Dual injector solvent elution and focussing technique for the on-line analysis of solid-phase extraction cartridges in HPLC

Summary A new dual injector solvent focussing and elution technique developed for high-performance liquid chromatography (HPLC) greatly improves chromatographic efficiency for the on-line analysis of C₁₈ solid-phase extraction (SPE) cartridges. Solutions containing three benzene homologs were used to characterize the dual injector analysis technique and to compare the chromatographic efficiency of this method with conventional SPE analysis methods. Sampling was performed off-line using a glass precolumn cartridge (3 mm i.d. × 30 mm) packed with 15–35 μm C₁₈ silica. On-line cartridge analysis was achieved with two injection valves in either serial or parallel configuration. The injection loop of the first valve contains the eluting solvent, and the cartridge holder is connected in place of the injection loop of the second valve. When an injection is made, both valves are turned to the inject position, and the solvent plug is forced through the cartridge, focussing the analyte at the solvent front as it elutes the cartridge. Solvent focussing at the head of the column, resulting from preconditioning of the column with a small plug of water during injection, further minimizes the variance of the injection plug and improves the chromatographic efficiency. The technique has potential applications to environmental and biological fluid analysis where analyte preconcentration and resolution from the sample matrix components may be difficult with current SPE methods.     

Polymer-coated synthetic fibers designed for miniaturized sample preparation process

Miniaturized sample preparation technique for complex sample matrices has been developed with a polymer-coated fibrous extraction medium. Several hundreds of fine fibrous materials were packed longitudinally into a fused-silica capillary followed by a polymeric coating on it to prepare the extraction capillary. The extraction capillary was installed in a liquid chromatograph as a sample loop of the injection valve. The on-line coupled sample preparation/separation system demonstrated a good validity for the analysis of phthalates in real river and wastewater samples. The lowest limits of quantification for several phthalates were less than 1 ng/ml. The effect of polymeric coating to the filaments on the extraction power was also investigated.     

Multiplexed detection of nitrate and nitrite for capillary electrophoresis with an automated device for high injection efficiency

A simple automated nanoliter scale injection device which allows for reproducible 5 nL sample injections from samples with a volume of <1 microL is successfully used for conventional capillary electrophoresis (CE) and Hadamard transform (HT) CE detection. Two standard fused silica capillaries are assembled axially through the device to function as an injection and a separation capillary. Sample solution is supplied to the injection capillary using pressure controlled with a solenoid valve. Buffer solution flows gravimetrically by the junction of the injection and separation capillaries and is also gated with a solenoid valve. Plugs of sample are pushed into the space between the injection and separation capillaries for electrokinectic injection. To evaluate the performance of the injection device, several optimizations are performed including the influence of flow rates, the injected sample volume and the control of the buffer transverse flow on the overall sensitivity. The system was then applied to HT-CE-UV detection for the signal-to-noise ratio (S/N) improvement of the nitric oxide (NO) metabolites, nitrite and nitrate. In addition, signal averaging was performed to explore the possibility of greater sensitivity enhancements compared to single injections.     

Automation for continuous analysis on microchip electrophoresis using flow-through sampling

Automation of electrophoretic microchips for sequential analysis of different samples is demonstrated. This system used an autosampler, which was on-line connected to the microchip and the whole process including sample loading and injection, analysis and data acquisition as well as washing were all automated. Rhodamin B at different concentrations was first loaded into a hydrodynamic flow stream by an autosampler, delivered to the microchip, and then sequentially injected into the electrophoretic microchannel for analysis and detection. Automation was achieved by running two independent programs, one for sample loading by an autosampler and the other one for electrophoretic injection by voltage switching, on the same computer. Using this sampling chip, each loaded volume (0.2-1 microL) can be injected for dozens of electrophoretic analyses (1-10 nL for each injection). The variances caused by the external connections, which did not affect the electrophoretic analysis but would cause band broadening of the loaded sample in the hydrodynamic flow stream, were theoretically deduced. Results indicate that the dead volume (approximately 300 nL) due to the connection fitting on the chip could lead to dilution of the loaded sample by a factor of one when 0.2 microL of sample was loaded. Such a design allows sequential analysis of a series of samples while the running buffer is continuously pumped into the connection capillary as well as microchannels for washing between two loaded samples to minimize cross contamination without human intervention. Using this sampling chip, the required sample amount and handling time can be greatly reduced compared to the manual method.     

