Analytical and Bioanalytical Chemistry (v.373, #6)

The continued evolution of hyphenated instruments by Jonathan V. Sweedler (321-322).

On-line coupling of miniaturized solid-phase extraction methods and microcolumn separation techniques are reviewed. A conventional solid-phase micro-extraction device and fiber-in-tube solid-phase extraction was employed as the sample preparation method. In the fiber-in-tube technique, a novel fibrous polymeric material was introduced as the extraction medium for the microscale sample preparation step which could be directly coupled with microcolumn liquid-phase separation systems, such as microcolumn liquid chromatography, capillary electrophoresis, and capillary electrochromatography. The applications of these hyphenated systems, consisting of the sample preparation and separation processes, are also reviewed.
Keywords: Fiber-in-tube solid-phase extraction (FIT-SPE) Sample preparation Microcolumn separation Hyphenated techniques Miniaturization

Capillary electrophoresis with wavelength-resolved laser-induced fluorescence detection by Xin Zhang; Jeffrey N. Stuart; Jonathan V. Sweedler (332-343).
Capillary electrophoresis (CE) enables rapid separations with high separation efficiency and compatibility with small sample volumes. Laser-induced fluorescence detection can result in extremely low limits of detection in CE. Single-channel fluorescence detection, however, furnishes little qualitative information about a species being detected, except for its CE migration time. Use of multidimensional information often enables unambiguous identification of analytes. Combination of CE with information-rich wavelength-resolved fluorescence detection is analogous with ultraviolet–visible diode-array detection and furnishes both qualitative and quantitative chemical information about target species. This review discusses recent advances in wavelength-resolved laser-induced fluorescence detection coupled with CE, with an emphasis on instrument design.
Keywords: Review Capillary electrophoresis Laser-induced fluorescence Wavelength-resolved fluorescence

The atomic-emission detector in gas chromatography is enormously versatile in applications in analytical chemistry. Its unique properties of high selectivity for most elements and low limits of detection combine to make it the preferred detector for many analytical problems. In this review the stress is laid on the possibility of using it for compound-independent calibration, for determination of the empirical formula of an unknown analyte, for isotope-selective detection, and on derivatization to give AED-active derivatives with advantageous detection properties. Both metals and non-metals are considered and examples of the use of atomic emission detection in real-world analysis are discussed.
Keywords: Atomic-emission detector Compound-independent calibration Empirical formula determination Isotope analysis Derivatization

Modulation techniques and applications in comprehensive two-dimensional gas chromatography (GC×GC) by Matthias Pursch; Kefu Sun; Bill Winniford; Hernan Cortes; Andy Weber; Terry McCabe; Jim Luong (356-367).
More than a decade after Phillips' first published work this article reviews recent developments in comprehensive two-dimensional gas chromatography (GC×GC). Special attention is devoted to the further development and diversity of modulation devices. These include heated sweepers, cryofocused modulators, and a variety of diaphragm valve-switching strategies. It is demonstrated that all modulation approaches can be very well suited to GC×GC, depending on the particular application. Diaphragm-valve modulation is very powerful for volatile organic compounds. Slotted heater and cryofocused modulation are preferred for samples that contain non-volatile components. Applications ranging from petroleum to environmental and biological samples are illustrated. Extension of the technique to GC×GC–mass spectrometry (MS) is also discussed and trends for future research activity are pointed out.
Keywords: Multidimensional gas chromatography High-speed analysis High resolution Comprehensive Complex mixtures

Electronic spectral detection in liquid chromatography by Wilton R. Biggs; John C. Fetzer (368-377).
The use of absorbance and fluorescence spectrometers as liquid chromatography detectors is described. These detectors offer sensitive detection that can also identify the peaks in a chromatogram. Many examples, mainly from the separation of polycyclic aromatic hydrocarbons, are given that show the selectivity and usefulness of these detectors.
Keywords: HPLC detection UV absorbance Fluorescence Full spectrum Diode array detector

Keywords: FTICR High resolution Proteomics Combinatorial libraries High through

On-line electrochemistry – MS and related techniques by Georg Diehl; Uwe Karst (390-398).
This review summarizes publications on the on-line coupling of electrochemistry with mass spectrometry. After a brief historic introduction it is divided into three parts, organized in order of increasing complexity of the experimental arrangement. The first section deals with the use of the electrospray ion source as an electrochemical reactor for oxidation or reduction reactions. It is followed by the second part which covers the hyphenation of different kinds of electrochemical flow cell with a variety of ionization interfaces. The last section focuses on the on-line coupling of chromatographic techniques with electrochemical flow cells and mass spectrometry.
Keywords: Electrochemistry Mass Spectrometry Hyphenated Techniques Liquid Chromatography Electrospray

