Headspace‐single drop microextraction with a commercial capillary electrophoresis instrument

Automated coupling of headspace‐single drop microextraction (HS‐SDME) and CE has been demonstrated using a commercial CE instrument. When a drop hanging at the inlet tip of a capillary for CE is used as the acceptor phase, HS‐SDME becomes a simple but powerful sample pretreatment technique for CE before injection to facilitate sample cleanup and enrichment. By combining HS‐SDME with an on‐line sample preconcentration technique, large volume sample stacking using an electroosmotic flow pump, the sensitivity can be improved further. The overall enrichment factors for phenolic compounds were from 1900 to 3400. HS‐SDME large volume sample stacking using an electroosmotic flow pump was successfully applied to a red wine sample to obtain an LOD of 4 nM (0.8 ppb) for 2,4,6‐trichlorophenol which is a precursor for 2,4,6‐trichloroanisole causing the foul odor in wine called cork taint.     

Improving sensitivity by large-volume sample stacking using the electroosmotic flow pump to analyze some nonsteroidal anti-inflammatory drugs by capillary electrophoresis in water samples

Large-volume sample stacking using the electroosmotic flow (EOF) pump (LVSEP) has been used to analyze some nonsteroidal anti-inflammatory drugs (NSAIDs) in water samples. With methanol as the run buffer solvent to suppress the EOF, sensitivity was enhanced by 80-100-fold. The sample for the analysis of real water sample was pretreated by solid-phase extraction (SPE). When the method was based on off-line SPE-LVSEP-CE, sensitivity improved by as much as 1000 times.     

Double sample preconcentration by in‐line coupled large volume single drop microextraction and sweeping in capillary electrophoresis

Single drop microextraction (SDME) is a convenient and powerful preconcentration method for CE before injection. By simple combination of sample‐handling sequences without modification of the CE apparatus, a drop of an aqueous acceptor phase covered with a thin organic layer was formed at the tip of a capillary; 10 min SDME of fluorescein and 6‐carboxyfluorescein from a donor phase of pH 1 to an acceptor phase of pH 9 provided 110‐fold enrichments without stirring the donor phase. To improve the concentration effect further, SDME was coupled with an on‐line (after injection) sample preconcentration method, sweeping, in which analytes in a long sample zone are accumulated at the boundary of a pseudostationary phase penetrating into the sample zone. It is thus necessary to inject a sample of much larger volume than that of a drop in typical SDME. A Teflon sleeve over the capillary inlet allowed a large volume drop to be held stably during extraction. By in‐line coupling 10 min SDME and sweeping of a 30 nL sample using a cationic surfactant dodecyltrimethylammonium, enrichment factors of the double preconcentration were increased up to 32 000.     

Micelle to solvent stacking of two alkaloids in nonaqueous capillary electrophoresis

This paper for the first time describes the development of micelle to solvent stacking (MSS) to nonaqueous capillary electrophoresis (NACE). In this proposed MSS-NACE, sodium dodecyl sulfate (SDS) micelles transport, release, and focus analytes from the sample solution to the running buffer using methanol as their solvent. After the focusing step, the focused analytes were separated via NACE. The focusing mechanism and influencing factors were discussed using berberine (BBR) and jatrorrhizine (JTZ) as model compounds. And the optimum condition was obtained as following: 50 mM ammonium acetate, 6% (v/v) acetic acid and 10 mM SDS in redistilled water as sample matrix, 50 mM ammonium acetate and 6% (v/v) acetic acid in pure methanol as the running buffer, -20 kV focusing voltage with 30 min focusing time. Under these conditions, this method afforded limits of detection (S/N=3) of 0.002 μg/mL and 0.003 μg/mL for BBR and JTZ, respectively. In contrast to conventional NACE, the concentration sensitivity was improved 128-153-fold.     

Large volume sample stacking in capillary electrophoresis of weakly acidic compounds using coated capillaries at high pH

Large volume stacking using the electroosmotic flow (EOF) pump (LVSEP) in capillary electrophoresis under a reverse potential is a convenient and straightforward approach for on-line concentration of dilute anionic sample solutions. LVSEP achieves automatic sample matrix removal and subsequent separation without intermediate polarity switching nor complicated instrumental setup. Since anionic analytes should move against the EOF in LVSEP, EOF needs to be suppressed. We extended the range of LVSEP up to pH 11 using various EOF suppression methods, such as dynamic coating by polymer pretreatment and permanent coating. Weakly acidic organic compounds (pK_a<5.2), chlorinated phenols (pK_a=7-9), and aromatic amino acids (pK_a2∼9.3) were concentrated and separated. By hydrodynamically filling the whole capillary of 27 cm long with the sample solution, fast and reliable injection was achieved and sensitivity enhancement factors as large as 170 were readily obtained in less than 8 min.     

