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.     

Simultaneous sample washing and concentration using a "trapping-and-releasing" mechanism of magnetic beads on a microfluidic chip

Simultaneous washing and concentration of functionalized magnetic beads in a complex sample solution were demonstrated by applying a rotational magnetic actuation system to a microfluidic chip under continuous flow conditions. The rotation of periodically arranged small permanent magnets close to the fluidic channel carrying a magnetic bead suspension allows trapping and releasing of the beads along the fluidic channel in a periodical manner. Each trapping and releasing event resembles one washing cycle. A purification efficiency of magnetic beads out of a mixed magnetic and non-magnetic bead sample solution of 83±4% at a flow rate of 0.5 μL min(-1), and a magnetic bead recovery or concentration efficiency of 91±5% were achieved using a flow rate of 0.2 μL min(-1). The detection performance of the device was experimentally evaluated with two different bioassays, using either streptavidin-coated magnetic beads in combination with biotinylated fluorescent isothiocyanate (FITC), or a mouse antigen (Ag)-antibody (Ab) system.     

气相色谱微体积热导检测器测量氙

气相色谱微体积热导检测器(μTCD)是与毛细管柱相连的浓度型检测器。为提高-TCD测量氙的能力,研究了色谱进样条件、分离条件和检测条件对氙响应影响。结果表明,当柱流量为1.5mL/min,1.0mL样品完全进样所需最短时间为0.4min;等量氙响应值随着柱流量的增加而增大,与色谱柱温度无关;在流量比(q)等于2.3时,等量氙响应值达到最大(0.98-V.s/hPa);且当q=2～3时,其均值为0.97-V.s/hPa;相对标准偏差(RSD)为1.2%。     

Solid-phase microextraction under controlled agitation conditions for rapid on-site sampling of organic pollutants in water

An electric drill coupled with a solid-phase microextraction (SPME) polydimethylsiloxane (PDMS) fiber or a PDMS thin film was used for rapid sampling of polycyclic aromatic hydrocarbons (PAHs) in aqueous samples. Laboratory experiments demonstrated that the sampling rates of SPME fiber and thin film can be predicted theoretically. Compared with the SPME fiber, the PDMS thin film active sampler exhibited a higher sampling rate and much better sensitivity due to its higher surface-to-volume ratio and its larger extraction phase volume. The amount of the analytes extracted by the thin film was around 100 times higher than those obtained by fiber, for both 5 min rapid sampling and equilibrium extraction. A new thin film active sampler was then developed for rapid on-site water sampling. The sampling kit included a portable electric drill, a copper mesh pocket, a piece of thin film, and a liner. Laboratory experiments indicated that the sampling remained in the linear uptake phase with this sampler to 8 min for the PAHs. Field test illustrated that this novel sampler was excellent for rapid on-site water sampling due to its short sampling period, high sampling efficiency and durability The thin film sampling kit facilitates on-site sampling, sample preparation, storage and transport. This new sampler is more user-friendly and easier to commercialize than previous samplers.     

Development of a push-pull microdialysis sampling technique for the quantitative determination of proteins

Summary An on-line push-pull sampling technique has been developed for continuous analysis of proteins of molecular weight from 5.7 to 67 kDa. The characteristics of the system include gradient elution with a total cycle time of 21 min, membrane stability, unattended automatic operation, and adjustment of the sampling mode and extraction fraction (the ratio of the concentration of analyte in the dialysate to that in the sample) by varying the effective dialysis length. The push and pull flow rates were adjusted in a manner which enabled three different modes of operation. When push-pull microdialysis was compared with conventional microdialysis sampling, significantly higher extraction fractions were obtained for all five model proteins studied. The technique has been applied to the quantification of proteins in cell samples. On-line fractionation enabled complementary MS identification of the proteins present.     

整体柱在线固相微萃取-高效液相色谱同步富集检测水中的苯氧羧酸类除草剂

采用C18毛细管整体柱作为固相微萃取整体柱,构建在线固相微萃取-高效液相色谱联用系统,同步富集检测环境水样中的5种苯氧羧酸类除草剂。详细考察了联用系统运行条件对富集检测的影响。联用系统运行最佳参数为:固相微萃取整体柱长度20 cm,进样流速0.04 mL/min,进样13 min,洗脱流速0.02 mL/min,洗脱5 min。在最佳条件下,5种苯氧羧酸类除草剂的检出限为:9 μg/L (苯氧丙酸)、4 μg/L (2-(2-氯)-苯氧丙酸)、4 μg/L (2-(3-氯)-苯氧丙酸)、5 μg/L (2,4-二氯苯氧乙酸)、5 μg/L (2-(2,4-二氯苯氧基)丙酸)。与HPLC系统直接进样对比,联用系统对5种检测对象表现出优良的富集能力。5种苯氧羧酸类除草剂的回收率在79.0%~98.0%之间(RSD≤3.9%)。该方法成功应用于水样中5种苯氧羧酸类除草剂的检测,结果令人满意。     