A complementary metal oxide semiconductor (CMOS)–capillary array electrophoresis (CAE) system has been used for DNA analysis. Because of its compactness and multiplex capability, the CAE-CMOS microchip is very suitable for the construction of a miniaturized high-throughput system for bioassays. Use of simultaneous laser-beam focusing on to the capillary array and a microscope objective contributed to the construction of the compact CMOS microchip–CAE system. To test the constructed system 100-base-pair (bp) DNA ladders and Hind III digest λ DNA were separated in poly(vinylpyrrolidone) (PVP) sieving matrix. The miniaturized and integrated CMOS microchip system used in this work had great potential for combination with a variety of microfabricated devices for biomedical research.
Keywords: CMOS microchip Capillary array electrophoresis Miniaturization DNA separation

Multidimensional approaches in biochemical speciation analysis by Joanna Szpunar; Ryszard Łobiński (404-411).
An understanding of the mechanisms controlling the essentiality and toxicity of trace elements in biological systems at the molecular level depends critically on the possibility of the identification, characterization, and quantification of chemical forms of these elements involved in life processes.Hyphenated techniques based on the combination of (electro)chromatography with ICP MS have become a routine tool for the analysis for metallospecies present in biological tissues. Finer analytical information on the true (down to individual species) speciation of trace elements in living organisms can be obtained by adding additional dimensions to the separation and detection steps, consisting of a sequential use of different HPLC separation mechanisms and capillary electrophoresis at the separation level, and of the use of electrospray MS, including collision induced dissociation MS, on the detection level. The value of the instrumental analytical data is decisively enhanced by the complementary use of molecular biology approaches involving gene identification, cloning and in vitro reproduction of the metal-controlled processes. A brief summary of the recent progress in biochemical speciation analysis is presented in the context of the latest research carried out in the authors' laboratory.
Keywords: Speciation Multidimensional chromatography Mass spectrometry Biological samples

Multielement trace-element speciation in metal-biomolecules by chromatography coupled with ICP–MS by C. Ferrarello; M. Fernández de la Campa; A. Sanz-Medel (412-421).
The state-of-the-art of multi-element speciation in biological materials by hybrid techniques with inductively coupled plasma–mass spectrometric (ICP–MS) detection is critically reviewed with special attention to multi-metal speciation in metallothioneins (MT) and metallothionein-like proteins (MLP). The most common separation techniques used for the purpose include conventional size-exclusion chromatography (SEC) (with off-line detection) and anion-exchange (AE) or reversed-phase (RP) HPLC coupled on-line to ICP–MS. Advantages and limitations of the different commercial element-selective ICP–MS detectors, including the quadrupole (Q), double focusing (DF), and time-of-flight (TOF) mass analysers, are discussed. Procedures reported for speciation analysis, by use of hybrid techniques, for multi-metal speciation in mussel MLP and rabbit liver MT are illustrated with special emphasis on work performed in the authors’ laboratory.
Keywords: Metal-biomolecules speciation Metallothionein-like proteins Inductively coupled plasma–mass spectrometry

The complexity of selenium speciation in the environment and in living organisms results in broad analytical challenges. The importance of the selective determination of the particular species of this element, to understand its metabolism and biological significance in clinical chemistry, biology, toxicology, and nutrition, calls for state-of-the-art analytical techniques. In this paper hyphenated techniques are evaluated with particular emphasis on interfaced separation with element-selective detection and identification of the selenium compounds detected.
Keywords: Selenium compounds Element-selective detection Hyphenated techniques HPLC-ICP-MS (IC)-ICP-MS GC-AED

Determination of butyltin compounds in environmental samples by isotope-dilution GC–ICP–MS by Ignacio J. Alonso; Jorge Encinar; Pablo González; Alfredo Sanz-Medel (432-440).
Isotope-dilution analysis in combination with GC–ICP–MS detection has been applied to the determination of butyltin species in environmental samples. Different spikes containing the isotopically labeled butyltin species have been synthesized in the laboratory after optimization of the reaction conditions. The isotopic compositions of the tin species in the different spike solutions were determined by GC–ICP–MS after derivatization by aqueous ethylation with sodium tetraethylborate. Reverse isotope-dilution analysis was used for quantitation of the spike solutions by means of natural MBT, DBT, and TBT standards. The mixed spikes were used for simultaneous analysis of MBT, DBT and TBT in the certified reference materials, PACS-2, CRM 462, and CRM 646, with satisfactory results. The excellent agreement of the different speciation results obtained by use of the different spikes is a good indicator of the precision, accuracy, and reliability which can be achieved by using isotope-dilution analysis for trace metal speciation.Application of a double spike containing 119Sn-enriched MBT (79.7 At%), 118Sn-enriched DBT (86.7 At%), and 119Sn-enriched TBT (83.1 At%) also enabled evaluation of the conditions resulting in quantitative extraction of the species from the solid matrix, in combination with possible alterations depending on the different extraction procedures used (mechanical shaking, ultrasounds, and microwaves). Mathematical equations used for this purpose computed the correct species concentrations directly and, additionally, the decomposition factors (from TBT to DBT and from DBT to MBT) after precise measurement of the 119Sn/120Sn and 118Sn/120Sn ratios for all butyltin species by GC–ICP–MS.
Keywords: Butyltin compounds Isotope-dilution analysis Gas chromatography ICP–MS

