Pseudostationary Phase Research Articles

Functionalized carbon nanotubes as the pseudostationary phase for capillary EKC separation of non‐steroidal anti‐inflammatory drugs

Functionalized multiwalled carbon nanotubes (f-MWCNTs) can serve as the pseudostationary phase (PSP) for the capillary EKC separation of non-steroidal anti-inflammatory drugs (NSAIDs). To increase their hydrophilicity, we treated MWCNTs, with a sonochemical process in a concentrated nitric/sulfuric acid mixture. The oxidized MWCNTs were then characterized by FT-IR, transmission electron microscopy, and X-ray photoelectron spectroscopy. We evaluated the potential of the PSP and the effects of buffer composition, pH, addition of organic modifier, and injection temperature on the NSAID separation. The PSP created a network structure of pi-pi interactions, hydrophobic forces, hydrogen bonding, and electrostatic interactions to separate NSAIDs, providing a different separation mode from SDS micelles. We achieved complete separation of six NSAIDs using a mixture of a borate buffer (75 mM, pH 10) with methanol (5%, v/v) containing 0.02 mg/mL f-MWCNTs, an applied voltage of +12 kV and detection at 214 nm. Better precision was obtained with a low injection temperature. The method was also satisfactorily applied to the analysis of NSAIDs spiked into a urine sample.

Determination of Phenytoin and its Major Metabolite in Human Serum by MEKC

Determination of phenytoin in biological fluids is necessary due to its non-linear pharmacokinetics and narrow therapeutic index. A simple and rapid micellar electrokinetic chromatographic method was developed for simultaneous determination of phenytoin and its main metabolite, 5-(4-hydroxyphenyl)-5-phenylhydantoin, in human serum. The separation was carried out using fused-silica capillary and UV detector at 214 nm. The background electrolyte consisted of borate buffer (10 mM, pH 10.5) with 50 mM sodium dodecyl sulphate as pseudostationary phase. The calibration graphs were linear at the concentration range of 5-30 mg/mL, and the detection limits for phenytoin and 5-(4-hydroxyphenyl)-5-phenylhydantoin added to blank serum were 1.8 and 1.5 mg/mL, respectively. The obtained results demonstrated that the proposed method is suitable for the serum phenytoin analysis in terms of selectivity and simplicity.

CE immunoassay with enhanced chemiluminescence detection of erythropoietin using silica dioxide nanoparticles as pseudostationary phase

The measurement of serum erythropoietin (EPO) is important for the detection of recombinant human EPO misuse in sports. In this paper, we have developed a sensitive and rapid CE immunoassay with enhanced chemiluminescence detection of EPO, in which silica dioxide nanoparticles (SiO2 NPs) were used as the pseudostationary phase to improve the separation efficiency of analytes. By adopting SiO2 NPs in the CE immunoassay, the separation can be successfully performed in neutral running buffer solution. However, running buffer of extreme pH was still necessary for the conventional CZE mode. Neutral phosphate buffer (pH 7.4) containing SiO2 NPs and poly(ethylene oxide) (PEO) was chosen as running buffer. The influences of SiO2 NPs, PEO and buffer concentration on the separation efficiency and EPO detection were investigated. EPO-horse radish peroxidase (HRP) and immunocomplex were baseline separated in a 10 mM phosphate buffer (pH 7.4) consisting of 0.08% PEO and 0.08% SiO2 NPs. The linear range for EPO was 1.8-158.0 ng/mL and the detection limit was 0.9 ng/mL. The assay was successfully applied for the quantification of EPO in human sera and the results correlated well with those obtained using chemiluminescence immunoassay kits, thus demonstrating that the present method was a potential powerful tool for EPO misuse detection and clinical diagnosis.

Cyclodextrin-MEEKC for the analysis of oxybutynin and its impurities

The development of a cyclodextrin-MEEKC method for the analysis of oxybutynin and five related impurities is described. Experimental design strategies were applied in order to reach baseline separation of the compounds in a short analysis time. Mixture design made it possible to find the best composition for the microemulsion acting as pseudostationary phase, which was constituted by 89.1% 10 mM borate buffer pH 9.2, 1.7% n-heptane, 9.2% SDS/ n-butanol in 1:2 ratio. The addition of (2-hydroxypropyl)-β-cyclodextrin to the background electrolyte was found to improve analysis performance. A Doehlert design, for the factors cyclodextrin concentration and voltage, was carried out and Derringer desirability function led to the identification of 18 mM and 29 kV as the optimal values. Applying the optimum conditions, separation of all the compounds, including the enantiomers of impurity 1, was obtained in less than 12 min. The method was validated according to ICH guidelines for drug assay and determination of impurities and was applied to oxybutynin tablet analysis.

