Chromatography using a water stationary phase and a carbon dioxide mobile phase

A novel chromatographic separation method is introduced which employs water (saturated with CO(2)) as a stationary phase and CO(2) (saturated with water) as a mobile phase. Since water and CO(2) have little miscibility, conditions can be attained that create a stationary phase of water lining the inside of an uncoated stainless steel capillary. Because altering temperature and pressure can change both the density of the mobile phase and the polarity of the stationary phase, these experimental parameters offer good flexibility for optimizing separations and allow for different gradient programmed separation options. Further, since this method is free of organic stationary and mobile phase components, it is environmentally compatible and allows the use of universal flame ionization detection. This system offers very good sample capacity, peak symmetry, and retention time reproducibility (～1% RSD run-to-run, ～4% RSD day-to-day). Analytes such as alcohols, carboxylic acids, phenols, and tocopherols are employed to investigate this relatively inexpensive and robust method. As an application, the system is used to quantify ethanol in alcoholic beverages and biofuel and to analyze caffeine levels in drinks. In all cases, quantitative results are obtained with quick throughput times and often little need for sample preparation.     

Effect of a variety of organic additives on retention and efficiency in micellar liquid chromatography

The effect of 21 organic additives (alkanols, alkane diols, dipolar aprotic solvents, alkanes) on the chromatographic behavior (retention, elution strength, efficiency) of probe solutes of widely differing hydrophobicity, such as benzene and 2-ethylanthraquinone, have been examined using a C18 stationary phase and sodium dodecyl sulfate (SDS) micellar mobile phases. The mobile-phase elution strength parallels the octanol-water partition coefficients of the additives or their ability to bind to the SDS micellar system, due to the increased solubility in the mobile phase and reduced affinity for the additive-modified surfactant-coated stationary phase. The comparison of the elution strength of micellar mobile phases with that of a reference acetonitrile-water system indicates that the elution strength is lower for micellar systems and depends on the nature of the eluted solute. The displacement of the solute-micelle and solute-stationary phase binding equilibria is quantified for several probe solutes eluted with micellar mobile phases in the presence of 1-propanol, 1-butanol, 1-pentanol, and acetonitrile. A correlation was also observed between the number of theoretical plates and the hydrophobicity of the alcohol additives: the efficiency initially increased steeply and reached a plateau. Compared to benzene, a more hydrophobic additive was needed to attain the maximum efficiency for the more hydrophobic 2-ethylanthraquinone analyte. Dipolar aprotic solvents appear to be somewhat more effective in enhancing the efficiency than alcohols. The results are rationalized in terms of the ability of the organic additives to alter the composition, structure, dynamics, and properties of the micelles and the surfactant-coated stationary phase.     

Simultaneous flame ionization and absorbance detection of volatile and nonvolatile compounds by reversed-phase liquid chromatography with a water mobile phase

A flame ionization detector (FID) is used to detect volatile organic compounds that have been separated by water-only reversed-phase liquid chromatography (WRP-LC). The mobile phase is 100% water at room temperature, without use of organic solvent modifiers. An interface between the LC and detector is presented, whereby a helium stream samples the vapor of volatile components from individual drops of the LC eluent, and the vapor-enriched gas stream is sent to the FID. The design of the drop headspace cell is simple because the water-only nature of the LC separation obviates the need to do any organic solvent removal prior to gas phase detection. Despite the absence of organic modifier, hydrophobic compounds can be separated in a reasonable time due to the low phase volume ratio of the WRP-LC columns. The drop headspace interface easily handles LC flows of 1 mL/min, and, in fact, compound detection limits are improved at faster liquid flow rates. The transfer efficiency of the headspace interface was estimated at 10% for toluene in water at 1 mL/min but varies depending on the volatility of each analyte. The detection system is linear over more than 5 orders of 1-butanol concentration in water and is able to detect sub-ppb amounts of o-xylene and other aromatic compounds in water. In order to analyze volatile and nonvolatile analytes simultaneously, the FID is coupled in series to a WRP-LC system with UV absorbance detection. WRP-LC improves UV absorbance detection limits because the absence of organic modifier allows the detector to be operated in the short-wavelength UV region, where analytes generally have significantly larger molar absorptivities. The selectivity the headspace interface provides for flame ionization detection of volatiles is demonstrated with a separation of 1-butanol, 1,1,2-trichloroethane (TCE), and chlorobenzene in a mixture of benzoic acid in water. Despite coelution of butanol and TCE with the benzoate anion, the nonvolatile benzoate anion does not appear in the FID signal, allowing the analytes of interest to be readily detected. The complementary selectivity of UV-visible absorbance detection and this implementation of flame ionization detection allows for the analysis of volatile and nonvolatile components of complex samples using WRP-LC without the requirement that all the components of interest be fully resolved, thus simplifying the sample preparation and chromatographic requirements. This instrument should be applicable to routine automated water monitoring, in which repetitive injection of water samples onto a gas chromatograph is not recommended.     

