The application of ‘Z’-shaped UV cells in open-tubular capillary column liquid chromatography

Summary Z-shaped UV cells for packed capillary columns cannot be used in open-tubular capillary column liquid chromatography (OT-LC) because of their relatively large volume. With post-column solvent make-up, a commercial Z-shaped cell (volume 100 nl) was used in our OT-LC system, resulting in 9-fold sensitivity increase against on-column UV detection and with little efficiency loss. A small volume (5 nl) Z-shaped cell allows the direct coupling of the cell to high efficiency columns. For dilute samples, 9-19-fold sensitivity enhancement could be obtained. However, column efficiency decreases with increasing signal level as a result of peak deformation.     

Capillary SFC-FTIR – effects of the flow-cell on chromatographic resolution

The quantitative effects on chromatographic resolution of FTIR flow-cells for detection in capillary SFC have been determined. A suitably designed cell with a volume of 500 nl was shown to cause only modest broadening of chromatographic peaks obtained from the surfactant mixture Triton X-100, showing that subsequent detection methods can therefore be used in a multi-hyphenated chromatographic ensemble. A larger-volume (980 nl) cell was found to give satisfactory results with 100 μ columns, but not with 50 μm columns. The practicability of a multi-hyphenated system was illustrated by a preliminary capillary SFC-UV-FTIR-FID study on a plant extract. Good infrared spectra were obtained, together with well-resolved UV and (subsequent) FID chromatograms.     

Signal enhancement in capillary electrophoresis by using a sleeve cell arrangement for optical detection

This study comparts signal enhancement and efficiency in CZK employing three modes of detection: the sleeve cell (a simple method for creating a region of extended path length for absorption detection), the bubble cell (extended light path capillaries), and on-column detection in 75 μm i.d. capillary. Flow profile in the sleeve cell was monitored under a microscope. An abrupt change in capillary diameter in the sleeve cell region (from 50 μm to 220 μm) did not produce extensive band broadening. The sleeve-cell detection arrangement delivered a 3.5 fold increase in corrected peak area when compared with an oil-column detection in 75 μm i.d. column.     

A remote flow cell for UV absorbance detection with capillary HPLC based on a single strand fiber optic

A remote flow cell based on a single strand of fused-silica fiber optic was built for UV absorbance detection with a packed capillary HPLC system, using commercially available pumps, detection electronics (Shimadzu) and fittings. This 'off-column' flow cell design is applicable to both pressure and electro-osmotically driven systems. The goals were to minimize the linearity and light leakage problems that often limit the performance of UV absorbance detection with capillary chromatography. A linear dynamic range of 10(3) (reserpine, lambda = 220 nm), and a concentration detection limit of 5.1 x 10(-8) mol l-1 were observed. Baseline noise was measured at 3.5 x 10(-5) absorbance units (AU), with a standard deviation of 1.7 x 10(-5) AU. The illuminated volume of approximately 3 nl was optimized for capillaries with inner diameters in the range 50-100 microns, and flow rates from 100 nl min-1 to 1 microliter min-1. These modifications of readily available instrumentation have allowed the construction of a practical system for fractionating complex mixtures of peptides in small amounts, prior to mass spectrometry or additional wet chemistry steps.     

Experimental investigation of the flow induced by artificial cilia

The fluid transport produced by rectangular shaped, magnetically actuated artificial cilia of 70 μm length and 20 μm width was determined by means of phase-locked Micro Particle Image Velocimetry (μPIV) measurements in a closed microfluidic chamber. The phase-averaged flow produced by the artificial cilia reached up to 130 μm s(-1) with an actuation cycle frequency of 10 Hz. Analysis of the measured flow data indicate that the present system is capable of achieving volume flow rates of V[combining dot above](cilia) = 14 ± 4 μl min(-1) in a micro channel of 0.5 × 5 mm(2) cross-sectional area when no back pressure is built up. This corresponds to an effective pressure gradient of 6 ± 1 Pa m(-1), which equals a pressure difference of 0.6 ± 0.1 mPa over a distance of 100 μm between two rows of cilia. These results were derived analytically from the measured velocity profile by treating the cilia as a thin boundary layer. While the cilia produce phase-averaged velocities of the order of O(10(2)μm s(-1)), time-resolved measurements showed that the flow field reverses two times during one actuation cycle inducing instantaneous velocities of up to approximately 2 mm s(-1). This shows that the flow field is dominated by fluid oscillations and flow rates are expected to increase if the beating motion of the cilia is further improved.     