Loop-type interface for concurrent solvent evaporation in coupled HPLC-GC. Analysis of raspberry ketone in a raspberry sauce as an example

Presently, two coupling techniques are used for directly introducing HPLC fractions into capillary GC: The retention gap technique (involving negligible or partially concurrent solvent evaporation) and fully concurrent solvent evaporation. While the former involves use of a conventional on-column injector, it is now proposed that concurrent solvent evaporation technique be carried out using a switching valve with a built-in sample loop. The technique is based on the concept that the carrier gas pushes the HPLC eluent into the GC capillary against its own vapor pressure, generated by a column temperature slightly exceeding the solvent boiling point at the carrier gas inlet pressure. Further improvement of the technique is achieved by flow regulation of the carrier gas (accelerated solvent evaporation) and backflushing of the sample valve (improved solvent peak shape). Concurrent solvent evaporation using the loop-type interface is easy to handle, allows transfer of very large volumes of HPLC eluent (exceeding 1 ml), and renders solvent evaporation very efficient, allowing discharge of the vapors of 1 ml of solvent through the column within 5–10 min.     

Application of programmed-temperature split/splitless injection to the trace analysis of aliphatic hydrocarbons by gas chromatography.

The dependence of the programmed-temperature solvent split sampling technique using a PSS (programmed-split/splitless) injection mode on different variables affecting the introduction of large sample volumes for a mixture of alkanes in capillary GC was evaluated. Apart from the studies found in the literature on different factors such as speed of injection. presence of adsorbent in the liner, internal diameter of the liner, initial and final injector temperature, split flow-rate and initial split time, affecting the chromatographic signal of different compounds, others were studied whose influence has not been considered until now. They include length of the microsyringe needle, adsorbent distribution in the liner, injection volume on analyte discrimination, speed of injector heating, time which the column stays at the initial temperature and time that the injector stays at the final temperature. Once finalised, the study of the PSS injection mode was compared with the conventional mode of gas chromatography splitless injection, and found that the proposed method increases sensitivity in GC trace analysis. Finally, the application of both injection modes in the determination of aliphatic hydrocarbons was tested in an atmospheric particulate sample.     

Large volume splitless injection with concurrent solvent recondensation: keeping the sample in place in the hot vaporizing chamber

An injector liner packed with a plug of glass wool is compared with a laminar and a mini laminar liner for large volume (20-50 microL) splitless injection with concurrent solvent recondensation (CSR-LV splitless injection). Videos from experiments with perylene solutions injected into imitation injectors show that glass wool perfectly arrested the sample liquid and kept it in place until the solvent had evaporated. The sample must be transferred from the needle to the glass wool as a band, avoiding 'thermospraying' by partial solvent evaporation inside the needle. The liquid contacted the liner wall when the band was directed towards it, but from there it was largely diverted to the glass wool. In the laminar liners, part of the liquid remained and evaporated at the entrance of the obstacle, while the other proceeded to the center cavity. Vapors formed in the center cavity drove liquid from the entrance of the obstacle upwards, but the importance of such problems could not be verified in the real injector. Some liquid split into small droplets broke through the obstacle and entered the column. Breakthrough through the laminar liners was confirmed by a chromatographic experiment. An improved design of a laminar liner for large volume injection is discussed as a promising alternative if glass wool causes problems originating from insufficient inertness.     

Loop-column technique: a new sample clean-up method for the determination of gallopamil from biological matrix

Summary A new sample clean-up method for the HPLC determination of gallopamil from biological matrix has been developed. In this method the loop capillary of the injection valve was replaced by a loop column filled with Nucleosil particles. This technique offers reduced extraction time (<1 min) and high sensitivity with a limit of detection as low as 0.1 ng/ml.     

