Improved partition efficiency with threaded cylindrical column in vortex counter-current chromatography

Type-I coil planet centrifuge produces a uniformly circulating centrifugal force field to produce vortex motion of two immiscible solvent phases in a cylindrical cavity of the separation column to perform efficient countercurrent chromatography. The partition efficiency obtained from the original vortex column was substantially improved by threading the cylindrical cavity to increase the area of mass transfer between the two phases. Partition efficiency of the threaded column was evaluated by three different two-phase solvent systems with a broad range of hydrophobicity each with a set of suitable test samples. Overall results of the present studies indicated that the threaded cylindrical column substantially improves the partition efficiency in terms of theoretical plate number, peak resolution, and height equivalent of one theoretical plate. The results also indicated that higher peak resolution is produced by eluting either the upper phase in the head to tail direction or the lower phase in the reversed direction. When there is a choice in the mobile phase, a better separation is achieved by using the less viscous phase as the mobile phase. Since the present system gives extremely low column pressure, it may be a potential alternative to the conventional type-J HSCCC system for a large-scale preparative separation.     

Golden rules and pitfalls in selecting optimum conditions for high-speed counter-current chromatography

This paper aims to be an aid to those chemists who are interested in utilizing high-speed counter-current chromatography (HSCCC), which is free of irreversible adsorption and offers high resolution comparable to column chromatography. It explains the selection of HSCCC conditions step by step including the selection of two-phase solvent systems, determination of partition coefficient (K) of analytes, preparation of two-phase solvent system and sample solution, selection of elution mode, flow rate, rotation speed, and on-line monitoring of the eluate. The paper covers both standard HSCCC and pH-zone-refining CCC techniques. Technical terms (italic) unfamiliar to the beginner are comprehensively explained in Glossary. Various examples of two-phase solvent systems used in HSCCC are listed in Appendices A and B. The commercial sources of HSCCC and other CCC instruments are described in detail in the study edited by Berthod [A. Berthod (Ed.), Counter-current Chromatography, Elsevier, Amsterdam, 2003].     

Separation of Photosynthetic Pigments by High-performance Liquid Chromatography: Comparison of Column Performance,Mobile Phase,and Temperature

High-performance liquid chromatography (HPLC) has been commonly used as method of separating and identifying photosynthetic pigments such as chlorophylls and carotenoids because of such advantages as speed, high resolution and sensitivity. In this technique, high separation relies largely on the type of column material. This study compared the efficiency of five reverse-phase columns, C8, C18, C18 monolithic, π-NAP, and cholester, for separation of photosynthetic pigments at several fixed conditions of mobile phase and temperature. This investigation also analysed the parameters of Δt_R and t_R ratio for selected pigments and resolution for structural isomers, such as α- and β-carotene. Among above columns tested, cholester column is suitable for separation of pigments not only for a broad range of polarity, but also for hydrophobic pigments in a simple mobile phase. This finding can help in the selection of column and HPLC parameters in separating photosynthetic pigments.     

一种用于大容量逆流色谱制备的立式多层螺旋管行星式离心机

Owing to no complications caused by solid supports, such as adsorptive sample loss and deactivation, tailing of solute peaks, and contamination, countercurrent chromatography (CCC) has been an area of intense research since the first introduction of CCC in 1970,[1] and various apparatus and broad applications have been advanced[2,3]. For these developments, the type-J synchronous planet centrifuge has received considerable attention, which relies not only on its relatively simple mechanic design, but also on its high partition efficiency and short elution time caused by mixing and settling for the efficient chromatographic separations. In the past, however, almost all of type-J centrifuges rotated slowly were disposed horizontally due to the original design and some experiments that gravis plays an important role at a low rotary speed as similar to type-V rotating multilayer helical tube in unit gravity[4-9]. In fact,we discovered that the upright apparatus holds more retention of stationary phase than the horiziontal aparatus when large standard tubings were used as mutilayer coil column and the aparatus was operated under same contions. We report here a new coil planet centrifuge with four upright cylindrical columns for large scale countercurrent chromatographic preparation. The design principle and apparatus of UCCC is as samilar to type-J multilayer coil planet centrifuge. Four uptight cylindrical column holders are symmetrically arranged around the centrifuge axis as similar to the type-J HSCCC with three horizontal multilayer coils connected in series[8] . A series of experiments indicat that upright CCC has many advantages over the horizontal CCC when using a large-bore tube as multilayer coil column for large scale countercurrent chromatographic separation.Upright CCC provide a versatile countercurrent chromatographic method for large-scale preparation from very crude sample. It has good preparative capacity and flexible suitability to various sample and two-phase system.The present apparatus not only can be operated at a high speed as similar as commonly used HSCCC for the system having short settling time and but also can be run at a low speed for the system having relative long settling time. Because of automatical control and seal-free flow through device, the uptight CCC apparatus may be readily scaled up to industrial preparation.     

