Separation Methodologies Research Articles

Zooming in: Fractionation strategies in proteomics

The recent development of mass spectrometry, i.e., high sensitivity, automation of protein identification and some post-translational modifications (PTMs) significantly increased the number of large-scale proteomics projects. However, there are still considerable limitations as none of the currently available proteomics techniques allows the analysis of an entire proteome in a single step procedure. On the other hand, there are several successful studies analyzing well defined groups of proteins, e.g., proteins of purified organelles, membrane microdomains or isolated proteins with certain PTMs. Coupling of advanced separation methodologies (different prefractionation strategies, such as subcellular fractionation, affinity purification, fractionation of proteins and peptides according to their physicochemical properties) to highly sensitive mass spectrometers provides powerful means to detect and analyze dynamic changes of low abundant regulatory proteins in eukaryotic cells on the subcellular level. This review summarizes and discusses recent strategies in proteomics approaches where different fractionation strategies were successfully applied.

Free‐Standing Gold Nanoparticle Membrane by the Spontaneous Reduction of Aqueous Chloroaurate Ions by Oxyethylene‐Linkage‐Bearing Diamine at a Liquid–Liquid Interface

The one‐step synthesis of a free‐standing, robust gold nanoparticle membrane (see Figure) via the spontaneous reduction of aqueous chloroaurate ions by oxyethylene‐bearing diamine molecules in chloroform at the liquid–liquid interface is described. Such simply assembled membranes may have important applications in separation methodologies and as surfaces for enzyme, DNA, and cell immobilization.

Recent advances in capillary electrophoresis and capillary electrochromatography of pollutants.

Recent advances in capillary electrophoresis (CE) and capillary electrochromatography (CEC) separation, detection and sample preparation methodologies applied for the determination of a variety of pollutants is overviewed. The reviewed literature illustrates the wide range of CE applications, indicating the continuing interest in CE and CEC in the environmental field.

Results of the GEP-ISFG collaborative study on two Y-STRs tetraplexes: GEPY I (DYS461, GATA C4, DYS437 and DYS438) and GEPY II (DYS460, GATA A10, GATA H4 and DYS439)

A collaborative exercise was carried out by the Spanish and Portuguese ISFG Working Group (GEP-ISFG) in order to evaluate the performance of two Y-chromosome STR PCR tetraplexes, which include the loci DYS461, GATA C4, DYS437 and DYS438 (GEPY I), and DYS460, GATA A10, GATA H4 and DYS439 (GEPY II). The participating laboratories were asked to type three samples for the eight markers, using a specific amplification protocol. In addition, two control samples, with known haplotypes, were provided. The results obtained by the 13 different participating laboratories were identical, except for two laboratories that failed to type correctly the same two samples for GATA C4. By sequence analyses, two different GATA C4 allele structures were found. One control sample (allele 21) and two questioned samples (allele 22, correctly typed by all the laboratories, and allele 25) presented the following repeat structure: (TCTA) 4(TGTA) 2(TCTA) 2(TGTA) 2(TCTA) n , but different from the one found for allele 26 in one sample included in this exercise, as well as in the second control sample (allele 23), namely (TCTA) 4(TGTA) 2(TCTA) 2(TGTA) 2(TCTA) 2(TGTA) 2(TCTA) n . The collaborative exercise results proved that both Y-tetraplexes produce good amplification results, with the advantage of being efficiently typed using different separation and detection methodologies. However, since GATA C4 repeat presents a complex structure, with alleles differing in sequence structure, efficient denaturing conditions should be followed in order to avoid typing errors due to sizing problems.

Platinum Group Metals in Catalysis: Fabrication of Catalysts and Catalyst Precursors

This short review article presents an overview of the preparation of platinum group metal complexes that are utilized as catalysts or catalyst precursors. The organization of this article begins with a description of the sources of the platinum group metals, goes through their basic refining and separation methodologies, and then into each individual metal's elaboration into value-added coordination and organometallic complexes that have demonstrated applications in homogeneous catalysis. The objective of this article is to explain how these catalysts are synthesized and the wide variety of organic transformations that are catalyzed by platinum group metal complexes. One conclusion that can be drawn from the article is that the further elaboration of the complexity of platinum group metal catalysts goes hand-in-hand with the selectivity of the particular organic transformation being catalyzed.

