Fundamentals and practice for ultrasensitive laser-induced fluorescence detection in microanalytical systems

Laser-induced fluorescence is an extremely sensitive method for detection in chemical separations. In addition, it is well-suited to detection in small volumes, and as such is widely used for capillary electrophoresis and microchip-based separations. This review explores the detailed instrumental conditions required for sub-zeptomole, sub-picomolar detection limits. The key to achieving the best sensitivity is to use an excitation and emission volume that is matched to the separation system and that, simultaneously, will keep scattering and luminescence background to a minimum. We discuss how this is accomplished with confocal detection, 90 degrees on-capillary detection, and sheath-flow detection. It is shown that each of these methods have their advantages and disadvantages, but that all can be used to produce extremely sensitive detectors for capillary- or microchip-based separations. Analysis of these capabilities allows prediction of the optimal means of achieving ultrasensitive detection on microchips.     

Rapid Separation of Proteins with Monodisperse Porous Polystyrene-Divinylbenzene(PS/DVB) Chromatographic Packings

It has been recognized for half a century that stagnant mobile phase mass transfer is a dominant limitation in liquid chromatography. The resolution is seriously affected at high mobile phase velocity. One approach to solve this problem is to eliminate the pores. Through the use of 1~2 μm non-porous particles it has been possible to carry out protein separations an order of magnitude faster,albeit at the expense of diminished loading capacity and high operating pressure. A second alternative is to cause liquid to flow through the particles. Because convective transports is much more rapid than that achieved by diffusion, these materials can also be used at an order of magnitude higher velocity for mobile phase in protein separations.     

Theoretical description of a new analytical technique: comprehensive online multidimensional fast Fourier transform separations

Comprehensive multidimensional separations are today dominated by systems that are fundamentally limited to highly asymmetrical online separations sacrificing separation space, or to lengthy, time consuming offline separations. With the exception of pulse-modulated methods, separations have thus been limited to two dimensions. It is proposed that some of the limitations and shortcomings of these methods may be ameliorated or overcome by employing multi-dimensional detection whereby each analyte is effectively labelled in the frequency domain by a series of pulsed-injections, and a symmetrical, comprehensive online analysis performed with the resulting signal processed by sequential Fourier analysis. A semi-empirical computer model of this system was developed and its feasibility positively demonstrated in simulations of high-efficiency separations in two dimensions. Separations of higher dimensionality were shown to be possible but involved signal-processing challenges beyond the present work. By eliminating wrap-around effects and enabling the separation of physically unseparated peaks, the technique facilitates significant improvements in peak capacity per unit of analysis time as well as greatly improved signal to noise ratios. Because these comprehensive online multidimensional Fourier transform separations depend heavily upon the practical lifetime of imposed injection pulses, it is envisaged that this method will leverage emerging high-efficiency micro- and nanoscale separations technologies.     

DNA separations in microfabricated devices with automated capillary sample introduction

A novel method is presented for automated injection of DNA samples into microfabricated separation devices via capillary electrophoresis. A single capillary is used to electrokinetically inject discrete plugs of DNA into an array of separation lanes on a glass chip. A computer-controlled micromanipulator is used to automate this injection process and to repeat injections into five parallel lanes several times over the course of the experiment. After separation, labeled DNA samples are detected by laser-induced fluorescence. Five serial separations of 6-carboxyfluorescein (FAM)-labeled oligonucleotides in five parallel lanes are shown, resulting in the analysis of 25 samples in 25 min. It is estimated that approximately 550 separations of these same oligonucleotides could be performed in one hour by increasing the number of lanes to 37 and optimizing the rate of the manipulator movement. Capillary sample introduction into chips allows parallel separations to be continuously performed in serial, yielding high throughput and minimal need for operator intervention.     

Considerations on HILIC and polar organic solvent-based separations: use of cyclodextrin and macrocyclic glycopetide stationary phases

There is a natural tendency in science to prefer straightforward, logical classification systems. The use of mobile phase-stationary phase combinations that do not fit neatly into the standard "normal phase" or "reversed-phase" categories has been going on for over 50 years. The term "hydrophilic interaction chromatography" (HILIC) is sometimes being used as a general category for these "other" separations. In some cases, it may be appropriate and in others, not. Indeed the mechanistic constrains used to define the method seem to be varying with time. Given the name HILIC, it is assumed that water is not only present in the mobile phase, but also plays an essential role in the retention mechanism. However, there is residual water present in all organic solvents. Regardless, the number of reported separations in this alternative mode has increased tremendously in the last two decades. This is due to the advent of new stationary phases and an emphasis on polar, biologically important molecules. We discuss the relationships between HILIC and other chromatographic modes. We then examine two classes of stationary phases that have played a major role in these separations. These particular stationary phases can be used to provide appreciable mechanistic information as well.     