On-column complexation and simultaneous separation of vanadium(IV) and vanadium(V) by capillary electrophoresis with direct UV detection

An on-column complexation method has been developed for the simultaneous determination of V(IV) and V(V). Vanadium species were chelated with aminopolycarboxylic acids to form anionic complexes which were separated by capillary zone electrophoresis (CZE) with direct UV detection. Ethylenediaminetetraacetic acid (EDTA), diethylenetriaminepentacetric acid (DTPA), nitrilotriacetic acid (NTA), and N-2-hydroxyethylethlendiaminetriacetric acid (HEDTA) were investigated as both ligand and running electrolyte. Of the ligands studied the complexes of EDTA with V(IV) and V(V) resulted in the highest selectivity and UV response.The conditions used for on-column complexation and separation, including pH, and electrolyte ligand concentration, were examined to achieve reasonable separation selectivity and detection sensitivity. The optimum separation of the anionic forms of V(IV) and V(V) was obtained by use of CZE with UV detection at 185 nm and an electrolyte containing 5 mmol L(-1) EDTA at pH 4.0. Linear calibration plots were obtained in the concentration range10-300 micro mol L(-1); detection limits were 3 micro mol L(-1) for V(IV) and 1 micro mol L(-1) for V(V). The proposed method was demonstrated for the determination of vanadium in groundwater spiked with V(IV) and V(V).     

Sample introduction in high speed capillary gas chromatography; input band width and detection limits

The speed of analysis in capillary gas chromatography can be substantially increased by reduction of the column inner diameter. However, special demands are then posed upon instrumental design. In particular, the sampling system is highly critical because it has to be capable of delivering extremely small injection band widths which must be compatible with the column inside diameter. This study focuses on the evaluation of two potentially suitable sample introduction systems with respect to input band width and detection limits and their compatibility with small bore (≦ 100 μm) columns in capillary gas chromatography. One of them allows liquid on-column injection, based on liquid splitting, of only a few nl onto small bore (≦ 100 ?m) fused silica columns. For gases, input band widths as low as 1 ms are obtained with this system. The other one is part of a miniaturized gas chromatograph with extremely low dead volume interfaces and detector volumes. It allows input band widths for gases of a few ms. Without any preconcentration ppm concentrations are measured in gaseous samples with a 80 ?m thick film capillary column. It will be shown that a further reduction of the minimum detectable amount and analysis time is possible with this equipment.     

Construction and analytical application of an on-column photo reactor for improved detection of iron-species as plant metabolites in capillary flow injection and capillary electrophoresis

An on-column photo reactor for CE, which is constructed from UV-transparent capillaries and a small Pen-Ray UV lamp, is applied to the analysis of small, non-covalent iron-species. These iron-species, e.g. phytosiderophores (PS) in grasses and the non-protein amino acid nicotianamine (NA), play an important role in plant metabolism. The photo reactor is placed directly in front of the on-column absorbance detector, illuminating only some centimeters of the capillary. The photo reactor is used for capillary electrophoresis (CE) and also for capillary flow injection analysis (CFIA). Photoinduced sensitivity changes for model iron-species and for plant extracts are investigated, using UV detection and capacitively coupled contactless conductivity detection (C(4)D). The detection sensitivity for iron-species is enhanced in CFIA; the enhancement factor depends on the type of iron-species. In CE, the sensitivity of iron-species is kinetically dependent on the type of iron-species, decreasing with both detectors, but a photo reaction product is detectable. The relationship between irradiation window length and sensitivity is investigated quantitatively using EDTA-Fe(III). Pure ligands without iron are little affected by the photo reaction in both CFIA and CE. In CE, the detector signals of iron-species in real plant samples are selectively decreased with the proposed photo reactor, thus enabling a simple screening method for such photoactive iron-species.     

