On-line Sample Concentration Technique Research Articles

The increase of detection sensitivity of micellar electrokinetic capillary chromatography method of stamp pad inks components by applying a sample stacking mode for the purpose of questioned document examination

The study focused on comparison of on-line sample concentration techniques, i.e. sample stacking modes, applied in capillary electrophoresis in order to improve the detection of components of stamp pad inks so that destruction of analysed document could be minimized. In particular, such techniques were taken into consideration as: normal stacking mode/field amplified sample stacking (NSM/FASS), high-salt concentration, sweeping, electrokinetic injection + FASS, and field enhanced sample injection (FESI). At first, two mixtures of 4 red dyes and 4 violet/blue dyes were examined. As a result, in case of red dyes two environments: ACN/1 mg mL−1 aqueous NaCl (95:5, v/v) and 10-fold water dilution of BGE preceded by water plug (6 s, 0.7 psi) injection, were chosen as the most promising for further examination. Regarding the blue/violet dyes, the 100-fold water dilution of BGE was selected as giving the best increase of height of peaks. Ultimately, nine stamp pad inks in three colours (red, blue and violet) produced by different manufacturers were examined under the optimal conditions. Only 5 dots cut-out from inked paper were sampled. Almost all examined samples were possible to be differentiated basing on the obtained electrophoretic profiles. In conclusion, the developed sample stacking mode of MECC–based method was found as a useful tool in the differentiation of stamp pad inks for the purposes of forensic questioned document analysis.

Combination of large-volume sample stacking with an electroosmotic flow pump with field-amplified sample injection on cross-channel chips.

A combination of two online sample concentration techniques, large-volume sample stacking with an electroosmotic flow (EOF) pump (LVSEP) and field-amplified sample injection (FASI), was investigated in microchip electrophoresis (MCE) to achieve highly sensitive analysis. By applying reversed-polarity voltages on a cross-channel microchip, anionic analytes injected throughout a microchannel were first concentrated on the basis of LVSEP, followed by the electrokinetic stacking injection of the analytes from a sample reservoir by the FASI mechanism. As well as the voltage application, a pressure was also applied to the sample reservoir in LVSEP-FASI. The applied pressure generated a counter-flow against the EOF to reduce the migration velocity of the stacked analytes, especially around the cross section of the microchannel, which facilitated the FASI concentration. At the hydrodynamic pressure of 15 Pa, 4520-fold sensitivity increase was obtained in the LVSEP-FASI analysis of a standard dye, which was 33-times higher than that obtained with a normal LVSEP. Furthermore, the use of the sharper channel was effective for enhancing the sensitivity, e.g., 29 100-fold sensitivity increase was achieved with the 75-μm wide channel. The developed method was applied to the chiral analysis of amino acids in MCE, resulting in the sensitivity enhancement factor of 2920 for the separated d-leucine.

Ionic liquids based on imidazole for online concentration of catecholamines in capillary electrophoresis

Potential possibilities of long-chain ionic liquids based on imidazole (1-dodecyl-3-methylimidazolium chloride and 1-cetyl-3-methylimidazolium chloride) for online sample concentration techniques (field-amplified sample stacking, head-column field-amplified sample stacking, and sweeping) of catecholamines were studied in both capillary zone electrophoresis and micellar electrokinetic chromatography. The use of a high-conductivity sample matrix in sweeping was found to significantly increase the separation efficiency of analyte up to 2×106 theoretical plates per meter and remarkably reduce limits of detection for catecholamines up to 50ng/mL. This approach was shown to be suitable for the determination of trace amounts of catecholamines in biological fluids.

Multidimensional capillary electrophoresis.

