An investigation of glasses for capillary chromatography

An investigation was conducted of various glasses, other than soda lime or borosilicate, for use in glass capillary gas chromatography. The work has uncovered some unique chromatographic qualities in the use of potash soda lead and fused silica glasses as materials for making glass capillary columns. The fused silica proved to be an ideal material for capillary column construction, being inherently more inert than glass containing metal oxides. It has been shown that through the use of thin wall capillary tubing of high flexibility many of the mechanical problems associated with glass capillary columns, such as fragility and column straightening, can be avoided.     

Referenced Capacitively Coupled Conductivity Detector for Capillary Electrophoresis

A dual capacitively coupled contactless conductivity detector for capillary electrophoresis was developed. The two channels are arranged in a bridge configuration so that one of them acts as a reference whose signal is subtracted. This effectively compensates for the baseline conductivity of the separation buffer so that the electronic zero setting is not necessary. Changes in the buffer composition are automatically accounted for, as are temperature drifts. The system is demonstrated for the detection of inorganic model cations in capillary electrophoresis. Besides the use with two separate capillaries, one of which solely serves as reference, it was also found possible to use a single capillary which is looped back through the reference cell.     

An inexpensive thread-based system for simple and rapid blood grouping

This study investigates the use of thread as a flexible and low-cost substrate for the rapid grouping of blood. The use of a capillary substrate such as thread for blood grouping utilises the sensitivity of the flow resistance of large particles in narrow capillary channels to separate agglutinated red blood cells (RBCs) from plasma. Large and discrete particles formed in a continuous liquid phase do not provide capillary wicking driving force and fall behind the capillary wicking front, leading to their separation from the wicking liquid. The capillary substrate therefore provides a very promising but different mechanism for the separation of the agglutinated RBCs and the blood serum phase compared to most existing blood grouping methods. The principle of chromatographic separation is also exploited in this study via the use of suitable dyes to enhance the visual detection of the agglutinated RBCs and the serum phase; surprising and encouraging outcomes are obtained. Using a thread-based device, the ABO and Rh groups can be successfully determined with only 2 μL of whole blood from a pricked finger tip within 1 min and without pre-treatment of the blood sample. It is hoped that a new, inexpensive, rapid and simple method may provide an easy-to-use blood grouping platform well suited to those in developing or remote regions of the world.     

Incorporation of Disposable Screen‐Printed Electrodes for Use in Capillary Electrophoresis End‐Column Amperometric Detection System

The development and performance of an end‐column amperometric detection system integrated with disposable screen‐printed electrodes for capillary electrophoresis is presented. In this system, the electrode and capillary can be easily replaced and the capillary/electrode alignment procedure is straightforward. The use of easily replaceable screen‐printed electrodes offers a tremendous benefit for capillary electrophoresis applications requiring frequent replacement of the working electrode due to fouling. This simple and convenient system is very attractive for routine analyses by capillary electrophoresis with electrochemical detection. The separation and determination of uric acid in human urine is presented.     

Integrated electroosmotically-driven on-line sample purification system for nanoliter DNA sequencing by capillary electrophoresis

An integrated on-line system is developed for DNA sequencing at the nanoliter scale. The technique involves the use of a nanoreactor for small-volume cycle-sequencing reaction, capillary zone electrophoresis (CZE) for purification of the sequencing fragments, and capillary gel electrophoresis (CGE) for separation of the purified DNA fragments. The nanoreactor and CZE are integrated into one capillary, where a 100-nl dye-labeled terminator cycle-sequencing reaction is carried out followed by CZE to separate excess dye-labeled terminators from the sequencing fragments. On-line electrokinetic injection of the purified DNA fragments into the CGE system is accomplished at a small-volume tee connector by which the CZE capillary is interfaced to the CGE system. The utility of the system is demonstrated in sequencing nanoliter volumes of single-stranded DNA (M13mp18) and double-stranded DNA (pGEM). The use of voltage to drive both CZE and CGE makes it feasible for automation and future adaptation of the whole system to a microchip.     