Enantioseparation by MEKC using a ligand exchange‐based chiral pseudostationary phase

In this paper, a new ligand-exchange -MEKC mode, based on the design of a unique lipohilic species (4'-octadecylneamine derivative), which served both as micelle-forming surfactant (by its hydrophobic part) and central ion-complexing ligand (by its hydrophilic part) is described. The CMC of the used lipophilic neamine derivative was first determined by surface tension measurements. Subsequent NMR experiments were performed in order to investigate the Cu(II) binding properties of the neamine micellar phase. The enantioseparation properties of both the octadecylneamine derivative-Cu(II) MEKC and the native neamine-Cu(II) CE systems were evaluated and compared using the tryptophan racemate as a probe analyte. The effects of several different electrophoretic conditions on the enantiomer migration behavior in the ligand-exchange-MEKC mode were examined. The developed methodology was also applied to the enantioseparation of other analytes such as 1-methyl-tryptophan, 3,5-diiodo-tyrosine and 1-naphtyl-alanine.

The effect of co‐surfactant‐modified micelles on chiral separations in EKC

The use of chiral pseudostationary phases in EKC provides high efficiencies and excellent resolution for enantiomeric separations. The chiral pseudostationary phases of interest in this study are alcohol-modified ("swollen") micelles, in which a co-surfactant (medium chain-length alcohol) is added with the surfactant. In this study, the chiral surfactant, dodecoxycarbonylvaline (DDCV), along with the co-surfactant, 2-hexanol, has been prepared as swollen micelle in order to investigate the chiral separation of enantiomeric pairs. Three sets of experiments were investigated in which swollen micelle systems contained: chiral surfactant and racemic co-surfactant; chiral surfactant and chiral co-surfactant; and phase ratio increases, in which both chiral surfactant and chiral co-surfactant were employed. In the first two sets of experiments, co-surfactant concentration was held constant and the surfactant concentration was increased. In the third set of experiments, both surfactant and chiral surfactant concentrations were increased proportionally. The chromatographic figures of merit for each enantiomeric pair were investigated and compared with various chiral aggregate systems. In swollen micelle compositions using constant racemic 2-hexanol concentration, when DDCV concentration increased, enantioselectivity and resolution increased; whereas, efficiency remained constant for most of the test compounds. Compositions using constant S-2-hexanol concentration reached a maximum in all chromatographic figures of merit when DDCV concentration was increased from 2 to 3%. An increase in both surfactant and co-surfactant concentrations led to noisy baselines and chiral aggregates that were generally unstable in solution.

Investigation of interaction between the drug and cell membrane by capillary electrophoresis

By introducing cell membrane into electrophoretic buffer as pseudo-stationary phase, a novel capillary electrophoresis method was established to explore the interaction between drugs and cell membrane, where the interaction between citalopram and rabbit red blood cell membrane was used as an example. A series of concentrations of cell membrane were suspended into the running buffer by peak-shift method. The binding constant of citalopram to rabbit red blood cell membrane of 0.977 g−1·L was obtained after treatment of Scatchard plot. This method could provide not only a new way for the investigation on the interactions between drugs and cell membrane, but also a new approach for high throughput screening of the drug membrane permeability, biological activity, and evaluating drugs in vivo.

