Liquid exclusion-adsorption chromatography: new technique for isocratic separation of nonionic surfactants. I. Retention behaviour of fatty alcohol ethoxylates

A new technique of liquid chromatography is described, which allows a baseline separation of fatty alcohol ethoxylates with 15–20 ethylene oxide units under isocratic conditions. The new method is based on a combination of two different chromatographic modes for the individual structural units: size exclusion for the poly(oxyethylene) chain and adsorption interaction for the hydrophobic end fragments. A theory is provided for this mixed exclusion–adsorption mode of liquid chromatography. Chromatographic data are found to be in a good agreement with this theory.     

Rapid separation of basic drugs by nonaqueous capillary electrophoresis

Summary Nonaqueous capillary electrophoresis (NACE) has been used to achieve rapid separations of basic drugs. A high electric field was obtained by using short capillaries. Baseline separations of basic drugs, including amphetamines, tropane alkaloids and local anesthetics, were achieved in 1 min by selection of the appropriate organic solvent and electrolyte composition. Thus, high-throughput analyses can be performed. Peak efficiency up to 9154 theoretical plates s⁻¹ was achieved in a separation performed at 923V cm⁻¹. No discernible loss in resolution was observed when a conventional capillary (64.5cm) was replaced by a short (32.5 cm) capillary.     

Large-volume sample on-line concentration of angiotensins in non-aqueous capillary electrophoresis

A single step on-line concentration and separation method for peptides in non-aqueous capillary electrophoresis was developed. ACN containing 50 mM tetraethylammonium perchlorate was used as the electrophoretic medium; angiotensins I-IV were separated as a result of the differences in the magnitudes of their interactions with perchlorate anions. When the sample solution (ACN containing 0.5% trifluoroacetic acid and angiotensins) was injected as a large-volume plug, the analytes were concentrated at the inlet end of the capillary by both sweeping and stacking mechanisms; the separation procedure then started automatically without any operations such as polarity change. It was found that the concentration of analytes, injection period, and concentration of tetraethylammonium perchlorate in the electrophoretic medium were important factors for both separation and concentration efficiencies. The angiotensins were concentrated and separated with the large-volume injection of up to 80% of the effective capillary length.     

Sample-stacking techniques in non-aqueous capillary electrophoresis

In sample-stacking techniques, the detection limit cannot be improved by simply increasing the length of the sample solution, because the individual electrophoretic parameters must be optimized. In an attempt to increase the amount of sample injected, as well as to focus them onto a small zone, two novel methods are proposed. One of these employs an "ultra-high conductivity zone", which was inserted between the sample zone and background solution to build an unequal conductivity gradient. The other employs a "low temperature bath". A portion of the capillary (near the junction between the sample solution and the background solution) was immersed in a low temperature bath, which served as a "pseudo-high-conductivity zone" due to the fact that conductivity would increases when the temperature is decreased. As a result, a large volume of sample injection can be achieved. Using 3,4-methylenedioxymethamphetamine as a model compound, the detection limit was determined to be 1.6 x 10(-6) M (S/N = 3) by means of normal non-aqueous capillary electrophoresis (NACE). This could be improved to 3.0 x 10(-8) M, 4.8 x 10(-9) M and 5.0 x 10(-9) M, respectively, when the normal stacking, ultra-high conductivity zone NACE-stacking and the low-temperature zone NACE-stacking methods were applied.     

Analysis of neutral surfactants by non-aqueous capillary electrophoresis using an electroosmotic flow reversal