A thermodynamic study of the temperature-dependent elution order of cyclic alpha-amino acid enantiomers on a copper(II)-D-penicillamine chiral stationary phase

The reversal of the elution order of cyclic alpha-amino acid enantiomers as a function of the temperature on a copper(II)-N,S-dioctyl-D-penicillamine ligand-exchange column is described. The thermodynamic parameters accounting for the retention and the separation of analytes were determined by means of van't Hoff plots. The influence of different chromatographic conditions on these parameters was investigated, showing little effect of the Cu(II) concentration in the eluent but strong influence of the organic modifier content on the separation. Further, the pH of the mobile phase was found to be a determining factor for the retention of the analytes. Based on these findings, a separation mechanism is postulated comprising the importance of complex formation for primary docking at the stationary phase, while hydrophobic interactions are crucial for chiral discrimination.     

Temperature-responsive chromatographic separation of amino acid phenylthiohydantions using aqueous media as the mobile phase

Recently, green chemistry has become one of the most important subjects of science for environmental pollution prevention. Here, we report development of a novel chromatographic technology for phenylthiohydantoin (PTH)-amino acid analyses in which only aqueous solution is used as the mobile phase. We have devised HPLC adsorbents (stationary phase) by modifying the surfaces of microparticulate silica gel using functional polymers. The thermoresponsive copolymer, poly(N-isopropylacrylamide-co-n-butyl methacrylate) (IBc) was used to modify the silica stationary phase surfaces. This polymer-grafted surface exhibits temperature-regulated hydrophilic/hydrophobic property changes in water. PTH-amino acid interactions with this surface are readily modulated by changing the column temperature using an isocratic aqueous mobile phase. Increasing hydrophobic interactions between more hydrophobic PTH-amino acids with hydrophobized polymer-grafted surfaces at elevated mobile phase temperatures is used for the effective separation of PTH-amino acids in aqueous solution. This study is aimed at the development of novel separation processes, which are also environmentally benign, for use with biochemical substances in order to meet the growing needs of the life sciences and biotechnology. The method is useful for various separations in life science so that proteins can maintain their biological activity and enzymes, their enzymatic activity.     

pH gradient reversed-phase HPLC

pH gradient HPLC is reported, which is a new original mode of reversed-phase high-performance liquid chromatography applicable to ionogenic analytes. The method consists of programmed increase during the chromatographic run of the eluting strength of the mobile phase with respect to the acid/base analytes separated. Unlike the well-established conventional gradient HPLC, where the eluting power of the mobile phase is increased with time due to the increasing content of organic modifier, in the pH gradient HPLC that is realized by linearly increasing (in the case of acids) or decreasing (in the case of bases) the pH of the eluent of a fixed organic modifier content, thus providing functional increase in the degree of analyte dissociation and, hence, a decrease in its retention. The pH gradient mode has typical features of gradient HPLC, such as reduced peak width and minimized peak-tailing due to peak compression, which is especially advantageous in the case of organic base analytes. It may be of special value for separation of those analytes which are susceptible to the higher concentrations of organic solvents, as many bioanalytes are. A theory of the pH gradient HPLC has been elaborated, and its full mathematical formalistic is presented step by step in a comprehensive manner. Although fundamental relationships at the basis of pH gradient HPLC are more complex than in the case of the organic gradient variant, the resulting mathematical model is easily manageable. Its applicability to predict changes in retention and separation of test mixtures of analytes accompanying the changes in chromatographic conditions has been demonstrated experimentally in both gradient and isocratic HPLC. The proposed model supplies a rational basis for modifications of eluent pH aimed at optimization of separations and for convenient assessment of chromatographically relevant physicochemical parameters of analytes, such as pK(a).     