Large volume sample introduction using temperature programmable injectors: Implications of liner diameter

Temperature programmable injectors with liner diameters ranging from 1 to 3.5 mm are evaluated and compared for solvent split injection of large volumes in capillary gas chromatography. The liner dimensions determine whether a large sample volume can be introduced rapidly or has to be introduced in a speed controlled manner. The effect of the injection technique used on the recovery of n-alkanes is evaluated. Furthermore the influence of the liner diameter on the occurrence of thermal degradation during splitless transfer to the analytical column is studied. Guidelines are given for the selection of the PTV liner internal diameter best suited for specific applications.     

Coupling of sequential injection analysis and capillary electrophoresis - Laser-induced fluorescence via a valve interface for on-line derivatization and analysis of amino acids and peptides

The on-line coupling of sequential injection analysis (SIA) and capillary electrophoresis (CE) via an in-line injection valve is presented. The SIA system is used for automated derivatization of amino acids and peptides. Dichlorotriazinylaminofluorescein serves as the derivatization agent, thus enabling sensitive laser-induced fluorescence detection of the derivatized analytes. The SIA procedure includes the following steps: (a) introduction of reagent and sample zones in a holding coil, (b) sample and reagent mixing in a reaction coil, (c) stop-flow step for increase of the reaction time, and (d) delivery of derivatized sample into the loop of the micro-valve interface. A small portion of the analyte zone is introduced electrokinetically in the separation capillary via the valve interface and CE analysis is performed. Factors affecting the CE separation, such as pH, the borate and sodium dodecyl sulphate concentration of the background electrolyte have been optimized. The derivatization conditions have been studied to obtain a high reaction yield in a relative short time. The transfer of a part of the reaction plug into the loop of the valve interface has been optimized. Using des-Tyr(1)-[Met]-enkephalinamide as test compound, it is demonstrated that after automated derivatization, on-line electrophoretic analysis could be achieved. Glycine has been selected as the internal standard in order to correct for variations in reaction time and filling of the injection loop. For the enkephalin, good reproducibility (RSD<4.5% calculated by the ratio of the peak areas) and linearity (0.5-5 microg mL(-1), R(2)>or=0.994) are obtained with a detection limit of 30 ng mL(-1) (S/N=3).     

改变相比/顶空气相色谱法测定变压器油中溶解气体的分配常数

采用改变相比/顶空气相色谱法测定了甲烷、乙炔、乙烯、乙烷和丙烷在变压器油中的分配常数。顶空瓶中的气体样品经石英毛细管送到气相色谱仪的六通进样阀样品管中,然后进行分离和定量。采用标准曲线法定量,通过测定5个不同相比时轻烃组分的顶空浓度,计算顶空浓度倒数与相比之间的线性回归方程,测定了20 ℃和50 ℃时烃类气体在变压器油中溶解气体的分配常数。除甲烷外,计算所得的分配常数与文献值基本吻合,油中溶解气体浓度的实验值与实际值之间的相对误差小于4.14%,表明用此方法可以测定不同温度下变压器油中溶解气体的分     

大体积进样技术在环境分析中的应用

在毛细管气相色谱法(CGC)中,采用大体积进样技术(LVI),即使用能够容纳大体积样品的进样装置以及增加可控时间的溶剂蒸汽放空装置,可以满足环境样品中超痕量组分的分析要求,简化样品浓缩步骤以及实现液相色谱(LC)与CGC的在线联用。针对分析物的性质、毛细管柱的规格和分析的目的已发展了多种LVI。本文总结了几种常见的LVI,包括柱头进样(OCI)和程序升温进样(PTV),以及近年来发展的一些新技术,如在柱同时溶剂浓缩进样、样品直接引入进样/复杂基质进样和同时溶剂冷凝无分流进样,阐述了各种进样技术的基本原理及其与样品提取、LC纯化在线联用的方法在环境分析应用中的一些最新研究进展。     

An improved reaction valve for flow injection analysis

Summary A new type reaction valve for flow injection analysis has been developed. In a FIA system with this valve the injected sample is once passed into a loop of the valve, thereafter the valve is rotated to the next position. Therefore, the sample is retained in the loop in a stop-state for a long time and the next sample is introduced into another loop in the next position. When the valve is rotated for one round, the stopped sample in the fist loop is again passed into the detector flow cell. Thus, an analysis, which needs a long reaction time, can be performed with this valve with high precision.