The separation of KM 20, that is in fact a mixture of non-ionic surfactants, was carried out by non-aqueous capillary electrophoresis. This complex mixture resulting from the condensation of ethylene oxide with fatty alcohols does not have chromophoric moieties. So, we analysed it after derivatization by means of 3,5-dinitrobenzoyl chloride. The proposed approach is based both on the formation of complexes with alkaline or ammonium cations in methanol and on the utilisation of a positively charged capillary. From a comparative study on the capillary treatment procedure, we used hexadimethrine bromide as electroosmotic flow reverser in order to obtain both repeatable analyses and good resolutions of the largest KM 20 oligomers. Then, among the five cations used to form complexes with KM 20, we pointed out that ammonium cation led to the best resolutions. Moreover, we evidenced that the counter-ion of this cation had a great influence on resolution because it modified the magnitude of electroosmotic flow. Ion pair formation that is more or less strong between ammonium and its counter-ion was involved in this variation of electroosmotic flow. So, we calculated the association constants for various ammonium salts in methanol. Then, using ammonium chloride as background electrolyte, we optimised the concentration of this salt, in methanol, in order to reach the optimal separation of KM 20 oligomers. Thus, a baseline separation was obtained by using 6 x 10(-2) mol/L NH4Cl as running electrolyte. In these conditions, we separated, in about 30 min, more than 30 oligomers of KM 20. The distribution of these oligomers that was determined from the optimal separation, appeared consistent with that obtained from HPLC analyses. Indeed, we determined that the mean ethoxylation number was equal to 18 while its real value is equal to 20.     

Separation of open-cage fullerenes using nonaqueous capillary electrophoresis

In this study, nonaqueous capillary electrophoresis (NACE) was used to separate three open-cage fullerenes. Trifluoroacetic acid (TFA) was used as the nonaqueous background electrolyte to change the analytes’ mobilities. The selectivity and separation efficiency were critically affected by the nature of the buffer system, the choice of organic solvent, and the concentrations of TFA and sodium acetate (NaOAc) in the background electrolyte. The optimized separation occurred using 200 mM TFA/20 mM NaOAc in MeOH/acetonitrile (10:90, v/v), providing highly efficient baseline separation of the open-cage fullerenes within 5 min. The migration time repeatability for the three analytes was less than 1% (relative standard deviation). Thus, NACE is a rapid, useful alternative to high-performance liquid chromatography for the separation of open-cage fullerenes.     

反向电渗流非水毛细管电泳法快速测定微量赤霉素

赤霉素是一类重要的植物激素,是结构相似的弱酸性二萜类化合物。针对毛细管电泳法分离、测定赤霉素速度慢、效率不佳等问题,该文发展了一种可快速测定微量活性赤霉素的非水毛细管电泳-紫外检测法。以聚环氧乙烷动态涂布毛细管,利用正离子使电渗流反向,将含10 mmol/L醋酸铵的甲醇-水(95∶5,v/v)作为缓冲体系,在0.08%(v/v)醋酸含量下,分离8种内源性赤霉素。结果表明,赤霉素出峰时间的相对标准偏差(RSD)≤2.1%(日内)或≤4.3%(日间),峰面积的RSD≤4.5%(日内)或≤6.9%(日间),检出限(S/N=3)为1.04~2.20 mg/L,相关系数为0.998 2~0.999 3,回收率为87.2%~93.5%。该方法简单、快速、稳定,以含醋酸铵的甲醇水溶液为缓冲体系,预先考虑了质谱测定的需要,可用于实际样品如麦芽中赤霉素的分析,具有一定的应用价值。     

非水毛细管电泳法测定藤黄中藤黄酸的含量

藤黄酸(gambogic acid, GA)等环氧杂蒽酮类化合物的水溶性差,可通过非水毛细管电泳(non-aqueous capillary electrophoresis, NACE)分析。本文系统地考察了添加20%~60%(v/v)的甲醇或乙腈的运行电解质溶液对藤黄提取液中藤黄酸分离的影响。比较了不同的运行电解质溶液、运行电解质溶液浓度、pH、添加剂 β-环糊精的浓度、分离温度及分离电压的影响,建立了测定藤黄药材中藤黄酸含量的非水毛细管电泳方法。在40%乙腈、10 mmol/L β-环糊精、20 mmol/L四硼酸钠(pH 9.86)为运行电解质溶液、分离电压为10 kV、分离温度为30 ℃、检测波长为280 nm的条件下进行测定。结果表明,藤黄酸在2~2000 mg/L范围内线性关系良好,相关系数为0.9996,检出限(S/N=3)为2 mg/L。将本方法应用于越南、泰国、缅甸、印度4个产地的藤黄药材中藤黄酸的含量测定,测得含量为1.67~472.40 mg/g(相对标准偏差(RSD)为1.12%~2.60%),其中越南产藤黄中藤黄酸含量低,其他产地藤黄中藤黄酸的含量高。实际藤黄样品中藤黄酸的加标回收率为95.2%~105.6%。非水毛细管电泳方法简单、快速、重现性好,可用于藤黄药材中藤黄酸的含量测定。     

