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Analytical and Bioanalytical Chemistry (v.382, #3)

Advances in separation science by Stephen A. Wise; Kiyokatsu Jinno; Toyohide Takeuchi (pp. 533-534).

Sample preparation for peptides and proteins in biological matrices prior to liquid chromatography and capillary zone electrophoresis by N. F. C. Visser; H. Lingeman; H. Irth (pp. 535-558).

The determination of peptides and proteins in a biological matrix normally includes a sample-preparation step to obtain a sample that can be injected into a separation system in such a way that peptides and proteins of interest can be determined qualitatively and/or quantitatively. This can be a rather challenging, labourious and/or time-consuming process. The extract obtained after sample preparation is further separated using a compatible separation system. Liquid chromatography (LC) is the generally applied technique for this purpose, but capillary zone electrophoresis (CZE) is an alternative, providing fast, versatile and efficient separations. In this review, the recent developments in the combination of sample-preparation procedures with LC and CZE, for the determination of peptides and proteins, will be discussed. Emphasis will be on purification from and determination in complex biological matrices (plasma, cell lysates, etc.) of these compounds and little attention will be paid to the proteomics area. Additional focus will be put on sample-preparation conditions, which can be ‘hard’ or ‘soft’, and on selectivity issues. Selectivity issues will be addressed in combination with the used separation technique and a comparison between LC and CZE will be made.

Keywords: Sample preparation; Proteins; Peptides; Biological matrix; CZE; LC

Analysis of cyanobacterial pigments and proteins by electrophoretic and chromatographic methods by Christa L. Colyer; Christopher S. Kinkade; Pertti J. Viskari; James P. Landers (pp. 559-569).

Cyanobacteria are a diverse and ubiquitous group of prokaryotes with several unifying features. Amongst these is the macromolecular structure known as the phycobilisome, which is composed of water-soluble phycobiliproteins covalently bound by linker peptides or proteins in a configuration designed to optimize energy transfer to the photosynthetic reaction center of the organism. Phycobiliproteins are highly fluorescent by virtue of their covalently bound, linear tetrapyrrole chromophores known as bilins. Analysis of these prosthetic pigments, along with other non-water soluble pigments, such as the chlorophylls and carotenoids, can provide insight into microbial diversity. The effects of environmental growth conditions and stresses can also be probed by measuring pigment and protein concentrations. This review will focus, therefore, on applications of various chromatographic and electrophoretic methods for the analysis of cyanobacterial pigment and protein constituents. Although the greatest emphasis will be placed on the measurement of bilins and phycobiliproteins, this review will also consider other pigments and proteins important to cyanobacterial growth and survival, such as chlorophyll a, carotenoids, ectoenzymes, linker and membrane proteins, and extracellular proteins.

Keywords: Cyanobacteria; Chromatography; Electrophoresis; Phycobiliproteins; Bilins; Ectoenzymes; Linker peptides; Phycobilisomes

Probing affinity via capillary electrophoresis: advances in 2003–2004 by Melissa Gayton-Ely; Theron J. Pappas; Lisa A. Holland (pp. 570-580).

This review addresses recent advances in capillary electrophoresis of biological-based molecular interaction from a broader perspective, based on applications reported during the period 2003–2004. These capillary electrophoresis-based studies of molecular interactions include affinity capillary electrophoresis, electrokinetic chromatography, and free zone electrophoresis. The review is written as a general synopsis of applications and does not cover the theory or protocol involved in the implementation of the analyses.

Keywords: Review; ACE; Binding; Affinity; Capillary electrophoresis; Molecular interaction

Recent advances in the application of capillary electrophoresis to neuroscience by Paula R. Powell; Andrew G. Ewing (pp. 581-591).

With fast separation times (seconds to minutes), minimal sample requirements (nanoliters to femtoliters), and excellent mass detection limits (femtomole to zeptomole), capillary electrophoresis (CE) is ideally suited for in vitro and in vivo sampling of neurological samples with a high degree of spatial resolution. Advances in extracellular fluid analysis employing improved microdialysis and push–pull perfusion sampling methodologies has enabled the resolution of neurotransmitters present in limited amounts using CE. Great progress has been made to resolve complex neuropeptides, amino acids, and biogenic amines in tissue and cell cultures. Finally, owing largely to the small volume sampling abilities of CE, investigations of single nerve cells, both invertebrate and mammalian, have been accomplished. These applications of CE to the advancement of neuroscience are presented.

Keywords: Capillary electrophoresis; Neuroscience; Microdialysis; Single-cell analysis; Extracellular fluid analysis; Biogenic amines

Recent advances in peptide chiral selectors for electrophoresis and liquid chromatography by Louis Bluhm; Junmin Huang; Tingyu Li (pp. 592-598).

