The gradient volume principle for fast evaluation of optimum conditions for isocratic analysis

Summary The paper describes by simple experiments in a pragmatical way by easy rules of thumbs gradient optimization. Besides selection of the stationary phase and initial and final conditions the two other important variables are program time and eluent flow rate. It is demonstrated, that when the product of both, the gradient volume, is kept constant, the solutes are always eluted with the same eluent composition at column outlet. At constant gradient volume, peak broadening depends on flow rate and on the eluent properties (viscosity) at which the solutes elute, and on the time the solutes spend in the column. Because peak broadening increases with increasing gradient volume, the peak capacity in gradient elution shows an optimum at gradient volumes around 15 empty column volumes (program times 45 to 60 min at flow rates of 1 ml/min with standard columns).Gradient elution can also be used for fast evaluation of optimum eluent composition for isocratic analysis. This procedure requires a calibration of the equipment for determination of eluent composition at column outlet. The sample is chromatographed in a standard gradient run of 10 to 15 empty column volumes. The eluent composition at which the solute of interest elutes during the gradient is used for isocratic analysis, where the k' value of this solute will then be around 2.Part of Ph. D. Thesis H. Elgass, Saarbrücken, 1978, present address Hewlett-Packard, Waldbronn, FRG. In part presented at Eastern Analytical Symposium, New York, 1982.     

Dispersion effects of laminar flow and spray chamber volume in capillary electrophoresis-inductively coupled plasma-mass spectrometry: a numerical and experimental approach

Band broadening related to laminar flow and spray chamber dead volume is a potential problem in flow injection (FI)-inductively coupled plasma-mass spectrometry (ICP-MS). We studied these two dispersion effects with a sheath flow capillary electrophoresis (CE)-ICP-MS interface. A numerical model was used to simulate advection diffusion processes in the CE-capillary and dispersion in the spray chamber. Experimental results of FI with this CE-ICP-MS interface agree well with numerical modeling results. Dispersion due to laminar flow depends strongly on capillary diameter and analyte diffusion coefficient and to a lesser extent on laminar velocity and capillary length and typically amounts to one order of magnitude peak width increase. Three spray chambers of 5, 20 and 150 ml dead volume showed an increase in band broadening and peak tailing with increasing dead volume. The use of standard Scott-type spray chambers (>90 ml volume) increases peak widths by 5-10 s regardless of injection time. The use of a low dead volume spray chamber is recommended for experiments where resolution is critical. The modeling approach can be extended to the coupling of other flow injection techniques, like micro-LC and nano-LC with ICP-MS.     

Radiochemical detection for packed capillary liquid chromatography-mass spectrometry

For the identification of trace level organic molecules, such as drug or pesticide metabolites, there is need of a practical method to do packed capillary liquid chromatography-mass spectrometry (LC-MS) with radiochemical detection. This problem has been successfully solved by use of a post column flow-splitter, with coaxially transported makeup flow that increases the split flow rate to a flow compatible with commercially available radiochemical flow cells. To test the device, 14C-labeled azoxystrobin, a commercial fungicide, was analyzed by liquid chromatography-radiochemical activity monitor-mass spectrometry (LC-RAM-MS) using a 0.32 mm i.d. packed capillary column. Azoxystrobin could be detected at 500 pCi with good signal/noise. The method is general and can be used with capillary LC columns of smaller diameters. Column efficiency of about 20,000 theoretical plates/m was achieved using either radiochemical or mass spectrometric data, thus demonstrating the lack of band broadening using the described method for radiochemical detection. The simple hardware described allows the routine use of packed capillary LC with radiochemical detection.     

An investigation of peak-broadening effects arising when combining CE with MS

In this study, peak-broadening effects caused by nebulizing gas flow and lack of temperature control have been investigated for separation capillaries with three different inner diameters. The study was performed with serial UV/ESI-MS detection in an effort to distinguish between peak broadening arising in the separation and peak broadening arising in the ion source. The nebulizing gas was found to significantly affect both migration time and separation efficiency when using capillaries with 50 and 75 microm id. If the nebulizing gas is on during injection, the injection volume increases to such an extent that significant peak broadening is induced. Reducing the id to 25 microm minimizes the parabolic flow induced by the nebulizing gas. Results indicate that the nebulizing gas pressure can be optimized to minimize peak broadening in the ion source. A decrease in detection sensitivity, possibly related to the orthogonal design of the interface, was observed when the nebulizing gas pressure was increased. A tapered capillary tip was found to provide superior separation efficiency as well as sensitivity.     