天冬酰胺合成酶B抑制剂高效液相色谱筛选方法的建立与应用

建立了一种快速、高效测定天冬酰胺合成酶B(AS-B)酶活性的反相高效液相色谱法(RP-HPLC)。酶反应体系中的氨基酸经2,4-二硝基氟苯(DNFB)柱前衍生,通过RP-HPLC测定酶反应体系前后底物及产物的变化来分析酶的活性。采用的色谱柱为Agilent C18柱(250 mm×4.6 mm,5 μm),以50 mmol/L醋酸钠缓冲液(pH 6.2)-乙腈(体积比为15:85)为流动相,流速为1.0 mL/min,柱温为30 ℃,检测波长365 nm,于6 min内实现了各组分的基线分离。通过该方法测定反应动力学参数进行AS-B的抑制定量分析。将已知AS-B抑制剂L-谷氨酸-γ-甲酯作用于酶反应体系,测得的抑制剂的抑制常数与文献值相接近,证明该方法可用于AS-B抑制剂的筛选。     

An automatic sampling device for capillary zone electrophoresis

An automatic sampling device has been developed for capillary zone electrophoresis. The sample is introduced to the column electrokinetically by rapidly exchanging buffer for sample in a narrow channel drilled in a plexiglass block. The autosampler is capable, under computer control, of performing multiple sample injections from a large volume sample source (such as a reaction vessel) as the sample concentration changes, and thus presents the possibility of analyzing time-varying processes by CZE. Peak area reproducibility in electropherograms obtained after use of the sampler is less than 1% and efficiency is more than 2.2 × 10⁵ theoretical plates.     

A microfluidic-based enzymatic assay for bioactivity screening combined with capillary liquid chromatography and mass spectrometry

The design and implementation of a continuous-flow microfluidic assay for the screening of (complex) mixtures for bioactive compounds is described. The microfluidic chip featured two microreactors (1.6 and 2.4 microL) in which an enzyme inhibition and a substrate conversion reaction were performed, respectively. Enzyme inhibition was detected by continuously monitoring the products formed in the enzyme-substrate reaction by electrospray ionization mass spectrometry (ESI-MS). In order to enable the screening of mixtures of compounds, the chip-based assay was coupled on-line to capillary reversed-phase high-performance liquid chromatography (HPLC) with the HPLC column being operated either in isocratic or gradient elution mode. In order to improve the detection limits of the current method, sample preconcentration based on a micro on-line solid-phase extraction column was employed. The use of electrospray MS allowed the simultaneous detection of chemical (MS spectra) and biological parameters (enzyme inhibition) of ligands eluting from the HPLC column. The present system was optimized and validated using the protease cathepsin B as enzyme of choice. Inhibition of cathepsin B is detected by monitoring three product traces, obtained by cleavage of the substrate. The two microreactors provided 32 and 36 s reaction time, respectively, which resulted in sufficient assay dynamics to enable the screening of bioactive compounds. The total flow rate was 4 microL min-1, which a 25-fold decrease was compared with a macro-scale system described earlier. Detection limits of 0.17-2.6 micromol L-1 were obtained for the screening of inhibitors, which is comparable to either microtiter plate assays or continuous-flow assays described in the literature.     

Consideration of some sampling problems in the on-line analysis of batch processes by low-field NMR spectrometry

A low-field medium-resolution NMR spectrometer, with an operating frequency of 29 MHz for 1H, has been assessed for on-line process analysis. A flow cell that incorporates a pre-magnetisation region has been developed to minimise the decrease in the signal owing to incomplete polarisation effects. The homogeneous esterification reaction of crotonic acid and 2-butanol was monitored using a simple sampling loop; it was possible to monitor the progression of the reaction through changes in CH signal areas of butanol and butyl crotonate. On-line analysis of heterogeneous water-toluene mixtures proved more challenging and a fast sampling loop system was devised for use with a 5 L reactor. The fast sampling loop operated at a flow rate of 8 L min(-1) and a secondary sampling loop was used to pass a sub-sample through the NMR analyser at a slower (mL min(-1)) rate. It was shown that even with super-isokinetic sampling conditions, unrepresentative sampling could occur owing to inadequate mixing in the reactor. However, it was still possible to relate the 1H NMR signal obtained at a flow rate of 60 mL min(-1) to the composition of the reactor contents.     