Multidimensional separation where two or more orthogonal displacement mechanisms are combined is a promising approach to increase peak capacity in CE. The combinations allow dramatic improvement of analytical performance since the total peak capacity is given by a product of the peak capacities of all methods. The initial reports were concentrated on the construction of effective connections between capillaries for 2D analysis. Today, 2D and 3D CE systems are now able to separate real complex biological or environmental mixtures with good repeatability, improved resolution with minimal loss of sample. This review will present the developments in the field of multidimensional CE during the last 15 years. The endeavors in this specific field were on the development of interfaces, interface-free techniques including integrated separations, microdevices, and on-line sample concentration techniques to improve detection sensitivity.

Toward 10 000‐fold sensitivity improvement of oligosaccharides in capillary electrophoresis using large‐volume sample stacking with an electroosmotic flow pump combined with field‐amplified sample injection

A combination of two online sample concentration techniques, large-volume sample stacking with an electroosmotic flow pump (LVSEP) and field-amplified sample injection (FASI), was investigated in CE to achieve highly sensitive oligosaccharide analysis. In CE with LVSEP-FASI, analytes injected throughout the capillary were concentrated on the basis of LVSEP, followed by an electrokinetic introduction of concentrated analytes from the inlet vial by the FASI mechanism. After switching the inlet vial solution from the sample to running buffer, the concentrated analytes were then separated by CZE. In the present LVSEP-FASI-CZE, pressure was applied to the capillary inlet until the inlet vial solution was exchanged. The applied pressure generated a counterflow against the EOF. It kept the stacked sample zone within the capillary, minimizing loss of concentrated analytes. Fluorescein was first analyzed by LVSEP-FASI-CZE to optimize preconcentration condition. Up to 110 000-fold sensitivity increase was obtained with 200 μL of sample, compared to normal CZE with sample injection of 0.3psi for 3 s (ca. 1.7 nL). From the results, the pressure application improved the efficiency of the FASI-mode concentration significantly at total concentration time longer than 10 min. In the analysis of maltoheptaose, a 10 000-fold sensitivity increase was achieved, which is the highest concentration efficiency ever reported in CE of oligosaccharides. The relative standard deviations of the detection time and peak height were 2.4 and 11%, respectively. In the analysis of glucose oligomer, up to 8600-fold sensitivity increases were achieved without reducing the separation performance of conventional CZE.

Sweeping of alprenolol enantiomers with an organic solvent and sulfated β‐cyclodextrin in capillary electrophoresis

Sweeping, an on-line sample concentration technique in CE, is the picking and accumulation of analytes by the pseudostationary phase or complexing additive. In the presence of an electric field, the analytes concentrated at the additive front that initially penetrated the sample zone. Here, we describe the sweeping of cationic alprenolol enantiomers using sulfated β-CD and organic solvent. The separation solution contained the anionic additive while ACN was in the sample solution. With fused silica capillaries, positive polarity, and solutions buffered at pH 3, the direction of the enantiomers' effective electrophoretic mobility was the same as the electrophoretic mobility (or electrophoretic mobility without additive). When the amount of ACN in the sample was increased (i.e. 60%), the interaction between the analytes and additive became negligible. This caused the sweeping boundary to shift from the electrophoretically moving β-CD front to the zone between the sample and separation solution. The equation that described the narrowing of injected sample zone was derived. The performance of sweeping with 60% ACN in the sample was then studied under different operating conditions (e.g. type of injection, injection time, and CD concentration). The low interaction between enantiomers and additive gave only moderate increases in sensitivity (approximately tenfold), but was improved when field enhancement was used during electrokinetic injection. With a conductivity difference (separation/sample solution) of 70 and a short injection time of 30 s at 20 kV, peak improvements of >100-fold was easily achieved.