A microchip device to enhance free flow electrophoresis using controllable pinched sample injections

Micro free flow electrophoresis (µFFE) is a valuable technique capable of high throughput rapid microscale electrophoretic separation along with mild operating conditions. However, the stream flow separation nature of free flow electrophoresis affects its separation performance with additional stream broadening due to sample stream deflection. To reduce stream broadening and enhance separation performance of µFFE, we presented a simple microfluidic device that enables injection bandwidth control. A pinched injection was formed in the reported µFFE system using operating buffer at sample flow rate ratio (r) setting. Initial bandwidth at the entrance of separation chamber can be shrunk from 800 to 30 µm when r increased from 1 to 256. Stream broadening at the exit of separation chamber can be reduced by about 96% when r increased from 4 to 128, according to both theoretical and experimental results. Moreover, the separation resolution for a dye mixture was enhanced by a factor of 4 when r increased from 16 to 128, which corresponded to an 80% reduction in sample initial bandwidth. Furthermore, a similar enhancement on amino acids separation was obtained by using injection control in the reported µFFE device and readily integrated into online/offline sample preparation and/or downstream analysis procedures.     

Corrections for Instrumental and Secondary Broadening in the Chromatographic Analysis of Linear Copolymers

Abstract

The effects of instrumental broadening and of polymer/solvent/packing interactions (secondary broadening) are separately investigated in the context of chromatography experiments, for the characterization of linear copolymers with two types of repeating units. The problems associated to the estimation of: a) the joint molecular weight distribution - chemical composition distribution (MWD-CCD) through orthogonal chromatography; and of b) the average MWD - average CCD through standard size exclusion chromatography (SEC) with dual detection are considered. The main difficulty with the secondary broadening correction, is the calibration for this effect. In the case of standard SEC with dual detection, a simple solution was found to the instrumental broadening problem, that involves a direct extension of the calibration and deconvolution techniques developed for linear homopolymers and mass detectors.     

Direct determination of band broadening in size exclusion chromatography

A simple method to correct the measured extent of band broadening in size exclusion chromatography for the contribution of narrow (polydisperse) standards is presented. It is based on the assumptions that commercial polymer standards can be described by a Poisson distribution and the additivity of peak variances. Two sets of standards (polystyrene from two suppliers) were investigated under normal working conditions, i.e. a combination of four columns with different porosities and a flow rate of 1 ml/min. Furthermore, the polystyrene standards were used to determine the extent of band broadening for four additional combinations of columns (varying in their separation range and porosities) as a function of the elution volume. The assumption of a constant peak variance for band broadening turned out to be a (very) rough approximation for some combinations of columns, but all results taken together demonstrate that this assumption is not generally applicable. Qualitative agreement between theory and experiment was found with a rearranged van Deemter equation.     

High-speed gas chromatography: the importance of instrumentation optimization and the elimination of extra-column band broadening

This review provides a summary of chromatographic theory as it applies to high-speed gas chromatography. A novel method for determining the optimal linear flow velocity, u (opt), from specific experimental parameters, is discussed. An in-depth theoretical understanding of u (opt) and its relation to experimental parameters is presented, in the absence of extra-column band broadening, as a means of method evaluation and optimization. Recent developments in high-speed GC are discussed, in the context of the theory presented within this review, to ascertain the influence of extra-column band broadening. The theory presented herein can be used as a means of evaluating the various areas of GC instrumentation (injection, separation, detection, etc.) that need further development to further minimize the effects of extra-column band broadening. The theoretical framework provided in this review, can be, and is, readily used to evaluate high-speed GC results presented in the literature, and thus, the general practitioner may more readily select a specific capillary length and/or internal diameter for a given application. For example, it is theoretically shown, and prior work cited, that demonstrates a peak width of approximately 1 ms is readily achievable in GC, when extra-column band broadening is eliminated.     

