Band broadening of DNA fragments isolated by polyacrylamide gel electrophoresis in capillary electrophoresis

Polyacrylamide gel electrophoresis (PAGE) is used frequently for isolation and purification of DNA fragments. In the present study, DNA fragments extracted from polyacrylamide gels showed significant band broadening in capillary electrophoresis (CE). A pHY300PLK (a shuttle vector functioning in Escherichia coli and Bacillus subtilis) marker, which contained nine fragments ranging from 80 to 4870 bp, was separated by PAGE, and each fragment was isolated by phenol/chloroform extraction and ethanol precipitation. After extraction from the polyacrylamide gel, the peaks of the isolated DNA fragments exhibited band broadening in CE, where a linear poly(ethylene oxide) was used as a sieving matrix. The theoretical plate numbers of the DNA fragments contained in the pHY300PLK marker were >10⁶ for all the fragments before extraction. However, the DNA fragments extracted from the polyacrylamide gel showed decreased theoretical plate numbers (5–20 times smaller). The degradation of the theoretical plate number was significant for middle sizes of the DNA fragments ranging from 489 to 1360 bp, whereas the largest and smallest fragments (80 and 4870 bp) had no obvious influence. The band broadening was attributed to contamination of the DNA fragments by polyacrylamide fibers during the separation and extraction process.     

Broadening of peaks eluted before the solvent in capillary GC Part II: The role of phase soaking

Summary Phase soaking is a solvent effect which tends to reconcentrate peaks eluted after and to broaden peaks eluted before the solvent. The principles of the phase soaking effect on peaks eluted before the solvent are discussed. The broadening effect is distinguished from the broadening effect occurring in the flooded column inlet by partial solvent trapping. It was found that in most cases broadening by partial solvent trapping strongly predominated over broadening by phase soaking. Phase soaking was noticeable only in the neighbourhood of the solvent peak.Phase soaking does not broaden peaks eluted before the solvent as much as it re-concentrates those eluted after it. The two phase soaking effects on the front and the rear of the solvent band (that is, of the soaked zone) differ strongly from each other.Peak broadening by phase soaking is negligible for non-trapped components, because such solutes start their chromatography before a significant quantity of solvent enters the column. Phase soaking only broadens solute bands which were retained by the solvent in the column inlet, that is, bands already broadened by partial solvent trapping.     

Study of the peak broadening due to detection in the electrophoretic separation of DNA by CE and microchip CE and the application of image sensor for ultra‐small detection cell length

Narrow peaks are important to high‐resolution and high‐speed separation of DNA fragments by capillary electrophoresis and microchip capillary electrophoresis. Detection cell length is one of the broadening factors, which is often ignored in experiments. However, is it always safe to neglect detection cell length under any condition? To answer this question, we investigated the influence of detection cell length by simulation and experiments. A parameter named as detection cell length ratio was proposed to directly compare the detection cell length and the spatial length of sample band. Electrophoretic peaks generated by various detection cell length ratios were analyzed. A simple rule to evaluate the peak broadening due to detection cell length was obtained. The current states of the detection cell length of detection system and their reliabilities in capillary electrophoresis and microchip capillary electrophoresis were analyzed. Microchip capillary electrophoresis detection with an ultra‐small detection cell length of 0.36 μm was easily achieved by using an image sensor.     

Band broadening effects in capillary column recycle gas chromatography with valves

Sources leading to band broadening and peak distortion have been studied in a capillary column recycle system, based on a commercial microvalve. The ferrule system was a major contributor to band broadening. It was found that the quality of connections is very critical but the internal volume of the valve is not important. The pressure pulses resulting from each switching operation produced some band broadening with substances that had just been transferred through the valve but no adverse effects were noticed when they had travelled some distance into the column. Flow into valve cavities and through tees also produced a drop in efficiency. It is not clear whether this is solely due to problems in column connection or if changes in flow pattern also contribute. The activity of the valve toward polar substances was also assessed. No attempt was made to deactivate valve surfaces. The components of the Grob test mixture were almost unaffected but highly polar and acidic compounds such as nitrophenol showed severe losses. No evidence for catalysis was found.     

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.     