Simultaneous determination of p-hydroxybenzoic acid and parabens by capillary electrophoresis with improved sensitivity in nonaqueous media

New methods based on nonaqueous capillary electrophoresis (NACE) were developed as promising alternatives for the simultaneous separation and determination of p-hydroxybenzoic acid (PHBA) and a group of parabens (methyl, ethyl, propyl, butyl and benzyl p-hydroxybenzoates), with good resolution and excellent sensitivity. As an effective on-line preconcentration technique, large-volume sample stacking (LVSS) was successfully combined with NACE allowing significant sensitivity enhancement. Identification and quantification of the analytes were performed by diode array detection (DAD). The influence of different parameters, such as buffer apparent pH, concentration of electrolyte, temperature, applied voltage and sample volume, on the efficiency, resolution and sensitivity of the electrophoretic separation was studied. The analytical performance was evaluated, and both NACE-DAD and LVSS-NACE-DAD methods showed good linearity, precision and instrumental LODs at low ng/mL levels. These LODs were compared with those described in the literature, and it was found that NACE-DAD method was comparable to GC-MS, while LVSS-NACE-DAD procedure achieved sensitivity similar to LC-MS, LC-MS/MS and GC-MS/MS, even using conventional ultraviolet-visible absorption detection. To test their suitability, proposed methods were evaluated for the analysis of PHBA and parabens at low and sub-ng/mL levels in environmental water samples.     

Solid-phase extraction and large-volume sample stacking with an electroosmotic flow pump in capillary electrophoresis for determination of methotrexate and its metabolites in human plasma

This paper describes approaches for large-volume sample stacking (LVSS) with an EOF pumpin CE for the determination of methotrexate (MTX) and its metabolites in human plasma. After pretreatment of plasma through a SPE cartridge, a large sample volume was loaded by hydrodynamic injection (3 psi, 70 s) into the capillary filled with phosphate buffer (70 mM, pH 6.0) containing 0.01% polyethylene oxide. Following removal of a large plug of sample matrix from the capillary using polarity switching (-25 kV), the separation of anionic analytes was subsequently performed without changing polarity again, achieving an improvement of sensitivity of around a 100-fold. The method was applied to therapeutic drug monitoring of MTX in one acute lymphoblastic leukemia patient. This study is one of very few applications showing the feasibility of LVSS in analysis of biological samples by CE.     

On-line preconcentration of perfluorooctanoic acid and perfluorooctanesulfonic acid by nonaqueous capillary electrophoresis

Separation of major environmental pollutants as perfluorooctanoic acid (PFOA) and perfluorooctanesulfonic acid (PFOS) by capillary electrophoresis is reported for the first time. It is not possible to resolve the solutes in an aqueous media. However, the use of methanol and acetonitrile as the background electrolyte (BGE) solvents allowed their rapid separation in an uncoated capillary. A major effort was put into BGE optimization in respect to both separation efficiency and detection for further on‐line preconcentration. 5 mmol.L^?1 naphthalene‐1‐sulfonic acid and 10 mmol.L^?1 triethylamine dissolved in ACN/MeOH (50:50 v/v) provided best separation and detection conditions. Next, the large‐volume sample stacking and the field‐amplified sample injection were applied and compared. Large‐volume sample stacking improved limits of detection (LODs) with regard to the standard injection by 69 times for PFOA and 143 times for PFOS with LODs of 280 and 230 nmol.L^?1, respectively. Field‐amplified sample injection improved LODs 624 times for PFOAand 806 times for PFOS with LODs 31 and 40 nmol.L^?1, respectively. Both preconcentration methods showed repeatabilities of migration times less than 1.2% RSD intraday and 6.6% RSD interday. The method was applied on PFOA and PFOS analysis in a sample of river water treated with solid‐phase extraction, which further improved LOD toward 5.6 × 10^?10 mol.L^?1 for PFOS and 6.4 × 10^?10 mol.L^?1 for PFOA and allows the method to be used for river water contamination screening or decomposition studies.     

