromatography with hydroxypropyl‐β‐cyclodextrin (HP‐β‐CD) as chiral mobile phase additive for studying the stereoselective skin permeation of suprofen, ketoprofen, naproxen, indoprofen, fenoprofen, furbiprofen, ibuprofen and carprofen. The effects of the mobile phase composition, concentration of HP‐β‐CD and column temperature on retention and enantioselective separation were investigated. With 2‐APA NSAIDs as acidic analytes, the retention times and resolutions of the enantiomers were strongly related to the pH of the mobile phase. In addition, both the concentration of HP‐β‐CD and temperature had a great effect on retention time, but only a slight or almost no effect on resolutions of the analytes. Enantioseparations were achieved on a Shimpack CLC‐ODS (150 × 4.6 mm i.d., 5 μm) column. The mobile phase was a mixture of methanol and phosphate buffer (pH 4.0–5.5, 20 mM) containing 25 mM HP‐β‐CD. This method was flexible, simple and economically advantageous over the use of chiral stationary phase, and was successfully applied to the enantioselective determination of the racemic 2‐APA NSAIDs in an enantioselective skin permeation study. Copyright © 2009 John Wiley & Sons, Ltd.