Recent developments in the coupling of highly selective separation techniques such as capillary electrophoresis (CE) and high-performance liquid chromatography (HPLC) to element-specific and molecule-specific detectors, such as inductively-coupled plasma mass spectrometry (ICP-MS) and electrospray ionization-tandem mass spectrometry (ESI-MS/MS) for the characterization and quantification of metallothioneins (MTs) are critically reviewed and discussed. This review gives an update based on the literature over the last five years. The coupling of CE to ICP-MS is especially highlighted. As a result of progress in new interface technologies for CE-ICP-MS, research topics presented in the literature are changing from "the characterization of interfaces by metallothioneins" to the "characterization of metallothioneins by CE-ICP-MS". New applications of CE-ICP-MS to the analysis of MTs in real samples are summarized. The potential of the on-line isotope dilution technique for the quantification of MTs and for the determination of the stoichiometric composition of metalloprotein complexes is discussed. Furthermore, a selection of relevant papers dealing with HPLC-ICP-MS for MT analysis are summarized and compared to those dealing with CE-ICP-MS. In particular, the use of size-exclusion (SE)-HPLC as a preliminary separation step for metallothioneins in real samples prior to further chromatographic or electrophoretic separations is considered. Additionally, the application of electrospray ionisation-tandem mass spectrometry (ESI-MS/MS) for the identification of metallothionein isoforms following electrophoretic or chromatographic separation is discussed.
Keywords: Metallothionein isoforms; Capillary electrophoresis; High-performance liquid chromatography; Inductively-coupled plasma-mass spectrometry; Electrospray ionization-tandem mass; Hyphenated techniques

HPLC–ICP–MS determination of selenium distribution and speciation in different types of nut by Sasi S. Kannamkumarath; Kazimierz Wrobel; Katarzyna Wrobel; Anne Vonderheide; Joseph A. Caruso (454-460).
In addition to determination of total selenium in nuts, the element distribution among different fractions (lipid extract, low molecular weight, and protein fractions), and speciation analysis were studied. Improved precision for total selenium determination was observed after elimination of lipids. Because selenium was not detected in any of the lipid extracts obtained from the different types of nuts (ICP–MS), in each determination and/or speciation procedure used in this work lipids were extracted (chloroform–methanol, 2:1) and discarded before analysis. In agreement with previously reported data, high selenium levels were found in Brazil nuts (those purchased without shells contained approximately a quarter the content than those purchased with shells) and significantly lower levels in walnuts, cashews, and pecans nuts. Low-molecular-weight compounds were extracted with perchloric acid (0.4 mol L–1) to furnish a fraction containing 3 to 15% of the total selenium in different types of nuts. The proteins were isolated from nut samples by dissolution in 0.1 mol L–1 sodium hydroxide and subsequent precipitation with acetone. They were then dissolved in phosphate buffer pH 7.5. Analysis of protein fractions focused on selenium in two possible states – weakly and firmly bound to proteins. Results obtained for Brazil nuts by size-exclusion chromatography with on-line ICP–MS detection, in the absence and in the presence of β-mercaptoethanol, showed that approximately 12% of total selenium was weakly bound to proteins. To obtain information about firmly bound selenium, the protein extracts were hydrolyzed enzymatically with proteinase K. Speciation was performed by means of ion-pairing HPLC–ICP–MS. The primary species found in all types of nuts was Se-methionine (19–25% of total selenium for different types of nuts).
Keywords: HPLC–ICP–MS Nuts Proteinase K Selenomethionine Speciation

Rapid separation of elemental species by multicapillary GC by B. Rosenkranz; J. Bettmer (461-465).
Multicapillary gas chromatography has been applied to the speciation of organomercury compounds. Basic investigation of the fundamental properties of multicapillary columns, to evaluate their potential and limitations as a rapid separation unit, are presented. For analysis of methylated and ethylated mercury compounds a complete separation can be performed within 45 s under isothermal conditions. The adaptation of the technique for use with a purge and trap system and with an element-selective plasma-emission detector results in a compact and effective system for mercury speciation. Results from analysis of certified reference materials were in good agreement with certified values within the significance levels.
Keywords: Metal speciation Multicapillary gas chromatography Atomic spectrometry