The application of peptides in chiral separations using techniques such as capillary electrophoresis (CE), electrokinetic capillary chromatography (EKC) and liquid chromatography is the focus of this review. Methods for finding peptide selectors using combinatorial library approaches are discussed, as well as recent advances in the use of peptides as general chiral selectors for electrophoresis and liquid chromatography. One example shows the effectiveness of polymeric dipeptide surfactants as general chiral selectors for electrophoresis. Another example shows the versatility of oligoproline chiral stationary phases, exhibiting resolution for a number of racemic analytes comparable to other well-established chiral stationary phases.

Keywords: Peptide chiral selector; Chiral separation; Combinatorial library; Chiral stationary phase; Electrophoresis; Chiral chromatography

Stereoselective peptide analysis by Christoph Czerwenka; Wolfgang Lindner (pp. 599-638).

The stereochemistry of a peptide determines its spatial features and can profoundly influence its chemical properties and biological activity. Thus, the analysis of the stereochemical properties of a peptide is an important aspect of its characterisation. For such investigations a “selector” that engages in stereoselective interactions with the peptide analytes is often used. A substantiated knowledge of the underlying molecular recognition mechanism will therefore be helpful in understanding existing and developing new stereoselective analysis systems. After a short introduction concerning the fundamentals of peptide stereoisomers and their biological implications, the stereoselective peptide analysis methods described in the literature are comprehensively reviewed. The characteristics and applications of the employed methods based on various techniques including chromatography (pressure- and electrokinetically driven), capillary electrophoresis, nuclear magnetic resonance spectroscopy and mass spectrometry are discussed. The various selectors that have been utilised to discriminate peptide enantiomers and/or diastereomers are described concurrently. The review concludes with an overview of combinations and comparisons of techniques that have been applied to the analysis of peptide stereoisomers and constitute a trend for further developments.

Keywords: Review; Stereoselective analysis; Peptides; Stereoisomers; Enantiomers; Diastereomers; HPLC; Capillary electrophoresis; NMR spectroscopy; Mass spectrometry

Enantioselective separations by packed column subcritical and supercritical fluid chromatography by Karen W. Phinney (pp. 639-645).

Enantioselective separations have been one of the most successful applications of supercritical fluid chromatography (SFC). Although analytical scale separations have dominated the literature, the use of SFC for preparative chiral separations is growing. Both analytical and preparative scale SFC separations seek to take advantage of the high efficiency, high throughput, and rapid method development associated with the technique. This review will cover recent developments in the application of SFC to enantioseparations.

Keywords: Chiral separation; Chiral stationary phase; Enantiomers; Method development; Preparative; SFC

Order and disorder in alkyl stationary phases by Lane C. Sander; Katrice A. Lippa; Stephen A. Wise (pp. 646-668).

Covalently modified surfaces represent a unique state of matter that is not well described by liquid or solid phase models. The chemical bond in tethered alkanes imparts order to the surface in the form of anisotropic properties that are evident in chromatographic and spectroscopic studies. An understanding of the structure, conformation, and organization of alkyl-modified surfaces is requisite to the design of improved materials and the optimal utilization of existing materials. In recent years, the study of alkyl-modified surfaces has benefited from advances in modern analytical instrumentation. Aspects of alkyl chain conformation and motion have been investigated through the use of nuclear magnetic resonance spectroscopy, Fourier transform infrared spectroscopy, Raman spectroscopy, fluorescence spectroscopy, and neutron scattering studies. Chromatography provides complementary evidence of alkyl chain organization through interactions with solute probes. Computational simulations offer insights into the structure of covalently modified surfaces that may not be apparent through empirical observation. This manuscript reviews progress achieved in the study of the architecture of alkyl-modified surfaces.

Keywords: Conformational order; Stationary phase; Alkyl-modified surface; Raman spectroscopy; Nuclear magnetic resonance; Molecular dynamics simulation

The development of the DIGE system: 2D fluorescence difference gel analysis technology by Rita Marouga; Stephen David; Edward Hawkins (pp. 669-678).