Highly sensitive detection of chiral amino acids by CE based on on‐line stacking techniques

We report a novel means for chiral separation and stacking of amino acids by MEKC with LED-induced fluorescence detection. Naphthalene-2,3-dicarboxaldehyde, hydroxypropyl-beta-cyclodextrin (Hp-beta-CD), SDS, and poly(ethylene oxide) (PEO) serve as a derivatized agent, chiral selector, pseudostationary phase, and concentrated medium, in sequence. To improve speed, resolution, and stacking efficiency, the analysis of chiral amino acids was performed under discontinuous conditions--the capillary was filled with a solution of 100 mM Tris-borate, 150 mM SDS, and 50 mM Hp-beta-CD, whereas buffer vials contain 20 mM Tris-borate, 150 mM SDS, 50 mM Hp-beta-CD, and 0.5% w/v PEO. A solution of nonionic PEO enters the capillary with the help of EOF during the separation. Through interaction of SDS micelles/Hp-beta-CD and chiral amino acid, the negatively charged complexes migrated into the PEO solution and stacked at the boundary between the sample zone and the PEO solution. Compared with normal sample injection (10 nL sample volume), a several hundred-fold sensitivity improvement for chiral amino acids was obtained under the injection of 270 nL sample volume (30% of the capillary volume). Meanwhile, the LOD at S/N of three for DL-amino acids were in the range of 0.18-0.22 nM. The proposed method has been applied for the determination of DL-leucine in urine and plasma samples.

Combination of On-Chip Field Amplification and Bovine Serum Albumin Sweeping for Ultrasensitive Detection of Green Fluorescent Protein

We report a highly effective on-chip preconcentration method by combining field-amplified sample injection (FASI) and bovine serum albumin (BSA) sweeping for ultrasensitive detection of green fluorescent protein (GFP) on a simple cross-channel microchip device. With the formation of a stagnant sample/running buffer boundary by balancing the hydrodynamic flow and the electro-osmotic flow (EOF), GFP molecules can be continuously injected into the sample loading channel and stacked. We have also demonstrated that BSA is a very effective pseudo-stationary phase for sweeping concentration of proteins in comparison to the commonly used micelles. The combination of FASI and BSA sweeping yields a concentration factor of 3570 and a limit of detection of 8.4 pM for GFP. Using this method, we have separated GFP and GFP-insulin-like growth factor-I (GFP-IGF-I) fusion protein. The entire assay (GFP concentration, matrix elimination, and electrophoretic separation) can be completed within <5 min. Furthermore, we have successfully applied this method for the detection of GFP expression of E. coli cells and the GFP content in single E. coli cells.

Characterisation of mixed lithium dodecyl sulphate/lithium perfluorooctanesulphonate pseudo‐stationary phases in MEKC

Lipophilicity and methylene selectivity of mixed pseudo-stationary phases (PSPs) (containing lithium dodecyl sulphate (LDS) and lithium perfluorooctanesulphonate (LiPFOS) in different molar ratios) applied in MEKC have been investigated. Micellar proportion (t(prop,mic), a quantity expressing that how much time is spent by the analyte in the micellar phase related to its whole migration time), CLOGP(50) value (showing the value of hydrophobicity of a molecule spending exactly 50% of its migration time in the PSP) and methylene selectivity have been determined for different LDS/LiPFOS mixed phases. Values of the above-mentioned parameters have been determined for analytes with different chemical structures (alkylbenzene and alkylphenone homologous series, alcohols). Good linear correlation was obtained between either the micellar proportion, CLOGP(50), or methylene selectivity and the phase composition for the mixed phases. Lipophilicity and methylene selectivity of the mixed LDS/LiPFOS PSPs can be calculated and can continuously be changed by mixing the two single phases (LDS and LiPFOS) in the appropriate (and calculable) portion.

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.

Multivariate approach for the enantioselective analysis in MEKC‐MS: II. Optimization of 1,1′‐binaphthyl‐2,2′‐diamine in positive ion mode

Enantiomeric separation and detection of 1,1'-binaphthyl-2,2'-diamine (BNA) has been successfully optimized by MEKC-ESI-MS using a polymeric surfactant polysodium N-undecenoxycarbonyl-L-leucinate (poly-L-SUCL) as a pseudostationary phase. In the first step, MEKC conditions were optimized by a five-factor three-level central composite design (CCD) of experiment. All five MEKC factors (buffer pH, percentage of ACN in the running buffer, concentration of surfactant, concentration of ammonium acetate (NH4OAc), and voltage) were found significant to the responses (measured as the chiral resolution and analysis time). The interactions between MEKC factors were further evaluated using a quadratic model equation which allowed the generation of 3-D response surface image to reach the optimum conditions. To obtain the best S/N, sheath liquid composition and spray chamber parameters were successfully optimized using the same strategy. Baseline enantiomeric resolution in less than 20 min and optimum MS signal of BNA enantiomers (S/N = 45 at 0.4 mg/mL) were ultimately achieved at the optimized conditions. The adequacy of the model was validated by experimental runs at the optimal predicted conditions. The predicted results were found to be in good agreement with the experimental data.