Three-phase solvent systems for comprehensive separation of a wide variety of compounds by high-speed counter-current chromatography

Three-phase solvent systems were efficiently utilized for high-speed counter-current chromatography (HSCCC) to separate multiple components with a wide range of hydrophobicity. The compositions of three-phase systems were optimized according to their physical parameters such as volume ratio, viscosity and specific gravity of upper (UP), middle (MP) and lower (LP) phases. The three-phase systems composed of n-hexane-methyl acetate-acetonitrile-water (4:4:3:4, v/v/v/v) was selected for HSCCC separation of a mixture of 15 standard compounds with a wide range in hydrophobicity from beta-carotene to tryptophan. The separation was initiated by filling the column with a mixture of MP and LP both as a stationary phase followed by elution with UP to separate the hydrophobic compounds. Then the mobile phase was switched to MP to elute the moderately hydrophobic compounds, and finally the polar compounds still retained in the column were fractionated by eluting the column with LP. The system successfully resolved all 15 compounds in one-step operation in 70 min.     

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.     

Simultaneous separation of hydrophilic and hydrophobic compounds by using an online HILIC-RPLC system with two detectors

In the present study, we developed a novel online hydrophilic interaction chromatography (HILIC)-RPLC separation system for simultaneous separation of both hydrophilic and hydrophobic solutes in a complex sample with one injection. A HILIC Si column and a C18 column were hyphenated with an interface including two electronic 2-position valves, a solvent pump, and a solute transfer column. By using column-switching technique, the nonretained hydrophobic solutes were eluted out of the HILIC column and transferred into the C18 column to perform further separation. The hydrophilic solutes were separated on the HILIC column at the same time. One detector was equipped for each column to record the individual chromatograms. By separating a standard mixture and a traditional Chinese medicine (TCM) extract, the developed HILIC-RPLC system demonstrates its potential for "entire-components" separation of complex samples with improved peak capacity and throughput compared with the common single-column LC.     

Direct method for determination of Sudan I in FD&C Yellow No. 6 and D&C Orange No. 4 by reversed-phase liquid chromatography

A reversed-phase liquid chromatographic method was developed to determine parts-per-million and higher levels of Sudan 1, 1-(phenylazo)-2-naphthalenol, in the disulfo monoazo color additive FD&C Yellow No. 6 and in a related monosulfo monoazo color additive, D&C Orange No. 4. Sudan I, the corresponding unsulfonated monoazo dye, is a known impurity in these color additives. The color additives are dissolved in water and methanol, and the filtered solutions are directly chromatographed, without extraction or concentration, by using gradient elution at 0.25 mL/min. Calibrations from peak areas at 485 nm were linear. At a 99% confidence level, the limits of determination were 0.008 microg Sudan I/mL (0.4 ppm) in FD&C Yellow No. 6 and 0.011 microg Sudan I/mL (0.00011%) in D&C Orange No. 4. The confidence intervals were 0.202 +/- 0.002 microg Sudan I/mL (10.1 +/- 0.1 ppm) near the specification level for Sudan I in FD&C Yellow No. 6 and 20.0 +/- 0.2 microg Sudan I/mL (0.200 +/- 0.002%) near the highest concentration of Sudan I found in D&C Orange No. 4. A survey was conducted to determine Sudan I in 28 samples of FD&C Yellow No. 6 from 17 international manufacturers over 3 years, and in a pharmacology-tested sample. These samples were found to contain undetected levels (16 samples), 0.5-9.7 ppm Sudan I (0.01-0.194 microg Sudan I/mL in analyzed solutions; 11 samples including the pharmacology sample), and > or =10 ppm Sudan I (> or = 0.2 microg Sudan I/mL; 2 samples). Analyses of 21 samples of D&C Orange No. 4 from 8 international manufacturers over 4 years found Sudan I at undetected levels (8 samples), 0.0005 to < 0.005% Sudan I (0.05 to < 0.5 microg Sudan I/mL in analyzed solutions; 3 samples, including a pharmacology batch), 0.005 to <0.05% Sudan I (0.5 to <5 microg Sudan I/mL; 9 samples), and 0.18% Sudan I (18 microg Sudan I/mL; 1 sample).     