Development of a capillary electrophoresis-mass spectrometry method using polymer capillaries for metabolomic analysis of yeast

Metabolomics is an emerging field in analytical biochemistry, and the development of such a method for comprehensive and quantitative analysis of organic acids, carbohydrates, and nucleotides is a necessity in the era of functional genomics. When a concentrated yeast extract was analyzed by CE-MS using a successive multiple ionic-polymer layer (SMIL)-coated capillary, the adsorption of the contaminants on the capillary wall caused severe problems such as no elution, band-broadening, and asymmetric peaks. Therefore, an analytical method for the analysis of anionic metabolites in yeast was developed by pressure-assisted CE using an inert polymer capillary made from poly(ether etherketone) (PEEK) and PTFE. We preferred to use the PEEK over the PTFE capillary in CE-MS due to the easy-to-use PEEK capillary and its high durability. The separation of anionic metabolites was successfully achieved with ammonium hydrogencarbonate/formate buffer (pH 6.0) as the electrolyte solution. The use of 2-propanol washing after every electrophoresis run not only eliminated wall-adsorption phenomena, but allowed for good repeatability to be obtained for migration times in the metabolomic analysis.     

A new capillary electrophoresis end-column amperometric detection system without the need for capillary/electrode alignment

A self-aligning end-column amperometric detection system for capillary electrophoresis was constructed. In this system, the electrode and capillary were exchanged easily and the capillary/electrode alignment procedure is not required. Gold, gold/mercury amalgam, copper and carbon fiber could be used as the working electrode. The principle is in the use of two disk holders with the capillary and the electrode in the center, so that by inserting the disk holders into a groove in the working electrode port, the capillary and the electrode are automatically aligned and the distance between the capillary and the electrode is assured at 0.24 mm. The relative standard deviation obtained using five different gold/mercury amalgam microdisk electrodes for determination of cysteine was 1.5% for the migration time and 3.3% for the electrophoretic peak current. The simple and convenient system was attractive for the routine analysis by capillary electrophoresis with electrochemical detection. The system was applied to the determination of promethazine hydrochloride in human serum.     

Wall deactivation with fluorosurfactants for capillary electrophoretic analysis of biomolecules

This paper describes the use of fluorosurfactants as buffer additives for capillary electrophoretic separation of proteins and peptides. Due to fluorosurfactant bilayer formation at the capillary inner wall, the surface charge can be adjusted and even reversed. If the running buffer pH is kept at a level where the proteins have the same sign of charge as the wall, electrostatic repulsion will be obtained. The protein wall adsorption can therefore be reduced and the separation performance can be noticeably increased. The separation performance can also be further improved by including mixtures of different types of fluorosurfactants in the running buffer. The buffer system can accordingly be adapted for a certain separation problem. Mechanisms for the use of fluorosurfactants for wall deactivation in capillary electrophoretic protein separations is discussed in the present work and some examples of applications are also presented.     

On-line isotachophoretic sample focusing for loadability enhancement in capillary electrochromatography-mass spectrometry

The use of isotachophoretic (ITP) sample focusing to improve the detection limits for the analysis of charged compounds in capillary electrochromatography (CEC) is described. A coupled-column set-up was used with a 220-microm inner diameter capillary, in which counterflow ITP focusing was performed, connected via a T-junction to a 75-microm inner diameter CEC capillary. As is illustrated, the use of ITP focusing resulted in a dramatic reduction of the sample concentration detection limits. To demonstrate the performance of the ITP-CEC combination, several cationic low-molecular mass compounds in a plasma and urine matrix are analysed using UV-absorbance and mass spectrometric detection. A linear calibration curve was constructed over three decades and detection limits in the low nmol/l range were found for academic samples, using UV-absorbance detection.     