Preparation and Chromatographic Application of Macroporous Silicate in a Capillary

Continuous macroporous silica gel networks were prepared in a fused silica capillary, and evaluated in reversed-phase liquid chromatography. Under pressure-driven conditions, considerable dependence of column efficiency on the linear velocity of the mobile phase was observed in spite of the small size of the silica skeletons. A major source of band broadening in the pressure-driven mode was found in the A-term of van Deemter equation. The performance of the continuous silica capillary column in the electro-driven mode was much better than that in the pressure-driven mode.     

Divergent dispersion behavior of ssDNA fragments during microchip electrophoresis in pDMA and LPA entangled polymer networks

Resolution of DNA fragments separated by electrophoresis in polymer solutions ("matrices") is determined by both the spacing between peaks and the width of the peaks. Prior research on the development of high-performance separation matrices has been focused primarily on optimizing DNA mobility and matrix selectivity, and gave less attention to peak broadening. Quantitative data are rare for peak broadening in systems in which high electric field strengths are used (>150 V/cm), which is surprising since capillary and microchip-based systems commonly run at these field strengths. Here, we report results for a study of band broadening behavior for ssDNA fragments on a glass microfluidic chip, for electric field strengths up to 320 V/cm. We compare dispersion coefficients obtained in a poly(N,N-dimethylacrylamide) (pDMA) separation matrix that was developed for chip-based DNA sequencing with a commercially available linear polyacrylamide (LPA) matrix commonly used in capillaries. Much larger DNA dispersion coefficients were measured in the LPA matrix as compared to the pDMA matrix, and the dependence of dispersion coefficient on DNA size and electric field strength were found to differ quite starkly in the two matrices. These observations lead us to propose that DNA migration mechanisms differ substantially in our custom pDMA matrix compared to the commercially available LPA matrix. We discuss the implications of these results in terms of developing optimal matrices for specific separation (microchip or capillary) platforms.     

Two dimensional separations of human urinary protein digest using a droplet-interfaced platform

For highly complex mixtures, coelution is a common phenomenon in chromatography. A great deal of resolution is hidden in coelution, and lost due to inevitable molecular diffusion during sample transfer. The molecular diffusion may lead to band broadening and remix of separated peaks, which cause degradation of achievable resolution. In this study, we introduced droplet microfluidics as a high performance sample transfer tool in two dimensional nanoflow liquid chromatography–capillary electrophoresis separation of a human urine sample. The fine fractionation capability and sampling completeness enabled by the droplet-interface demonstrated the 2D system’s usefulness in high-resolution mapping of real world biological samples.     

Behavior of heavy solutes on a precolumn in on-column gas chromatography

The behavior of heavy solutes present in a sample injected on the precolumn has not been clarified in on-column injection onto gas chromatograph having a capillary column coupled to an uncoated precolumn in an oven. To investigate this point, an on-column gas chromatograph has been developed which is constructed with a heatable precolumn outside the oven. Some experiments were carried out in order to evaluate the performance. As a result, it was found that heavy solutes reach the first part of the main capillary column after the solvent has gone, resulting in sharp peaks without solvent effects. The reason why sharp peaks appear for the heavy solutes is also discussed. The cold-trapping effect has been shown to play an important role in narrowing the band width of the heavy solutes. Some of the advantages of the gas chromatograph developed are also presented.     

Performance evaluation of ion‐exchange chromatography in capillary format

The performance of a recently introduced capillary ion‐exchange chromatography system was explored. Experiments were conducted in isocratic mode with a commercial capillary anion‐exchange column (id = 0.4 mm, L = 15 cm) using a five‐anion standard mixture. The achieved results were compared to the performance of a standard bore ion‐exchange system (id = 4 mm, L = 15 cm), which was considered as a reference. The first‐generation capillary columns exhibited a minimal reduced plate‐height value below two witnessing a good packing quality and system performance. However, compared to the standard bore system the capillary system displayed an increased apparent C‐term which could be due to a difference in packing morphology and/or possible external band‐broadening contributions. For fast separations, the standard bore system outperformed the capillary system, while for complex separations both systems performed nearly equally well. In addition, the retention characteristics of the capillary system were investigated. To illustrate the suitability of the capillary system, the analysis of real‐world water samples originating from two local Belgian rivers was demonstrated.     

