Application of counter-current chromatography as a new pretreatment method for the determination of polycyclic aromatic hydrocarbons in environmental water

Counter‐current chromatography (CCC) was investigated as a new sample pretreatment method for the determination of trace polycyclic aromatic hydrocarbons (PAHs) in water environmental samples. The experiment was performed with a non‐aqueous binary two‐phase solvent system composed of n‐heptane and acetonitrile. The CCC column was first filled with the upper stationary phase, and then a large volume of water sample was pumped into the column while the CCC column was rotated at 1600 rpm. Finally, the trace amounts of PAHs extracted and enriched in the stationary phase were eluted out by the lower mobile phase and determined by gas chromatography–flame ionization detector (GC‐FID) or gas chromatography–mass spectrometry (GC‐MS). The enrichment and cleanup of PAHs can be fulfilled online by this method with high recoveries (84.1–103.2%) and good reproducibility (RSDs: 4.9–12.2%) for 16 EPA PAHs under the optimized CCC pretreatment conditions. This method has been successfully applied to determine PAHs in lake water where 8 PAHs were detected in the concentration of 40.9–89.9 ng/L. The present method is extremely suitable for the preparation of large volume of environmental water sample for the determination of trace amounts of organic pollutants including PAHs as studied in this paper.     

Using the liquid nature of the stationary phase in counter-current chromatography V. The back-extrusion method

The main feature of counter-current chromatography (CCC) is that the stationary phase is a liquid as well as the mobile phase. The retention volumes of solutes are directly proportional to their distribution coefficients K(D) in the biphasic liquid system used in the CCC column. Solutes with high K(D) coefficients are highly retained in the column. The back-extrusion method (BECCC) uses the fact that the liquid stationary phase, that contains the retained solutes, can be easily moved. Switching the column inlet and outlet ports without changing the liquid phase used as the mobile phase causes the rapid collapse of the two immiscible liquid phases inside the column, the previously stationary phase being gathered at the new column outlet. Then this previously stationary liquid phase is extruded outside the CCC column carrying the retained solutes. The back-extrusion method is tested with a standard mixture of five compounds and compared with the recently described elution-extrusion method. It is shown that the chromatographic resolution obtained during the back-extrusion step is good because the solute band broadening is minimized as long as the solute is located inside the "stationary" phase. However, a major drawback of the BECCC method is that all solutes are split between the liquid phases according to their distribution ratios when the CCC column equilibrium is broken. The change of flowing direction should be done after a sufficient amount of mobile phase has flushed the column in the classical mode, eluting solutes with small and medium distribution ratios. Otherwise, a significant portion of the solutes will stay in the mobile phase inside the column and produce a broad peak showing after the stationary phase extrusion.     

pH-zone-refining counter-current chromatography: Origin,mechanism, procedure and applications

Since 1980, high-speed counter-current chromatography (HSCCC) has been used for separation and purification of natural and synthetic products in a standard elution mode. In 1991, a novel elution mode called pH-zone refining CCC was introduced from an incidental discovery that an organic acid in the sample solution formed the sharp peak of an acid analyte. The cause of this sharp peak formation was found to be bromoacetic acid present in the sample solution which formed a sharp trailing border to trap the acidic analyte. Further studies on the separation of DNP-amino acids with three spacer acids in the stationary phase revealed that increased sample size resulted in the formation of fused rectangular peaks, each preserving high purity and zone pH with sharp boundaries. The mechanism of this phenomenon was found to be the formation of a sharp trailing border of an acid (retainer) in the column which moves at a lower rate than that of the mobile phase. In order to facilitate the application of the method, a new method was devised using a set of retainer and eluter to form a sharp retainer rear border which moves through the column at a desired rate regardless of the composition of the two-phase solvent system. This was achieved by adding the retainer in the stationary phase and the eluter in the mobile phase at a given molar ratio. Using this new method the hydrodynamics of pH-zone-refining CCC was diagrammatically illustrated by three acidic samples. In this review paper, typical pH-zone-refining CCC separations were presented, including affinity separations with a ligand and a separation of a racemic mixture using a chiral selector in the stationary phase. Major characteristics of pH-zone-refining CCC over conventional HSCCC are as follows: the sample loading capacity is increased over 10 times; fractions are highly concentrated near saturation level; yield is improved by increasing the sample size; minute charged compounds are concentrated and detected at the peak boundaries; and elution peaks are monitored with a pH flow meter for compounds with no chromophore. Since 1994, over 70 research papers on pH-zone-refining CCC have been published with the trends increasing in the recent years.     