Validation of the Willems badge diffusive sampler for nitrogen dioxide determinations in occupational environments

The Willems badge, a diffusive sampler for nitrogen dioxide, has previously been validated for ambient air measurements. This paper describes the laboratory and field validation of the Willems badge for personal sampling under working environment conditions. The mean sampling rate in the laboratory tests was 46 ml min(-1), with an RSD of 12%. No statistically significant effects on sampling rate of the sampling time, concentration of NO2 or relative humidity were found. A slightly decreased sampling rate was observed at low wind velocity. This was also confirmed during static sampling, which makes the sampler less appropriate for static sampling indoors. No back diffusion was observed. Storage of the samplers for two weeks before or after exposure did not affect the sampling rate. Our analysis is based on a modified colorimetric method, performed by FIA (flow injection analysis). This technique was compared to ion chromatography analysis. The use of ion chromatography lowered the detection limit from 11 to 2 microg m(-3) for an 8 h sample, and furthermore enabled the detection of other anions. In conclusion, the diffusive sampler was found to perform well for personal measurements in industrial environments.     

High-efficiency headspace sampling of volatile organic compounds in explosives using capillary microextraction of volatiles (CMV) coupled to gas chromatography–mass spectrometry (GC-MS)

A novel geometry configuration based on sorbent-coated glass microfibers packed within a glass capillary is used to sample volatile organic compounds, dynamically, in the headspace of an open system or in a partially open system to achieve quantitative extraction of the available volatiles of explosives with negligible breakthrough. Air is sampled through the newly developed sorbent-packed 2 cm long, 2 mm diameter capillary microextraction of volatiles (CMV) and subsequently introduced into a commercially available thermal desorption probe fitted directly into a GC injection port. A sorbent coating surface area of ～5?×?10^?2 m² or 5,000 times greater than that of a single solid-phase microextraction (SPME) fiber allows for fast (30 s), flow-through sampling of relatively large volumes using sampling flow rates of ～1.5 L/min. A direct comparison of the new CMV extraction to a static (equilibrium) SPME extraction of the same headspace sample yields a 30 times improvement in sensitivity for the CMV when sampling nitroglycerine (NG), 2,4-dinitrotoluene (2,4-DNT), and diphenylamine (DPA) in a mixture containing a total mass of 500 ng of each analyte, when spiked into a liter-volume container. Calibration curves were established for all compounds studied, and the recovery was determined to be ～1 % or better after only 1 min of sampling time. Quantitative analysis is also possible using this extraction technique when the sampling temperature, flow rate, and time are kept constant between calibration curves and the sample.     

Milli-free flow electrophoresis: I. Fast prototyping of mFFE devices

We coin a term of milli-free flow electrophoresis (mFFE) to describe mid-scale FFE with flow rates intermediate to macro-FFE and micro-FFE (μFFE). Introduced decades ago, mFFE did not find practical applications. We revive mFFE, as we view it as a viable purification complement to continuous synthesis in capillary reactors with product flow rates of ～5 to 2000 μL/min, too small for macro-FFE but too large for μFFE. The development of the tandem of continuous synthesis/purification will require the production and evaluation of a large number of prototypes of mFFE devices. As the first step, we developed a fast (<24 h) and economical (～$10) method for prototyping mFFE devices using a robotic milling machine. mFFE prototypes are constructed from two machined matching poly(methyl methacrylate) (PMMA) substrates, which are bonded in 10 min using dichloromethane to provide a strong and irreversible seal. Using the developed prototyping technology, we designed and evaluated 25 prototypes of mFFE devices. By optimizing the feed rates and rotational speeds of the drills, the depth of the electrode channels, the dimensions of the entrance and exit reservoirs, the sample flow rate, and the diameter and position of the sample input, we were able to achieve indefinitely long operation of the device with cycles of alternating 15-min electrophoresis and 0.5-min regeneration (bubble removal). The test analytes, rhodamine B and fluorescein, were baseline resolved by mFFE for flow rates ranging from 10 to 600 μL/min. These results prove that our prototyping approach is suitable for the challenging task of multi-parameter optimization of mFFE devices.     