亲水相互作用色谱柱串联C18柱高效分析饮品中的胭脂红

建立了亲水相互作用色谱柱(HILIC)串联C18柱的方法,高效分离实际样品中的胭脂红,并对色谱柱的串联顺序以及色谱分离条件进行优化。结果表明:当HILIC柱在前C18柱在后,流动相为乙腈-乙酸铵(5 mmol/L)=91∶9(V/V),流速为0.9 mL/min,柱温为25℃,检测波长为508 nm时,此时胭脂红的分离效果最佳。在最佳的色谱分离条件下,选用两种市售饮品进行实际样品的测定,回收率在81.2%~119%。方法适用于水相样品中的强极性物质的高效分离与分析。     

Characterisation of the surface Lewis acid-base properties of poly(butylene terephthalate) by inverse gas chromatography

Two modes of high-speed counter-current chromatography (HSCCC) were applied to separate 3- and 4-sulfophthalic acid from a mixture. Conventional HSCCC was useful for the separation of up to several hundred milligram quantities of these positional isomers, while pH-zone-refining CCC was implemented successfully to separations at the multigram level. The conventional HSCCC separations were performed with a standard J-type HSCCC system that has a superior resolution but a lower level of retention of the stationary phase of the biphasic solvent system used (acidified n-butanol-water). The pH-zone-refining CCC separations were performed with an X-type HSCCC system (a cross-axis system) that has a higher capability for retention of the stationary phase. The purified positional isomers (over 99% pure as determined by HPLC) were characterized by 1H NMR and negative ion electrospray ionization mass spectrometry.     

Electrochromatographic performance of conventional and polar-embedded C16 silica monolithic stationary phases

A novel monolithic silica column that has a polar‐embedded amide‐secondary amine group linking with C16 functionality for RP‐CEC is described. The amide‐secondary aminealkyloxysilane was synthesized by the reaction of 3‐(2‐aminoethylamino) propyltrimethoxysilane with hexadecanoyl chloride. Then, the silylant agent was bonded to the silica monolith matrix to produce hexadecanamide‐secondary amine bonded silica (HDAIS) monolithic column. The electrochromatographic performance of HDAIS monolithic column for the separation of neutral, basic and polar solutes was studied, which was compared to that using the hexadecyl bonded silica monolithic column. The HDAIS monolithic column displayed reduced hydrophobic retention characteristics in the separation of five hydrophobic n‐alkylbenzenes and four polar phenols when compared to the hexadecyl bonded silica monolithic column. A very much reduced silanol activity of HDAIS monolithic column was observed in the separation of test basic mixture including four aromatic amines, atenolol and metoprolol with 10 mM borate buffer (pH 7.5) containing 30% v/v ACN as the mobile phase. The comparison results indicate good performance for both polar and basic mixtures on HDAIS monolithic column in RP‐CEC, and also show promising results for further applications.     

Isolation of all-trans lycopene by high-speed counter-current chromatography using a temperature-controlled solvent system

The effect of solvent system, partition coefficient, retention of stationary phase, column, revolution speed, and flow rate of mobile phase are well known parameters to effect HSCCC (high-speed counter-current chromatography) separations. Temperature effects on chromatographic techniques like HPLC and GC are well studied, but the influence of temperature on CCC solvent systems is hardly investigated. This paper presents the influence of temperature on several key parameters (partition coefficient, settling time, volume ratios) in the hydrophobic HSCCC solvent system hexane:dichloromethane:acetonitrile (30:11:18, v/v/v) used for the isolation of lycopene from tomato paste at 10, 15, 20 and 25 degrees C.     