Using the liquid nature of the stationary phase in countercurrent chromatography. IV. The cocurrent CCC method

The retention volumes of solutes in countercurrent chromatography (CCC) are directly proportional to their distribution coefficients, K(D) in the biphasic liquid system used as mobile and stationary phase in the CCC column. The cocurrent CCC method consists in putting the liquid "stationary" phase in slow motion in the same direction as the mobile phase. A mixture of five steroid compounds of widely differing polarities was used as a test mixture to evaluate the capabilities of the method with the biphasic liquid system made of water/methanol/ethyl acetate/heptane 6/5/6/5 (v/v) and a 53 mL CCC column of the coil planet centrifuge type. It is shown that the chromatographic resolution obtained in cocurrent CCC is very good because the solute band broadening is minimized as long as the solute is located inside the "stationary" phase. Pushing the method at its limits, it is demonstrated that the five steroids can still be (partly) separated when the flow rate of the two liquid phases is the same (2 mL/min). This is due to the higher volume of upper phase (72% of the column volume) contained inside the CCC column producing a lower linear speed compared to the aqueous lower phase linear speed. The capabilities of the cocurrent CCC method compare well with those of the gradient elution method in HPLC. Continuous detection is a problem due to the fact that two immiscible liquid phases elute from the column. It was partly solved using an evaporative light scattering detector.     

Preparative separation of quaternary ammonium alkaloids from Corydalis yanhusuo W. T. Wang by pH-zone-refining counter-current chromatography

The optimal extraction condition for extracting quaternary ammonium alkaloid dehydrocorydaline from Corydalis yanhusuo W. T. Wang was investigated using orthogonal experimental design. pH‐zone‐refining counter‐current chromatography (CCC) with normal phase elution was successfully applied to preparative separation of alkaloids from the crude extract of Corydalis yanhusuo. The separation was performed with a biphasic solvent system composed of chloroform (CHCl₃)–methanol (MeOH)–water (2:1:1, v/v), in which the lower organic phase containing 10 mM of triethylamine was used as the mobile phase, while the upper aqueous phase containing 10 mM of hydrochloric acid was used as the stationary phase. The separation mechanism of quaternary ammonium alkaloids using pH‐zone‐refining CCC was discussed in comparison with standard high‐speed CCC. In the present study, the separation of 1.200 g of crude sample yielded 129 mg of dehydrocorydaline and 12 mg of palmatine at a high purity of 94 and 92%, respectively. Recovery for dehydrocorydaline and palmatine was 85 and 86%, respectively.     

Comprehensive analysis of pharmaceutical products using simultaneous mixed‐mode (ion‐exchange/reversed‐phase) and hydrophilic interaction liquid chromatography

Liquid chromatographic assays were developed using a mixed‐mode column coupled in sequence with a hydrophilic interaction liquid chromatography column to allow the simultaneous comprehensive analysis of inorganic/organic anions and cations, active pharmaceutical ingredients, and excipients (carbohydrates). The approach utilized dual sample injection and valve‐mediated column switching and was based upon a single high‐performance liquid chromatography gradient pump. The separation consisted of three distinct sequential separation mechanisms, namely, (i) ion‐exchange, (ii) mixed‐mode interactions under an applied dual gradient (reversed‐phase/ion‐exchange), and (iii) hydrophilic interaction chromatography. Upon first injection, the Scherzo SS C₁₈ column (Imtakt) provided resolution of inorganic anions and cations under isocratic conditions, followed by a dual organic/salt gradient to elute active pharmaceutical ingredients and their respective organic counterions and potential degradants. At the top of the mixed‐mode gradient (high acetonitrile content), the mobile phase flow was switched to a preconditioned hydrophilic interaction liquid chromatography column, and the standard/sample was reinjected for the separation of hydrophilic carbohydrates, some of which are commonly known excipients in drug formulations. The approach afforded reproducible separation and resolution of up to 23 chemically diverse solutes in a single run. The method was applied to investigate the composition of commercial cough syrups (Robitussin®), allowing resolution and determination of inorganic ions, active pharmaceutical ingredients, excipients, and numerous well‐resolved unknown peaks.     