Advances in greener separation processes ? case study: recovery of chlorinated aromatic compounds

Effective waste management must address waste avoidance, waste reuse, waste recovery and, as the least progressive option, waste treatment. Separation methodologies fit well within a modern green processing ethos, serving to isolate, concentrate and recover valuable material from toxic waste (notably aqueous) streams. An overview is provided of the established separation techniques that focuses on organic contaminants and the associated remediation objectives. The Case Study chosen to illustrate the role separation has to play in environmental pollution control deals with the removal of toxic chloroarenes from wastewater. This is a topic of growing concern as governmental legislation focuses on a substantial reduction in the emission of a broad range of chlorinated compounds. The sources and environmental impact associated with chloro-emissions and the existing treatment technologies are presented and discussed. Adsorption represents the most widely applicable non-destructive control technology, offering the possibility of recovery/recycle. Taking adsorption on activated carbon as a standard, the performance of novel catalytically generated structured carbon is evaluated and shown to outperform activated carbon in batch and semi-batch operation. The level of uptake is markedly higher on carbon surfaces that have been functionalised by acid washing and partial oxidation. The carbon nanofilament adsorbent bed exhibits lower back pressures when treating a continuous contaminant flow, separation of the liquid from the solid in batch operation is more facile and a chloroarene recovery/concentration is possible in polar solvents, a combination of factors that facilitates cleaner separation/recycle.

Selenium in plants by mass spectrometric techniques: developments in bio-analytical methods : Plenary Lecture

Various plants can accumulate Se up to the thousands of ppm. These are called accumulators and they have potential to remediate areas contaminated with this metalloid. Some of them, like Arabidopsis thaliana or Brassica juncea (Indian mustard) have been investigated in terms of the Se metabolic pathway. To date few studies have been done for selenium speciation with most studies reporting total selenium concentration in various parts of the plant. This present report summarizes some of the studies carried out in terms of: extraction of Se species, cleaning procedures, separation methodologies and mass spectrometric techniques employed. The use of inductively coupled plasma mass spectrometry (ICP-MS) in conjunction with liquid or gas chromatographic techniques (HPLC and GC) as separation techniques provide an attractive methodology for determining Se species in plants. Some of the species produced by the plant, such as Se-methylselenocysteine or Se-methylselenomethionine can be identified at ppb levels by RP-HPLC-ICP-MS, while others needed to be further characterized by ES-MS. The coupling of GC-ICP-MS using solid phase microextraction (SPME) as sample preparation system is also evaluated for the determination of the Se/S volatile species in the head-space of Brassica juncea seedlings. Detection limits on the ppt level (7–300 ppt depending on the species) and adequate precision for Se and S species suggest GC-ICP-MS as an excellent methodology for determination of volatiles with minimum sample preparation.

Potential of Chemiluminescence and Bioluminescence in Organic Analysis

The possibilities offered by chemiluminescence (CL)-based analytical techniques apparently still remain unfamiliar to the average analytical researcher. Some of the advantages are that only minimal analytical instrumentation is required and because no external light source is needed, the optical system is quite simple. Hence strong background light levels are excluded leading to improved detection limits by this so-called dark-field technique. CL is observed in solid-, gas- and liquid-phase reactions and all have been used analytically. In solution, the most frequently used systems involve the organic reagents: luminol and its derivatives, acridinium esters, lophine, peroxyoxalates, etc. Many important applications have been established in recent years for the determination of organic compounds, biomolecules, drugs and carcinogenics in a variety of environmental and clinical matrices. As in fluorescence detection, by applying derivatization techniques it is possible to convert a molecule, which does not possess the ability to participate in a CL system, into another specie featuring this property. The rapid development of immobilization techniques has considerably enhanced the applications of CL, especially in flow injection analysis (FIA), in immunoassay and in the development of CL-based sensors. Also, the combination of CL-based detection employing prior separation methodologies such as high performance liquid chromatography (HPLC) or capillary electrophoresis (CE) has proved to offer promising analytical possibilities, providing excellent analytical sensitivity and selectivity, and allowing the resolution and quantification of various analytes in a complex mixture. This review intends to show general principles and characteristics of the technique which make it very useful in organic analysis.