Micellar electrokinetic chromatography: A review of methodological and instrumental innovations focusing on practical aspects

This review article addresses recent methodological and instrumental innovations in MEKC with emphasis on practical aspects. Like its predecessors, this review is intended to provide an updated overview covering work on the most salient methodological contributions to enhancing sensitivity and resolution in MEKC-based determinations published over the past two years. The most widespread approaches to enhancing sensitivity, which include improving "classical" online sample concentration techniques, combinations of on- and off-line sample concentration protocols and recent developments are discussed, and so are modifications of existing MEKC systems with various micellar phases, the use of BGE additives (organic modifiers, chiral selectors, gold nanoparticles) and coated capillaries, and the implementation of 2D separations and chemometric methods to enhance resolution. Instrumental approaches such as MS and LIF are also discussed, and proposals for overcoming the problems typically encountered in directly coupling MEKC with MS, and the recent inception of quantum dots with a great potential for LIF detection in MEKC, are also dealt with. Finally, foreseeable developments on potential future directions are also expressed.

Micelle to solvent stacking of organic cations in micellar electrokinetic chromatography with sodium dodecyl sulfate

The on-line sample concentration technique, micelle to solvent stacking (MSS), was studied for small organic cations (quaternary ammonium herbicides, β-blocker drugs, and tricyclic antidepressant drugs) in reversed migration micellar electrokinetic chromatography. Electrokinetic chromatography was carried out in fused silica capillaries with a background solution of sodium dodecyl sulfate (SDS) in a low pH phosphate buffer. MSS was performed using anionic SDS micelles in the sample solution for analyte transport and methanol or acetonitrile as organic solvent in the background solution for analyte effective electrophoretic mobility reversal. The solvent also allowed for the separation of the analyte test mixtures. A model for focusing and separation was developed and the mobility reversal that involved micelle collapse was experimentally verified. The effect of analyte retention factor was observed by changing the % organic solvent in the background solution or the concentration of SDS in the sample matrix. With an injection length of 31.9 cm (77% of effective capillary length) for the 7 test drugs, the LODs (S/N = 3) of 5–14 ng/mL were 101–346-fold better when compared to typical injection. The linearity ( R 2, range = 0.025–0.8 μg/mL), intraday and interday repeatability (%RSD, n = 10) were ≥0.988, <6.0% and <8.5%, respectively. In addition, analysis of spiked urine samples after 10-fold dilution with the sample matrix yielded LODs = 0.02–0.10 μg/mL. These LODs are comparable to published electrophoretic methods that required off-line sample concentration. However, the practicality of the technique for more complex samples will rely on dedicated sample preparation schemes.

Separation and sweeping of metal ions with EDTA in CZE‐ESI‐MS

Ethylenediaminetetraacetic acid (EDTA) in the background electrolyte (BGE) of capillary zone electrophoresis coupled to an electrospray ionization mass spectrometer is presented as an approach for the determination of metal ions. Significant signals for the metal-ligand complexes were observed even when EDTA was continually eluted from the capillary during the entire electrophoretic run. The signal-to-noise ratio was improved by the addition of ammonia to the sheath liquid and by using an acquisition m/z range above the m/z of EDTA. The LODs for the test metal ions (i.e. calcium(II), manganese(II), and zinc(II)) with conventional injection were around 1-2 mg/L with corrected peak areas that are linear from 8 to 100 mg/L. The presence of EDTA in the BGE was critical not only for the separation but also for sweeping via complexation as an on-line sample concentration technique. The peak height of the test metal ions was improved at least tenfold with sweeping via EDTA complexation and yielded LODs in the μg/L range.

Application of simple on-line sweeping sample concentration technique coupled micellar electrokinetic chromatography for simultaneous analysis of estrogen and androgen epimer