Monolithic column with zwitterionic stationary phase for capillary electrochromatography

A capillary electrochromatography (CEC) monolithic column with zwitterionic stationary phases was prepared by in situ polymerization of butyl methacrylate, ethylene dimethacrylate, methacrylic acid, and 2-(dimethyl amino) ethyl methacrylate in the presence of porogens. The stationary phases have zwitterionic functional groups, that is, both tertiary amine and acrylic acid groups, so the ionization of those groups on the zwitterionic stationary phase was affected by the pH values of the mobile phase, and further affects the strength and direction of the electroosmotic flow (EOF). Separations of alkylbenzenes and polycyclic aromatic hydrocarbons based on the hydrophobic mechanism were obtained. Separation of various types of polar compounds, including phenols, anilines, and peptides, on the prepared column were performed under CEC mode with anodic and cathodic EOF, and different separation selectivities of those polar analytes were observed on the monolithic capillary column by using mobile phases with different pH values.     

Effect of the temperature and mobile phase composition on the retention behavior of nitroanilines on ligand-exchange stationary phase

This paper deals with the separation of isomers of nitroaniline by liquid chromatography using the ligand-exchange technique. The chromatographic separations were performed on the ligand-exchanger sporopollenin. The sporopollenin used as support of stationary phase was modified with carboxylated-ethylenediamine matrix and was loaded with cobalt(II) ions. Using the column packed with cobalt(II) loaded carboxylated diaminoethyl sporopollenin [Co(II)-CDAE-S], the retention behavior of 3- and 4-nitroanilines was investigated. The mobile phase used, was a mixture of 0.05 M NH(4)OH in ethanol-water. The resolution was strongly affected by the presence of ammonium hydroxide in the mobile phase and a concentration of 0.05 M was shown to be necessary for the separation of analytes. To study the effects of temperature on the resolution, column runs were also performed at various temperatures (15-60 degrees C). With increasing temperature, a decreased interaction between the solutes and the ligand-exchanger was observed. Consequently, the best results were obtained using a mixture of 0.05 M NH(4)OH in ethanol-water (10:90, v/v) as the mobile phase at a column temperature of 35 degrees C. Ligand-exchange chromatography on the Co(II)-CDAE-S could be a useful alternative method for the separation of nitroaniline.     

Off-line coupling of subcritical water extraction with subcritical water chromatography via a sorbent trap and thermal desorption

In this study, the off-line coupling of subcritical water extraction (SBWE) with subcritical water chromatography (SBWC) was achieved using a sorbent trap and thermal desorption. The sorbent trap was employed to collect the extracted analytes during subcritical water extraction. After the extraction, the trap was connected to the subcritical water chromatography system, and thermal desorption of the trapped analytes was performed before the SBWC run. The thermally desorbed analytes were then introduced into the subcritical water separation column and detected by a UV detector. Anilines and phenols were extracted from sand and analyzed using this off-line coupling technique. Subcritical water extraction of flavones from orange peel followed by subcritical water chromatographic separation was also investigated. The effects of water volume and extraction temperature on flavone recovery were determined. Because a sorbent trap was used to collect the extracted analytes, the sensitivity of this technique was greatly enhanced as compared to that of subcritical water extraction with solvent trapping. Since no organic solvent-water extractions were necessary prior to analysis, this technique eliminated any use of organic solvents in both extraction and chromatography processes.     