Two-dimensional (2D) gel electrophoresis is a powerful technique enabling simultaneous visualization of relatively large portions of the proteome. However, the well documented issues of variation and lack of sensitivity and quantitative capabilities of existing labeling reagents, has limited the use of this technique as a quantitative tool. Two-dimensional difference gel electrophoresis (2D DIGE) builds on this technique by adding a highly accurate quantitative dimension. 2D DIGE enables multiple protein extracts to be separated on the same 2D gel. This is made possible by labeling of each extract using spectrally resolvable, size and charge-matched fluorescent dyes known as CyDye DIGE fluors. 2D DIGE involves use of a reference sample, known as an internal standard, which comprises equal amounts of all biological samples in the experiment. Including the internal standard on each gel in the experiment with the individual biological samples means that the abundance of each protein spot on a gel can be measured relative (i.e. as a ratio) to its corresponding spot in the internal standard present on the same gel. Ettan DIGE is the system of technologies that has been optimized to fully benefit from the advantages provided by 2D DIGE.

Keywords: 2D DIGE; CyDye DIGE fluors; DeCyder differential analysis software; Fluorescent labeling; Proteomics

Nuclear magnetic resonance and high-performance liquid chromatographic evaluation of polymer-based stationary phases immobilized on silica by Christoph Meyer; Urban Skogsberg; Norbert Welsch; Klaus Albert (pp. 679-690).

Three poly(ethylene-co-acrylic) acid copolymers (–CH₂CH₂–)_x[CH₂CH(CO₂H)–]_y with different chain lengths and mass fractions of acrylic acid were covalently immobilized as stationary phases on silica via two variants of spacer molecules (3-aminopropyltriethoxysilane and 3-glycidoxypropyltrimethoxysilane). Different mobilities of the alkyl chains in the stationary phases were observed using ¹³C solid-state NMR spectroscopy. The stationary phases with more rigid trans-ordered alkyl chains had better selectivity for geometric β-carotene and xanthophyll isomers (provitamin A derivatives). Also, all the separations of the analytes were affected by polar interactions with the chromatographic sorbent. This was further proved by separating more polar cis/trans retinoic acid isomers (vitamin A derivatives). ¹³C high-resolution/magic-angle spinning (HR/MAS) NMR measurements of the chromatographic sorbents suspended in the mobile phase confirmed a dependence of molecular shape recognition ability on alkyl chain conformation.

Keywords: Polymer-based stationary phases; Solid- and suspended-state magic angle spinning nuclear magnetic resonance spectroscopy; High-performance liquid chromatography; Provitamin and vitamin A

Raman spectroscopic study of the conformational order of octadecylsilane stationary phases: effects of electrolyte and pH by Christopher J. Orendorff; Jeanne E. Pemberton (pp. 691-697).

This study investigates effects of the electrolyte, of acidic and basic compounds, and of pH on the rotational and conformational order of octadecylsilane stationary phases with surface coverages of 3.09 and 6.45 μmol/m². Both phases exhibit an increase in alkyl chain rotational and conformational order in 5–200 mM aqueous electrolyte solutions relative to water. These stationary phases are effectively “salted-out” of aqueous electrolyte solutions, thereby causing alkyl chain intermolecular interactions to increase with a concomitant increase in alkyl chain order. Although the presence of acidic and basic compounds generally has no effect on the conformational order of either stationary phase as a function of pH, the higher coverage stationary phase does exhibit pH-dependent changes in aqueous solutions of benzoic acid. At pH values below the pK_a of benzoic acid, the conformational order of this stationary phase is unchanged relative to that observed in the same pH solution in the absence of benzoic acid. In light of independent evidence that such monosubstituted aromatics interact with the octadecylsilane stationary phase under these conditions, the absence of a measurable effect on alkyl chain order for these conditions is attributed to benzoic acid self-association at the stationary phase-mobile phase interface. In contrast, at pH values above the pK_a of benzoic acid, slight disordering of the alkyl chains is observed and is attributed to repulsive interactions between retained benzoate anions.

Selectivity of long chain stationary phases in reversed phase liquid chromatography by Catherine A. Rimmer; Lane C. Sander; Stephen A. Wise (pp. 698-707).

A series of commercial monomeric and polymeric C₁₈, C₂₇, and C₃₀ stationary phases were compared with immobilized poly(ethylene-co-acrylic acid) stationary phases synthesized in-house. The columns were characterized on the basis of methylene selectivity, silanol activity, metal activity, pore size, shape selectivity, and the ability to separate tocopherol isomers and carotenoid isomers. Monomeric and polymeric C₃₀ phases were shown to yield excellent separations of the tocopherol isomers while the polymeric C₃₀ and polyethylene phases were more appropriate to the separation of carotenoids.

Keywords: Stationary phases; LC; Shape selectivity; Carotenoids; Tocopherols

Chromatography in silico, basic concept in reversed-phase liquid chromatography by Toshihiko Hanai (pp. 708-717).