Application of reversed phase high performance liquid chromatography for subproteomic analysis of cardiac muscle

The application of protein separation methodologies, such as reversed phase chromatography, should allow differential separation of the proteome, or at least specific subproteomes, comparable to that achieved by two-dimensional electrophoresis (2-DE). A rapid sequential protein extraction method (termed "IN Sequence") was developed to isolate three distinct subproteomes of cardiac muscle. Two subproteomes, those enriched for the cytoplasmic or myofilament proteins, can be separated by either reversed phase high performance liquid chromatography (RP-HPLC) or 2-DE. Reversed phase HPLC of the myofilament protein enriched extract was optimized for resolution and peak numbers by altering flow rate, gradient rate and the organic modifiers, isopropanol and acetronitrile. The myofilament protein enriched extract from failing swine heart, due to coronary artery ligation (LAD), was compared to the extract from a sham operated animal (SHAM). The HPLC chromatograms of these extracts were similar, but distinctive in many regions. The HPLC fractions, collected within some of these distinct regions of the chromatograms were analyzed using peptide mass fingerprinting - mass spectrometry and immunoblot analysis. Two myofilament proteins, troponin T and myosin heavy chain, were identified and found differentially modified in the SHAM and LAD hearts. Both troponin T and myosin heavy chain are problematic proteins for 2-DE, but yet they were resolved by reversed phase chromatography. Therefore, RP-HPLC can be used in conjunction with 2-DE to enhance protein separation of myofilament protein subproteome.

Plastic Microfluidic Systems for High-Throughput Genomic Analysis and Drug Screening

Plastic Microfluidic Systems for High-Throughput Genomic Analysis and Drug Screening

PCR template preparation for capillary DNA sequencing.

Fluorescence-based capillary DNA sequencing has facilitated the early completion of several complex sequencing projects. While capillary systems offer great benefits in terms of ease of use and automation, we find that they are sufficiently different from slab gel separation methodologies, demanding re-examination of the protocols used to generate and use DNA sequencing templates. We have recently initiated a large-scale Human Open Reading Frame EST project involving 30 laboratories feeding 11 MegaBace 1000 capillary sequencers. The group has already produced more than 300,000 valid sequences. The most successful template preparation protocol we have found is described here. We have found that a crucial step is the standardization of the quantity and quality of the templates, which have been achieved by overnight bacterial culture followed by PCR using limiting amounts of primers. Using this protocol, there is no need for post-PCR purification, and the final preparation cost is US $0.09/template. After sequencing 10,848 templates using this protocol, 78% of the reads were accepted (after discarding vectors without inserts and inserts smaller than 100 nucleotides), and 85% of the total number of bases had Phred scores of 15 or above.

Plastic Microfluidic Systems for High-Throughput Genomic Analysis and Drug Screening

Genomic analysis and drug discovery depend increasingly on rapid, accurate analysis of large sets of sample and extensive compound collections at relatively low cost. By capitalizing on advances in microfabrication, genomics, combinatorial chemistry, and assay technologies, new analytical systems are expected to provide order-of-magnitude increases in analysis throughput along with comparable decreases in per-sample analysis costs. ACLARA’s single-use, plastic LabCard systems, which transport fluids between reservoirs and through interconnected microchannels using electrokinetic mechanisms, are intended to address these analytical needs. These devices take advantage of recent developments in microfluidic and microfabrication technologies to permit their application to DNA sequencing; genotyping and DNA fragment analysis, as well as pharmaceutical candidate screening, and preparing biological samples for analysis. In a parallel effort, ACLARA has developed a new class of reporter molecules that are particularly well suited to capillary electrophoretic analysis. These electrophoretic mobility tags, called eTag reporters, can be used to uniquely label multiplexed sets of oligonucleotide recognition probes or proteins, thereby permitting traditionally homogeneous biochemical reporter assays to be multiplexed for CE analysis. Biochemical multiplexing is key to achieving new thresholds in analytical throughput while maintaining economically viable formats in many application areas. ACLARA’s microfluidic, lab-on-a-chip concept promises to revolutionize chemical analysis, similar to the way miniaturization revolutionized computing, making tools continually smaller, more integrated, less expensive, and higher performing. Microfluidic devices are made up of interconnected networks of microchannels and tiny volume reservoirs in which all the processes required for single analyses may be miniaturized, integrated, and automated within a single substrate the size of a human hand or smaller. However, LabCard devices are more than tiny replicates of existing equipment. They are characterized by high speed, parallel analysis using designs devised to eliminate sample cross contamination while automating processes that are otherwise undesirably cumbersome at the macro laboratory scale. Electrophoresis is one of the most widely used analytical separation methodologies for life science research. Electrophoresis refers to the movement of a charged molecule under the influence of an electric field. Electrophoresis can be used to separate molecules that have different charge-to-mass ratios such as proteins; or, molecules that have similar charge-to-mass ratios but different masses such as DNA fragments. In recent years, the development of capillary electrophoresis (CE), which is performed in a fused silica capillary filled with a buffer or polymer solution, has increased the speed of analytical separations. Planar, microchannel devices offer further improvements over capillary electrophoresis. In general, both electrophoresis and electroosmosis occur when a high electric field is applied along a microchannel (Figure 1). In practice, these effects can be comparable in magnitude, in which case ions of one charge move rapidlyat velocities of millimeters per second-while those of opposite charge move backward or slowly forward, depending upon which of the two effects dominates. The amount of fixed charge on the inner surface of a channel can be controlled by pH, specific adsorption of charged species onto the surface, or surface chemical modification. Therefore, the contribution of electroosmosis (the pumping mechanism) can be tuned relative to electrophoresis (the separating mechanism). The capability to transport and separate exceptionally small liquid volumes with precise electrical control is a powerful tool, which is complemented by an additional benefit of using microchannels with micrometer cross-sections. While electrophoresis channels are of similar cross section in both capillaries and microchannel devices, the planar format of the microchannel devices enables structures more complex than a single, non-intersecting channel to be used. Two or more channels can intersect to