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Verbessertes Reaktionsventil für die Flie\injektionsanalyse

     

Development of a novel high volume band compression injector for the analysis of complex samples like toxaphene pesticide

A new type of injector has been developed for gas chromatographic analysis. The injector has high volume and band compression (HVBC) capabilities useful for the analysis of complex samples. The injector consists essentially of a packed liner operated at room temperature while a narrow heated zone is used to axially scan the liner selectively desorbing the compounds of interest. The scanning speed, distance and temperature of the zone are precisely controlled. The liner is connected to an interface which can vent the solvent or any undesirable compounds, and transfer the analytes to an analytical column for separation and quantification. The injector is designed to be compatible with injection volumes from 1 to more than 250microL. At a low sample volume of 1microL, the injector has competitive performances compared to those of the "on-column" and "split/splitless" injectors for the fatty acid methyl esters and toxaphene compounds tested. For higher volumes, the system produces a linear response according to the injected volume. In this explorative study, the maximum volume injected seems to be limited by the saturation of the chromatographic system instead of being defined by the design of the injector. The HVBC injector can also be used to conduct "in situ" pretreatment of the sample before its transfer to the analytical column. For instance, a toxaphene sample was successively fractionated, using the HVBC injector, in six sub-fractions characterized by simpler chromatograms than the chromatogram of the original mixture. Finally, the ability of the HVBC injector to "freeze" the separation in time allowing the analyst to complete the analysis at a later time is also discussed.     

In-Valve Sample Preparation Cartridge Designed for Microcolumn Liquid Chromatography

Miniaturized sample preparation technique for complex sample matrices has been developed with a polymer-coated fibrous extraction medium. By using extraction cartridge packed longitudinally with polymer-coated fiber into a short PEEK capillary, the extraction efficiency was significantly improved. The extraction cartridge was installed in an injection valve as a sample loop and the on-line coupling with microcolumn liquid chromatography (micro-LC) was demonstrated. This on-line coupled sample preparation/separation system showed a good validity for the analysis of phthalates and tricyclic antidepressant drugs in typical water samples. The lowest limits of quantification for several phthalates and tricyclic antidepressants (TCA) drugs were less than 1 ng mL^?1. The effect of polymeric coating on the filament surface on the extraction power was also investigated.     

On-line preparation of microsamples prior to CE

An experimental setup is presented here for the automated analysis of microsamples, based on the on-line coupling of a capillary SPE module and a CE unit using a two-position six-port valve, an open-closed valve to isolate electrically the sample preparation from the CE unit and a "T" interface. A C18 trapping microcolumn (dimensions 2.5 cm x 100 microm id x 360 microm od) was used for the SPE step. The utility of the proposed experimental setup was demonstrated by applying it to the determination of quinolone antibiotics in serum microsamples, which was efficiently carried out in less than 20 min (4 min for protein denaturation and 15 min for analytes preconcentration and CE-UV separation-determination). A complete optimization study was performed for preconcentration and cleanup of quinolones, the coupling of sample preparation module to the CE unit and electrophoretic separation of quinolones. A preconcentration factor of 10.4 was achieved. The volume injected with the proposed method was 125 nL versus 160 nL introduced by hydrodynamic injection. The volume required for the analysis was 2 microL, which makes the proposed experimental setup very useful for the analysis of microsamples in fields of current interest such as metabolomics or proteomics.     