Measurement of partition coefficients in waterless biphasic liquid systems by countercurrent chromatography

Countercurrent chromatography (CCC) is a chromatographic separation technique that uses a liquid as a stationary phase. Centrifugal forces are used to immobilize the liquid stationary phase when the liquid mobile phase is pushed through it. In CCC, the solutes are separated according to their liquid-liquid partition coefficients. The solutes studied were the alkylbenzene homologues from benzene to hexylbenzene and some polyaromatic hydrocarbons (PAHs) from naphthalene to coronene. Their liquid-liquid partition coefficients were measured in the five waterless biphasic systems formed by heptane, as the apolar liquid phase of the five biphasic systems, and four dipolar aprotic solvents, dimethyl sulfoxide, dimethylformamide, furfural, and N-methylpyrrolidone, and the polar proton-donor solvent methanol. The coefficients were compared to the corresponding capacity factors obtained by classical liquid chromatography on octadecyl-bonded silica. For the five biphasic solvent systems studied, linear relationships were found between the partition coefficients and the sp(3) and sp(2) hybridized carbon atom number for the alkylbenzene and PAH series, respectively. The sp(2) and sp(3) transfer energies were estimated, and their ratio was used to quantify the solvent selectivity toward aromatic extraction.     

Determination of pKa by pH gradient reversed-phase HPLC

pH gradient reversed-phase HPLC consists of a programmed increase during the chromatographic run of the eluting power of the mobile phase with regard to ionizable analytes. On the analogy of the conventional organic modifier gradient RP HPLC, in the pH gradient mode, the eluting strength of the mobile phase increases due to its increasing (with acid analytes) or decreasing (with basic analytes) pH, whereas the content of organic modifier is kept constant. We have shown previously that the pH gradient separations are technically possible using standard chromatographic equipment. Here we demonstrate that the method is uniquely suitable to determine pK(a) values of analytes. A strict theoretical model is proposed to determine pK(a) values based on the retention data from a pH gradient RP HPLC run. The pK(a) data so obtained are discussed in relation to the concentration of methanol in the mobile phase, the type of stationary phase, and the duration of the gradient. The pK(a) values determined by the pH gradient method are related to the respective data obtained conventionally in a series of isocratic experiments. A close similarity of the two types of chromatographically determined pK(a) data is demonstrated. The HPLC-derived pK(a) parameters correlate to the literature pK(a) values determined by titrations in water. The chromatographically derived and the reference pK(a) values are not identical, however. That is probably due to the effects on the chromatographic pK(a) of the specific sites of interactions with analytes on the surfaces of the HPLC stationary phases. Nonetheless, the proposed pH gradient HPLC method may supply in a fast and convenient manner comparable acidity parameters for larger series of drug candidates, including those available in only minute amounts, without need of their purification, and also when the compounds are provided as complex mixtures, like those produced by combinatorial chemistry.     

Covalently bound ionene polyelectrolyte-silica gel stationary phases for HPLC

Micelle-mimetic ionene-based stationary phases for high-performance liquid chromatography (HPLC) are prepared by attaching [3,16]- and [3,22]-ionenes to aminopropyl silica through a carbon-nitrogen bond. These [x,y]-ionenes are polyelectrolytic molecules consisting of dimethylammonium charge centers interconnected by alternating alkyl chain segments containing x and y methylene groups, some of which can form aggregate species whose properties mimic those of conventional surfactant micelles. These ionene-bonded stationary phases were characterized using different recommended HPLC test mixtures. Test solute chromatographic behavior on the ionene phases was found to be similar to that of intermediate oligomeric or polymeric C-18 and/or phenyl phases, depending upon the specific test mixture employed. In addition, the phases exhibit significant solute shape recognition ability. The ionene stationary phases were successfully employed for the separation of the components of the recommended ASTM reversed-phase test mixture, as well as for ortho-, meta- and para-disubstituted benzenes and other positional or geometric isomeric compounds. The ionene materials allow for chromatographic separations under either reversed-phase or ion-exchange conditions. The retention mechanism on these multimodal phases can occur by hydrophobic partitioning or electrostatic interactions, depending upon the characteristics of the components of the analyte mixture (neutral or anionic). The effects of alteration of the percent organic modifier, flow rate and temperature of the mobile phase on chromatographic retention and efficiency on these phases were briefly examined.     