Basic phenomena in reversed-phase liquid chromatography have been quantitatively analyzed using a computational chemical calculation. Pyridine interacted with an ionized silica surface under neutral conditions. Alkyl-chain length affected the contact surface area with an analyte. Steric hindrance was demonstrated using a model graphitic carbon phase and unsaturated alkenes. Quantitative structure–retention relationships in reversed-phase liquid chromatography were demonstrated for phenolic compounds and acidic and basic drugs. The correlations between predicted and measured retention factors were satisfactory. Dissociation constants were derived from the atom partial charge and used to predict retention factors of partially ionized compounds.

Keywords: QSRR; Computational chemistry; Liquid chromatography; Molecular mechanics; Atom partial charge; pK_a

Theoretical opportunities and actual limitations of pH gradient HPLC by Roman Kaliszan; Pawel Wiczling (pp. 718-727).

In a series of reports published recently by our laboratory comprehensive theory and experimental conditions were established for reversed-phase high-performance liquid chromatography (RP HPLC) employing the programmed pH gradient of mobile phase. A procedure was developed providing, rapidly and conveniently, the acidity (pK_a) of weak acids and bases and their lipophilicity (hydrophobicity) log k_w. The basis of the double-gradient RP HPLC, employing simultaneous gradients of organic modifier content and mobile phase pH, was also elaborated. The fundamentals of the approach are presented briefly and systematically and its advantages and limitations are discussed. It is demonstrated that the newly introduced pH gradient method increases the analytical versatility of RP HPLC and our understanding of its physicochemical basis.

Keywords: Gradient HPLC; pH gradient HPLC; Double pH/organic gradient HPLC; Lipophilicity; log k_wAcidity constant; pK_a

Retention characteristics of a new butylimidazolium-based stationary phase by Y. Sun; B. Cabovska; C. E. Evans; T. H. Ridgway; A. M. Stalcup (pp. 728-734).

A new HPLC stationary phase has been synthesized based on the ionic liquid n-butylimidazolium bromide. Imidazolium was covalently immobilized on a silica substrate through an n-alkyl tether and the retention characteristics of the resulting stationary phase were evaluated systematically. Using 28 small aromatic test solutes and reversed phase conditions, the linear solvation energy relationship approach was successfully used to characterize this new phase. The retention characteristics of the test solutes show remarkable similarity with phenyl stationary phases, despite the presence of a positive charge on the new imidazolium phase. Operated in the reversed phase mode, this new stationary phase shows considerable promise for the separation of neutral solutes and points to the potential for a truly multi-modal stationary phase.

Keywords: Butylimidazolium bromide; LSER; Reversed phase; HPLC; Ionic liquids; Retention mechanisms

Liquid chromatographic properties of aromatic sulfur heterocycles on a Pd(II)-containing stationary phase for petroleum analysis by Kishore Sripada; Jan T. Andersson (pp. 735-741).

Polycyclic aromatic sulfur heterocycles (PASHs) can show very poor reactivities in catalytic hydrodesulfurization processes in refineries, especially those in high-boiling fractions and distillation residues. An insight into the structural features of the most recalcitrant PASHs is essential for developing more efficient catalysts and improving refinery processes. The very high complexities of such mixtures necessitate fractionation of the samples into smaller subsets according to defined criteria. A stationary phase containing a palladium(II)-complex was previously shown to be efficient for separating PASHs in lighter petroleum fractions. Here we characterize this ligand exchange chromatographic phase using a large number of sulfur aromatic model compounds that were synthesized for the purpose. In general, compounds containing thiophene rings that are not condensed with other aromatic systems are weakly retained and elute in a first fraction with the polycyclic aromatic hydrocarbons. Thiophene rings condensed with other aromatic rings are more strongly retained and elute in a later fraction with a more polar eluent. If the sulfur is in a non-aromatic ring, the compound is irreversibly retained by the Pd(II) ions. Some steric effects are seen in compounds with alkyl or aryl substituents close to the sulfur atom but in general they do not interfere strongly with the complexation. Thus it seems possible to separate groups of aromatic sulfur compounds according to their complexation properties. For instance, such fractionated samples can be studied much more easily by mass spectrometric techniques.

Keywords: Pd-ACDA-silica gel; Ligand exchange chromatography; Polycyclic aromatic sulfur heterocycles (PASHs); Polycyclic aromatic hydrocarbons (PAHs); Vacuum residue; Hydrodesulfurization

Prediction of clozapine metabolism by on-line electrochemistry/liquid chromatography/mass spectrometry by Suze M. van Leeuwen; Bertrand Blankert; Jean-Michel Kauffmann; Uwe Karst (pp. 742-750).