Ultrahigh pressure liquid chromatography using elevated temperature

Fast liquid chromatographic (LC) methods are important for a variety of applications. Reducing the particle diameter (d(p)) is the most effective way to achieve fast separations while preserving high efficiency. Since the pressure drop along a packed column is inversely proportional to the square of the particle size, when columns packed with small particles (<2 microm) are used, ultrahigh pressures (>689 bar) must be applied to overcome the resistance to mobile phase flow. Elevating the column temperature can significantly reduce the mobile phase viscosity, allowing operation at higher flow rate for the same pressure. It also leads to a decrease in retention factor. The advantage of using elevated temperatures in LC is the ability to significantly shorten separation time with minimal loss in column efficiency. Therefore, combining elevated temperature with ultrahigh pressure facilitates fast and efficient separations. In this study, C6-modified 1.0 microm nonporous silica particles were used to demonstrate fast separations using a temperature of 80 degrees C and a pressure of 2413 bar. Selected separations were completed in 30 s with efficiencies as high as 220,000 plates m(-1).     

Use of Centrifugal Partition Chromatography and Proteins in The Preparative Separation of Amino Acid Enantiomers

Abstract

The growth of analytical methodologies for the separation of enantiomers has been impressive. Attention is now turning to the large scale separation of enantiomers. Often scaling-up sensitive analytical separations is ineffective and inefficient. Centrifugal partition chromatography (CPC) may be a viable alternative for the preparative separation of racemic mixtures in some cases. The use of proteins as chiral selectors in CPC is examined. Attention was focused on proteins that previously were used as bonded phases in analytical LC columns. The enzymatic properties of α-chymotrypsin allowed it to be used as a bioreactor in conjunction with CPC. When proteins are used as components of the stationary or mobile phase there can be problems with denaturation. However, when used in external incubation processes or as column bioreactors coupled with CPC, effective gram-scale separations can be performed. Tryptophan methyl ester was used as a model compound to evaluate this approach.     

粘度对聚合物共混物相分离动力学的影响研究

模拟了粘度反差二元流体混合物的相分离,考察了粘度对相分离动力学的影响,发现相区域的增长主要由粘度较大的组分所控制,合理地解释了粘度效应所导致的一些实验现象.证明了即使组分间的粘度比很大,也没有出现反转相结构,说明在不施加剪切流场情况下,粘度反差不是形成反转相的原因.     

高效毛细管电泳分离的优化策略

本文提出一种新的优化策略，综合分析表面活性剂浓度、缓冲液ＰＨ值、有机添加剂浓度，缓冲溶液浓度及操作电压等实验因素对高效毛细管电泳分离的影响，用多种优化目标函数考察分离度和峰分布均匀性。     

Atmospheric pressure surface sampling/ionization techniques for direct coupling of planar separations with mass spectrometry

Planar separations, which include thin layer chromatography and gel electrophoresis, are in widespread use as important and powerful tools for conducting separations of complex mixtures. To increase the utility of planar separations, new methods are needed that allow in situ characterization of the individual components of the separated mixtures. A large number of atmospheric pressure surface sampling and ionization techniques for use with mass spectrometry have emerged in the past several years, and several have been investigated as a means for mass spectrometric read-out of planar separations. In this article, we review the atmospheric pressure surface sampling and ionization techniques that have been used for the read-out of planar separation media. For each technique, we briefly explain the operational basics and discuss the analyte type for which it is appropriate and some specific applications from the literature.     

An inexpensive microslab gel DNA electrophoresis system with real-time fluorescence detection

In this paper, we describe the construction of a simple yet powerful gel electrophoresis apparatus that can be used to perform size-selective separations of DNA fragments in virtually any laboratory. This system employs a microslab gel format with a novel gel casting technique that eliminates the need for delicate combs to define sample loading wells. The compact size of the microslab gel format allows rapid separations to be performed at low voltages using submicroliter sample volumes. Real time fluorescence detection of the migrating DNA fragments is accomplished using an inexpensive digital microscope that directly connects to any PC with a USB interface. The microscope is readily adaptable for this application by replacing its white light source with a blue light-emitting diode (LED) and adding an appropriate emission filter. Both polyacrylamide and agarose gels can be used as separation matrices. Separation performance was characterized using standard dsDNA ladders, and correct sizing of a 191 bp PCR product was achieved in 15 min. The low cost and simplicity of this system makes it ideally suited for use in a variety of laboratory and educational settings.     

