Femtoliter Gradient Elution System for Liquid Chromatography Utilizing Extended Nanofluidics.

A gradient system was developed for the separation of proteins on a femtoliter scale utilizing nanofluidic channels. In the history of chromatography, miniaturization of the separation column has been important for efficient separation and downsizing of instruments. Previously, our group developed a small and highly efficient chromatography system utilizing nanofluidic channels, although a flexible design of the gradient was difficult and separation of proteins was not achieved. Here, we propose a flexible gradient system using standard HPLC pumps and an auxiliary mixer with a simple sample injection system. In contrast to our previous sample injection system using pressure balance, the system enables a femtoliter-scale sample injection which is compatible with gradient elution using HPLC pumps. The system was carefully designed, verified for sample injection and gradient elution, and finally applied to the separation of proteins from model and real samples. This femtoliter-scale, efficient separation system will contribute to omics studies at the single-cell level.

Capillary separation: micellar electrokinetic chromatography.

Micellar electrokinetic chromatography (MEKC), a separation mode of capillary electrophoresis (CE), has enabled the separation of electrically neutral analytes. MEKC can be performed by adding an ionic micelle to the running solution of CE without modifying the instrument. Its separation principle is based on the differential migration of the ionic micelles and the bulk running buffer under electrophoresis conditions and on the interaction between the analyte and the micelle. Hence, MEKC's separation principle is similar to that of chromatography. MEKC is a useful technique particularly for the separation of small molecules, both neutral and charged, and yields high-efficiency separation in a short time with minimum amounts of sample and reagents. To improve the concentration sensitivity of detection, several on-line sample preconcentration techniques such as sweeping have been developed.

Micellar electrokinetic chromatography for high-performance analytical separation.

Capillary electrophoresis (CE) is a relatively new method of analytical separation having the advantages of high separation efficiency, requirement of a small sample amount, low operating cost, and fast separation time. CE is a separation method where the analyte migrates under an electric field due to a charge on the analyte. Hence, CE was unable to separate neutral analytes until the advent of micellar electrokinetic chromatography (MEKC). MEKC is performed with an addition of ionic micelles to an electrophoretic medium, where a portion of the analyte is incorporated into the micelle and has an apparent charge, which can be subject to electrophoretic separation. The migration velocity of the neutral analyte in MEKC depends on what portion of the analyte is incorporated into the micelle. Thus, the separation principle of MEKC is similar to that of chromatography, although the micelle corresponding to the stationary phase in chromatography is not stationary inside the capillary. The fundamental characteristics and theoretical treatments of the behavior of the analyte in MEKC were studied extensively by the author's group. MEKC has been established as one of the most popular separation modes in CE. This review describes how MEKC was developed and how it is useful as a method of analytical separation.

On-line selective enrichment and ion-pair reaction for structural determination of sulfated glycopeptides by capillary electrophoresis-mass spectrometry.

We describe a new capillary electrophoresis-mass spectrometry (CE-MS)-based technique for analyzing sulfated glycopeptides. The proposed method performs selective enrichment of sulfated glycopeptides from a complex mixture of peptides based on field-enhanced sample injection and ion-pair reaction with a basic ion-pair reagent (Lys-Lys-Lys; KKK) at the exit end of a capillary in a single analysis, which permits successful fragmentation of sulfated glycopeptides in positive-ion mode at the MS/MS stage for comprehensive structural analysis. In this study, the method was verified using a model sulfated monosaccharide, N-acetyl-d-galactosamine 4-sulfate (GalNAc 4S). As an example of an application of this method, sulfated glycopeptides were selectively enriched from the enzymatic digest of thyroid stimulating hormone, affording approximately 500-fold sensitivity enhancement, and structural information was successfully obtained via on-line ion-pair complexation reaction.

Integration of various stacking processes in carrier ampholyte-based capillary electrophoresis.

Field-enhanced sample stacking, field-enhanced sample injection as well as electrokinetic supercharging have been successfully integrated in carrier ampholyte-based capillary electrophoresis. Through the analysis of different test sample mixtures, it has been shown that the carrier ampholyte-based background electrolytes, in spite of their very low conductivity, allow efficient online preconcentration of analytes by field-amplified techniques. Sensitivity enhancement factors of the same magnitude as those usually encountered with classical conductive background electrolytes have been obtained in such carrier ampholyte-based buffers. Depending on the online preconcentration method that has been integrated, sensitivity enhancement factors between 50 and several thousands have been reached.