Combining electrochemical conversion, liquid chromatography and electrospray ionization mass spectrometry (EC/LC/ESI-MS) on-line allows the rapid identification of possible oxidation products of clozapine (CLZ) in the absence and in the presence of glutathione. CLZ is, depending on the applied potential, oxidized to various products in an electrochemical flow-through cell using a porous glassy carbon working electrode. Several hydroxylated and demethylated species are detected on-line using LC/MS. While hydroxy-CLZ is most abundant at a potential of 400 mV, demethylation occurs more readily at higher potentials (at around 700 mV versus Pd/H₂ reference). In the presence of glutathione (GSH), various isomeric glutathione adducts and respective products of further oxidation can be identified. The thioadducts are characterized by tandem MS. Mono-GSH and bis-GSH derivatives can be seen in the chromatograms. The results correlate well with the cyclic voltammetric profile of CLZ. The data are relevant from a pharmacological point of view, since similar metabolites (phases I and II) have been reported in the literature. The EC/LC/MS and EC/MS methods should be valuable tools that can be used to anticipate and understand the metabolization patterns of molecules of pharmacological interest and to point out reactive intermediates.

Keywords: Clozapine; Oxidation; Electrochemistry; LC/MS; Metabolism

Combined solid-phase extraction and 2D LC–MS for characterization of the neuropeptides in rat-brain tissue by Anders Holm; Espen Storbråten; Albena Mihailova; Bartosz Karaszewski; Elsa Lundanes; Tyge Greibrokk (pp. 751-759).

A comprehensive two-dimensional capillary liquid chromatographic (2D LC) method has been established for determination of neuropeptides in rat brain tissue. Rats were exposed to different levels of stress before sacrificing and the aim of this study was to design a powerful separation and detection technique capable of characterizing differences between cerebral neuropeptide expression as a function of stress level. Rat brain samples were homogenized and subjected to clean-up by solid-phase extraction (SPE) on both a reversed-phase (C₁₈) and a weak cation-exchange (CBA) cartridge. The samples were divided in two fractions (A and B) depending on retention on the CBA column. Subsequently, 50 μL of the sample were injected on to a strong cation exchanger (SCX) at a mobile phase pH of 3, which enabled preconcentration of positively charged compounds. The trapped compounds were eluted using step gradients of ammonium formate in water–ACN (90:10, v/v). Before enrichment in the second dimension, the eluate from the first dimension was diluted with water containing 0.1% TFA. The compounds eluting from the first dimension were trapped in the second dimension using a dual precolumn system consisting of two short capillary columns packed with Kromasil C₁₈, 10 μm particles. Subsequently, the trapped compounds were backflushed on to a 10 cm long, 320 μm I.D. analytical column packed with Kromasil C₁₈ 3.5 μm particles, on which they were efficiently separated. Detection was performed using an ion-trap mass spectrometer (ITMS) in both the MS and the MS–MS mode. Comparison of base-peak chromatograms (BPC) from MS analysis of stressed and non-stressed rats clearly revealed several differences in neuropeptide expression. The MS–MS data obtained combined with Mascot software were employed for peptide identification.

Keywords: Multidimensional liquid chromatography; Neuropeptides; Ion-trap mass spectrometry; Capillary liquid chromatography

Detection of N-monomethyl-lysine generated by metabolic transmethylation by Huba Kalász; Imre Klebovich; Katalin Balogh-Nemes; Ágnes Szilágyi; Mária Tihanyi; Tibor Szarvas; József Lengyel (pp. 760-764).

Administration of radiolabelled deprenyl to rats resulted in the urinary elimination of a ¹⁴C-labelled N^ε-monomethyl-lysine. An increased level of N^ε-monomethyl-lysine was found following an oral dose of another drug, also containing an N-methyl group. The urine sample was treated with 9-fluorenylmethoxycarbonyl chloride and then subjected to high-performance liquid chromatography (HPLC); the radioactive fraction was identified as N^ε-monomethyl-lysine by using HPLC-MS in electrospray mode. Identification of N^ε-monomethyl-lysine in the radioactive fraction gives experimental proof of transmethylation from a well-known drug to an endogenous compound.

Keywords: HPLC; HPLC/MS/MS; MethylationN-Monomethyl-lysine; Deprenyl; Urinary

Verification of statistical-overlap theory in micellar electrokinetic chromatography by Siyuan Liu; Joe M. Davis (pp. 765-776).

