Analytical Separation Techniques Research Articles

Application of silica-based hyper-crosslinked sulfonate-modified reversed stationary phases for separating highly hydrophilic basic compounds

The separation and determination of hydrophilic basic compounds are of great importance in many fields including clinical and biological research, pharmaceutical development and forensic analysis. However, the most widely used analytical separation technique in these disciplines, reversed-phase liquid chromatography (RPLC), usually does not provide sufficient retention for several important classes of highly hydrophilic basic compounds including catecholamines, many drug metabolites and many drugs of abuse. Commonly eluents having little or no organic modifier and/or strong ion pairing agents must be used to achieve sufficient retention and separation. Use of highly aqueous eluents can lead to column failure by dewetting, resulting in poor retention, low selectivity and irreproducibility and slow recovery of performance. The use of a strong ion pairing agent to increase retention renders the separation incompatible with mass spectrometric detection and complicates preparative separations. This paper describes the successful applications of a novel type of silica-based, hyper-crosslinked, sulfonate-modified reversed stationary phase, denoted as −SO 3–HC–C 8–L, for the separation of highly hydrophilic cations and related compounds by a hydrophobically assisted cation-exchange mechanism. Compared to conventional reversed-phases, the −SO 3–HC–C 8–L phase showed significantly improved retention and separation selectivity for hydrophilic amines. Concurrently, due to the presence of both cation-exchange and reversed-phase retention mechanisms and the high acid stability of hyper-crosslinked phases, the separation can be optimized by changing the type or concentration of ionic additive or organic modifier, and by varying the column temperature. In addition, gradients generated by programming the concentration of either the ionic additive or the organic modifier can be applied to reduce the analysis time without compromising resolution. Furthermore, remarkably different chromatographic selectivities, especially toward cationic solutes, were observed upon comparing the −SO 3–HC–C 8–L phase with conventional reversed-phases. We believe that the combination of these two types of stationary phases will be very useful in two-dimensional liquid chromatography.

Two Glutaraldehyde‐Immobilized Trypsin Preparations for Peptide Mapping by Capillary Zone Electrophoresis, Liquid Chromatography, and Mass Spectrometry

Trypsin was immobilized using glutaraldehyde either by covalent attachment to aminopropyl controlled pore glass (CPG) or by direct crosslinking without a carrier. As peptide mapping is a comparative method, reproducibility of the analytical separation techniques (liquid chromatography, HPLC, and capillary zone electrophoresis, CZE) and the proteolyses resulting from both enzyme preparations were evaluated. Elution time reproducibilities of 0.3 and 0.6% were found for HPLC and CZE maps, respectively. Proteolysis reproducibility was tested for each trypsin preparation and compared with solution phase proteolysis. Sequence coverages of ca 65% were obtained from matrix‐assisted laser desorption/ionization−time‐of‐flight (MALDI‐TOF) mass spectral mapping for the two solid phase preparations.