Rapid and preparative separation of traditional Chinese medicine Evodia rutaecarpa employing elution-extrusion and back-extrusion counter-current chromatography: Comparative study

Traditional Chinese medicines (TCMs) have attracted much attention in recent years. Elution-extrusion and/or back-extrusion counter-current chromatography (EECCC/BECCC) both take full advantage of the liquid nature of the stationary phase. They effectively extend the solute hydrophobicity window that can be studied and rendered the CCC technique particularly suitable for rapid analysis of complex samples. In this paper, a popular traditional Chinese medicine, Evodia rutaecarpa, was used as the target complex mixture for extrusion CCC separations. With a carefully selected biphasic liquid system (n-hexane/ethyl acetate/methanol/water, 3/2/3/2, v/v) and optimized conditions (V_CM = V_C, mobile phase flow rate: 3 mL/min in descending mode, sample loading: 100 mg), five fractions could be obtained in only 100 min on a 140-mL capacity CCC instrument using both elution- and back-extrusion methods. Each fraction was analyzed and identified compared with the data of major standards using LC/MS. Moreover, the performance of both extrusion protocols was systematically compared and summarized. EECCC could be operated continuously and was found extremely suitable for high-throughput separation; however, post-column addition of a clarifying reagent is recommended to smooth the UV-signal during the extrusion process. Considering BECCC, the practical operation is very simple by just switching a 4-port valve to change the flow direction. The change of flowing direction should be done after a sufficient amount of mobile phase has flushed the column in the classical mode so that solutes with small and medium distribution constants have been eluted. Otherwise, a significant portion of the solutes will stay in the mobile phase inside the column, mix together and produce a broad peak showing in the mobile phase eluting after the stationary phase extrusion. Compared with classical CCC or other preparative separation tools, extrusion CCC approaches exhibit distinguished superiority in the modernization process of traditional Chinese medicines.     

Mathematical model of computer-programmed intermittent dual countercurrent chromatography applied to hydrostatic and hydrodynamic equilibrium systems

Dual high-speed countercurrent chromatography (dual CCC) literally permits countercurrent flow of two immiscible solvent phases continuously through the coiled column for separation of solutes according to their partition coefficients. Application of this technique has been successfully demonstrated by separation of analytes by gas–liquid and liquid–liquid two-phase systems. However, the method cannot be directly applied to the system with a set of coiled columns connected in series, since the countercurrent process is interrupted at the junction between the columns. However, this problem can be solved by intermittent dual CCC by eluting each phase alternately through the opposite ends of the separation column. This mode of application has an advantage over the conventional dual CCC in that the method can be applied to all types of CCC systems including hydrostatic equilibrium systems such as toroidal coil CCC and centrifugal partition chromatography. Recently, the application of this method to high-speed CCC (hydrodynamic system) has been demonstrated for separation of natural products by Hewitson et al. using a set of conventional multilayer coil separation columns connected in series. Here, we have developed a mathematical model for this intermittent dual CCC system to predict retention time of the analytes, and using a simplified model system the validity of the model is justified by a series of basic studies on both hydrodynamic and hydrostatic CCC systems with a computer-programmed single sliding valve. The present method has been successfully applied to spiral tube assembly high-speed CCC (hydrodynamic system) and toroidal coil CCC (hydrostatic system) for separation of DNP-amino acid samples with two biphasic solvent systems composed of hexane–ethyl acetate–methanol–0.1 M hydrochloric acid (1:1:1:1 and 4:5:4:5, v/v).     

GLC multiresidue analysis of postharvest fungicides in citrus fruit

Summary A simple GLC multiresidue method for the analysis of fenpropimorph, thiabendazole, imazalil, propiconazole and prochloraz residues in citrus fruit, with a limit of detection of 0.1 ppm, is reported.The fungicides are extracted with hexane: ethyl acetate (90:10, v/v) after pH-adjustment of the fruit homogenate. The GLC-separation is carried out using a bonded phase, SE-54, fused silica capillary column and fungicides are determined with a nitrogen-phosphorus detector.Recovery was always higher than 80% with standard deviation equal or lower than 7%. The method is suitable to determine residues in citrus, whole fruit or peel, treated with these fungicides at usual concentrations to avoid citrus decay.