Theory of Gradient Elution Liquid Chromatography with Linear Solvent Strength: Part 2. Peak Width Formation

The process of formation of the width (σ _b) of a solute band migrating along a column and its effect on the width (σ) of a corresponding peak in a chromatogram are quantified along with the extra-column contributions (Δσ _b and Δσ) to these parameters due to insufficiently narrow injection plugs. Previously unknown expressions for σ _b and Δσ _b as functions of the band migration distance and time were found. The negative gradients in the solvent strength cause the fronts of the solute bands to travel slower than their tails. This compresses the bands (reduces their widths). Previously unknown expressions describing the band compression process as functions of the band migration distance and time are found. The band compression tends to narrow the peaks. However, as shown here, the gradients that compress the bands also reduce their elution speeds. This tends to broaden the peaks (typically ignored phenomenon) and, as shown here, can cause a slight net peak broadening under normal conditions (in spite of general expectations that the gradients should narrow the peaks). On the other hand, as shown here, the gradients can significantly suppress the harmful effect of the extra-column peak broadening.     

Concurrent solvent evaporation for on-line coupled HPLC-HRGC

A technique is proposed which allows introduction of very large volumes of liquid (10 ml were tested) into capillary columns equipped with short (1–2 m long) retention gaps. It is based on concurrent solvent evaporation, i.e. evaporation of the solvent during introduction of the sample. The technique presupposes high carrier gas flow rates (at least during sample introduction) and column temperatures near the solvent boiling point. The major limitation of the method is the occurrence of peak broadening for solutes eluted up to 30°, in some cases up to 100°, above the injection temperature. This is due to the absence of solvent trapping and a reduced efficiency of phase soaking. Therefore, use of volatile solvents is often advantageous. Application of the concurrent solvent evaporation technique allows introduction of liquids which do not wet the retention gap surface. However, the method is still not very attractive for analysis of aqueous or water-containing solutions (reversed phase HPLC).     

Performance of an in-plane detection cell with integrated waveguides for UV/Vis absorbance measurements on microfluidic separation devices

A microfluidic device with integrated waveguides and a long path length detection cell for UV/Vis absorbance detection is presented. The 750 microm U-cell detection geometry was evaluated in terms of its optical performance as well as its influence on efficiency for electrophoretic separations in the microdevice. Stray light was found to have a strong effect on both, the sensitivity of the detection and the available linear range. The long path length U-cell showed a 9 times higher sensitivity when compared to a conventional capillary electrophoresis (CE) system with a 75 microm inner diameter (ID) capillary, and a 22 times higher sensitivity than with a 50 microm ID capillary. The linear range was comparable to that achieved in a 75 microm ID capillary and more than twice as large as in a 50 microm ID capillary. The use of the 750 microm U-cell did not contribute significantly to band broadening; however, a clear quantification was made difficult by the convolution of several other band broadening sources.     

Peak broadening in microchip electrophoresis: a discussion of the theoretical background

A review on peak (band, zone) broadening in electromigration separation methods is presented, mainly covering articles published between the begining of 2000 and middle of 2002. Most attention is drawn to work dealing with microchip electrophoresis performed in micrototal analysis systems (microTAS) or the lab-on-a-chip, but many of the results are significant for capillary zone electrophoresis in general. The paper reviews the theoretical background of the peak dispersion due to the geometry of the separation channel, the transversal nonhomogeneity of the electroosmootic flow, and electromigration dispersion (sample overload) connected with the occurrence of the system zones (system peaks, system eigenpeaks).     

Capillary action liquid chromatography

Capillary action LC (caLC) is introduced as a technique using capillary action as the driving force to perform LC in capillary columns packed with HPLC type microparticulate materials. A dry packing method with centrifugal force was developed to prepare capillary columns in parallel (10 columns per 3 min) to support their disposable use in caLC. Using a digital microscope for real‐time imaging and recording separations of components in a dye mixture, caLC was found to have flow characteristics similar to TLC. Based on the investigation of microparticulate HPLC silica gels of different size (1.5–10 μm) and a typical TLC grade irregular medium, Merck 60G silica, the van Deemter curves suggested molecular diffusion as the major contribution to band broadening in caLC. With Waters Xbridge 2.6 μm silica, plate heights down to 8.8 μm were obtained, comparable to those achievable in HPLC. Assisted by an image‐processing method, the visual caLC separation was converted to a classical chromatogram for further data analysis and such a facility confirmed the observation of highly efficient bands.     