Capillary electrophoresis in mixed aqueous-organic media: Effect of tetrahydrofuran on mobilities, dissociation constants and separation of a series of quinolones

The influence of the composition of aqueous-tetrahydrofuran mixtures on the electrophoretic behaviour of nine quinolones was studied in order to predict the optimum conditions for their separation. The effect of pH, pK a and activity coefficients on electrophoretic behaviour was established. Standard pH values for buffer solutions of the NIST scale as previously determined in THF-water were used in accordance with IUPAC rules. pH was therefore measured for these media as for water. The pK a values of quinolones were determined from the electrophoretic mobilities-pH data pairs in different THF-water mixtures. The electrophoretic resolution between pairs of substances was then predicted in order to obtain the optimal experimental conditions for separation methodologies.

Bioanalytical Applications of Accelerator Mass Spectrometry for Pharmaceutical Research

Accelerator mass spectrometry (AMS) is a mass spectrometric method for quantifying isotopes. It has had great impact in the geosciences and is now being applied in the biomedical fields. AMS measures radioisotopes such as 14C, 3H, 41Ca, and 36Cl, and others, with attomole sensitivity and high precision. Its use is allowing absorption, distribution, metabolism and elimination studies, as well as detailed pharmacokinetics, to be carried out directly in humans with very low chemical or radiological hazard. It is used in combination with standard separation methodologies, such as chromatography, in identification of metabolites and molecular targets for both toxicants and pharmacologic agents. AMS allows the use of very low specific activity chemicals (< 1 mCi/mmol), creating opportunities to use compounds not available in a high specific activity form, such as those that must be biosynthesized, produced in combinatorial libraries, or made through inefficient synthesis. AMS is allowing studies to be carried out with agents having low bioavailability, low systemic distributions, or high toxicity where administered doses must be kept low (<1 microg/kg). It may have uses in tests for idiosyncratic metabolism, drug interaction, or individual susceptibility, among others. The ability to use very low chemical doses, low radiological doses, small samples and conduct multiple dose studies may help move drug candidates into humans faster and safer than before. The uses of AMS are growing and its potential for drug development is only now beginning to be realized.

Prediction of electrophoretic mobilities in non-aqueous capillary electrophoresis: Optimal separation of quinolones in acetonitrile–water media

A model of electrophoretic behaviour is used to predict the optimum conditions for the separation of a series of quinolones zwitterionic substances in mixtures of acetonitrile–water up to 30% (w/w) of acetonitrile. The effect of pH, p K a, the electrophoretic mobility of protonated and anionic species, and activity coefficients on the electrophoretic behaviour of quinolones is considered. The model proposed allows the resolution between substances in acetonitrile–water mixtures to be predicted from a few experimental data and thus permits one to obtain the best experimental conditions for separation methodologies.

Recent advancements in amino acid analysis using capillary electrophoresis

Recent advances in the analysis of amino acids using capillary electrophoresis are addressed. This area of research continues to receive increased attention as is evident from the 62 references reviewed. This review discusses current detection strategies including UV absorbance, laser-induced fluorescence, electrochemical, and others. Separation methodologies for both derivatized and underivatized amino acids are reviewed. Both direct and indirect enantiomeric resolution of amino acids are addressed. Applications utilizing capillary electrophoresis for the analysis of amino acids are discussed. This review covers literature published in 1997 and 1998.