Incorporation of single-wall carbon nanotubes into an organic polymer monolithic stationary phase for mu-HPLC and capillary electrochromatography

Single-wall carbon nanotubes (SWNT) were incorporated into an organic polymer monolith containing vinylbenzyl chloride (VBC) and ethylene dimethacrylate (EDMA) to form a novel monolithic stationary phase for high-performance liquid chromatography (HPLC) and capillary electrochromatography (CEC). The retention behavior of neutral compounds on this poly(VBC-EDMA-SWNT) monolith was examined by separating a mixture of small organic molecules using micro-HPLC. The result indicated that incorporation of SWNT enhanced chromatographic retention of small neutral molecules in reversed-phase HPLC presumably because of their strongly hydrophobic characteristics. The stationary phase was formed inside a fused-silica capillary whose lumen was coated with covalently bound polyethyleneimine (PEI). The annular electroosmotic flow (EOF) generated by the PEI coating allowed peptide separation by CEC in the counterdirectional mode. Comparison of peptide separations on poly(VBC-EDMA-SWNT) and on poly(VBC-EDMA) with annular EOF generation revealed that the incorporation of SWNT into the monolithic stationary phase improved peak efficiency and influenced chromatographic retention. The structures of pretreated SWNT and poly(VBC-EDMA-SWNT) monolith were examined by high-resolution transmission electron microscopy, Raman spectroscopy, scanning electron microscopy, and multipoint BET nitrogen adsorption/desorption.     

Systematic Approach to Links between Separations in MEKC and Reversed-Phase HPLC

Retention factors and partition coefficients in micellar electrokinetic chromatography (MEKC) and reversed-phase high-performance liquid chromatography (RP-HPLC) are compared for a series of alkylbenzenes and substituted phenols. In both techniques, separations are based on partitioning between an aqueous phase and an alkyl phase. In MEKC, this was an SDS (C12) micellar pseudostationary phase, and in RP-HPLC an ODS 2 (C18) stationary phase. A nonporous silica (Micra 1.5-μm NPS), which has a low carbon loading, was used rather than a standard porous silica to avoid excessive retention in HPLC and to allow identical mobile phase conditions to be used in both separation modes. The average ratio of analyte retention factors, k(MEKC):k(HPLC), was found to be equal to the ratio β(MEKC):β(HPLC), where β is the phase ratio. This implies that partition coefficients, P, are similar in both MEKC and HPLC, since P = k/β, and that the dominant contribution to stability within each alkyl phase arises from hydrophobic interactions which are common to both separation media. Since partition coefficients are similar in MEKC and HPLC under aqueous buffer conditions, information on retention in one technique may be transferred to the other, provided that the phase ratios are known. In MEKC and HPLC, linear correlations of log octanol-water partition coefficients, K(ow), vs log k for the test compounds were transformed, knowing the phase ratio, to give log P values as a function of log K(ow). This allows quantitative links between MEKC and HPLC to be extended to include octanol-water partitioning. The addition of acetonitrile as an organic modifier over the concentration range 0-20% (v/v) was found to have a greater effect on k in HPLC than in MEKC. This could be a result of a decrease in the MEKC phase ratio due to an increase in the critical micelle concentration.     