Enhancement of sensitivity in capillary supercritical fluid chromatography through optimization of injection and detection techniques

The reproducibility of peak areas as a function of the technique used for sample injection was investigated in capillary supercritical fluid chromatography (SFC). An injection technique has been developed to increase the volume of sample introduced into the capillary column. Using a modified time-split injection technique, long injection duration times were successfully applied to achieve lower detection limits. Analytes were effectively focused at the head of the analytical column using a unique pressure trap program. Because this on-column focusing was performed only by pressure and temperature programming, no instrumental modifications were necessary. Up to 1.0 μL of sample solution was injected onto 50 μm i.d. columns using this technique, with no observable peak splitting. Dual detection using ultraviolet (UV) absorption and flame ionization detection (FID) was performed in series, thereby avoiding the necessity of splitting the column effluent. For the compounds investigated (five nitroaromatics and one phthalate ester), the absolute sensitivity of the UV detector was significantly greater than that of the FID.     

On the minimization of the band-broadening contributions of a modern, very high pressure liquid chromatograph

The contributions of the volume of sample injected, the mobile phase flow rate, the inner diameter of the needle seat capillary and that of the connector capillary, the heat exchanger, and the detector cell volume to the widths of bands eluted from the 1290 Infinity HPLC instrument were investigated in depth. Four sample volumes (0.16, 0.80, 4.0, and 20 μL), three flow rates (0.04, 0.4, and 4.0 mL/min), two needle seat capillary I.D. (100 mm × 115 and 140 μm), three sets of connector capillary I.D. (350 mm × 80, 115, and 140 μm placed upstream the column, and 220 mm × 80, 115, and 140 μm downstream the column), two UV detector cell volumes (0.8 and 2.4 μL), and the presence/absence of the heat exchanger (1.6 μL) between the inlet connector capillary tube and the column were combined to generate up to 4 × 3 × 2 × 3 × 2 × 2=288 system configurations for this instrument. For each configuration, 5 consecutive injections were performed in order to assess the injection-to-injection repeatability, providing 1440 elution band profiles which are analyzed. The results demonstrate that the band broadening contribution of the instrument depends mostly on the detector cell volume and on the inner diameter of the needle seat capillary tube. The impact of these two contributions is particularly important at high flow rates (4 mL/min). Best efficiencies are obtained with a small sample volume, below 1 μL, which avoids volume overload of the instrument, or with large sample volumes, which maximize the radial concentration gradients of the sample across the instrument channels, in the vicinity of the anfractuosities of the channel walls. The injection of large sample volumes reveals the imperfection of current injection systems, the performance of which is remote from the one expected to provide an ideal rectangular injection (～+4 μL(2)). Although the present behavior of the instrument is satisfactory, serious improvements would become necessary to operate the next generation of more efficient columns that might be commercialized soon.     

Micellar electrokinetic chromatography with on-line Fourier transform infrared detection