A reliable, convenient, and sensitive on-line sweeping-MEKC sample concentration technique has been applied for the simultaneous separation of six steroids including two pairs of epimer with 10 mM phosphate buffer (pH 7.0) that contains 80 mM sodium dodecyl sulfate (SDS), 14 mM β-cyclodextrin (β-CD), and 4% (v/v) methanol. The column length was 105 cm (effective length, 90 cm). Samples were hydrostatically injected for 600 s. The separation was performed at ambient temperature under an applied voltage of 25 kV. The external standard calibration curves of the six steroidal hormones proved good linearity ( r 2 = 0.9785–0.9941) within the concentration range 0.025–1.0 μg mL −1. The limit of detections of the on-line sweeping-MEKC with the ultraviolet detector at 220 nm for estrone, α-estradiol, β-estradiol, androstenedione, epitestosterone, and testosterone were 10, 24, 28, 53, 73, and 11 ng mL −1 and were 240, 125, 93, 47, 32, and 200 times more sensitive than MEKC, respectively. The Saccharomyces cerevisiae mediated simultaneous stereoselective reduction of estrone and androstenedione exhibited a 100% stereoselectivity toward β-estradiol and testosterone. The accuracy and precision achieved for the spiking experiments of the sweeping-MEKC were 95–98% and less than 3.8% (RSD), respectively.

Potential of long chain ionic liquids for on-line sample concentration techniques: Application to micelle to solvent stacking

The performance of micelle to solvent stacking (MSS) in capillary zone electrophoresis (CZE) was improved for anionic analytes using the long chain ionic liquid type cationic surfactant 1-dodecyl-3-methylimidazolium tetrafluoroborate (C 12-MIM-BF 4). The peak heights and corrected peak areas of the test profens and herbicides were enhanced up to 59 and 110-fold, respectively when compared to typical injection. These were up to 10 times better compared to the surfactant cetyltrimethyl ammonium bromide as MSS carrier. This performance was attributed to the properties of C 12-MIM-BF 4. MSS requires micelles in the sample for transport of bound analytes to a stacking boundary that contains an organic solvent for effective electrophoretic mobility reversal. The ionic liquid micelles provided better analyte transport properties that resulted from its hydrophobic and pi–pi interaction capabilities. The good solubility of the ionic liquid in high percentages of organic solvent also facilitated a more effective reversal of mobility. The LODs obtained for the test analytes were from 0.06 to 0.12 μg/mL. The linearity R 2 values in terms of peak height and corrected area were ≥0.99. The interday repeatabilities (%RSD, n = 10,) were 0.5–2.2% for retention time, 1.9–4.7% for corrected areas and 4.1–6.4% for peak heights.

On-line sample concentration of organic anions in capillary zone electrophoresis by micelle to solvent stacking

Micelle to solvent stacking (MSS) is a new on-line sample concentration technique for charged analytes in capillary zone electrophoresis (CZE). Sample concentration in MSS mainly relies on the reversal in the effective electrophoretic mobility of the analyte at the boundary zone between the sample solution (S) and CZE background solution (BGS) inside the capillary. The basic condition for MSS is that the S is prepared in a matrix that contains an additive (i.e., micelles) which interacts with and has an opposite charge compared to the analytes. In addition, the BGS must contain a sufficient percentage of organic solvent. MSS was first reported for organic cations using anionic dodecyl sulfate micelles as additive in the S and methanol or acetonitrile as organic solvent in the BGS. Here, theoretical and experimental studies on MSS are described for organic anions using cationic cetyltrimethyl ammonium micelles as additive in the S and methanol as organic solvent in the BGS. Up to an order of magnitude improvement in concentration sensitivity was obtained for the test hypolipidaemic drugs using MSS in CZE with UV detection. The optimized method was also evaluated to the analysis of a spiked wastewater sample that was subjected to a simple extraction step.

Sample stacking and sweeping in microemulsion electrokinetic chromatography under pH-suppressed electroosmotic flow

Two on-line sample concentration techniques, sample stacking and sweeping under pH-suppressed electroosmotic flow, were evaluated in microemulsion electrokinetic chromatography. The concept of stacking with anion selective electrokinetic injection and a water plug in a reverse-migrating microemulsion (SASIW-RMME) was brought forward in this article. Six flavonoids were concentrated using a microemulsion consisting of 80 mM sodium dodecyl sulfate, 1.2% (v/v) ethyl acetate, 0.6% (v/v) 1-butanol, 10% acetonitrile (v/v) and 50 mM phosphoric acid (pH* 1.8). Significant detector response improvements were achieved. The limits of detection were in the low ng/ml level. Finally, the sample of Fructus aurantii Immaturus was analyzed using sweeping technique.