Separation and sequencing of isomeric oligonucleotide adducts using monolithic columns by ion-pair reversed-phase nano-HPLC coupled to ion trap mass spectrometry

An ion-pair reversed-phase nano-high-performance liquid chromatography (IP-RP-nano-HPLC) method using a monolithic poly(styrene-divinylbenzene) (PS-DVB) column coupled to nanoelectrospray ionization mass spectrometry (nano-ESI-MS) was evaluated to separate and identify isomeric oligonucleotide adducts derived from the covalent binding of (+/-)-anti-7r,8t-dihydroxy-9t,10-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene [(+/-)-anti-BPDE] to double-stranded (ds) 5'-PO4--ACCCGCGTCCGCGC-3'/5'-GCGCGGGCGCGGGT-3' oligonucleotide. The influence of three different nanospray emitters on electrospray signal was evaluated in terms of analyte ion sensitivity. The best nanoelectrospray performance for the oligonucleotides was observed with the distal metal-coated emitter. The performance of three different stationary phases was also investigated. The chromatographic separation performance of the polymeric monolithic PS-DVB stationary phase significantly surpassed that of columns packed with the microparticulate sorbents C18 or PS-DVB. Different mobile phase organic solvents and ion-pairing reagents were also evaluated. An optimized mobile phase consisting of methanol and 25 mM triethylammonium bicarbonate resulted in the best chromatographic resolution and increased MS sensitivity of the oligonucleotides. By using a monolithic PS-DVB stationary phase fabricated in a nanocolumn, four positional isomeric (+/-)-BPDE-oligonucleotide adducts were separated and identified. In addition to four of the possible five positional isomers, three positional isomers were also resolved to several diastereoisomers, although their stereostructures could not be identified in the absence of reference standards.     

Determination of Anionic, Neutral, and Cationic Species of Arsenic by Ion Chromatography with ICPMS Detection in Environmental Samples

Ion chromatographic methods developed to separate either cationic, neutral, and anionic arsenic species or soluble and suspended arsenic species were successfully used in DORM-2 standard reference material and in water samples of environmental interest. The most effective separation of the analytes within 10 min was achieved with a nitric acid gradient elution using a strong anion-exchange stationary phase with additional capacity for hydrophobic interactions (IonPac AS7). The elemental-specific detection mode allows the sensitive determination of the arsenic species in the submicrogram per liter range. The calibration results were compared with those obtained by an alkaline water-methanol mixed eluent combined with a weak anion-exchange column (IonPac AS4A-SC). Differences in sensitivities were eclipsed by the low level of the baseline and the noise when using nitric acid. The gradient method was used to determine arsenic species in highly ferrous/ferric-contaminated leachates of lignite spoil. The companion elements underwent parallel screening to explain the interactions of arsenic species with the major elements.     

RP-HPLC法丙交酯纯度的定量测定

建立了高效液相色谱(HPLC)测定丙交酯纯度的方法。解决了分析过程中丙交酯水解不稳定的问题。在HPLC分析过程中,无水的有机流动相不能实现丙交酯与其它杂质的分离,有机溶剂和水的混合溶液作流动相能有效改善丙交酯与其它杂质的分离效果。通过对实验条件的摸索,并对丙交酯水解动力学进行研究,发现丙交酯在选定的色谱条件下,在流动相中的水解反应具有准一级反应动力学的特征,由此可对丙交酯进行定量分析。实验表明,相对标准偏差RSD<1.0%,方法切实可行。     

Water-induced coacervation of alkyl carboxylic acid reverse micelles: phenomenon description and potential for the extraction of organic compounds