On-line sample preconcentration in capillary electrophoresis. Fundamentals and applications.

On-line preconcentration is one of the aspects of analytical method development using capillary electrophoretic techniques. The choice of the sample matrix alone can significantly alter both method sensitivity and separation efficiency. The recent trend to detect samples in narrower separation vessels also necessitates the need to improve detection sensitivity. The desire to detect very low levels of analytes using limited amounts of sample from biological specimens and the high separation efficiency obtainable using very large injections compared to classical small size injections also adds to this list. Indeed, one of the rich areas of research in the capillary electrophoresis field is on on-line sample preconcentration. More than 400 published research articles gathered from the http://www.webofscience.com from the year 2000 described a form of on-line preconcentration in capillary electrophoresis. This review provides a comprehensive table listing the applications of on-line preconcentration in capillary electrophoresis.

Online preconcentration of arsenic compounds by dynamic pH junction-capillary electrophoresis.

An online preconcentration technique by dynamic pH junction was studied to improve the detection limit for anionic arsenic compounds by CE. The main target compound is roxarsone, or 3-nitro-4-hydroxyphenylarsonic acid, which is being used as an animal feed additive. The other inorganic and organoarsenic compounds studied are the possible biotransformation products of roxarsone. The arsenic species were separated by a dynamic pH junction in a fused-silica capillary using 15 mM phosphate buffer (pH 10.6) as the BGE and 15 mM acetic acid as the sample matrix. CE with UV detection was monitored at a wavelength of 192 nm. The influence of buffer pH and concentration on dynamic pH junction were investigated. The arsenic species focusing resulted in LOD improvement by a factor of 100-800. The combined use of C18 and anion exchange SPE and dynamic pH junction to CE analysis of chicken litter and soils helps to increase the detection sensitivity. Recoveries of spiked samples ranged between 70 and 72%.

Multiple enzyme linked immunosorbent assay system on a capillary-assembled microchip integrating valving and immuno-reaction functions.

Multiple enzyme linked immunosorbent assay (ELISA) chip is developed by using capillary-assembled microchip (CAs-CHIP) technique, which involves simple embedding of 2-3mm length of square capillaries possessing valving and immuno-reaction functions into the microchannels fabricated on a PDMS substrate. In contrast to the previously reported ELISA chips, our system enables not only the flexible design of the multi-ELISA chip required for many different diagnostic purposes, but also the valving operation required for a reliable analysis. Here, a thermo-responsive polymer-immobilized capillary was used together with a small Peltier device, as a valving part, and different antibody-immobilized capillaries were used as immuno-reaction part. Sample solution and detecting reagent solutions were sequentially introduced through the valving capillary, and the valve is closed to completely stop the solution flow inside the immuno-reaction capillaries and detected using thermal lens microscope (TLM). Different anti-IgGs (human, goat, chicken) were immobilized and used as ELISA parts of CAs-CHIP. Sequential introductions of the mixed IgG solution, mixed enzyme-antibody solution and substrate solution facilitated the multiple determinations of 0.1 ng mL(-1) IgGs (human, goat, chicken) with total analysis time of about 30 min. The valve-integrated multi-ELISA chip developed here can be applied for many different diagnostic purposes by using different immuno-reaction capillaries necessary for a specific clinical diagnostic application.

Characterization of electrokinetic gating valve in microfluidic channels.

Electrokinetic gating, functioning as a micro-valve, has been widely employed in microfluidic chips for sample injection and flow switch. Investigating its valving performance is fundamentally vital for microfluidics and microfluidics-based chemical analysis. In this paper, electrokinetic gating valve in microchannels was evaluated using optical imaging technique. Microflow profiles at channels junction were examined, revealing that molecular diffusion played a significant role in the valving disable; which could cause analyte leakage in sample injection. Due to diffusion, the analyte crossed the interface of the analyte flow and gating flow, and then formed a cometic tail-like diffusion area at channels junction. From theoretical calculation and some experimental evidences, the size of the area was related to the diffusion coefficient and the velocity of analytes. Additionally, molecular diffusion was also believed to be another reason of sampling bias in gated injection.