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GLC-Verfahren zur Bestimmung mehrerer Fungicide in Citrusfrüchten nach der Ernte

     

Determination of low levels of an antioxidant in polyolefins by large-volume injection temperature-programmed packed capillary liquid chromatography

Sub-ambient column temperatures, promoting strong interactions between the analyte and the stationary phase material, were utilized to focus large volumes of the polyolefin antioxidant Irganox 1076 [benzenepropanoic acid, 3.5-bis(1,1-dimethylethyl)-4-hydroxy-, octadecyl ester] on the column inlet, using pure acetonitrile as sample solvent and mobile phase. Injection volumes up to 100 microl were successfully employed on a 50 cm x 320 microm I.D. capillary column packed with 5 microm Kromasil 100 ODS particles. Irganox 1076 was eluted after completed injection by temperature programming, using a temperature program from 7 to 90 degrees C, in 3 degrees C min(-1). UV detection, using a low-dispersion "U"-shaped flowcell, was performed at 280 nm. The method was applied for the determination of Irganox 1076 that was extracted from low-density polyethylene (0.6 ppm, w/w). Both Soxhlet and microwave-aided solvent extractions were performed, using chloroform and acetonitrile as solvents, respectively. The microwave-aided extraction with acetonitrile was found to give approximately the same yield as the standard Soxhlet reference method. Consequently, small volumes of acetonitrile could be used both as extraction solvent, sample solvent and mobile phase, simplifying the analysis process. The mass limit of detection of the method was found to be 3.3 ng, corresponding to a concentration limit of detection of 33 ng ml(-1), utilizing an injection volume of 100 microl. The within and between day precision of retention times displayed relative standard deviations below 1.2%.     

Performance comparison using the GUESS mixture to evaluate counter-current chromatography instruments

Comparing the performance of different counter-current chromatography (CCC) J-type centrifuges has and will always be difficult. This is due to the number of variables such as speed of rotation, swung radius, β-value, column bore, column length that can be chosen during the design of an instrument. This situation is further complicated by the implication that some of these variables are intrinsically linked, such that if one is changed another or others can also change. The chromatographer has no influence on these hardware parameters once the instrument designer has chosen them. However, the chromatographer wants a CCC column that retains the most liquid stationary phase in order to achieve the best separation of the components in a mixture. What matters most is column performance in terms of: sample loading per injection, speed of separation, purity of target and yield of target. The instrument that has the best performance in all these areas is called a “high-performance” CCC system. This paper demonstrates to the modern chromatographer that a “high-performance” CCC instrument has shorter, lower volume columns that are rotated faster to provide quicker separations, even with the same sample loading.     

Separation of aldicarb,aldicarb sulfoxide,and aldicarb sulfone on unmodified silica with reverse-phase eluents

Summary Aldicarb, aldicarb sulfoxide, and aldicarb sulfone were chromatographed on an octyl-silica bonded-phase column and on an unmodified silica column using acetonitrile/water mobile phases. The elution order of the analytes from the silica column was different from that using the octyl-silica bonded phase and allowed confirmation of residues of aldicarb sulfoxide in citrus nectar. Isocratic elution of the unmodified silica column allowed rapid sample throughput.     

Closed-Loop Recycling Dual-Mode Counter-Current Chromatography with Specified Sample Loading Durations: Modeling of Preparative and Industrial-Scale Separations

We previously reported on a new counter-current chromatography (CCC) operating mode called closed-loop recycling dual-mode counter-current chromatography (CLR DM CCC), which incorporates the advantages of closed-loop recycling (CLR) and dual-mode (DM) counter-current chromatography and includes sequential separation of compounds in the closed-loop recycling mode with the mobile x-phase and in the inverted-phase counter-current mode with the mobile y-phase. The theoretical analysis of several implementations of this separation method was carried out under impulse sample injection conditions. This study is dedicated to the further development of CLR DM CCC theory applied to preparative and industrial separations, where high-throughput operation is required. Large sample volumes can be loaded via continuous loading within a specified time. To simulate CLR DM CCC separations with specified sample loading durations, equations are developed and presented in “Mathcad” software.     