逆流法对凝胶色谱峰加宽效应的研究

在以无孔玻璃珠为填料的色谱柱上研究了流动相中的分离能力和峰加宽效应。得到一定的分离能力。根据Kelley和Billmeyer描述流动相中峰加宽效应的理论,塔板高度和溶液的分子量有关而和分子量分布无关。但实验表明,对于具有相同分子量,但分子量分布不同的样品峰宽不同。苯的逆流峰加宽因子h稍大于直流峰加宽因子h′。产生这个差别的原因可以用在逆转过程中流速场受到干扰来解释。 在以多孔硅胶为填料的柱中对一系列不同分子量、分子量分布和化学结构的样品考察了逆流峰加宽因子,h,和淋出体积,Ve,之间的关系。实验表明h和Ve之间的关系具有普适性,这个结果和Tung的一致。在多孔填料柱上对苯同样得到h>h′。这个结果意味着用逆流法得到的h来校正GPC体系造成的峰加宽稍嫌不够。 只有对于分子量分布很窄的样品,其逆流淋出曲线的形状才非常接近高斯分布。     

用逆流法研究热场流分级中峰加宽效应和测定分子量分布

验证了逆转法在场流分级这种单相色谱中的可行性。用逆转法消除样品的多分散性对塔板高度的贡献,以便研究在热场流分级柱槽中其它实验条件的影响。测定了几种聚苯乙烯样品的分子量分布。     

High Performance Size Exclusion Chromatography with Microparticulate Porous Silica Spheres. Influence of Flow Rate,Salute Mass Transfer and Polydispersity of Samples

Abstract

The contributions to peak broadening in Size Exclusion Chromatography with microparticles of porous silica spheres having narrow size distributions have been determined by measuring the plate height dependence on flow rate for toluene and for polystyrene standards covering a wide range of molecular weights. From these contributions, the diffusion coefficients of the macromolecules in the pore matrix and the polydispersities of the samples could be evaluated. It is shown that for permeating polymers the band broadening is determined by the eddy diffusion in the mobile phase, by the slow mass transfer of the solute in the stationary phase and by the polydispersity of the standards. In properly packed columns the eddy diffusion term is of minor importance compared to the other effects, whereas the solute mass transfer, which is a velocity dependent process, can be minimized only at extremely low flow rates.     

Peak broadening from an electrothermal vaporization sample introduction source into an inductively coupled plasma

Signal broadening using electrothermal vaporization with inductively coupled mass spectrometry (ETV-ICPMS) occurs at a rate much faster than would be predicted by simple longitudinal diffusion. A Monte Carlo simulation that focused on particle motion within the transport tubing was created to elucidate the causes of this dispersion within ETV-ICPMS. Several parameters, including the diffusion coefficient, tube diameter, transport tube length, and flow rate were varied to discern their role in signal broadening. Using typical instrumental parameters, the parabolic flow profile generated by laminar flow of the carrier gas was shown to be the primary cause of dispersion. Manipulating the aforementioned variables to lessen the effects of laminar flow led to a decrease in dispersion. Conversely, increasing the role of laminar flow promoted broadening. The broadening processes should be applicable to any transient introduction system where material must be transported to a detection system. Due to the difference in the rate of broadening expected for particles of different sizes, the simulation was used to calculate the average size of particles generated in the ETV using different mass amounts of sample. No change in particle size (∼1 nm) was seen for mass amounts ranging from 10–10 000 pg, which suggests that the particle number is increased with increasing sample mass rather than the average particle size. Using this method of determining particle size, it might be possible to further evaluate the mechanisms of physical ‘carrier’ action.     

Development and characterization of an integrated silicon micro flow cytometer

This paper describes an innovative integrated micro flow cytometer that presents a new arrangement for the excitation/detection system. The sample liquid, containing the fluorescent marked particles/cells under analysis, is hydrodynamically squeezed into a narrow stream by two sheath flows so that the particles/cells flow individually through a detection region. The detection of the particles/cells emitted fluorescence is carried out by using a collection fiber placed orthogonally to the flow. The device is based on silicon hollow core antiresonant reflecting optical waveguides (ARROWs). ARROW geometry allows one to use the same channel to guide both the sample stream and the fluorescence excitation light, leading to a simplification of the optical configuration and to an increase of the signal-to-noise ratio. The integrated micro flow cytometer has been characterized by using biological samples marked with standard fluorochromes. The experimental investigation confirms the success of the proposed microdevice in the detection of cells. An erratum to this article can be found at     

Dispersion of discrete sample signals within aerosol spray chambers: preliminary investigations

A barrel-type spray chamber with a baffle system and various other structures were used for characterization of band-broadening phenomena occurring within aerosol spray chambers, with an ICP-AES detector used in this case. Absolute values of aerosol particle size distributions from nebulizer/spray chamber systems were measured and found to be affected by the relative positions of impact surfaces with respect to the nebulizer. Smaller particles, but less total mass was observed as the baffle was moved closer to the nebulizer tip. However, based on measurements herein, it does not appear that particle sizes in any case will be small enough within the spray chamber for diffusion to influence band broadening for the bulk of the aerosol mass, as particularly important for mass sensitive detectors such as ICP-AES, although diffusion is likely to be important to particle number sensitive detectors.