Simple on-line sample preconcentration technique for peptides based on dynamic pH junction in capillary electrophoresis-mass spectrometry.

We report an on-line sample preconcentration technique based on dynamic pH junction in capillary electrophoresis-mass spectrometry (CE-MS). For peptide analysis, the samples were dissolved in a solution with higher pH than the background solution (BGS), and were injected into the capillary as a long plug. The pH difference between the sample matrix and BGS caused changes in analytes' mobilities during electrophoresis, resulting in narrowing of their bands at the boundary. Around 550-1000-fold sensitivity enhancement could be achieved in terms of peak intensity without degrading peak shape and resolution. This technique is easy to perform and will be useful for peptide mass fingerprinting in protein analysis.

Integration of multianalyte sensing functions on a capillary-assembled microchip: simultaneous determination of ion concentrations and enzymatic activities by a "drop-and-sip" technique.

A general and simple implementation of simultaneous multiparametric sensing in a single microchip is presented by using a capillary-assembled microchip (CAs-CHIP) integrated with the plural different reagent-release capillaries (RRCs), acting as various biochemical sensors. A novel "drop-and-sip" technique of fluid handling is performed with a microliter droplet of a model sample solution containing proteases (trypsin, chymotrypsin, thrombin, elastase) and divalent cations (Ca2+, Zn2+, Mg2+) that passes through the microchannel with the aid of a micropipette as a vacuum pump, concurrently filling each RRC via capillary force. To avert the evaporation of the nanoliter sample volume in each capillary, PDMS oil is dropped on the outlet hole of the CAs-CHIP exploiting the capillary force that results in spontaneous sealing of all the RRCs. In addition, this high-speed sample introduction alleviates the possibility of protein adsorption and capillary cross-contamination, allowing a reliable and multianalyte determination of a sample containing many different proteases and divalent cations by using the fluorescence image analysis. Presented results suggested the possible application of this microchip in the field of drug discovery and systems biology.

Sample enrichment techniques in capillary electrophoresis: focus on peptides and proteins.

Compared to chromatography-based techniques, the concentration limits of detection (CLOD) associated with capillary electrophoresis are worse, and these have largely precluded their use in many practical applications. To overcome this limitation, researchers from various disciplines have exerted tremendous efforts toward developing strategies for increasing the concentration sensitivities of capillary electrophoresis (CE) systems, via the so-called sample enrichment techniques. This review highlights selected developments and advances in this area as applied to the analyses of proteins and peptides in the last 5 years.

Capillary-assembled microchip as an on-line deproteinization device for capillary electrophoresis.

A capillary-assembled microchip (CAs-CHIP), prepared by simply embedding square capillaries in a lattice polydimethylsiloxane (PDMS) channel plate with the same channel dimensions as the outer dimensions of the square capillaries, has been used as a diffusion-based pretreatment attachment in capillary electrophoresis (CE). Because the CAs-CHIPs employ square-section channels, diffusion-based separation of small molecules from sample solutions containing proteins is possible by using the multilayer flow formed in the square section channel. When a solution containing high-molecular-weight and low-molecular-weight species makes contact with a buffer solution, the low-molecular-weight species, which have larger diffusion coefficients than the high-molecular-weight species, can be collected in a buffer-solution phase. The collected solution containing the low-molecular-weight species is introduced into the separation capillary to be analyzed by CE. This type of system can be used for CE analysis in which pretreatment is required to remove proteins. In this work a fluorescently labeled protein and rhodamine-based molecules were chosen as model species and a feasibility study was performed.

Protein tryptic digests analyzed by carrier ampholyte-based capillary electrophoresis coupled to ESI-MS.

In this study, narrow pH cuts of carrier ampholytes have been used as buffers in CE for the analysis of protein tryptic digests. Their low conductivity allows very efficient separations under high electric field strength without inducing any significant Joule heating. In this study, the capabilities of narrow pH cuts of carrier ampholytes for the separation of protein tryptic digests have been assessed. Three proteins of different molecular masses have been studied: cytochrome C (horse heart), beta-lactoglobulin B (bovine) and human transferrin. Efficient, rapid and repeatable separations of the peptides resulting from the tryptic digestion have been achieved in this buffer. Moreover, the feasibility of the coupling of carrier ampholyte-based capillary electrophoresis with ESI-MS has been demonstrated through the study of the cytochrome C tryptic digest.