The limited peak capacity of neutral compounds in micellar electrokinetic chromatography (MEKC) causes peak overlap in a simple 38-compound sample that is predicted by statistical-overlap theory (SOT). The low-concentration sample was prepared in-house from several compound classes to span the entire migration-time range and was resolved partially in a pH=7 phosphate buffer containing 50 mM sodium dodecyl sulfate. Peaks, singlets, doublets, and other multiplets were identified on the basis of known migration times and were counted at 13 voltages spanning 4 – 26 kV. These numbers agreed well with predictions of a simple SOT based on the assumption of an inhomogeneous Poisson distribution of migration times. Because the dispersion theory of MEKC is simple, the standard deviations of single-component peaks were modeled theoretically. As part of a new way to implement SOT, probability distributions of the numbers of peaks, singlets, and so on, were computed by Monte Carlo simulation. These distributions contain all theoretical information on peak multiplicity predictable by SOT and were used to evaluate the agreement between experiment and theory. The peak capacity of MEKC was calculated numerically and substituted into the simplest equations in SOT, affirming that peak overlap arises from limited peak capacity.

Keywords: Micellar electrokinetic chromatography; Statistical-overlap theory

Performance and selectivity of polymeric pseudostationary phases for the electrokinetic separation of amino acid derivatives and peptides by Shannon Schulte; Anup K. Singh; Erika Rauk; Christopher P. Palmer (pp. 777-782).

Two polymeric pseudostationary phases, one an acrylamide polymer and the second a siloxane polymer, have been investigated for the separation of naphthalene-2,3-dicarboxaldehyde (NDA)-derivatized amino acids and small peptides. The dervatized amino acids were detected by UV absorbance and laser-induced fluorescence (LIF) detection. The polymers provided very high efficiency and good selectivity for the separation of the amino acids. The separation selectivity using the polymers was significantly different from that of SDS micelles, and there were subtle differences in selectivities between the polymers. Although very good detection limits were obtained with LIF detection, a significant background signal was observed when the polymers were not washed to remove fluorescent impurities. The polymers did not separate the peptides very well. It is postulated that the fixed covalent structure of the polymers prevents them from interacting strongly or efficiently with the peptides, which are large in relation to the analytes typically separated by electrokinetic chromatography using polymers.

Keywords: Polymer; Pseudosstationary phase; Electrokinetic chromatography; Amino acids; Peptides

The transitional isoelectric focusing process by Tiemin Huang; Zhen Liu; Janusz Pawliszyn (pp. 783-788).

The transitional isoelectric focusing (IEF) process (the course of pH gradient formation by carrier ampholytes (CAs) and the correlation of the focusing time with CA concentration) were investigated using a whole-column detection capillary isoelectric focusing (CIEF) system. The transitional double-peak phenomenon in IEF was explained as a result of migration of protons from the anodic end and hydroxyl ions from the cathodic end into the separation channel and the higher electric field at both acidic and basic sides of the separation channel. It was observed that focusing times increase logarithmically with CA concentration under a constant applied voltage. The correlation of focusing time with CA concentration was explained by the dependence of the charge-transfer rate on the amount of charged CAs within the separation channel during focusing.

Keywords: Isoelectric focusing; Carrier ampholytes; Transitional IEF process; Focusing time

Fast electrophoresis in conventional capillaries by employing a rapid injection device and contactless conductivity detection by Andrea Rainelli; Peter C. Hauser (pp. 789-794).

A purpose-made set-up featuring an automated fast injector allowed the easy optimization of the injected amount and the adjustment of the separation length of conventional capillaries from a minimum of 5 cm upward. It was found that a compromise in capillary length for separation efficiency and analysis time also has to take into account the injected amount, which in turn affects the sensitivity and hence the detection limit. The versatility of the system was demonstrated by the analysis of the major cations and anions in natural water samples in less than 1 min, the concurrent determination of a mixture of amino acids and carbohydrates in 160 s, and of three active ingredients in a pharmaceutical preparation in 40 s. Plate numbers were typically around 50,000 and detection limits down to 1 μM could be achieved.

Keywords: Capillary electrophoresis; Fast separation; Automated injection; Contactless conductivity detection

The use of etched, chemically modified, rectangular capillaries as a separation medium for open tubular capillary electrochromatography by Joseph J. Pesek; Maria T. Matyska; Kelley Freeman; Gabriella Carlon (pp. 795-803).

The use of etched, chemically modified, capillaries with a rectangular inner channel for open tubular electrochromatography is investigated. Comparisons of separation capabilities are made between circular and rectangular capillaries undergoing the same etching and chemical modification processes. With the long dimension of the rectangular column aligned in the direction of the optical light path, the relative sensitivities of the two capillary geometries are evaluated. The electrochromatographic properties of two catechins found in tea are investigated on the rectangular etched octadecyl-modified capillary.