Rapid determination of carbamate pesticides in food using dual counter-current chromatography directly interfaced with mass spectrometry

Dual counter-current chromatography (dual CCC)-tandem mass spectrometry (MS/MS) was successfully performed with a newly designed spiral column for dual CCC. The small column capacity required for directly coupling with electrospray MS/MS was accomplished by forming a rectangular spiral groove on a plastic disk and sealing it with a PTFE sheet. This novel dual CCC-MS/MS technique was successfully applied for the rapid determination of methomyl, fenobucarb and carbaryl pesticides in food. A two-phase solvent system of n-hexane-acetonitrile-0.1% formic acid (45:45:10) was suitable for both good dual CCC separation and sufficient ionization of pesticides. Recoveries of these three pesticides from mandarin orange and spinach samples fortified at 0.05 mg/kg were in the range of 93-107% with relative standard deviations of 2.4-3.8%.     

Application of high-speed counter-current chromatography for the isolation of 9'-cis-neoxanthin from fresh spinach

Preparative HSCCC (high-speed counter-current chromatography) could be applied for the isolation of 9'-cis-neoxanthin from a crude carotenoid extract of fresh spinach leaves. The separation was performed on a Pharma-Tech Research Corp. CCC 1000 with a solvent system composed of hexane:ethanol:water at a volume ratio of 5:5:4.5 at a flow rate of 3 mL/min and at 850 rpm, using the lower phase as mobile phase. 9'-cis-neoxanthin with a purity of up to 94% could be obtained with a single HSCCC purification step of the crude carotenoid extract.     

柱切换直接进样高效液相色谱法测定蜂蜜中3种抗生素的残留量

建立了柱切换反相高效液相色谱法直接进样分离、测定蜂蜜中3种四环素族抗生素(土霉素OTC、四环素TC、金霉素CTC)残留量的分析方法。方法包括：用缓冲溶液溶解样品、直接进样、二次蒸馏水作流动相在C18预柱上在线富集和净化，然后用柱切换阀将预柱与一个C18分析柱接通，草酸溶液-乙腈-甲醇作流动相、紫外检测器在350nm处检测。各组分回收率均大于85％；相对标准偏差小于10％；标准曲线的相关系数在0．9983～0．9991之间；最低检出浓度(≤0．02mg／kg)，满足欧盟和日本等国要求(0．05mg／kg)。     

High-Performance Liquid Chromatography of Fungicides in Citrus Fruits

Abstract

The determination of three common citrus fungicides, diphenyl (DP), o-phenylphenol (oPP) and thiabendazole (TBZ), was investigated with reversed-phase high-performance liquid chromatography. DP and oPP were successfully chromatographed and quantitated by utilizing a reversed-phase Unisil Q C₁₈ column after extraction with an essential oil distillation apparatus. An acetonitrile-0.1M H₃PO₄ (55:45) mobile phase was used. TBZ was chromatographed by using a Unisil Q C₈ column with a mobile phase of acetonitrile-0.1M H₃PO₄ (80:20) after the extraction with ethyl acetate. The fungicides were detected with fluorescence detection at typical residue levels on citrus. The     

高效液相色谱法测定柑橘皮中柠檬苦素

提出了高效液相色谱法测定柑橘皮中柠檬苦素含量的方法。样品用体积分数为70%乙醇溶液于50℃超声提取75min。以Sinochrom ODS-BP(250mm×4.6mm,10μm)为分离柱,以乙腈-水(45+55)溶液作为流动相,用紫外检测器在波长210nm处进行测定。柠檬苦素的质量浓度在12.0～384mg.L-1范围内与其峰面积呈线性关系,方法的平均回收率为99.9%,相对标准偏差(n=5)为1.8%。     

Indirect conductimetric detection of amino acids after liquid chromatographic separation

A liquid chromatographic method using indirect conductimetric detection is proposed for the determination of low levels of organic compounds, which does not require any special functional characteristics of the analyte. The signal detected is proportional to the molar concentration of the analyte and independent of its nature. The detector response is linearly dependent on analyte concentrations over at least three orders of magnitude. The basis of the detection is to create a conducting background, which will decrease on elution of the organic compounds. The theory of the method is discussed, with special reference to the quantitative displacement of the conducting species of the mobile phase from the column by the analyte on sample injection. The proposed method has been applied to study the chromatographic behaviour of twenty-one amino acids, where a 5 -μm Econosil CN column was used as the stationary phase with a mixture of water-acetonitrile-tetrahydrofuran (70:20:3) containing 1 mM perchloric acid or trichloroacetic acid as the mobile phase. The proposed method allows as little as 10 ng of each amino acid to be determined.