Preparative capillary zone electrophoresis using a dynamic coated wide-bore capillary

Preparative capillary zone electrophoresis separations of cytochrome c from bovine and horse heart are performed efficiently in a surfactant-coated capillary. The surfactant, dimethylditetradecylammonium bromide (2C(14)DAB), effectively eliminated protein adsorption from the capillary surface, such that symmetrical peaks with efficiencies of 0.7 million plates/m were observed in 50-microm id capillaries when low concentrations of protein were injected. At protein concentrations greater than 1 g/L, electromigration dispersion became the dominant source of band broadening and the peak shape distorted to triangular fronting. Matching of the mobility of the buffer co-ion to that of the cytochrome c resulted in dramatic improvements in the efficiency and peak shape. Using 100 mM bis(2-hydroxyethyl)imino-tris(hydroxymethyl)methane phosphate buffer at pH 7.0 with a 100-microm id capillary, the maximum sample loading capacity in a single run was 160 pmol (2.0 microg) of each protein.     

A new interface for combining capillary electrophoresis with inductively coupled plasma-mass spectrometry

This paper describes the development and design of a new, efficient, simple and robust interface for coupling capillary electrophoresis (CE) with inductively coupled plasma-mass spectrometry. The interface is based on a modified microconcentric nebulizer which permits a low flow rate of about 6 μL/min in the free aspiration mode. This interface construction provides an electrical connection for stable electrophoretic separations and adapts the flow rate of the electro-osmotic flow inside the CE capillary to the flow rate of the nebulizer for efficient transport of the analytes into the plasma. By optimization of the fluid mechanical properties the interface prevents the nebulizer from causing any laminar flow in the CE capillary and thus the high resolution power of CE can be preserved. Furthermore, this new device permits independent optimization of the nebulization from the CE whereby exact positioning of the CE capillary is not necessary, thus enabling fast exchange. A low dead volume spraychamber has been constructed which circumvents any band broadening of the sharp CE signals. Peak widths down to 3.5 s comparable to CE with UV detection are possible. Received: 5 February 1999 / Revised: 21 April 1999 / Accepted: 23 April 1999     

A new interface for combining capillary electrophoresis with inductively coupled plasma-mass spectrometry

This paper describes the development and design of a new, efficient, simple and robust interface for coupling capillary electrophoresis (CE) with inductively coupled plasma-mass spectrometry. The interface is based on a modified microconcentric nebulizer which permits a low flow rate of about 6 μL/min in the free aspiration mode. This interface construction provides an electrical connection for stable electrophoretic separations and adapts the flow rate of the electro-osmotic flow inside the CE capillary to the flow rate of the nebulizer for efficient transport of the analytes into the plasma. By optimization of the fluid mechanical properties the interface prevents the nebulizer from causing any laminar flow in the CE capillary and thus the high resolution power of CE can be preserved. Furthermore, this new device permits independent optimization of the nebulization from the CE whereby exact positioning of the CE capillary is not necessary, thus enabling fast exchange. A low dead volume spraychamber has been constructed which circumvents any band broadening of the sharp CE signals. Peak widths down to 3.5 s comparable to CE with UV detection are possible. Received: 5 February 1999 / Revised: 21 April 1999 / Accepted: 23 April 1999     

Rydberg or Valence? The Long‐Standing Question in the UV Absorption Spectrum of 1,1′‐Bicyclohexylidene

The electronic excited states of the olefin 1,1′‐bicylohexylidene (BCH) are investigated using multiconfigurational complete active space self‐consistent‐field second order perturbation theory in its multi‐state version (MS‐CASPT2). Our calculations undoubtedly show that the bulk of the intensity of the two unusually intense bands of the UV absorption of BCH measured with maxima at 5.95 eV and 6.82 eV in the vapor phase are due to a single ππ* valence excitation. Sharp peaks reported in the vicinity of the low‐energy feature in the gas phase correspond to the beginning of the π3s_R Rydberg series. By locating the origin of the ππ* band at 5.63 eV, the intensity and broadening of the observed bands and their presence in solid phase is explained as the vibrational structure of the valence ππ* transition, which underlies the Rydberg manifold as a quasi‐continuum.     

场流分级的机内浓缩和再进样方法

<正> 除制备色谱外,一般色谱和场流分级的进样量都是很小的,而溶质层在经过色谱柱或场流分级柱槽后由于分离以及加宽效应浓度又要大大降低,因此,所得级分中溶质的含量极少。如需把所得级分做再进样,则必须在同一条件下重复收集同一集分,经浓缩后再进