In‐line coupled single drop liquid–liquid–liquid microextraction with capillary electrophoresis for determining fluoroquinolones in water samples

A simple in‐line single drop liquid–liquid–liquid microextraction (SD‐LLLME) coupled with CE for the determination of two fluoroquinolones was developed. The method is capable to quantify trace amount of analytes in water samples and to improve the sensitivity of CE detection. For the SD‐LLLME, a thin layer of organic phase was used to separate a drop of 0.1 M NaOH hanging at the inlet of the capillary from the aqueous donor phase. By this way, the analytes were extracted to the acceptor phase through the organic layer based on their acidic/basic dissociation equilibrium. The drop was immersed into the organic phase during 10 min for extraction and then it is directly injected into the capillary for the analysis. Parameters such as type and volume of organic solvent phase, aqueous donor, and acceptor phases and extraction time and temperature were optimized. The enrichment factor was calculated, resulting 40‐fold for enrofloxacin (ENR) and sixfold for ciprofloxacin (CIP). The linear range were 20–400 μg/L for ENR and 60–400 μg/L for CIP. The detection limits were 10.1 μg/L and 55.3 μg/L for ENR and CIP, respectively, and a good reproducibility was obtained (4.4% for ENR and 5.6% for CIP). Two real water samples were analysed applying the new method and the obtained results presented satisfactory recovery percentages (90–100.3%).     

Selective preconcentration of amino acids and peptides using single drop microextraction in-line coupled with capillary electrophoresis

Single drop microextraction (SDME) can be in-line coupled with capillary electrophoresis by attaching a drop to the tip of a capillary. With a 2-layer drop comprised of an aqueous basic acceptor phase covered with a thin organic layer, acidic analytes in an aqueous acidic donor phase can be extracted into the organic layer and then back-extracted into the acceptor phase. However, preconcentration of amino acids and peptides by SDME is difficult since their zwitterionic properties prevent them from being partitioned in the middle organic phase. When amino acids were derivatized with 4-fluoro-7-nitro-2,1,3-benzoxadiazole (NBD-F), amino acids without a charged side chain were converted to carboxylic acids. In the acidic donor phase, those NBD-amino acids were predominantly neutral and they were successfully concentrated into the basic acceptor phase. In the meantime, amino acids with a charged side chain after NBD-F derivatization were not concentrated via SDME. With this selective SDME, we were able to extract acidic and neutral amino acids obtaining several hundred-fold enrichments within 5 min at 25 °C, while leaving basic amino acids—Arg, Lys, and His—in the acidic donor phase. Furthermore, detection sensitivity was enhanced by employing laser-induced fluorescence detection. We then applied this technique to the selective concentration of peptides.     

Sensitive analysis of amino acids with carrier-mediated single drop microextraction in-line coupled with capillary electrophoresis

In order to analyze amino acids sensitively without derivatization, we have developed carrier-mediated single drop microextraction (SDME). Nonane-1-sulfonic acid was added to an acidic sample donor solution as a carrier to form neutral ion pair complexes with amino acids. The ion pair complexes were extracted to the organic phase, covering a drop of an aqueous basic acceptor phase hanging at the tip of a capillary, and then back-extracted to the basic acceptor phase, where both the amino acids and the carrier have negative charges and the ion pair complexes are broken. The resulting extract of enriched amino acids was injected into the capillary and analyzed by capillary electrophoresis. With 20-min SDME with agitation of the donor phase, enrichment factors of four aromatic amino acids were up to 120-fold, yielding the LOD of 70-500 nM. The linear dynamic ranges for corrected peak areas were 1-100 μM with linear correlation coefficients larger than 0.9959. With internal standardization, the intraday RSDs of migration times and corrected peak areas were 0.01-0.04% and 2.0-3.7%, respectively. The capabilities of sample cleanup including desalting and preconcentration of carrier-mediated SDME were demonstrated with the analysis of human urine after minimal pretreatment of acidification and centrifugation.     