Use of a parallel path nebulizer for capillary-based microseparation techniques coupled with an inductively coupled plasma mass spectrometer for speciation measurements

A low flow, parallel path Mira Mist CE nebulizer designed for capillary electrophoresis (CE) was evaluated as a function of make-up solution flow rate, composition, and concentration, as well as the nebulizer gas flow rate. This research was conducted in support of a project related to the separation and quantification of cobalamin (vitamin B-12) species using microseparation techniques combined with inductively coupled plasma mass spectrometry (ICP-MS) detection. As such, Co signals were monitored during the nebulizer characterization process. Transient effects in the ICP were studied to evaluate the suitability of using gradients for microseparations and the benefit of using methanol for the make-up solution was demonstrated. Co signal response changed significantly as a function of changing methanol concentrations of the make-up solution and maximum signal enhancement was seen at 20% methanol with a 15 μl/min flow rate. Evaluation of the effect of changing the nebulizer gas flow rates showed that argon flows from 0.8 to 1.2 l/min were equally effective. The Mira Mist CE parallel path nebulizer was then evaluated for interfacing capillary microseparation techniques including capillary electrophoresis (CE) and micro high performance liquid chromatography (μHPLC) to inductively coupled plasma mass spectrometry (ICP-MS). A mixture of four cobalamin species standards (cyanocobalamin, hydroxocobalamin, methylcobalamin, and 5′ deoxyadenosylcobalamin) and the corrinoid analogue cobinamide dicyanide were successfully separated using both CE-ICP-MS and μHPLC-ICP-MS using the parallel path nebulizer with a make-up solution containing 20% methanol with a flow rate of 15 μl/min.     

Microfabricated passive vapor preconcentrator/injector designed for microscale gas chromatography

The design, fabrication, and preliminary testing of a micromachined-Si passive vapor preconcentrator/injector (μPPI) are described. Intended for incorporation in a gas chromatographic microsystem (μGC) for analyzing organic vapor mixtures, the μPPI captures vapors from the air at a known rate by means of passive diffusion (i.e., without pumping) and then desorbs the vapor sample thermally by means of an integrated heater and injects it downstream (with pumping). The μPPI chip comprises a 1.8 μL deep reactive-ion-etched (DRIE) Si cavity with a resistively heated membrane floor and a DRIE-Si cap containing >1500 parallel diffusion channels, each 54 × 54 × 200 μm. The cavity is packed with 750 μg of a commercial graphitized carbon adsorbent. Fluidic and heat-transfer modeling was used to guide the design process to ensure power-efficient sample transfer during thermal desorption. Experiments performed with toluene at concentrations of ~1 ppm gave a constant sampling rate of 9.1 mL min(-1) for up to 30 min, which is within 2% of theoretical predictions and corresponds to a linear dynamic mass uptake range of ~1 μg. The cavity membrane could be heated to 250 °C in 0.23 s with 1 W of applied power and, with 50 mL min(-1) of suction flow provided by a downstream pump, yielded >95% desorption/injection efficiency of toluene samples over an 8-fold range of captured mass.     

On-Line Coupling of Separation Techniques to NMR

The hyphenation of chromatographic separation techniques with NMR spectroscopy is one of the most powerful and time-saving methods for the separation and structural elucidation of unknown compounds and molecular compositions of mixtures. Most of the routinely used NMR flow-cells have detection volumes between 40–180 μL for conventional separations with analytical columns, and the newest designs employ detection volumes in the order of 200 nL for capillary separations. The low flow rates used in capillary chromatography permit the use of deuterated solvents. Unequivocal structural assignment of unknown chromatographic peaks is possible by two-dimensional stopped-flow capillary HPLC-NMR experiments.     

Characterization of various geometric arrangements of “air-assisted” flow gating interfaces for capillary electrophoresis

For connecting flow-through analytical methods with capillary electrophoresis, a chip working in the air-assisted flow gating interface regime is cast from poly(dimethylsiloxane). In the injection space, the exit from the delivery capillary is placed close to the entrance to the separation capillary. Prior to injecting the sample into the separation capillary, the background electrolyte is forced out of the injection space by a stream of air. In the empty space, a drop of the sample with a volume of <100 nL is formed between the exit from the delivery capillary and the entrance into the separation capillary, from which the sample is injected hydrodynamically into the separation capillary. After injection, the injection space is filled with BGE, and the separation can be begun. Three geometric variants for the mutual geometric arrangement of the delivery and separation capillaries were tested: the delivery capillary is placed perpendicular to the separation capillary, from either above or below, or the capillaries are placed axially, that is, directly opposite one another. All of the variants are equivalent from the analytical and separation efficiency viewpoints. The repeatability expressed by RSD is up to 5%. The tested flow gating interface variants are also suitable for continuous and discontinuous sampling at flow rates of the order of units of μL/min. The developed instrument for sequential electrophoretic analysis operates fully automatically and is suitable for rapid sequential monitoring of dynamic processes.     