Application of Nanomaterials in Environmental Analysis and Monitoring

Recent advances in the development and application of nanomaterials in analytical chemistry for environmental analysis and monitoring are reviewed, with focus on sensors, separation and extraction techniques, including the use of nanomaterials as transducer elements for electrochemical sensors, as nanolabels for optical sensors, as stationary phases for liquid chromatography and gas chromatography, as pseudostationary phases or buffer additives in capillary electrophoresis, capillary electrochromatography, and microchip electrophoresis, as well as extraction materials for enrichment prior to chromatographic analysis. Examples of different nanomaterials-based analytical techniques for the detection of major families of environmental pollutants, i.e., organic contaminants, heavy metals and air pollutants, are presented.

Determination of antihyperglycemic drugs in nanomolar concentration levels by micellar electrokinetic chromatography with non-ionic surfactant

A method for the separation of six selected antihyperglycemic (antidiabetic) drugs (tolbutamide, gliclazide, glimepiride, glibenclamide, repaglinide, and glipizide) was developed with use of micellar electrokinetic chromatography. Two non-ionic poly(ethylene glycol)-based surfactants Genapol X-080 and Triton X-114 (reduced) were studied as neutral pseudostationary phases. High alkaline pH 10.0 was used to obtain negative charges of separated antidiabetic drugs and non-ionic surfactants were employed for selectivity alteration. Both non-ionic surfactants provided good selectivity at concentration 0.2% (v/v) in sodium borate buffer and the separation of six drugs was obtained within 5 min. An on-line preconcentration method based on reversed electrode polarity switching was employed for the determination of antihyperglycemic drugs in blood serum after acetonitrile protein precipitation. The limits of detection ranged from 20.8 nmol L −1 for tolbutamide to 6.5 nmol L −1 for glibenclamide, respectively.

1-Hexadecyl-3-methylimidazolium Ionic Liquid as a New Cationic Surfactant for Separation of Phenolic Compounds by MEKC

The ionic liquid 1-hexadecyl-3-methylimidazolium bromide ([C16MIm]Br) has been used as a novel cationic surfactant for separation of phenolic compounds, including quinol, phloroglucinol, resorcinol, phenol, p-cresol, and m-nitrophenol, by micellar electrokinetic capillary chromatography (MEKC). The effects of buffer concentration and pH, concentration of [C16MIm]Br, and applied potential were studied. Use of the optimized buffer (25 mmol L−1 NaH2PO4), 10 mmol L−1 [C16MIm]Br, and an applied potential of −15 kV enables optimum separation with regard to resolution and migration time. The phenolic compounds were detected at 214 nm. The micelle of this long-alkyl-chain imidazolium ionic liquid acts as a pseudo-stationary phase in this MEKC separation.

Retention Behavior of Highly Hydrophobic, Structurally Related Corticosteroids with SDS Based Microemulsion

The present work describes the retention behavior of structurally related corticosteroids separated with SDS based microemulsion. The analytes include cortisone acetate (CA), hydrocortisone (H), hydrocortisone acetate (HA), prednisolone (P), prednisolone acetate (PA), and prednisone (Ps), which are not easily separated by the conventional MEKC. Surfactants with linear and planar structures have been compared for the separation of these analytes. By fine tuning the parameters, the results revealed that complete baseline separation could be achieved with a microemulsion consisted of 0.8% (w/w) n-octane, 3.6% (w/w) SDS, 6.6% (w/w) 1-butanol, and 89% (w/w) phosphate buffer (40 mM, pH 8.0), under an applied voltage of +7 kV and a column dimension of 53 cm (effective length, 30 cm) × 75 μm id. Although the composition of the microemulsion has a high recognition property for these structurally related compounds, the high affinity of these analytes with the pseudostationary phase made the analysis time very long. The effects of the addition of acetonitrile, methanol, and isopropanol have also been studied. Acetonitrile was found to have the greatest impact on this separation.