Dispersion within the turbulent gas-phase regions of spray chambers appears to be relatively small. Geometries which introduce stagnant gas flow regions appear to have larger effects on band-broadening. The smoke tests utilized in these studies were found to be useful aids for visualization of spray chamber flow phenomena that influence dispersion of discrete signals.

Recirculation phenomena can also play a role in band broadening. Recirculation of aerosol re-entrained into the nebulizer jet appears to have an effect on dispersion, as represented by the full width at half maximum values. On the other hand, recirculation renebulization leads to substantial peak tailing.     

Flow-through radioactivity detection of 3H- and 14C-labelled compounds in reversed-phase liquid chromatography by a liquid scintillation counting technique based on post-column extraction

A method for flow-through liquid scintillation counting in reversed-phase liquid chromatography (RP-LC) based on the continuous extraction of aqueous column eluates with a water-immiscible liquid scintillator is evaluated in terms of sensitivity, reproducibility and extra-column band broadening. ³H- and ¹⁴C-labelled phenylthiohydantoinamino acid derivatives of widely different polarity serve as model compounds. For extractable derivatives, counting efficiencies of over 30 and 80% can be obtained for ³H and ¹⁴C, respectively. The reproducibility and extra-column band broadening depend on the mixing ratio of scintillator and LC eluent; relative standard deviations (peak areas) of less than 3% can be obtained. The sensitivity of flow-through counting can be increased at least 150-fold by storing the segmented scintillator/eluate stream in a capillary storage loop. After the separation is complete, the stream is re-introduced into the radioactivity detector at reduced flow-rates to increase the mean residence time, i.e., the counting time, in the detector.     

Comparison of μm and mm sized disk electrodes for end-column electrochemical detection in capillary electrophoresis

End-column electrochemical detection based on either the use of a 25 μm microdisk electrode or a 0.5 mm macrodisk electrode has been compared with respect to performance and influence on non-aqueous capillary electrophoretic separations. Despite the much higher coulometric efficiency obtained with the larger disk electrode, the microdisk electrode configuration offers comparable limits of detection (LOD) for the neutral and positively charged ferrocene compounds employed in conjunction with a non-aqueous acetonitrile-based buffer. The LODs for ferrocene were found to be 4.0 × 10^–8 M and 6.7 × 10^–8 M for the microdisk and macrodisk detector, respectively. In addition, both detector arrangements showed different relative responses for neutral and positively charged analytes. The macroelectrode-based detector introduced additional zone broadening while this was not found to be the case with the microelectrode arrangement. Using the microelectrode detector, the band broadening in an electro-osmotically driven flow system was compared to that in a gravity flow-based system. It was demonstrated that the zone broadening under gravity flow conditions was approximately twice as large as under electro-osmotic flow conditions for a typical set of experimental parameters. Received: 1 June 1998 / Revised: 20 August 1998 / Accepted: 24 August 1998     

Achieving high peak capacity production for gas chromatography and comprehensive two-dimensional gas chromatography by minimizing off-column peak broadening