Coacervates made up of alkanoic (C8-C16) and alkenoic (C18) acid reverse micelles were described for the first time, and their potential for the extraction of organic compounds prior to liquid chromatography was examined. The coacervation process occurred in miscible binary mixtures of water and a variety of protic and aprotic solvents. The phase behavior of alkyl carboxylic acids was found to be a function of both the Hildebrand solubility parameter, delta, and the hydrogen-bonding capability of the solvent. The best solvents for analytical extractions were those featuring the lowest delta values. The phase behavior of alkyl carboxylic acid/water/tetrahydrofuran (THF) ternary systems as a function of component concentration, pH, ionic strength, and temperature was investigated. The efficiency and the time required for phase separation depended on the experimental procedure used (i.e., standing, centrifugation, stirring, and sonication). The formation of alkyl carboxylic acid reverse micelles in THF was proven using both hydrophilic fluorescent probes and scattered light measurements. The structure of the coacervates consisted of spherical droplets dispersed in a continuous phase. Phase volume ratios were a function of both alkyl carboxylic acid and THF concentration. The low volume obtained (e.g., 1.5 microL per mg of decanoic) compared to that obtained by other coacervates (e.g., 5.1 microL per mg of dodecane sulfonic acid and 11.3 microL per mg of Triton X-114) greatly improved the concentration factors reached by coacervation-based extractions. Parameters affecting the extraction efficiency were assessed. Analytes in a wide range of polarity were efficiently extracted on the basis of the hydrophobic (e.g., PAHs) and hydrogen bond (e.g., chlorophenols, bisphenols, pesticides, phthalates, nonionic surfactants, dyes, and photographic developers) interactions that reverse micelles can establish. The coacervates were compatible with the chromatographic determination of analytes following UV or MS detection. They were successfully applied to the extraction of alkylphenol ethoxylates (octyl and nonyl) and alcohol ethoxylates (C12-C16) from influent and effluent wastewater and river water samples. Nonionic surfactants in the coacervate were directly separated and quantified by liquid chromatography-ion trap mass spectrometry. Concentration factors were around 160. The recovery of nonionics in the environmental water samples ranged from 90 to 104%.     

猪肝中克伦特罗的测定-反相高效液相色谱法

文中建立一种简单、快速测定猪肝中克伦特罗的液相色谱检测方法。鲜猪肝中克伦特罗在碱性条件下（pH—pKa〉2）,乙酸乙酯匀浆萃取并酸化乙酸乙酯有机基质后水反萃取,SCXSPE柱净化及氨化甲醇洗脱,氮吹近干后挥干,甲醇定容后测定。色谱分离采用ShisedoCapcellPAKSCXUGSO150mm×4．6mm柱,乙腈、磷酸二：氢钾为流动相,210nm波长处检测。该方法工作标准曲线的线性范围为185．6ng·ml～46．4μg·m^-1,相关系数r=O．99995,其检出限为0．15μg·ml^-1,回收率为92．4～95．2％。该方法的准确性和精密度满足检测要求需要。     

Elution-extrusion countercurrent chromatography. Use of the liquid nature of the stationary phase to extend the hydrophobicity window

Countercurrent chromatography (CCC) is a liquid chromatography technique with a liquid stationary phase. Taking advantage of the liquid nature of the stationary phase, it is possible to perform unique operations not possible in classical liquid chromatography with a solid stationary phase. It is easy to avoid any solute-irreversible absorption in the CCC column. If the retention volumes of solutes become too high, the dual mode will be used. The roles of the phases are reversed. The stationary phase becomes the mobile phase, and the CCC column is started again. The solutes elute rapidly in what was previously the stationary phase. The theoretical basis of the dual-mode method is recalled. The dual-mode method is a discontinuous method. The separation should be stopped when the phase switch is performed. The elution-extrusion procedure is another way to avoid any irreversible adsorption of solutes in the column. The method uses the fact that the liquid volumes occupied by the solutes highly retained inside the column can be orders of magnitude lower than the mobile-phase volume that would be needed to elute them. The elution-extrusion method also has two steps: the first step is a regular CCC chromatogram. Next, the stationary phase containing the partially separated hydrophobic solutes is extruded out of the column in a continuous way using the liquid stationary phase. The theory of the process is developed and compared to the dual-mode theory. Alkylbenzene homologues are experimentally used as model compounds with the heptane/methanol/water biphasic liquid system to establish the theoretical treatment and compare the performance of two types, hydrodynamic and hydrostatic, of CCC columns. It is shown that the method can dramatically boost the separation power of the CCC technique. An apparent efficiency higher than 20 000 plates was obtained for extruded octylbenzene and a 160-mL hydrodynamic CCC column with less than 500 plates when conventionally used.