Micellar electrokinetic chromatography (MEKC) was successfully coupled to Fourier transform infrared (FTIR) detection, using a micromachined IR-transparent flow cell with an optical path length of 15 micro m for the on-line detection of five neutral analytes. Tight connections between the flow cell and the capillaries were achieved by creating a small O-ring of UV-curing epoxy adhesive on the sharply cut capillary ends. The background electrolyte consisted of 15 mM phosphate buffer at pH 7 and 40 mM sodium dodecyl sulfate (SDS). Five analytes (paracetamol, caffeine, p-nitro benzyl alcohol, m-nitrophenol and p-nitrophenol) were successfully separated, yielding detailed IR stack plots that could be used for quantification and identification. Linear calibration graphs were obtained for each individual analyte present in mixtures at concentrations up to 10 mM. The limit of detection (3 S/N) ranged between 1.1 and 1.5 mM (1.2-1.8 ng). Analytes were identified by comparing spectra obtained during the MEKC separation with those resulting from completely filling the capillary with each individual analyte dissolved in the micelle-containing electrolyte. Information on the specific functional groups of all analytes could be elucidated from the spectra. Since FTIR is a nondestructive detection technique, a conventional on-line UV detector was introduced directly after the developed IR flow cell to test the system's performance and to demonstrate that tandem FTIR and UV detection is feasible.     

Large-volume sample stacking for on-capillary sample enrichment in the determination of naphthalene- and benzenesulfonates in real water samples by capillary zone electrophoresis

We investigated the on-line preconcentration of a test mixture of 15 substituted and unsubstituted naphthalene(NSs) and benzenesulfonates (BZSs) by large-volume sample stacking (LVSS). Analyses were carried out by capillary zone electrophoresis (CZE) with on-column UV detection. In particular, we focused on how experimental variables such as the inside diameter of the capillary, the volume of sample introduced and polarity switching influenced the enrichment procedure. The best results were obtained when 300 nl were injected and stacked using a bubble cell capillary. Under these conditions, LVSS increased the detector response of conventional hydrodynamic injection by a factor of 40. The limits of detection of the method were between 5 and 10 microg l(-1). Determinations were reproducible, in terms of peak area and migration time, under such conditions. The performance of the method was examined by determining NS and BZS in real samples, such as tap, river and surface waters and inflow/outflow waters from a water treatment plant. Real samples were injected directly into the CZE column with little or no preparation.     

Miniaturized on-line digestion system for the sequential identification and characterization of protein analytes

A miniaturized on-column digestion system constructed for the sequential analysis of semi-purified protein analytes is presented. By utilizing fused silica capillary (diameter 150microm) packed with a zone of trypsin-modified Eupergit C beads and a second zone of reversed-phase C18 material, a linear column set-up was constructed. The protein analytes (pmol amounts) were first digested in the 600nl trypsin reactor portion of the system. Next, the generated peptides were trapped in the C18 column shaped as an electrospray emitter. Finally, after washing the matrix free from salts and other hydrophilic impurities present in the sample, peptides were eluted. A stepwise increased concentration profile of organic solvent, created by a dual syringe pump system, promoted the release of bound peptides, which were identified by electrospray ionization MS/MS. This approach proved to be very efficient, achieving almost complete digestion of the proteins studied, with suitable operational stability maintained for more than 1 week. Further, a small nebulizer was designed and fitted to the electrospray emitter. A significant improvement of the spray stability was observed and droplet build-up on the capillary was avoided, even at flow rates well above 1500nl/min. The proteins chloroperoxidase, staphylococcal enterotoxin B and protein A (injection volume 0.3microl, salt concentration 0.2-1M) were sequentially digested, desalted, eluted, detected and conclusively identified by bioinformatics web tools with an analytical cycle time of 10min.     

Micro-impedance cytometry for detection and analysis of micron-sized particles and bacteria

The sensitivity of a microfluidic impedance flow cytometer is governed by the dimensions of the sample analysis volume. A small volume gives a high sensitivity, but this can lead to practical problems including fabrication and clogging of the device. We describe a microfluidic impedance cytometer which uses an insulating fluid to hydrodynamically focus a sample stream of particles suspended in electrolyte, through a large sensing volume. The detection region consists of two pairs of electrodes fabricated within a channel 200 μm wide and 30 μm high. The focussing technique increases the sensitivity of the system without reducing the dimensions of the microfluidic channel. We demonstrate detection and discrimination of 1 μm and 2 μm diameter polystyrene beads and also Escherichia coli. Impedance data from single particles are correlated with fluorescence emission measured simultaneously. Data are also compared with conventional flow cytometry and dynamic light scattering: the coefficient of variation (CV) of size is found to be comparable between the systems.     