Purification and Recovery of Bulky Hydrophobic DNA Adducts

For many years 32P postlabeling has detected DNA adducts at very low levels and yet has not been able to identify unknown adducts. Mass spectrometry offers substantially improved identification powers, albeit at some loss in detection limits. With this ultimate utilization of mass spectrometry in mind, the current research presents a new method to quantitatively purify bulky hydrophobic DNA adducts at levels that are pertinent to ongoing DNA adduct research in human health and environmental fields. This method was demonstrated with benzo[a]pyrene adducts. Purification was accomplished with the use of small columns (7.5-mm frits) with an 11 mg bed of polystyrene–divinlybenzene beads which retained the adducts while permitting the nonadducted nucleotides to be washed out with water. Subsequently, the adducts were eluted with 50% MeOH and the sample was reduced in volume in an evacuated centrifuge. Purification was demonstrated at adduct levels ranging from 4 adducts in 106 nonadducted nucleotides to 4 in 108. For these levels, analyses by capillary electrophoresis with sample stacking and UV detection determined that recoveries ranged from 91 to 54%, respectively. The adduct quantities isolated should be sufficient to allow the use of current MS capabilities that are linked on-line to separation methodologies such as capillary electrophoresis, capillary electrochromatography, and high-pressure liquid chromatography.

Chromatographic behavior of ionizable compounds in liquid chromatography. Part 2. Standardization of potentiometric sensors and effect of pH and ionic strength on the retention of analytes using acetonitrile–water mobile phases

In this study several properties of acetonitrile–water (ACN–water) mixtures were examined which have an influence on the liquid chromatography (LC) behavior of ionizable compounds, such as Debye–Hückel parameters, pH scale, LC-useful pH range and solvatochromic parameters. Factor analysis techniques were applied to the correlation between standard pH S values of NIST buffer solutions and solvatochromic parameters in acetonitrile (ACN)–water. The relationships obtained allow the calculation of standard pH S values in any ACN–water mixture and thus permit correct pH measurement in these media. Through correct pH, p K a and activity coefficient values, a model describing the effect of pH on retention in LC is established and tested for several diuretic acids and peptide ampholytes using an octadecylsilica column and ACN–water mixtures of 7, 30, 35, 40, 45 and 50% (v/v) ACN as mobile phases. The proposed model uses the pH values in ACN–water mobile phases instead of in water, and takes into account the effect of activity coefficients. The usefulness of the proposed equations is twofold: they permit the determination of p K a of analytes in the hydroorganic media used as mobile phases, and also permit the prediction of the optimum pH for the separation methodologies using a minimum number of measurements. The proposed equations can be combined with the previously derived equation, which relates the retention to the composition of the mobile phase, in order to establish a general model relating elution behavior of the solutes with the significant mobile phase properties: composition, pH and ionic strength.

A new small particle packing for faster analysis with high resolution

The need for fast and efficient separations of complex samples such as pharmaceuticals and biologicals led to the development of fast, efficient, and reproducible 3.5 μm columns. Separations using 3.5 μm column are 30–50% faster at equal plate-count compared to 5 μm columns. The results show that the 3.5 μm columns (100 mm length) give the same efficiency and resolution of drug impurities as the 5 μm columns (150 mm length). For many analytical methods, switching to 3.5 μm columns saves time and reduces costs. Separation methodologies using 5 μm columns are easily modified to accommodate 3.5 μm columns of the same chemistry because efficiency, resolution and sensitivity remain the same. It is shown that 3.5 μm columns have lifetimes comparable to 5 μm columns of the same brand.

Developments in amino acid analysis using capillary electrophoresis.

The current status in the analysis of amino acids using capillary electrophoresis is addressed. This area of biological analysis has received increased attention with more than 200 articles being published in the last five years. This review discusses pre-, post-, and on-column derivatization techniques used to tag amino acids providing a detectable moiety. Several separation methodologies which provided resolution for large sets of amino acids are presented. An overview of advances in the enantiomeric resolution methodologies for amino acids is given. Both direct and indirect enantiomeric separation schemes are summarized. Recent advances in detection strategies for both derivatized and underivatized amino acids are presented. Applications utilizing amino acid analysis by capillary electrophoresis are described. This review covers articles published between 1991 and 1996.