Analytical methods for separating and isolating magnetic nanoparticles

Despite the large body of literature describing the synthesis of magnetic nanoparticles, few analytical tools are commonly used for their purification and analysis. Due to their unique physical and chemical properties, magnetic nanoparticles are appealing candidates for biomedical applications and analytical separations. Yet in the absence of methods for assessing and assuring their purity, the ultimate use of magnetic particles and heterostructures is likely to be limited. In this review, we summarize the separation techniques that have been initially used for this purpose. For magnetic nanoparticles, it is the use of an applied magnetic flux or field gradient that enables separations. Flow based techniques are combined with applied magnetic fields to give methods such as magnetic field flow fractionation and high gradient magnetic separation. Additional techniques have been explored for manipulating particles in microfluidic channels and in mesoporous membranes. Further development of these and new analytical tools for separation and analysis of colloidal particles is critically important to enable the practical use of these, particularly for medicinal purposes.     

A note on observability of fluctuation-induced structural interaction in a nematic mesophase

We discuss the observability of pseudo-Casimir interaction in nematic liquid crystals, and show that in physically relevant regimes the interaction is characterized by non-universal force profiles that depend strongly on the anchoring. Depending on the ratio of the anchoring strengths, the force may be either purely attractive at all separations or it may become repulsive at separations comparable to the geometric mean of the extrapolation lengths. Within the currently accessible experimental window, it appears that the 1/h ³ force could only be seen in very weakly anchored symmetric systems at separations no larger than a few 10 nm. We also find that while the conventional force measuring systems such as the atomic force microscope and surface force apparatus can provide some information on the fluctuationinduced force, alternative techniques, e.g. spinodal dewetting, could be used to obtain a more comprehensive insight extending over a wider range of separations.     

A note on observability of fluctuation-induced structural interaction in a nematic mesophase

We discuss the observability of pseudo-Casimir interaction in nematic liquid crystals, and show that in physically relevant regimes the interaction is characterized by non-universal force profiles that depend strongly on the anchoring. Depending on the ratio of the anchoring strengths, the force may be either purely attractive at all separations or it may become repulsive at separations comparable to the geometric mean of the extrapolation lengths. Within the currently accessible experimental window, it appears that the 1/h ³ force could only be seen in very weakly anchored symmetric systems at separations no larger than a few 10 nm. We also find that while the conventional force measuring systems such as the atomic force microscope and surface force apparatus can provide some information on the fluctuationinduced force, alternative techniques, e.g. spinodal dewetting, could be used to obtain a more comprehensive insight extending over a wider range of separations.     

An initial assessment of the use of gradient elution in microemulsion and micellar liquid chromatography

Novel microemulsion and micellar HPLC separations have been achieved using gradient elution and columns packed with reverse phase material. Initial attempts at gradient microemulsion liquid chromatography proved impossible on use of a microemulsion successfully used in capillary electrophoresis. Optimisation of the microemulsion composition allowed the generation of stable microemulsions to achieve separations in HPLC. The novel use of organic-solvent micellar chromatography in gradient elution mode was shown to give efficient separations. A range of efficient separations of pharmaceuticals and related impurities were obtained. Acidic, basic, and neutral solutes were resolved covering a wide range of water solubilities and polarities. Elution times were in the order of 4-15 minutes. Separations were briefly compared to those accomplished with a micellar HPLC system. It is proposed that gradient elution in both microemulsion and micellar HPLC can be regarded as a highly successful means of achieving resolution of complex mixtures and should be considered for routine analysis and further investigation.     

HPLC of biological macromolecules: The first decade

Summary During the past decade, HPLC has developed into a powerful new technique for the analysis of complex mixtures of biological macromolecules. Through the use of microparticulate supports of vastly improved mechanican strength, superior stationary phase chemistry, and advanced instrumentation, it is now possible to separate biological macromolecules more than 10 times faster and with greater resolution than in the classical SEC, IEC, HIC, bioaffinity, and hydroxyapetite chromatography columns. Additionally, the introduction of new separation modes such as RPC and metal chelate make it possible to carry out separations that were not possible with the classical gel-type media. It is anticipated that 1) expanded use of non-porous media, 2) development of new stationary phases for carbohydrates, 3) greater throughput and resolution in preparative separations, and 4) better understanding of retention mechanisms are a few of the areas of macromolecular separations in which advances can be expected in the next few years.     