Determination of nerve agent degradation products by capillary electrophoresis using field-amplified sample stacking injection with the electroosmotic flow pump and contactless conductivity detection

In the present study, field-amplified sample stacking injection using the electroosmotic flow pump (FAEP) was developed for the capillary electrophoretic separation of the four nerve agent degradation products methylphosphonic acid (MPA), ethyl methylphosphonic acid (EMPA), isopropyl methylphosphonic acid (IMPA) and cyclohexyl methylphosphonic acid (CMPA). Coupled to contactless conductivity detection, direct quantification of these non-UV active compounds could be achieved. Sensitivity enhancement of up to 500 to 750-fold could be obtained. The newly established approach was applied to the determination of the analytes in river water and aqueous extracts of soil. Detection limits of 0.5, 0.7, 1.4 and 2.7 ng/mL were obtained for MPA, EMPA, IMPA and CMPA, respectively, in river water and 0.09, 0.14, 0.44 and 0.22 μg/g, respectively, in soil.     

Novel approach to improve the detection of colchicine via online coupling of ionic liquid-based single-drop microextraction with capillary electrophoresis

A novel approach based on ionic liquid‐single‐drop microextraction (IL‐SDME) online coupling with capillary electrophoresis (CE) was used to determine a toxic alkaloid – colchicine. The IL‐SDME procedure was optimized by extraction solvent, drop volume controlling, sample volume and pH, extraction time, and ionic strength. Under optimum conditions, enrichment factor was as much as 41‐fold with a relative standard deviation of 2.8% (n=3). Linear range of response was observed from 1 to 100 μg/mL, with detection limit of 0.25 μg/mL and correlation coefficient (R²) of 0.9994. The extraction of colchicine from spiked Lanzhou lily sample was performed and obtaining good result with an average recovery rate of 102.4 and 98.8% at 5 and 50 μg/mL, respectively. Comparing with the previous methods, IL‐SDME‐CE is really a convenient, economical, and environmentally benign way for determining colchicine.     

Single-drop microextraction as a powerful pretreatment tool for capillary electrophoresis: A review

Single drop microextraction (SDME) is a convenient and powerful preconcentration and sample cleanup method for capillary electrophoresis (CE). In SDME, analytes are typically extracted from a sample donor solution into an acceptor drop hanging at the inlet tip of a capillary. The enriched drop is then introduced to the capillary for CE analysis. Since the volume of the acceptor drop can be as small as a few nanoliters, the consumption of solvents can be minimized and the preconcentration effect is enhanced. In addition, by covering the acceptor phase with an organic layer or by using an organic acceptor phase, inorganic ions such as salts in the sample solution can be blocked from entering the acceptor phase, providing desalting effects. Here, we describe the basic principles and instrumentation for SDME and its coupling with CE. We also review recent developments and applications of SDME-CE.     

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.     

The magnitude and reproducibility of the electroosmotic flow in silica capillary tubes

The influence of a number of factors, such as the nature of the buffer, the presence of additives therein, the manufacturing conditions, and the pretreatment of the capillary, on the magnitude and repeatability of the electroosmotic flow (EOF) in silica capillary tubes has been investigated. It was established that the nature of the buffer plays the most significant role in respect of the stability and magnitude of EOF, whereas the conditions used for manufacture and pretreatment of the tubing are less important. The EOF obtained using buffers consisting of tris(hydroxymethyl)aminomethane and ethylenediamine was more repeatable than that obtained using pure phosphate buffers. Buffer additives such as sodium dodecylsulfate, 1-butanol, sodium salts of 1-pentanesulfonic acid and 1-heptanesulfonic acid, altered the magnitude of the EOF only, not its repeatability.     

High-sensitivity capillary electrophoresis for speciation of organomercury in biological samples using hollow fiber-based liquid-liquid-liquid microextraction combined with on-line preconcentration by large-volume sample stacking

A hollow fiber-based liquid-liquid-liquid microextraction (HF-LLLME) combined with on-line large-volume sample stacking (LVSS) has been developed for the speciation of organomercury in biological samples by CE with UV detection. Separation was achieved in less than 11 min with an electrolyte consisting of 35 mM sodium tetraborate at pH 9.1. In LVSS, a reverse electrode polarity-stacking mode (REPSM) was applied as on-line preconcentration strategy. In HF-LLLME, the analytes were extracted from 12 mL volume of sample solution (pH adjusted to 3.0) into bromobenzene impregnated in the pores of the hollow fiber, and into an acceptor solution of L-cysteine (15 microL, 0.02% w/v) inside the hollow fiber. Under the optimized conditions, concentration factors of 2610-4580 were achieved and LODs in the range of 0.03-0.14 microg/L were feasible. The linearity was found to be over two orders of magnitude with correlation coefficient of 0.9991-0.9996. The developed method has been validated using a certified reference material (DORM-2, dogfish muscle), and the determined values coincided very well with the certified values. The method was also applied to the speciation of organomercury in three kinds of fish samples and human hair samples.     