Separation of phenylsuccinic acid enantiomers using biphasic chiral recognition high‐speed countercurrent chromatography

High‐speed countercurrent chromatography (HSCCC) combined with biphasic chiral recognition was successfully applied to the resolution of phenylsuccinic acid enantiomers. d ‐Isobutyl tartrate and hydroxypropyl‐β‐cyclodextrin were employed as lipophilic and hydrophilic selectors dissolved in the organic stationary phase and aqueous mobile phase, respectively. The two‐phase solvent system was made up of n‐hexane/methyl tert‐butyl ether/water (0.5:1.5:2, v/v/v). Impacts of the type and concentration of chiral selectors, the pH value of the aqueous phase solution as well as the temperature on the separation efficiency were investigated. By means of preparative HSCCC, pure enantiomer was obtained by separating 810 mg of racemate with a purity >99.5% and a recovery rate between 82 and 85%. The experimental results indicate that biphasic recognition HSCCC provide a promising means for efficient separation of racemates.     

Reversed-phase and weak anion-exchange mixed-mode silica-based monolithic column for capillary electrochromatography

A silica-based CEC monolithic column with mixed modes of RP and weak anion-exchange (WAX) was successfully prepared by using the sol-gel technique at mild temperature. The synthesizing procedure was optimized by changing the ratios of tetraethoxysilane (TEOS), aminopropyltriethoxysilane (APTES), and octyltriethoxysilane (C(8)-TEOS) in the mixture. While serving as WAX group, the amino group dominated the charge on the surface of the capillary column and generated an EOF from cathode to anode at low pH. At pH above 7.5, a cathodic EOF was observed due to the full ionization of silanol group and the suppression in the ionization of amino group. The morphology of monolithic columns was examined by SEM, and the performance of column was evaluated in detail by separating different kinds of compounds. As expected, the monolithic column exhibited RP chromatographic behavior for neutral solutes. Fast and efficient separation of six aromatic acids was obtained using acidic mobile phase with column efficiency up to 160,000 plates/m. Symmetrical peaks can be obtained for aromatic amines because positively charged amino groups on the surface can effectively minimize the adsorption of positively charged analytes to the stationary phase.     

Preparative separation of 1,3,6-pyrenetrisulfonic acid trisodium salt from the color additive D&C Green No. 8 (pyranine) by pH-zone-refining counter-current chromatography

In developing analytical methods for batch certification of the color additive D&C Green No. 8 (G8), the U.S. Food and Drug Administration needed the trisodium salt of 1,3,6-pyrenetrisulfonic acid (P3S) for use as a reference material. Since P3S was not commercially available, preparative quantities of it were separated from portions of a sample of G8 that contained ～3.5% P3S. The separations were performed by pH-zone-refining counter-current chromatography using dodecylamine (DA) as the hydrophobic counterion. The added DA enabled partitioning of the polysulfonated components into the organic stationary phase of the two-phase solvent system used, 1-butanol-water (1:1). Thus, a typical separation that involved 20.3g of G8, using sulfuric acid as the retainer acid and 20% DA in the stationary phase and 0.1M sodium hydroxide as the mobile phase, resulted in ～0.58 g of P3S of greater than 99% purity. The identification and characterization of the separated P3S were performed by elemental analyses, proton nuclear magnetic resonance, high-resolution mass spectrometry, ultra-violet spectra, and high-performance liquid chromatography.     

Determination of Sudan dyes in chili pepper powder by online solid‐phase extraction with a butyl methacrylate monolithic column coupled to liquid chromatography with tandem mass spectrometry

A poly(butyl methacrylate‐co‐ethylene dimethacrylate) monolithic column was fabricated and used as a novel sorbent for online solid‐phase extraction coupled to liquid chromatography with tandem mass spectrometry for the simultaneous determination of Sudan I–IV in chili pepper powder. The prepared columns were characterized by scanning electron microscopy, nitrogen adsorption‐desorption, and pressure drop measurements. Online solid‐phase extraction was performed on the synthesized monolithic column using 10 mM ammonium acetate solution as the loading solution with the aid of an online cleanup chromatography system. The desorption of Sudan I–IV was achieved with acetonitrile as the eluting solution at the flow rate of 0.5 mL/min. The extracted analytes were subsequently eluted into a C₁₈ analytical column for chromatographic separation using a mixture of 10% acetonitrile/90% formic acid (0.5%) solution as the mobile phase. Under the optimized conditions, the developed method had linear range of 1.0–50 μg/kg, a detection limit of 0.3 μg/kg, and a quantification limit of 1.0 μg/kg for each analyte. The intraday and interday recoveries of Sudan I–IV in chili pepper powder samples ranged from 94.8 to 100.9% and 94.9 to 99.4%, respectively. The intraday and interday precision were between 3.37–7.01% and 5.01–7.68%, respectively.     