Determination of fatty alcohol ethoxylates and alkylether sulfates by anionic exchange separation, derivatization with a cyclic anhydride and liquid chromatography

A method for the separation, characterization and determination of fatty alcohol ethoxylates (FAE) and alkylether sulfates (AES) in industrial and environmental samples is described. Separation of the two surfactant classes was achieved in a 50:50 methanol-water medium by retaining AES on a strong anionic exchanger (SAX) whereas most FAE were eluted. After washing the SAX cartridges to remove cations, the residual hydrophobic FAE were eluted by increasing methanol to 80%. Finally, AES were eluted using 80:20 and 95:5 methanol-concentrated aqueous HCl mixtures. Methanol and water were removed from the FAE and AES fractions, and the residues were dissolved in 1,4-dioxane. In this medium, esterification of FAE and transesterification of AES with a cyclic anhydride was performed. Phthalic and diphenic anhydrides were used to derivatizate the surfactants in industrial samples and seawater extracts, respectively. Separation of the derivatized oligomers was achieved by gradient elution on a C8 column with acetonitrile/water in the presence of 0.1% acetic acid. Good resolution between both the hydrocarbon series and the successive oligomers within the series was achieved. Cross-contamination of FAE with AES and vice versa was not observed. Using dodecyl alcohol as calibration standard, and correction of the peak areas of the derivatized oligomers by their respective UV-vis response factors, both FAE and AES were evaluated. After solid-phase extraction on C18, the proposed method was successfully applied to the characterization and determination of the two surfactant classes in industrial samples and in seawater.     

Separation of transition metals in nonaqueous media with capillary electrophoresis

The separation of transition metal Ni2+, Cu2+, Co2+, Zn2+, Cd2+ and Fe3+ in methanol was investigated by using different types of organic acids as complexing agents. In pure methanol, the weaker and simpler acetic, propionic, butyric and valeric acids could enhance metal ions selectivity by increasing acid concentration and metal ions could be separated with high efficiency. However, hydroxycarboxylic acids obviously made separation efficiency worse. The effect of mixed organic acids, mixture solvent (methanol-acetonitrile, methanol-water) on metal ions separation was discussed further. The advantages of using nonaqueous solvent over aqueous for metal ions separation were shown finally.     

非水介质毛细管电泳电导检测罗红霉素及其制剂

采用甲醇为分离介质，三(羟甲基)氨基甲烷-硼酸(Tris—H3BO3)为支持电解质，采用负高压，使用电导检测，对罗红霉素及其制剂进行了毛细管电泳分离检测，对电泳介质的种类、浓度、表观pH、以及操作电压和进样时问对分离的影响进行了研讨，在选定的条件下，罗红霉素的线性范围为19．0—142．0mg/L，检出限为0．8mg/L(S／N≥3)，峰面积的相对标准偏差RSD(n=6)为4．3％。3种供试品中罗红霉素的平均加标回收率分别为97．7％、94．8％、93．6％。     

Nonaqueous capillary electrophoresis using a titania-coated capillary

In this work, an ordered mesoporous titania film was introduced to coat a capillary by means of the sol-gel technique. Its electroosmotic flow (EOF) property was investigated in a variety of nonaqueous media (methanol, formamide and N,N'-dimethylformamide and mixtures of methanol and acetonitrile). The titania-coated capillary exhibited a distinctive EOF behavior, the direction and magnitude of which were strongly dependent on various parameters such as the solvent composition, apparent pH (pH*) and the electrolytes. The nonaqueous capillary electrophoresis separation of several alkaloids was investigated in the positively charged titania-coated capillary. Comparison of separation between coated and uncoated capillaries under optimal nonaqueous conditions was also carried out.     