Keywords: Silanization/hydrosilation; Tea analysis; Protein analysis

High benzene selectivity of mesoporous silicate for BTX gas sensing microfluidic devices by Yuko Ueno; Akiyuki Tate; Osamu Niwa; Hao-Shen Zhou; Takeo Yamada; Itaru Honma (pp. 804-809).

The gas selectivities of highly ordered mesoporous silicates and commercially-obtained porous silicates with respect to benzene, toluene and xylene were studied. After studying the porosities, pore uniformities, and surface silanol structures of the silicates and their relationships to gas selectivity in detail, we found that we could achieve high benzene selectivity by controlling the micropore size (less than 1 nm). Concluding that mesoporous silicate has a suitable micropore size and structure for benzene selectivity, we also observed that mesoporous silicate SBA-16 exhibited a high (>6) benzene selectivity from toluene and xylene even in a pseudo-atmospheric environment. A benzene detection limit of about 100 ppb was achieved by introducing SBA-16 into a microfluidic device originally developed for the separate detection of benzene, toluene, and xylene gases.

Keywords: Mesoporous silicate; Molecular selectivity; Micropore; Microfluidic device; BTX gas

Fabrication of an integrated PDMS microchip incorporating an LED-induced fluorescence device by Kyo Miyaki; Yanli Guo; Takuya Shimosaka; Tatsuro Nakagama; Hizuru Nakajima; Katsumi Uchiyama (pp. 810-816).

A microfluidic device with an integrated fluorescence detection system has been developed in order to miniaturize the entire analytical system. A blue or green light-emitting diode (LED) and an optical fiber were mounted in a polydimethylsiloxane-based microchip. The performance of this device was evaluated by microchip electrophoresis. When a green LED was used as the light source, the calibration curve of Sulforhodamine-101 was linear over the range 1–100 μM. The detection limit was found to be 600 nM (240 amol) for a S/N ratio of 3. When using a blue LED, the calibration curve of Fluorescein was linear over the range 0.2–100 μM. The detection limit was estimated to be 120 nM (50 amol) (S/N=3). The detection sensitivity per unit power was comparable to that of LIF. The RSD values for the migration time, peak height and peak area were 0.74, 7.18 and 9.45%, respectively. The integrated microfluidic device was successfully used to determine amino acid derivatives.

Keywords: Integrated PDMS microchip; Light-emitting diode; Fluorescence; Fabrication method; Microchannel electrophoresis

An electro-osmotic micro-pump based on monolithic silica for micro-flow analyses and electro-sprays by Zilin Chen; Ping Wang; Hsueh-Chia Chang (pp. 817-824).

A high-pressure electro-osmotic micro-pump fabricated by a sol–gel process is reported as a fluid-driving unit in a flow-injection analysis (FIA) system. The micro FIA system consists of a monolithic micro-pump on a glass slide (2.5×7.5 cm), a micro-injector, and a micro-sensor (2.5×1.5 cm). The monolithic silica matrix has a continuous skeleton morphology with micrometer-sized through-pores. The micrometer-size pores with a large negative surface charge density build up a large pressure under a DC electric field to drive fluid through the downstream units. A novel Nafion joint for the downstream cathode eliminates flow into the electrode reservoir and further enhances pressure build-up. The measured pump-pressure curve indicated a maximum pressure of 0.4 MPa at flow rate of 0.4 μL min⁻¹ at 6 kV. Despite the large voltage, the small current transmission area through the monolith produced a negligible current (less than 100 μA) that did not generate bubbles or ion contaminants. The flow rate can be precisely controlled in the range 200 nL to 2.5 μL min⁻¹ by varying the voltage from 1 to 6 kV. The high pump pressure and the large current-free DC field also enabled the pump to act as an electro-spray interface with a downstream analytical instrument.

Keywords: Electroosmotic micropump; Monolith; Sol–gel; Bio-sensor; Flow-injection analysis; Electrospray.

High-temperature separation with polymer-coated fiber in packed capillary gas chromatography by Yoshihiro Saito; Mitsuhiro Ogawa; Motohiro Imaizumi; Kazuhiro Ban; Akira Abe; Tsutomu Takeichi; Hiroo Wada; Kiyokatsu Jinno (pp. 825-829).