The benefit of the retrofitting of a conventional LC system to micro LC: a practical evaluation in the field of bioanalysis with fluorimetric detection

The interests in liquid micro-chromatography (higher column efficiencies, increase in sensitivity) are now well established. The enhancement of fluorimetric response induced by the reduction of the inner diameter of columns (4.6, 3.0, 1.0 and 0.3 mm respectively) coupled with adapted detection cells to control the loss of efficiency (8 micro L for the two first columns and 100 nL for the two smaller ones) has been studied in the bioanalytical field, using the plasma determination of native fluorescent antibacterial agents: fluoroquinolones. Ten-fold enhancement of the signal can easily be obtained when substituting a 0.3 mm i.d. column and 100 nL detection cell for a 4.6 mm i.d. column, and 8 micro L detection cell. In addition to inner diameter reduction, the detection cell geometry appears to be an essential parameter to obtain the best enhancement of the recorded signal. Hence, the enhancement of signal with micro-chromatography with fluorimetric detection appears to be a compromise between column inner diameter and flow cell volume reduction.     

Suitability of automated capillary and micro liquid chromatography for routine determination of drugs in human plasma samples from clinical pharmacokinetic investigations

One of the most widely acclaimed features of capillary and microcolumn LC, in comparison with conventional HPLC, is the enormous increase in mass sensitivity. Nevertheless, application of capillary and micro LC in quantitative trace bioanalysis, characterized by weak analyte concentrations in complex matrices, can only be of any practical utility if large sample volumes can be injected onto the columns without affecting chromatographic resolution and efficiency. Two applications of large volume injection in a non-eluting solvent (on-column focusing) for the quantitative analysis of drugs in biological fluids on both capillary and micro chromatographic systems are presented: the first example deals with a new selective H₁-antihistaminic drug, mizolastine, the second one with a well known calcium antagonist, diltiazem, and its main metabolites. For both compounds, results obtained on micro and capillary LC in comparison with conventional HPLC are reported. The results demonstrate that when conventional HPLC methods are transformed into either micro or capillary LC techniques, they gain in sensitivity. By means of an on-column focusing technique, it is possible to increase the sensitivity 3–5 fold in comparison to conventional HPLC methods, but not 50–60 fold as obtained on synthetic drug solutions. Column robustness, handiness, reproducibility, and suitability of micro systems for routine bioanalysis are discussed for both capillary and micro LC columns, as well as limits of the technique in trace organic analysis problems.     

Comparison of the performance characteristics of two tubular contactless conductivity detectors with different dimensions and application in conjunction with HPLC

Two tubular capacitively coupled contactless conductivity detection (C(4)D) cells with different geometric dimensions were evaluated with regard to their main analytical characteristics under non-separation and separation conditions in conjunction with liquid chromatography. A comparison of the performance of the tubular cells to a previously tested thin-layer detection cell was drawn. Additionally, using a theoretical model the experimental results were compared with sets of calculated values and partially enabled to model the complex behavior of C(4)D detection in combination with high-performance liquid chromatography (HPLC). While cell 1 is characterized by a geometric cell volume of 0.6 μL, a wall thickness of 675 μm, and an inner diameter of 125 μm, the respective values for cell 2 are 2.3 μL, 200 μm, and 250 μm. The main analytical parameters were evaluated using a potassium chloride (KCl) solution. The limits of detection were 0.4 μM KCl (5.7 × 10(-6) S m(-1)) for cell 1 and 0.2 μM KCl (3.2 × 10(-6) S m(-1)) for cell 2, which compares well to the previously found 0.2 μM for the thin-layer cell. A pair of linear ranges was found for both cells in a concentration interval ranging from 1 × 10(-6) to 1 × 10(-4) M (corresponding to 1.5 × 10(-5) to 1.5 × 10(-3) S m(-1)) KCl, respectively. Furthermore, the detector cells were applied to the HPLC separation of a model compound system consisting of benzoic acid, lactic acid, octanesulphonic acid, and sodium capronate. Separation of the compounds was achieved with a Biospher PSI 100 C18 column using 60% aqueous acetonitrile mobile phase. Calibration curves for the examined model system were well correlated (r2 > 0.997), and it was found that under separation conditions the arrangement with the lower cell volume (cell 1) yields higher sensitivity and respectively lower limits of detection for all model compounds. Compared with the thin-layer cell, the tubular cells show better overall performance in regard to the determined analytical characteristics.     