Sweeping and new on-line sample preconcentration techniques in capillary electrophoresis

Sweeping is a powerful on-line sample preconcentration technique that improves the concentration sensitivity of capillary electrophoresis (CE). This approach is designed to focus the analyte into narrow bands within the capillary, thereby increasing the sample volume that can be injected, without any loss of CE efficiency. It utilizes the interactions between an additive [i.e., a pseudostationary phase (PS) or complexing agent] in the separation buffer and the sample in a matrix that is devoid of the additive used. The accumulation occurs due to chromatographic partitioning, complexation or any interaction between analytes and the additive through electrophoresis. The extent of the preconcentration is dependent on the strength of interaction involved. Both charged and neutral analytes can be preconcentrated. Remarkable improvements--up to several thousandfold--in detection sensitivity have been achieved. This suggests that sweeping is a superior and general approach to on-line sample preconcentration in CE. The focusing mechanism of sweeping under different experimental conditions and its combination with other on-line preconcentration techniques are discussed in this review. The recently introduced techniques of transient trapping (tr-trapping) and analyte focusing by micelle collapse (AFMC) as well as other novel approaches to on-line sample preconcentration are also described.

Electrophoretic Separation of Proteins in Capillaries Filled with Poly(ethylene oxide)-stabilized Silver Nanoparticles

Poly(ethylene oxide)-stabilized silver nanoparticles (PEO-SSNPs) were synthesized and investigated for their potential use in capillary electrophoretic separation of proteins. It was found that the PEO-SSNPs were relatively stable against changes of the buffer ionic strength, while they began to aggregate with increase of buffer pH. Adding 0.024 g/L PEO-SSNPs to 30 mmol/L phosphate buffer (pH 2.66) led to obvious increase in the peak heights of albumin bovine and albumin egg. Also, the separation efficiency for all the standards enhanced due to the suppressed wall-adsorption of proteins in the presence of the nanoparticles. The above experiments suggest that the PEO-SSNPs are effective pseudostationary phase for CE separation of proteins.

Interactions between local anesthetics and lipid dispersions studied with liposome electrokinetic capillary chromatography

In the case of local anesthetic intoxication, intravenous administration of lipid-based Intralipid dispersion (Fresenius Kabi) can be used for the entrapment of hydrophobic drugs. Our long-term aim is to develop a sensitive, efficient, and non-harmful lipid-based formulation to specifically trap harmful substances. In this study liposome electrokinetic capillary chromatography (LEKC) was used to study the interactions between local anesthetics and Intralipid or liposome dispersions. Intralipid dispersion and extruded liposomes with different concentrations of 1-palmitoyl-2-oleyl- sn-glycerophosphatidylcholine (POPC), phosphatidylglycerol, cardiolipin, cholesterol, oleic acid, and linoleic acid were used as a pseudostationary phase in LEKC and their interactions with lidocaine, prilocaine, and bupivacaine were studied. POPC liposomes containing 1 mol% of palmitoyl-2-[12-[(7-nitro-2-1,3-benzoxadiazol-4-yl)amino]dodecanoyl]- sn-glycero-3-phosphocholine as a fluorescent marker were used for the first time in LEKC connected with laser-induced fluorescent detection in order to calculate the retention factor for anesthetics.

Comparison of aromatic and alkyl micelles for the electrokinetic determination of phthalates in virgin olive oil

Two electrokinetic methods for the separation of phthalates are proposed. One uses 25 mM sodium borate and 50 mM sodium taurodeoxycholate adjusted to pH 2.8, and the other uses 15 mM ammonium tetraborate, 100 mM SDS, 0.25% w:v hydroxypropylmethyl cellulose (HPMC) and 5% v:v methanol (MeoH) adjusted to pH 9. The BGE containing SDS micelles as the pseudo-stationary phase provided better analytical figures of merit, particularly as regards LOD (0.4-1.4 mg/L) and precision (1.1-6.5%). Adding MeoH and HPMC to the BGE proved essential in order to obtain narrow and symmetric peaks. The proposed method was successfully used to determine phthalates in virgin olive oil. Recoveries from spiked samples ranged from 96 to 106%. The precision obtained in the analysis of real samples, as RSD, was better than 6.8%.