By taking into consideration band broadening theory and using those results to select experimental conditions, and also by reducing the injection pulse width, peak capacity production (i.e., peak capacity per separation time) is substantially improved for one dimensional (1D-GC) and comprehensive two dimensional (GC×GC) gas chromatography. A theoretical framework for determining the optimal linear gas velocity (the linear gas velocity producing the minimum H), from experimental parameters provides an in-depth understanding of the potential for GC separations in the absence of extra-column band broadening. The extra-column band broadening is referred to herein as off-column band broadening since it is additional band broadening not due to the on-column separation processes. The theory provides the basis to experimentally evaluate and improve temperature programmed 1D-GC separations, but in order to do so with a commercial 1D-GC instrument platform, off-column band broadening from injection and detection needed to be significantly reduced. Specifically for injection, a resistively heated transfer line is coupled to a high-speed diaphragm valve to provide a suitable injection pulse width (referred to herein as modified injection). Additionally, flame ionization detection (FID) was modified to provide a data collection rate of 5kHz. The use of long, relatively narrow open tubular capillary columns and a 40°C/min programming rate were explored for 1D-GC, specifically a 40m, 180μm i.d. capillary column operated at or above the optimal average linear gas velocity. Injection using standard auto-injection with a 1:400 split resulted in an average peak width of ～1.5s, hence a peak capacity production of 40peaks/min. In contrast, use of modified injection produced ～500ms peak widths for 1D-GC, i.e., a peak capacity production of 120peaks/min (a 3-fold improvement over standard auto-injection). Implementation of modified injection resulted in retention time, peak width, peak height, and peak area average RSD%'s of 0.006, 0.8, 3.4, and 4.0%, respectively. Modified injection onto the first column of a GC×GC coupled with another high-speed valve injection onto the second column produced an instrument with high peak capacity production (500-800peaks/min), ～5-fold to 8-fold higher than typically reported for GC×GC.     

Evaluation of direct axial sample introduction for ion mobility detection after capillary gas chromatography

An ion mobility detector has been designed and constructed for direct axial interfacing with capillary gas chromatography. The principle advantages of this detector were the following: (1) Direct concentric introduction of the capillary column into the ionization region, eliminating peak broadening in the transfer line and improving the efficiency with which neutral molecules were swept from the detector. (2) A variable capillary insertion distance, providing a sensitivity/resolution interplay that could be modified in response to the needs of the assay. (3) An inert gas flow external to the drift cylinder, preventing atmospheric impurities from infiltrating the ultratrace detector. Qualitative and quantitative capabilities of the detector were evaluated using standard preparations of n-hexyl ether.     

Development of a microfluidic platform for single-cell secretion analysis using a direct photoactive cell-attaching method

A precise understanding of individual cellular processes is essential to meet the expectations of most advanced cell biology. Therefore single-cell analysis is considered to be one of possible approach to overcome any misleading of cell characteristics by averaging large groups of cells in bulk conditions. In the present work, we modified a newly designed microchip for single-cell analysis and regulated the cell-adhesive area inside a cell-chamber of the microfluidic system. By using surface-modification techniques involving a silanization compound, a photo-labile linker and the 2-methacryloyloxyethyl phosphorylcholine (MPC) polymer were covalently bonded on the surface of a microchannel. The MPC polymer was utilized as a non-biofouling compound for inhibiting non-specific binding of the biological samples inside the microchannel, and was selectively removed by a photochemical reaction that controlled the cell attachment. To achieve the desired single-macrophage patterning and culture in the cell-chamber of the microchannel, the cell density and flow rate of the culture medium were optimized. We found that a cell density of 2.0 × 10(6) cells/ml was the appropriate condition to introduce a single cell in each cell chamber. Furthermore, the macrophage was cultured in a small size of the cell chamber in a safe way for 5 h at a flow rate of 0.2 μl/min under the medium condition. This strategy can be a powerful tool for broadening new possibilities in studies of individual cellular processes in a dynamic microfluidic device.     

Separations using a porous-shell pillar array column on a capillary LC instrument

We investigated the achievable separation performance of a 9-cm-long and 1-mm-wide pillar array channel (volume = 0.6 μL) containing 5 μm diameter Si pillars (spacing 2.5 μm) cladded with a mesoporous silica layer with a thickness of 300 nm, when this channel is directly interfaced to a capillary LC instrument. The chip has a small footprint of only 4 cm × 4 mm and the channel consists of three lanes that are each 3 cm long and that are interconnected using low dispersion turns consisting of a narrow U-turn (10 μm), proceded and preceded by a diverging flow distributor. Measuring the band broadening within a single lane and comparing it to the total channel band broadening, the additional band broadening of the turns can be estimated to be of the order of 0.5 μm around the minimum of the van Deemter curve, and around some 1 μm (nonretained species) and 2 μm (retained species) in the C-term dominated regime. The overall performance (chip + instrument) was evaluated by conducting gradient elution separations of digests of cytochrome c and bovine serum albumin. Peak capacities up to 150 could be demonstrated, nearly completely independent of the flow rate.