Capillary electrophoresis (CE) mass spectrometry (MS), with its ability to separate compounds present in extremely small volume samples rapidly, with high separation efficiency, and with compound identification capability based on molecular weight, is an extremely valuable analytical technique for the analysis of complex biological mixtures. The highest sensitivities and separation efficiencies are usually achieved by using narrow capillaries (5–50 µm i.d.) and by using sheathless CE-to-MS interfaces. The difficulties in CE-to-MS interfacing and the limited loadability of these narrow columns, however, have prevented CE-MS from becoming a widely used analytical technique. To remedy these limitations, several CE-MS interfacing techniques have recently been introduced. While electrospray ionization is the most commonly used ionization technique for interfacing CE-to-MS, matrix assisted laser desorption ionization has also been used, using both on-line and off-line techniques. Moreover, the high concentration detection limit of CE has been addressed by development of several sample concentration and sample focusing methods. In addition, a wide variety of techniques such as capillary zone electrophoresis, capillary isoelectric focusing, and on-column transient isotachophoresis have now been interfaced to MS. These advances have resulted in a rapid increase in the use of CE-MS in the analysis of complex biological mixtures. CE-MS has now been successfully applied to the analysis of a wide variety of compounds including amino acids, protein digests, protein mixtures, single cells, oligonucleotides, and various small molecules relevant to the pharmaceutical industry.
Keywords: Capillary electrophoresis Electrospray ionization Mass spectrometry Protein analysis

High-performance polymers are complex mixtures of materials of different size and chemical composition and with different end groups and architecture. To determine the molecular heterogeneity of such systems, hyphenation of several techniques is required. The value of coupling mass spectrometry (MS) with separation techniques has already been recognized – such methods have proved to be among the most powerful for molecular characterization of complex polymer systems.The review focuses on matrix-assisted laser desorption/ionization (MALDI) and electrospray ionization (ESI) MS coupled with liquid chromatography (LC). Such hyphenation has been used for most polymer analysis by mass spectrometry coupled with separation techniques. The advantages and/or limitations of these techniques for polymer characterization are discussed. Future prospects are briefly outlined.
Keywords: Liquid chromatography Matrix-assisted laser desorption/ionization Electrospray ionization Polymers Hyphenation

A review is given dealing with commonly used adsorbent materials in ambient air analysis of volatile organic compounds (VOCs). The adsorbents covered in the paper are selected in consideration of their compatibility with thermal desorption. Initially, we discuss the requirements that an adsorbent should fulfill, and useful parameters for the selection and evaluation of an appropriate material. Then, the most important materials are presented considering their properties, advantages, and drawbacks. A few applications are given, but a complete review of sampling techniques and applications dealing with adsorptive enrichment and thermal desorption is beyond the scope of this paper.
Keywords: Adsorptive enrichment Thermal desorption Adsorbents Air analysis

Calmodulin (CaM) was used as an affinity tail to facilitate the purification of the green fluorescent protein (GFP), which was used as a model target protein. The protein GFP was fused to the C-terminus of CaM, and a factor Xa cleavage site was introduced between the two proteins. A CaM-GFP fusion protein was expressed in E. coli and purified on a phenothiazine-derivatized silica column. CaM binds to the phenothiazine on the column in a Ca2+-dependent fashion and it was, therefore, used as an affinity tail for the purification of GFP. The fusion protein bound to the affinity column was then subjected to a proteolytic digestion with factor Xa. Pure GFP was eluted with a Ca2+-containing buffer, while CaM was eluted later with a buffer containing the Ca2+-chelating agent EGTA. The purity of the isolated GFP was verified by SDS-PAGE, and the fluorescence properties of the purified GFP were characterized.
Keywords: Calmodulin GFP Affinity purification C-terminal fusion Factor Xa cleavage

Reversed-phase HPLC of polymer additives with multiple on-line spectroscopic analysis (UV, IR, 1H NMR and MS) by Dave Louden; Alan Handley; Eva Lenz; Ian Sinclair; Steve Taylor; Ian D. Wilson (508-515).
The reversed-phase chromatography of a number of polymer additives has been undertaken with on-line characterisation via a combination of diode array UV, 1H NMR, IR spectroscopy and mass spectrometry. This combination of spectrometers enabled the on-flow collection of full UV, 1H NMR, IR and mass spectra for a range of common polymer additives in amounts ranging from ca. 230 to 900 µg on-column. The practical difficulties associated with multiple hyphenation and potential future developments are discussed.
Keywords: HPLC Polymer additives Multiple hyphenation Spectroscopic characterisation