High-temperature gas chromatographic separation of several synthetic polymer mixtures with Dexsil-coated fiber-packed columns was studied. A bundle of heat-resistant filaments, Zylon, was longitudinally packed into a short metal capillary, followed by the conventional coating process with Dexsil 300 material. Prior to the packing process the metal capillary was deactivated by the formation of a silica layer. The typical size of the resulting column was 0.3-mm i.d., 0.5-mm o.d., 1-m length, and packed with about 170 filaments of the Dexsil-coated Zylon. The column temperature could be elevated up to 450°C owing to the good thermal stability of the fiber, Dexsil coating, and metal capillary; furthermore, this allowed the separation of low-volatile compounds to be studied.

Keywords: High-temperature separation; Zylon fiber; Dexsil; Metal capillary; Fiber-packed GC columns; Polymer analysis

The GC-MS detection and characterization of neopine resulting from opium use and codeine metabolism and its potential as an opiate-product-use marker by Abdel M. Al-Amri; Roger M. Smith; Babiker M. El-Haj (pp. 830-835).

Neopine, a minor opium alkaloid and an isomer of codeine (also known as β-codeine), has been detected in both the urine of opium users and pharmaceutical codeine users. The characterization of neopine was achieved by comparison of the mass spectra and GC retention times of the trimethylsilyl derivative. The presence of neopine in the urine of pharmaceutical codeine users was attributed to the metabolism of codeine through a double bond migration in ring C, from the 7–8 to the 8–14 position. The potential use of the alkaloid as a confirmation marker of opium and/or pharmaceutical codeine use and the ability to differentiate these from heroin use has been discussed.

Keywords: Neopine; Opium use marker; Codeine metabolite; Gas chromatography-mass spectrometry

Amino acid analysis by using comprehensive two-dimensional gas chromatography by Renuka Mayadunne; Thuy-Tien Nguyen; Philip J. Marriott (pp. 836-847).

The separation characteristics of alkylchloroformate-derivatised amino acids (AAs) by using comprehensive two-dimensional gas chromatography (GC×GC) is reported. The use of a low-polarity/polar column set did not provide as good a separation performance as that achieved with a polar/non-polar column set, where the latter appeared to provide less correlation over the separation space. The degree of component correlation in each column set was estimated by using the correlation coefficient (r²; for ¹t_R and ²t_R data) with the low-polarity/polar and polar/low-polarity sets returning correlation coefficients of 0.86, and 0.00 respectively, under the respective conditions employed for the experiments. The 1.5-m non-polar ²D column (0.1-mm ID; 0.1-μm film thickness) gave peak halfwidths of the order of 50–80 ms. Linearity of detection was good, over a three order of magnitude concentration range, with typical lower detection limit of ca. 0.01 mg L⁻¹, compared with 0.5 mg L⁻¹ for normal GC operation with splitless injection. The method was demonstrated for analysis of AAs in a range of food and beverage products, including wine, beer and honey. The major AA in these samples was proline. The Heineken beer sample had a relatively more complex and more abundant AA content compared with the other beer sample. The wine and honey samples also gave a range of AA compounds. Repetition of the sample preparation/analysis procedure for the honey sample gave acceptable reproducibility for individual AAs.

Keywords: Comprehensive two-dimensional gas chromatography; Amino acids; Derivatisation; GC/MS; GC×GC–TOFMS

In-capillary preconcentration of proteins for capillary electrophoresis using a cellulose acetate-coated porous joint by Xing-Zheng Wu; Rui Umeda (pp. 848-852).

This work describes the in-capillary preconcentration of proteins using a cellulose acetate-coated porous joint. The capillary wall near the inlet end of a capillary was made porous by HF etching. During the etching process, a voltage was applied across the capillary wall and the electric current across it was monitored. As the current passed through the capillary wall, it became porous. A solution of cellulose acetate in acetone was added to the etched porous joint. After the acetone was evaporated off, a cellulose acetate-coated porous joint was formed. To preconcentrate the protein ions, an electric voltage was applied between the inlet end of the capillary and the coated porous joint; the protein ions electromigrated to the porous joint but could not pass through it, while the buffer ions could pass easily through the joint. After allowing a certain amount of time for protein preconcentration, a separation voltage was applied across the two ends of the capillary, and normal capillary electrophoresis was carried out. The preconcentration factors for cytochrome c, lysozyme, ribonuclease, and chymotrypsinogen were 65, 155, 705, and 800, respectively. The cellulose acetate-coated porous joint was shown to be strong and stable over time, and was used to analyze trace proteins and macromolecules in biological samples.

Keywords: In-capillary preconcentration; Protein; Capillary electrophoresis; Porous joint; Cellulose acetate coating

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Analytical and Bioanalytical Chemistry (v.382, #3)

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Analytical and Bioanalytical Chemistry (v.382, #3)