On-column trace enrichment by sequential frontal and elution electrochromatography II. Enhancement of sensitivity by segmented capillaries with z-cell configuration--application to the detection of dilute samples of moderately polar and nonpolar pesticides

An on-column trace enrichment method for capillary electrochromatography of dilute samples is described. It involves the sequential use of frontal and elution electrochromatography on a segmented capillary column comprising of two contiguous segments each packed with a different sorbent. While the entering segment is for preconcentration by frontal electrochromatography the second segment is much longer and is meant for separation of the enriched analytes in the subsequent elution electrochromatography step. The preconcentration segment is usually packed with a sorbent that affords the highest affinity towards the solutes of interest while the separation segment is packed with a stationary phase that exhibits the highest selectivity and separation efficiency for the analytes. The detection is performed in the UV using a z-cell configuration for achieving an increased path length for detection. The effectiveness of this on-column trace enrichment is demonstrated on dilute samples of moderately polar solutes (e.g., carbamate insecticides) and nonpolar solutes (e.g., pyrethroid insecticides). Under optimal frontal and elution electrochromatography conditions. 817- and 1100-fold sensitivity increase are achieved for permethrin (a pyrethroid insecticide) and methiocarb (a carbamate insecticide), respectively, with a UV detector. The method is demonstrated with real water samples (e.g., tap and lake water samples) spiked with carbamate and pyrethroid insecticides. The limits of detection for the pesticides achieved in tap and lake waters reached 10(-8) to 10(-9) M.     

pH-independent large-volume sample stacking of positive or negative analytes in capillary electrophoresis

In capillary electrophoresis, the short optical path length associated with on-column UV detection imposes an inherent detection problem. Detection limits can be improved using sample stacking. Recently, large-volume sample stacking (LVSS) without polarity switching was demonstrated to improve detection limits of charged analytes by more than 100-fold. However, this technique requires suppression of the electroosmotic flow (EOF) during the run. This necessitates working at a low pH, which limits using pH to optimize selectivity. We demonstrate that LVSS can be performed at any buffer pH (4.0-10.0) if the zwitterionic surfactant Rewoteric AM CAS U is used to suppress the EOF. Sensitivity enhancements of up to 85-fold are achieved with migration time, corrected area, and peak height reproducibility of 0.8-1.6%, 1.3-3.7%, and 0.8-4.9%, respectively. Further, it is possible to stack either positively or negatively charged analytes using zwitterionic surfactants to suppress the EOF.     

新型纳升级毛细管刻蚀电喷雾质谱接口研究

对普通石英毛细管表面使用氢氟酸刻蚀技术进行刻蚀, 并与商品化鞘流液毛细管电泳-质谱接口(Sheathflow CE-MS interface)结合, 将其改装成一种新型的纳升级电喷雾质谱接口. 玻璃膜接口部分呈多孔结构, 壁厚约10 μm. 以细胞色素c对新型接口加以评价, 样品的流量最低可达到20 nL/min; 在50~500 nL/min流量范围内刻蚀接口具有较高的响应信号. 考察了接触电解质溶液对样品电离的影响; 比较微升级不锈钢接口和新型接口的蛋白质检测结果发现, 在流速为200 nL/min时, 检测灵敏度可以提高3.6倍.