Nonaqueous capillary electrophoresis of imatinib mesylate and related substances

In the present study, nonaqueous capillary electrophoretic separation of imatinib mesylate (IM) and related substances, N-(5-amino-2-methylphenyl)-4-(3-pyridyl)-2-pyrimidinamine (PYA), N-(4-methyl-3-(4-(pyridin-3-yl)pyrimidin-2-ylamino)phenyl)-4-((piperazin-1-yl)methyl) benzamide (NDI) and 4-chloromethyl-N-(4-methyl-3-((4-(pyridin-3-yl) pyrimidin-2-yl) amino) phenyl) benzamide (CPB) was developed. The influential factors affecting separation, including type and concentration of the electrolyte, applied voltage, and buffer modifier were investigated. Baseline separation of the studied analytes was obtained using a buffer of 50 mM Tris and 50 mM methanesulfonic acid in methanol at a apparent pH (pH*) of 1.65. To enhance the sensitivity, large-volume sample stacking was employed for online concentration. The strongest analytical signal with a suitable separation was achieved when the injection time was 100 s. The linearity ranges of PYA and NDI were 0.100-2.50 μg mL(-1) , and that of CPB was 0.125-2.50 μg mL(-1) , with good coefficients (r(2) > 0.9948). The relative standard deviations of intra- and interday were satisfactory. Under the optimized conditions, seven batches of the synthesized samples were analyzed and CPB was detected in two batches. Owing to its simplicity, effectiveness, and low price, the developed method is promising for quality control of IM.     

Enhanced detection of seven glucoconjugated and hydroxylated porphyrins and chlorins by nonaqueous capillary electrophoresis combined with stacking

The nonaqueous capillary electrophoresis mode which includes a preconcentration step based on a transient pseudo-isotachophoresis to the simultaneous separation of seven glucoconjugated and hydroxylated porphyrins and chlorins, exhibiting very close structures, is reported. A high methanol content, of the buffer solution, was necessary in order to prevent self-assembly of the compounds and to enhance their solubility during separation. With the addition of 66% (v/v) methanol and 1% (w/v) NaCl in the aqueous sample solution, large volumes could be injected (44% capillary volume) without a loss in resolution. Sensitivity of detection was therefore improved by a 100-fold factor with regard to the method employing normal injection (2% capillary volume). Optimum electrophoretic conditions, in terms of sensitivity and performance, were obtained by using 20 mM phosphoric acid buffer, pH 2.2 and 50% methanol. The method was validated and applied to qualitative analysis of glucoconjugates in serum samples.     

Electrostatic interaction mechanism on the separation of phenols by non-aqueous capillary electrophoresis

The electrostatic interaction between additive and analyte is of great importance to non-aqueous cap- illary electrophoresis(NACE)separation.Three tetraalkylammonium bromides and acetonitrile were applied as additives and running solvent respectively.The effect of alkyl chain length and concentra- tion of additive on electrostatic interactions was investigated by the separation of phenols.The sepa- ration ability was found to increase with decreasing alkyl chain length of the additive,and the resolu- tion values were increased with increasing additive concentration.The separation was seriously dete- riorated after a little amount of water was added in the running solution.Furthermore,the electrostatic interaction is strong under the conditions of low electron cloud density,weak steric hindrance and multi-interaction sites.Thus,the separation result can be predicted by theoretical analysis,which is helpful for the separation of other substances in NACE based on electrostatic interaction.     