Sampling and analysis of flue gases from a plasma incinerator

A system is described for monitoring flue gases from a plasma incinerator for polychlorinated biphenyls (PCBs), polychlorinated dibenzodioxins and polychlorinated dibenzofurans. The system is composed of three basic units: sampler/preconcentrator, gas chromatograph and mass-selective detector. The sampler operates by solid sorbent trapping and thermal desorption. The use of two adsorbers allows sampling at a high flow rate (～1 1 min^?1) and subsequent capillary gas chromatographic analysis without the need for cold traps. A sample trapped on the first adsorber is thermally desorbed and transferred by a carrier stream of 40 cm³ min^?1 to a second smaller adsorber and retrapped. It is then thermally desorbed and injected into the capillary column by a carrier gas at an appropriate flow rate. A sequential valve-minder activates the electric actuators of the two six-port valves used in the design and also controls the power required for heating the adsorbers. Operation of the sampler is automated and is initiated by a single push-button switch. In simulation, the system allowed the separation of the major compounds of interest from possible interferences in <15 min and afforded unambiguous identification of the hazardous compounds and their quantification. For a sample volume of 20 1, the minimum detectable concentration of PCBs is 25–50 ng m^?3.     

Characterization of microconcentric nebulizer uptake rates for capillary electrophoresis inductively coupled plasma mass spectrometry

There is demonstrated interest in combining capillary electrophoresis (CE) with inductively coupled plasma mass spectrometry (ICP-MS) for speciation determinations. When self-aspirating nebulizers are used for this application, it is important to offset the suction effect to avoid degradation of the separation. In this study, sample uptake rates for three microconcentric nebulizers of the same model, in combination with a cyclonic spray chamber, were characterized and compared for future utilization in CE–ICP-MS interfaces. The specific model studied was a MicroMist with a nominal uptake rate of 100 μl/min at 1 l/min argon gas flow rate per the manufacturer's specifications. Sample uptake rates at various nebulizer gas flows were measured by aspirating water from a weighed container and calculating the uptake rate in microliter per minute. The nebulizers studied provided good reproducibility from day to day, but a comparison of the different nebulizers reflected a significant difference in performance. A characteristic observed during the study was that uptake rates decreased with increasing nebulizer gas flow. This can be used for sample introduction for CE–ICP-MS. Interestingly, very different performance was observed when comparing the three different nebulizers of the same model. Uptake rates showed strong dependence on argon gas flow rates and the dimensions of the sample uptake tubing.     

Quantitative Determination by Using HPLC and GLC Methods for Cocaine HCl in Synthetic Binary Mixtures with Procaine HCl,Lidocaine HCl and Caffeine

ABSTRACT

Cocaine HCl is a substance which creates psychological and physical dependence. Usually it is available on the market being diluted by other substances, like local anesthetics, analeptics and CNS stimulating agents and inert substance.

In this study, HPLC and GLC methods were applied for qualitative and quantitative determinations of synthetic binary mixtures. In the HPLC determination, μ Bondopack C₁₈ 10 μm. column system, a mobile phase consisting of methanol-water-phosphoric acid-1% hexylamine (75:175:250:3,5) and U.V. detection by photodiodearray (196–600 nm) were used.

The linear concentration areas were found in a range of 2.5–25 μg/mL. The R.S.D percentages for cocaine HCl, procaine HCl, lidocaine HCl and caffeine were found as 0.922, 0.568, 1.18 and 1.04, respectively.

In the GLC determination, two different column systems, a 2% OV-17-Gas Chrom W-HP 100–200 mesh filled column and a 0.25 SE-52 fused silica capillary column, were used. Nitrogen was used in a filled column and helium was used in a capillary column. Mobile phase flow rates were set as 30 mL/min and a flame ionization detector was used with both column systems.

The linear concentration intervals were found in a range of 2–25 μL/mL in both methods. The R.S.D. for cocaine HCl, procaine HCl, lidocaine HCl, and caffeine were found to be 0.907, 0.948, 0.770, 0.901 in the filled column. For cocaine HCl, procaine HCl and caffeine R.S.D.'s of 0.774, 0.809, 0.814 were found, when a capillary column was used. In quantitative determinations, antipyrine was chosen as internal standard in the HPLC and GLC methods.