Discovery of surfactants for metal/semiconductor separation of single-wall carbon nanotubes via high-throughput screening

We report novel surfactants that can be used for the separation of metallic (M) and semiconducting (S) single-wall carbon nanotubes (SWCNTs). Among the M/S separation methods using surfactants in an aqueous solution, sodium dodecyl sulfate plays a key role in density gradient ultracentrifugation (DGU) and agarose gel separations. In this study, we screened 100 surfactants for M/S separation using a high-throughput screening system. We identified five surfactants, which could be used for both DGU and agarose gel separations, suggesting that the basic principle of these separations is common. These surfactants have relatively low dispersibilities, which is likely due to their common structural features, i.e., straight alkyl tails and charged head groups, and appeared to enable M- and S-SWCNTs to be distinguished and separated. These surfactants should stimulate research in this field and extend the application of electrically homogeneous SWCNTs not only for electronics but also for biology and medicine.     

Novel, trifunctional diamine for silica coating in capillary zone electrophoresis

A novel compound ?quaternarized piperazine [(N-methyl,N-4-iodobutyl)-N'-methylpiperazine] (QPzI)? for the coating of a silica capillary able to reduce or invert the electroosmotic flow (EOF) in capillary zone electrophoresis is reported. Unlike standard oligoamines (like spermine and tetraethylene pentamine) which are very efficient in quenching macromolecule interaction with the silica wall, but only in acidic pH ranges, QPzI acts all along the pH scale, including alkaline pH ranges. It is believed that QPzI behaves like a trifunctional derivative: it forms ionic bonds with dissociated silanols via its quaternary nitrogen, hydrogen bonds via its tertiary nitrogen and, most importantly, a covalent bond via alkylation of ionized silanols through the terminal iodine atom in the butyl chain. Excellent separations are obtained with a variety of organic compounds, such as aromatic carboxylic acids, tryptophan metabolites and arylalkanoic acids. Such separations could not be obtained in naked capillaries in the presence of oligoamines and on some occasions not even with capillaries coated with a covalent layer of neutral polymers. In separations taking place in alkaline media, QPzI is not added to the background electrolyte, but is used simply in the capillary pre-conditioning step, a unique feature strongly supporting the hypothesis of its covalent binding to the silica surface. In difficult separations, such as in the case of o-/p-OMe-phenylacetic acids or nicotinic/picolinic acid, which would not normally occur under standard conditions, it is believed that QPzI acts as a discriminator, thus playing an active role in the separation process, rather than simply modulating the EOF.     

A decade of capillary electrophoresis

Since the introduction of the first commercial capillary electrophoresis (CE) instrument a decade ago, CE applications have become widespread. Today, CE is a versatile analytical technique which is successfully used for the separation of small ions, neutral molecules, and large biomolecules and for the study of physicochemical parameters. It is being utilized in widely different fields, such as analytical chemistry, forensic chemistry, clinical chemistry, organic chemistry, natural products, pharmaceutical industry, chiral separations, molecular biology, and others. It is not only used as a separation technique but to answer physicochemical questions. In this review, we will discuss different modes of CE such as capillary zone electrophoresis, micellar electrokinetic chromatography, capillary gel electrophoresis, capillary isoelectric focusing, and capillary electrochromatography, and will comment on the future direction of CE, including array capillary electrophoresis and array microchip separations.     

Anion chromatography with a crown ether-based stationary phase and an organic modifier in the eluent

The unique ability of macrocyclic ligands, such as the crown ethers and cryptands, to selectively complex alkali metal cations can be used as the basis for chromatographic separations of anions. Specifically, macrocycles which are adsorbed onto a reversed-phase column, form positively charged anion-exchange sites when they combine with eluent cations. Previously we have demonstrated gradient anion separations based on changing the column capacity during the course of the separation by altering the eluent cation, temperature, or organic modifier content using cryptand-based columns. Herein we report that excellent separations can also be achieved using 18-crown-6 based columns. In this column, anion retention increases with increasing eluent strength and organic modifier content. This observation is in keeping with the relatively moderate affinity of crown ethers for alkali metals when compared to cryptands. The separation of anions achieved by optimizing mobile phase variables shows that isocratic separations of anions on the crown-based column are almost as good as separations achieved only under gradient conditions on cryptand-based columns. Cation gradients provide additional improvements on the separations using the crown-based column.