Anion‐selective exhaustive injection‐sweeping microemulsion electrokinetic chromatography

In this study, anion-selective exhaustive injection-sweeping (ASEI-sweeping) technique, which is a selective on-line sample concentration technique, was first proposed in microemulsion electrokinetic chromatography (MEEKC) for analyses of eight acidic phenolic compounds. In contrast to a capillary that is typically filled with nonmicellar background solution in conventional ASEI-sweeping MEKC method, in the proposed ASEI-sweeping MEEKC method, a capillary is filled with a low pH microemulsion solution (pH 2.0), and then with a short acid plug (pH 2.0, 1.9 cm) before field-amplified sample injection. This proposed design has two functions. First, the microemulsion solution that is present at the front of capillary column is able to avoid phase separation of microemulsion solution during MEEKC separation. Second, the presence of the short acid plug would effectively limit the partition behavior of acid analytes with the oil droplets in the microemulsion during field-amplified sample injection; otherwise, the stacking effect of acid analytes would be markedly reduced. This optimal ASEI-sweeping MEEKC method afforded about 96,000-fold to 238,000-fold increases in detection sensitivity in terms of peak areas without any separation efficiency loss when compared to normal MEEKC separation. Furthermore, trace levels (about 3 ng/g) of gallic acid and catechin in foods were also detected successfully by the proposed ASEI-sweeping MEEKC technique.

UV Light-Emitting Diode-Induced Fluorescence Detection Combined with Online Sample Concentration Techniques for Capillary Electrophoresis

The application of an ultraviolet (UV) light-emitting diode (LED) to on-line sample concentration/fluorescence detection in capillary electrophoresis (CE) is described. The utility of a UV-LED (peak emission wavelength at 380 nm, approximately 2 mW) for fluorescence detection was demonstrated by examining both a naturally fluorescent (riboflavin) compound and a nonfluorescent compound (tryptophan), respectively. The detection limit for riboflavin was determined to be 0.2 ppm by the normal MEKC mode, which was improved to 3-7 ppb when dynamic pH-junction technique was applied. On the other hand, the detection limit of the tryptophan derivative was determined to be 1.5 ppm using the MEKC mode, which was improved to 3 ppb when the sweeping-MEKC mode was applied. In an analysis of an actual sample, the concentrations of riboflavin in beer, and tryptophan in urine and milk samples were determined, respectively.

On‐line sample concentration techniques in capillary electrophoresis: Velocity gradient techniques and sample concentration techniques for biomolecules

Methods with a high sensitivity and high separation efficiency are goals in analytical separation techniques. On-line sample concentration techniques in capillary electrophoresis (CE) separations have rapidly grown in popularity over the past few years because they achieve this goal. This review describes the methodology and theory associated with a number of different techniques, including electrokinetic and chromatographic methods. For small molecules, several on-line concentration methods based on velocity gradient techniques are described, in which the electrophoretic velocities of the analyte molecules are manipulated by field amplification, sweeping, and isotachophoretic migration, resulting in the on-line concentration of the analyte zones. In addition, the on-line concentration methods for macromolecules are described, since the techniques used for macromolecules (DNAs and proteins), are different from those for small molecules, with respect to either mechanism or methodology. Recent studies relating to this topic are also discussed, including electrophoretic and chromatographic techniques on capillary or microchip.