Trace analysis of oxidized, nitrated, and chlorinated aromatic amino acids by capillary electrophoresis with electroosmotic flow modification allowing large-volume sample stacking

A capillary electrophoresis method has been developed for the simultaneous analysis of the oxidized, nitrated, and chlorinated aromatic amino acids, as well as their parent compounds. These modifications of the aromatic amino acids in proteins or free form are induced by the attack of reactive, mainly free radical species generated during cell stress, and these stable products may serve as biomarkers of cell damage. The analytes tyrosine, phenylalanine, dihydroxyphenylalanine, tryptophan, 3-nitrotyrosine, 3-chlorotyrosine, ortho-tyrosine, meta-tyrosine, 3-hydroxyphenylacetic acid (internal standard 1), and alpha-methyltyrosine (internal standard 2) were separated in their anionic forms in alkaline borate buffer. The polyamine spermine was used as electroosmotic flow (EOF) modifier. Adsorbing to the capillary wall, spermine can either suppress or even reverse the EOF depending on its concentration and the pH. The effects of the pH of the separation buffer, the spermine concentration, the temperature, and the applied field strength on the separation were examined. The modified aromatic amino acids are present in biological fluids in a much lower concentration than their parent compounds, thus high detection sensitivity of the analytical method is required. To achieve good detection sensitivity, field-amplified sample stacking of large injection volumes was applied. Omitting polyamine from the sample buffer allowed local reversal of the EOF, thus removal of the low conductivity sample buffer at the capillary inlet. In this way, 100% of the capillary to the detection window could be filled with the sample, and the detection limits achieved for the modified aromatic amino acids were in the range of 2.5-10 nM.     

基于离子液体的单滴微萃取-毛细管电泳在线联用测定水体中3种溴酚类化合物

建立了基于离子液体的单滴微萃取-毛细管电泳联用测定溴酚类化合物的方法。考察了萃取剂种类与体积、萃取时间、有机溶剂、盐浓度及萃取温度对萃取效率的影响。确定了最佳萃取条件为:以1-丁基-3-甲基咪唑六氟磷酸盐([C₄MIM]PF₆])离子液体作为萃取剂,萃取时间为8 min,样品溶液中NaCl浓度为10%(质量分数),萃取温度为20 ℃。在最佳条件下,3种溴酚(4-溴酚、2,6-二溴酚和2,4,6-三溴酚)在1~100 mg/L范围内呈良好的线性关系,线性相关系数为0.9939~0.9988;检出限为0.3 mg/L (S/N=3);该方法对3种溴酚的富集倍数分别为115.8、327.0和569.8; 6次平行测定的相对标准偏差为5.21%~6.47%;对本地区自来水、河水和湖水的加标回收率为87.8%~96.7%。结果表明,该方法稳定可靠,适合于水体中溴酚类污染物的测定。     

高效毛细管电泳大体积样品堆积在线富集法测定蒲公英中阿魏酸、绿原酸和咖啡酸

采用大体积样品堆积(LVSS)在线富集模式,建立了高效毛细管电泳(HPCE)测定蒲公英中阿魏酸、绿原酸和咖啡酸含量的方法。主要考察了在毛细管区带电泳(CZE)分离模式下,缓冲液的pH和浓度对分离效果的影响,以及在LVSS在线富集模式下,进样时间对富集效果的影响。在最优条件下阿魏酸、绿原酸和咖啡酸可在12 min内得到分离,3个成分在0.5~25.0μg/mL浓度范围内均有较好的线性关系(r2=0.999),平均加样回收率分别为104.9%,98.0%和100.1%,RSD(n=6)分别为3.6%,2.6%和1.0%。定量限(S/N=10)分别为0.10,0.10和0.03μg/mL,检出限(S/N=3)分别为0.03,0.03和0.01μg/mL。相对于常规CZE模式,本方法的富集效果倍数为17~19倍。建立的方法可用于蒲公英的日常检测与质量控制。