Separation of nonylphenol ethoxylates and nonylphenol by non-aqueous capillary electrophoresis

Capillary electrophoresis based on non-aqueous solvent background electrolytes was employed, with single and multiple wavelength UV detection, to evaluate discrimination among oligomer components of mixtures of non-ionic, long chain nonylphenol ethoxylates (NPnEO, with n = number of ethoxy units) and their lipophilic degradation products. The tested organic solvents included acetonitrile, methanol, ethanol, 1- and 2-propanol, 1-butanol and tetrahydrofurane in the presence of sodium acetate. A rational variation of composition of background electrolyte solvent mixtures allowed to modify the mobility of electroosmotic flow and the type and degree of interactions between the ionic additive (sodium acetate) and the components of the analyte mixtures. The physicochemical properties of the solvents, such as dielectric constant, viscosity and electron donor-acceptor ability regarding the additive, were considered to improve the resolution of lipophilic compounds with less than three ethoxy groups and the discrimination attainable for longer chain oligomers. The studied methodologies also allowed discerning between surfactants of similar (nominal) ethoxy chain lengths. This was demonstrated by the different peak distribution patterns observed for NPnEO compounds with n = 7.5 and 10, respectively.     

Determination of free fatty acids by capillary zone electrophoresis

Capillary zone electrophoresis was investigated for the separation of free fatty acids as an alternative to well established techniques such as GC or HPLC. The analysis was performed with indirect UV detection in a counterelectroosmotic flow mode using a diethylbarbiturate carrier electrolyte at a pH between 10 and 11 in a mixed aqueous-organic solvent. Separation of saturated and unsaturated fatty acids could be achieved after bromination of the double bonds. Problems with wall adsorption of fatty acids could be overcome by increasing the temperature and using a high concentration of a zwitterionic reagent to inactivate the silica surface. Increased sensitivity could be achieved after preconcentration on alumina. The method was applied to the determination of free fatty acids in dairy products. The advantages compared to traditional methods include short analysis times and simple preparation steps.Dedicated to Univ. Prof. Dr. Karl Winsauer on the occasion of his 70th birthday     

Nonaqueous capillary electrophoresis in coated capillaries: an interesting alternative for proteomic applications

This work brings together some contributions for the use of nonaqueous media for proteomic analysis, for both capillary electrophoresis (CE) separation and the preparation of tryptic digests. First, a ternary nonaqueous buffer consisting of 60/30/10 v/v methanol/acetonitrile/acetic acid with 12.5 mmol/L ammonium acetate was optimized for CE separation of the tryptic digest of lysozyme. Lysozyme was chosen as a model system for the protein digestion, which has also been prepared in an organic-rich medium with methanol/50 mmol/L NH(4)HCO(3), pH 8.0 (60/40 v/v). The separation results were compared to in silico (PeptideCutter program) digestion conditions, and high-efficiency peak separation (18 peaks) was obtained in 20 min with an electric field of 350 V/cm. In addition, we have evaluated the stability of a coated capillary with poly-N,N-dimethylacrylamide (60/30 cm total/effective length and 75 microm ID) for over 100 runs of tryptic digest with the nonaqueous background electrolyte solvent system. The migration times for ten selected peptide peaks presented 3-7% relative standard deviation.     

Peptide separations and dissociation constants in nonaqueous capillary electrophoresis: comparison of methanol and aqueous buffers

Nonaqueous capillary electrophoresis was evaluated for its potential to separate peptides in methanolic background electrolytes in comparison to aqueous-methanol (50% v/v) and water. Isomeric aspartyl dipeptides and Leu- and Met-enkephalin served as model compounds. pK(a) values were determined in the three solvent systems based on the apparent pH scale and in the case of methanol additionally based on the conventional pH scale. Changing from water to methanol led to an increase of the ionization constants describing the dissociation equilibria of the carboxyl group and the amino group, respectively. The pK(a) shift was more pronounced for the carboxylic acid function leading to a compression of the mobility-pH curve. As reported for aqueous buffers, efficient separations of the peptides were achieved in methanolic background electrolytes including the resolution of the diastereomers of the isomeric alpha- and beta-aspartyl dipeptides. In contrast to aqueous buffers, the separation of Leu- and Met-enkephalin could also be obtained in buffers in methanol at high pH.