Measurement of monomolecular binding constants of neutral phenols into the beta-cyclodextrin by continuous frontal analysis in capillary and microchip electrophoresis via a competitive assay.

Measurement of binding constant by chip electrophoresis is a very promising technique for the high throughput screening of non-covalent interactions. Among the different electrophoretic methods available that yield the binding parameters, continuous frontal analysis is the most appropriate for a transposition from capillary electrophoresis (CE) to microchip electrophoresis. Implementation of this methodology in microchip was exemplified by the measurement of inclusion constants of 2-naphtalenesulfonate and neutral phenols (phenol, 4-chlorophenol and 4-nitrophenol) into beta-cyclodextrin by competitive assays. The issue of competitor choice is discussed in relation to its appropriateness for proper monitoring of the interaction.

Integration of multiple-ion-sensing on a capillary-assembled microchip.

Multiple-ion-sensing functions are integrated on a capillary-assembled microchip (CAs-CHIP). Since the CAs-CHIPs are fabricated by embedding various chemically functionalized square capillaries onto a lattice PDMS channel plate having same channel dimensions as outer dimensions of square capillaries, integration of parallel multiple-ion-sensing is easily realized. Here, three ion-sensing capillaries are prepared and used for integrating these functions onto a single microchip. Ion-sensing square capillaries (sodium, potassium, calcium) are prepared by attaching ion-selective optode membranes to inner wall of capillaries, and are characterized in terms of response time, response range, and ion selectivity. Finally, fully characterized ion-sensing capillaries are embedded into PDMS channel plate in parallel to fabricate a multiple-ion-sensing chip. The CAs-CHIP-based strategy is promising for integrating multiple chemical sensing functions onto a single microchip.

Two-dimensional separation system of coupling capillary liquid chromatography to capillary electrophoresis for analysis of Escherichia coli metabolites.

A two-dimensional (2-D) separation system of coupling chromatography to electrophoresis was developed for profiling Escherichia coli metabolites. Capillary liquid chromatography (LC) with a monolithic silica-octadecyl silica column (500 x 0.2 mm ID) was used as the first dimension, from which the effluent fractions were further analyzed by capillary electrophoresis (CE) acting as the second dimension. Field-enhanced stacking was selectively employed as a concentration strategy to interface the two dimensions, which proved to be beneficial for the detection of metabolites. An artificial sample containing 118 standards, some of which lack chromophores or have weak UV absorbance, was used to optimize the 2-D separation system. Under the optimum conditions, 63 components in the artificial sample having absorbance at 254 nm could be well resolved and detected. The utility of the system was demonstrated by comprehensive analysis of E. coli metabolites. Comparing with the previous 2-D separation system we published in Anal. Chem. 2004, 76, 1419-1428, using a longer monolithic column in the first dimension improved the separation efficiency and offered the possibility of increasing the injection volume without compromising the separation efficiency. In the second dimension, field-enhanced stacking was used to improve the concentration sensitivity of the metabolites, and more metabolites in E. coli cell extract were detected and identified using the developed 2-D separation system. In addition, preliminary investigation for future CE-mass spectrometry coupling was also made in the study by using volatile buffers in the capillary LC and CE techniques.

Dynamic pH junction technique for on-line preconcentration of peptides in capillary electrophoresis.

A method based on the presence of a dynamic pH junction within the capillary to induce band narrowing for enhanced detection sensitivity for some peptides is presented. This technique is predicated on a sharp reduction in an analyte's migration velocity following a reversal of its electrophoretic direction from the acidic sample zone to the basic BGS zone. Larger-than-usual injection volumes of samples in relatively high-conductivity matrices were enabled, without degrading peak shape, resolution and efficiency. The size of the original sample plug was reduced by as much as 38-fold, and improvement in detector response in terms of peak height by as much as 124-fold was obtained. The effects of pH and concentration of the sample matrix, and the length of sample injection on the efficiency of the technique are discussed.