The bioactivity of 2,5-dimethoxy-4-ethylthiophenethylamine (2C-T-2) and its detection in rat urine by capillary electrophoresis combined with an on-line sample concentration technique

The bioactivity of 2,5-dimethoxy-4-ethylthiophenethylamine (2C-T-2) and its detection in rat urine by capillary electrophoresis combined with an on-line sample concentration technique

The bioactivity of 2,5-dimethoxy-4-ethylthiophenethylamine (2C-T-2) and its detection in rat urine by capillary electrophoresis combined with an on-line sample concentration technique

The bioactivity of 2,5-dimethoxy-4-ethylthiophenethylamine (2C-T-2) on nitric oxide (NO) production and the proliferation of spleen and thymus lymphocytes to mitogen stimulation in mice are reported for the first time. NO production by T and B lymphocytes in spleen and T cells in the thymus of mice decreased after the oral administration of 2C-T-2. This indicates that 2C-T-2 intake may perturb both neural and immune activity since a decrease in NO production is indicative of a weakened defense function. 2C-T-2 (the parent drug) in rat urine samples was detected by means of capillary electrophoresis/UV absorbance combined with an on-line sample concentration technique. When the CZE and MEKC modes were employed, the detection limit was found to be 4.5 and 5.0 μg/mL (at a 92.1% confidence level); whereas when on-line sample concentration methods, including stacking and sweeping-micellar electrokinetic chromatography were used, the detection limits were improved to 19.2 and 9.1 ng/mL, respectively. In an analysis of some actual samples from animal experiments, three male rats were administered 20 μg/g of body weight of 2C-T-2 by intra-peritoneal injection. The first- and second-day urine fractions were collected after the administration, for use in the analysis. As a result, 2.9 μg/mL and 0.25 μg/mL of 2C-T-2, respectively, were detected after ingestion of the doses.

Analysis of riboflavin in beer by capillary electrophoresis/blue light emitting diode (LED)-induced fluorescence detection combined with a dynamic pH junction technique

A simple, inexpensive and reliable method for the routine analysis of riboflavin in beer by capillary electrophoresis–light emitting diode (CE–LED) induced fluorescence detection is described. A simple and straightforward sample preparation is involved and the method is based on an inexpensive blue LED as the light source combined with an on-line sample concentration technique. For this detection system, using a normal micellar electrokinetic chromatography (MEKC), stacking-MEKC and dynamic pH junction techniques, the detection limits were found to be 480, 20 and 1 ng mL −1, respectively. In addition, the number of theoretical plates for riboflavin was determined to be 3.8×10 4 by means of a dynamic pH junction and this was improved to 3.2×10 6 when the dynamic pH junction-sweeping mode was applied. The concentrations of riboflavin in 12 samples of different types of commercial beer were found to be in the range of 130–280 ng mL −1.

Determination of corticosterone and 17-hydroxycorticosterone in plasma and urine samples by sweeping techniques using micellar electrokinetic chromatography

The analysis of corticosterone in mouse blood serum (metabolic-stress experiment) and 17-hydroxycorticosterone in human urine (exercise-stress experiment) samples by means of capillary electrophoresis/UV absorbance in conjunction with online sample concentration techniques is described. The use of normal MEKC had an analyte detection limit of 7 μg/ml (S/N=3); whereas when online sample concentration methods, including sweeping-micellar electrokinetic chromatography (Sweeping-MEKC) and cation-selective exhaustive injection-sweep-micellar electrokinetic chromatography (CSEI-sweep-MEKC) were used, the detection limits could be improved to 3 and 5 ng/ml, respectively. In the analysis of actual samples from animal metabolic-stress experiments (39 mouse), chronically stressed animals showed a higher level (552±152 ng/ml) and acute stressed animals showed an intermediate level (375±105 ng/ml). In comparison, normal animals show a lower concentration level of corticosterone (153±109 ng/ml). In addition, based on a human exercise-stress experiment (seven volunteers), the acute stressed humans (after exercise, 800 m of running) show a higher concentration of 17-hydroxycorticosterone (113±55 ng/ml for males; 128±25 for females) and the non-stressed humans (before exercise) show a lower concentration (63±37 ng/ml for male; 60±20 for female), respectively.