On-line determination of aliphatic amines in water using in-tube solid-phase microextraction-assisted derivatisation in in-valve mode for processing large sample volumes in LC

This paper describes a cost-effective procedure for the analysis of short-chain aliphatic amines in water samples using a solid-phase microextraction device. Analyte preconcentration and derivatisation were effected into a capillary column coated with 95% polydimethylsiloxane–5% polydiphenylsiloxane, which was used as the injection loop of a Rheodyne injection valve. The coating was previously loaded with the derivatisation reagent, 9-fluorenylmethyl chloroformate. A volume of 1 mL of samples was then drawn into the capillary column, and the extracted analytes were left to react on the capillary coating for 5 min. Next, the capillary column was cleaned by passing water. Finally, the injection valve was rotated, and the derivatives formed were dynamically desorbed and transferred to the analytical column into the mobile phase. Methylamine, ethylamine, propylamine, n-butylamine and n-pentylamine were selected as model compounds. Excellent sensitivity was achieved, being the limits of detection of 15–200 μg/L when using UV detection and of 0.1–0.4 μg/L by fluorescence.     

Single‐step CE for miniaturized and easy‐to‐use system

We developed a novel single‐step capillary electrophoresis (SSCE) scheme for miniaturized and easy to use system by using a microchannel chip, which was made from the hydrophilic material polymethyl methacrylate (PMMA), equipped with a capillary stop valve. Taking the surface tension property of liquids into consideration, the capillary effect was used to introduce liquids and control capillary stop valves in a partial barrier structure in the wall of the microchannel. Through the combined action of stop valves and air vents, both sample plug formation for electrophoresis and sample injection into a separation channel were successfully performed in a single step. To optimize SSCE, different stop valve structures were evaluated using actual microchannel chips and the finite element method with the level set method. A partial barrier structure at the bottom of the channel functioned efficiently as a stop valve. The stability of stop valve was confirmed by a shock test, which was performed by dropping the microchannel chip to a floor. Sample plug deformation could be reduced by minimizing the size of the side partial barrier. By dissolving hydroxyl ethyl cellulose and using it as the sample solution, the EOF and adsorption of the sample into the PMMA microchannel were successfully reduced. Using this method, a 100‐bp DNA ladder was concentrated; good separation was observed within 1 min. At a separation length of 5 mm, the signal was approximately 20‐fold higher than a signal of original sample solution by field‐amplified sample stacking effect. All operations, including liquid introduction and sample separation, can be completed within 2 min by using the SSCE scheme.     

Automated sample preparation based on the sequential injection principle. Solid-phase extraction on a molecularly imprinted polymer coupled on-line to high-performance liquid chromatography

A molecularly imprinted polymer (MIP) prepared using caffeine, as a template, was validated as a selective sorbent for solid-phase extraction (SPE), within an automated on-line sample preparation method. The polymer produced was packed in a polypropylene cartridge, which was incorporated in a flow system prior to the HPLC analytical instrumentation. The principle of sequential injection was utilised for a rapid automated and efficient SPE procedure on the MIP. Samples, buffers, washing and elution solvents were introduced to the extraction cartridge via a peristaltic pump and a multi-position valve, both controlled by appropriate software developed in-house. The method was optimised in terms of flow rates, extraction time and volume. After extraction, the final eluent from the extraction cartridge was directed to the injection loop and was subsequently analysed on HPLC. The overall set-up facilitated unattended operation, operation and improved both mixing fluidics and method development flexibility. This system may be readily built in the laboratory and can be further used as an automated platform for on-line sample preparation.     

Quantitation in miniaturized GC and SFC systems

Separations of high efficiency and/or speed can be achieved in capillary GC by capillary columns of lower internal diameter (< 50 μm). Sampling techniques for the analytical application of narrow bore fused silica columns have been evaluated with regard to quantitation. On-column injection cannot be applied. Therefore liquid samples have to be vaporized in external devices before they enter the chromatographic system. Sample introduction by syringe with subsequent splitting must and can be applied but requires special syringes with perfect piston sealing because of the high inlet pressures needed even with hydrogen as carrier gas. For general analytical applications, valve systems should be developed to eliminate both the syringe and the septum from instrumental GC set-up's. In SFC using either narrow bore capillary or packed microbore columns, time-controlled valve sampling with partial displacement of the sample from the loop seems to be an adequate technique because of the very high inlet pressures involved. Splitting in combination with valve operation can also be applied in capillary SFC at least to samples of good solubility in the mobile phase. A disadvantage of splitting in SFC is that another restriction for the adjustment of the split flow is necessary.