Interfacing microbore and capillary liquid chromatography to continuous-flow fast atom bombardment mass spectrometry for the analysis of glycopeptides

This work describes a system to interface either microbore or packed capillary gradient liquid chromatography (LC) to fast atom bombardment mass spectrometry (FAB-MS). The interface incorporates on-line ultraviolet detection and post-column matrix addition to enable independent optimization of both LC and FAB-MS. The glycopeptide antibiotic teicoplanin was chosen as a model system because this group of compounds places severe demands on the chromatographic separation and is difficult to analyze by FAB-MS. For both microbore and capillary LC, high-quality mass spectra of the major components in teicoplanin were obtained; however, the increased sensitivity of the capillary system allowed spectra to be obtained at low picomole concentrations. The sensitivity and ease of use make capillary LC the preferred system for use in LC-FAB-MS.     

Prevention of evaporation of small-volume sample solutions for capillary electrophoresis using a mineral-oil overlay.

The suppression of evaporation of water from small volumes of sample solutions or reagents for capillary electrophoresis by the use of a mineral-oil overlay was investigated in affinophoresis applications, in which the affinity constant of a mutant protein of recombinant human galectin-1 to a lactose affinophore, a triply negative charged ion having a lactoside as an affinity ligand, was determined. When an injection was carried out from a minimum of 20 microL of an aqueous solution beneath the oil overlay, no oil contamination inside the capillary was observed, provided the capillary was cleanly cut so that the end was flat, and the polyimide coating had been removed for a distance of about 2 mm from the end. Affinophoresis was carried out using 20 microL of an affinophore solution covered with an oil overlay. The abnormalities in the electropherograms as the result of the evaporation of the water from the solution during storage prior to use in an automatic operation of a capillary electrophoresis instrument were suppressed, with respect to the formation of a base line gap, an increase in the detection time of a marker ion and an increase in the initial current. A solution in a vial could be used repeatedly for a longer period of time when overlaid with mineral oil than in the absence of an overlay. The use of a mineral-oil overlay is a simple but very efficient technique for solving the problem of the evaporation of water from small volumes of aqueous solutions for use in capillary electrophoresis.     

New High-Performance Techniques for Ion-Exchange Separation

Results of the use of water-soluble cationic polymers for the synthesis of new anion exchangers for ion chromatography, capillary electrophoresis, and electrokinetic chromatography were summarized. The influence of functional groups in polymer molecules on the selectivity of the ion-chromatographic determination of ions and their mobility in capillary electrophoresis was considered.     

Novel design of multicapillary arrays for high-throughput DNA sequencing

A novel approach to design and optimize linear multicapillary arrays (LMCAs) for high-throughput DNA sequencing is proposed. A significant increase in the number of capillary lanes is obtained due to the use of composite insertions alternately placed between working capillaries of the array and a specific combination of refractive indices of the DNA separation matrix, capillary glass, the insertions and a medium which surrounds the capillary array. Theoretical and experimental studies showed that in conjunction with a dual-side laser illumination scheme, the proposed LMCA design allows a simultaneous uniform irradiation of as many as 550 working capillaries.     

Optimization of capillary electrophoretic enantioseparation for basic drugs with native beta-CD as a chiral selector

This study presents the advantages of the 20 microm inner diameter (id) capillary for the enantioseparation of ten basic drugs with native beta-CD as the chiral selector. The apparent binding constants of each enantiomeric pair were determined to calculate the optimum beta-CD concentration ([beta-CD]opt) and the optimization was subsequently carried out. Comparison of the 20 microm id with 50 microm id were made in terms of the results obtained in the optimization and detection limits. Applying the optimum conditions for each compound, reproducible results (RSD from 0-3; n>5) were obtained for the 20 microm id capillary. Although the sensitivity is lower in the 20 microm id capillary, the LOD determined using this capillary is still found to be acceptable for the ten basic drugs studied. Enhanced resolution and faster analysis times were the main advantages observed with the use of this capillary in enantioseparation.     