Preparation and characterization of monoliths covalently bonded chelating groups for capillary electrochromatographic separation of metal ions

In this work, a novel polymer-based monolithic column was prepared using an o-phthalaldehyde-l-phenylalanine Schiff base complex as the reactive center and a mixture of methanol and n-propanol as the porogen. The monolithic column was employed for the separation of a metal ion mixture including Pb(II), Mn(II), Cu(II), Ni(II), Cr(III), Fe(III) and Cr(VI). Tetrabutylammonium bromide (TBAB) was used as a mobile phase additive to enhance the separation efficiency of metal ions by EDTA precomplexation. Using a phosphate buffer (20 mM, pH 3.0), TBAB (10 mM), MeOH (15%, v/v), an applied voltage of −15 kV, and detection at 220 nm, the metal ion mixture was satisfactorily resolved. The average theoretical plate number was 17,900 plates/m. The separation was also carried out in the absence of TBAB, leading to dissimilar elution order and shorter retention time. The separation behavior of the monolithic column was also compared with that of the blank polymer. The unique properties of the monolithic column might be mediated by a combination of electrophoretic behavior and chromatographic retention involving hydrophobic and hydrophilic interactions, as well as ligand exchange.     

HPLC Separation of Different Groups of Small Polar Compounds on a Novel Amide-Embedded Stationary Phase

Retention behaviors of an amide-embedded silica base stationary phase, which was recently developed by our group, were studied by using six different groups of small polar compounds including phenolic compounds, substituted anilines, chlorinated herbicides, Sudan dyes and some nucleotides and nucleosides in HPLC. The chromatographic behaviors of the prepared stationary phase for these analytes were compared with those of a commercially available reversed-phase column ACE C18 under same conditions. Among the six groups of analytes studied, the amide-silica stationary phase showed enhanced selectivity towards phenolic compounds, substituted anilines, Sudan dyes and herbicides under reversed-phase conditions and satisfactory selectivity towards nucleosides and nucleotides which could not be separated with ACE C18 column under HILIC conditions. Experimental data provided some evidence that functional groups on the stationary phases might have certain degrees of influence on selectivity possibly through secondary interactions with the model compounds. The retentions of the moderately polar compounds such as phenolic acids, anilines and herbicides on the stationary phase are higher than highly polar compounds such as nucleotides and nucleosides due to both the hydrophobic and hydrophilic interactions between the stationary phase and analytes. The quantitative determination of Sudan dyes (I, II, III, and IV) in red chilli peppers was performed. Many red chilli peppers were screened and three of them contained Sudans dyes.     

基于二醛微晶纤维素功能化C18的反相/亲水色谱固定相的制备

通过十八烷基胺的氨基与二醛微晶纤维素的醛基共价键合,制备了基于二醛微晶纤维素(DMCC)官能化C₁₈的新型反相/亲水色谱固定相(C₁₈-DMCC/SiO₂),该色谱固定相被用于反相色谱(RPLC)和亲水相互作用色谱(HILIC)模式。C₁₈-DMCC/SiO₂色谱柱展现了良好的疏水选择性和芳香选择性,在反相色谱模式下可分离烷基苯和多环芳烃(PAHs)。苯胺类、酚类和糖苷类等极性化合物被用于评估该色谱柱在反相色谱模式下的极性选择性,商品C₁₈柱作对照柱,色谱评价结果令人满意。核酸碱基被用于评估C₁₈-DMCC/SiO₂色谱柱的亲水色谱性能。通过考察有机溶剂含量对分析物保留的影响,发现该新型色谱固定相具有反相/亲水色谱的典型特征。     

富勒烯C60的柱色谱分离法研究

本文提出了由Ｃ（６０）、Ｃ（７０）及少量高级富勒烯混合物（来自电弧汽化石墨所形成的碳灰）中分离出纯度为９９．９％Ｃ（６０）的两种改进的、简便价廉的柱色谱分离法．第一种方法用中性氧化铝作固定机以石油醚与甲苯（Ｖ／Ｖ＝９２．５／７．５）混合液作流动相；另一种方法以活性炭与硅胶作固定相，以甲苯作流动相，柱顶氮压约为０．０１ａｔｍ．本文尚将这两种方法与文献报道的两种相近的方法分别进行了比较．