Efforts to improve detection sensitivity for capillary electrophoresis coupled to atmospheric pressure photoionization mass spectrometry

Electrospray ionization performs best with volatile buffers. However, generally the best separation performance for capillary electrophoresis (CE) is achieved with non‐volatile buffers. Hyphenation of CE with mass spectrometry (MS) utilizing atmospheric pressure photoionization (APPI) enables use of a wider range of separation buffers without compromising detection sensitivity. As APPI is considered to be mass flow sensitive, the use of a larger inner diameter separation capillary (75 µm) allows larger volumes to be injected, without decreased separation performance, thus providing improved sensitivity (approx. a factor of 10), compared to the use of a 25 µm capillary. However, nebulizing gas flow and position of capillary tip in the sprayer have to be carefully optimized to prevent excessive band broadening. Further improvement in sensitivity (approx. a factor of 2) was obtained by decreasing the distance between the sprayer and ionization region, indicating that a specially designed CE/APPI‐MS interface for low flow rates will be favourable. Copyright © 2010 John Wiley & Sons, Ltd.     

Packing columns for capillary electrochromatography

Considering the current interest in capillary electrochromatography (CEC), performed in packed columns, we present the different methods used to pack capillary columns for use in CEC. General considerations on column packing are given and the column fabrication process is discussed in sufficient detail to allow instruction to those who are not experienced in the field. Five different packing methods are discussed to deliver packing material into the capillary column from a practical view point: slurry pressure packing, packing with supercritical CO2, electrokinetic packing, using centripetal forces, and packing by gravity. Entrapment of particulate material by sintering and sol-gel technology is also mentioned. Although slurry pressure packing procedures are most common, higher separation efficiencies are obtained using other packing approaches. Electrokinetic packing seems to be the simplest technique to deliver the packing material into the capillary columns. Nevertheless, as with the other packing techniques, skill and experience are required to complete all the steps involved in the fabrication of packed columns for CEC.     

Device for filling of small diameter capillaries with stationary phase solutions in liquefied gases

This paper describes a high pressure device for filling small diameter capillaries with stationary phase solutions is described. A liquid is forced into the capillary column with the help of high pressure syringe whose needle (provided with a side opening) is tightened in a PTFE seal. The device allows use of liquefied gas as solvent. A detailed procedure is given for filling the capillary with stationary phase solution. The performance of the device was evaluated by filling 12 m × 15 μm i. d. glass capillary with 6.5 % (w/v) SE-54.     

A fully automated linear polyacrylamide coating and regeneration method for capillary electrophoresis of proteins

Surface modification of the inner capillary wall in CE of proteins is frequently required to alter EOF and to prevent protein adsorption. Manual protocols for such coating techniques are cumbersome. In this paper, an automated covalent linear polyacrylamide coating and regeneration process is described to support long‐term stability of fused‐silica capillaries for protein analysis. The stability of the resulting capillary coatings was evaluated by a large number of separations using a three‐protein test mixture in pH 6 and 3 buffer systems. The results were compared to that obtained with the use of bare fused‐silica capillaries. If necessary, the fully automated capillary coating process was easily applied to regenerate the capillary to extend its useful life‐time.     

Monolithic silica column for in-tube solid-phase microextraction coupled to high-performance liquid chromatography

In-tube solid-phase microextraction (SPME) has successfully been coupled to capillary LC, and further an automated in-tube SPME system has been developed using a commercially available HPLC auto-sampler. However, an open tubular capillary column with a thick film of polymer (stationary phase) is unfavorable because the ratio of the surface area of coating layer contacted with sample solution to the volume of the capillary column is insufficient for mass transfer. A highly efficient SPME column is. therefore, required. We introduced a C18-bonded monolithic capillary column that was used for in-tube SPME. The column consisted of continuous porous silica having a double-pore structure. Both the through-pore and the meso-pore were optimized for in-tube SPME, and the optimized capillary column was connected to an HPLC injection valve for characterization. The results demonstrated that the pre-concentration efficiency is excellent compared with the conventional in-tube SPME. The novel method for both introduction and concentration of the samples was effective. satisfactory and suitable for use in the SPME medium.