Advances in HPLC detection—towards universal detection

High-performance liquid chromatography has already become one of themost widely applied analytical separation techniques because of its superior performance and reliability, especially in the pharmaceutical, environmental, forensic, clinical, food and flavor sciences. However, the relatively slow progress made in the field of HPLC detection has influenced the general progress and the extent of application of HPLC technology. Due to the lack of a universal detector that can be used for quantitative analysis without the need for an authentic standard, the information obtained from analytical HPLC is heavily limited. However, the needs of pharmaceutical companies (e.g. modern drug discovery research using highthroughput quantification methods for lead discovery) and other industries demand the development of a generic quantitative detection technique that can quickly determine the quantity of a chemical without the need to use an authentic standard and the tedious calibration procedure. Researchers are therefore focusing much of their activity into developing a truly “universal” detector for HPLC [1]. The HPLC detectors reported in peer-reviewed publications so far can be classified into [2]: elemental detectors (atomic absorption/emission, inductively coupled plasma–mass spectrometry and microwave-induced plasma); optical detectors (UV/visible, IR/Raman, optical activity, evaporative light scattering and refractive index); luminescent detectors (fluorescence/phosphorescence, chemiluminescence/bioluminescence); electrochemical detectors (potentiometry, novel material/modified electrodes, array electrodes and pulsed and oscillometric techniques); mass spectrometric detectors (time-of-flight/MALDI, Fourier transform ion cyclotron resonance mass spectrometry, electrospray/thermospray, atmospheric pressure ionization and particle beam); and other detection systems (nuclear magnetic resonance, radioactivity detectors, surface plasmon resonance). It is well known that NMR is a spectroscopic techniquewith a very high information content. Although the LC–NMR technique has been successfully applied to the characterization of drug impurities and synthetic compounds in the form of combinatorial libraries [3], analysts are aware of the very high cost of NMR instrumentation. For this reason, the LC–NMR technique has not gained widespread usage [4]. Attention has also focused on the design of LC–FTIR systems, which are either of the flow-cell or the solvent-elimination type. Problems facing this technique include the limited compatibility of the optimized separation and detection conditions, and the rather complicated interface required. Extensive investigations into the use of RI (refractive index) detectors [5–8], evaporative light scattering detectors (ELSDs) [9–12] and chemiluminescence nitrogen detectors (CLNDs) [13–31] as universal detectors for HPLC have been performed. The RI detector, which measures the change in the refractive index of the column effluent as it passes through the flow-cell, could be a universal detector in theory. The greater the difference in RIs between the sample and the mobile phase, the higher the sensitivity of the system. On the other hand, when analyzing complex mixtures, the sample components may cover a wide range of refractive index values, and the RIs of some of the sample components may closely match that of the mobile phase, making them “invisible” to the detector. The RI detector is a pure differential instrument, and any changes in the eluent composition require the rebalancing of the detector. This factor Anal Bioanal Chem (2008) 390:299–301 DOI 10.1007/s00216-007-1633-0

Theoretical and numerical analysis of temperature gradient focusing via Joule heating

We present a detailed theoretical and numerical analysis of temperature gradient focusing (TGF) via Joule heating-an analytical species concentration and separation technique relying upon the dependence of an analyte's velocity on temperature due to the temperature dependence of a buffer's ionic strength and viscosity. The governing transport equations are presented, analyzed, and implemented into a quasi-1D numerical model to predict the resulting temperature, velocity, and concentration profiles along a microchannel of varying width under an applied electric field. Numerical results show good agreement with experimental trials presented in previous work. The model is used to analyze the effects of varying certain geometrical and experimental parameters on the focusing performance of the device. Simulations also help depict the separation capability of the device, as well as the effectiveness of different buffer systems used in the technique. The analysis provides rule-of-thumb methodology for implementation of TGF into analytical systems, as well as a fundamental model applicable to any lab-on-a-chip system in which Joule heating and temperature-dependent electrokinetic transport are to be analyzed.

Quadrupole magnetic field-flow fractionation: A novel technique for the characterization of magnetic nanoparticles

Quadrupole magnetic field-flow fractionation (MgFFF) is an analytical separation and characterization technique for nano- and micro-sized magnetic particles. It fractionates particles according to their content of magnetite or other magnetic material. The potential and versatility of MgFFF for separation and characterization of magnetic nanoparticles, such as those used for immunospecific labeling of biological cells for magnetic separation, is demonstrated. A broadly polydisperse sample of dextran-coated magnetite nanoparticles was eluted under programmed field decay conditions, and quantitative data concerning the distribution of magnetite content were determined from the elution profile using a data reduction method.

Field-Flow Fractionation: Theory, Techniques, Applications and the Challenges

In this review, field-flow fractionation (FFF) is presented as a versatile and powerful analytical technique for separation and characterization of different sample types. The underlying principles of FFF separation theory including sources of the obstacles and the difficulties as well as the new approaches is also introduced. This paper describes FFF sub-techniques, presents its exemplary application and the future trends in development of FFF techniques.

Advances in the analysis of phenolic compounds in products derived from bees

Honey and propolis are rich in phenolic compounds, which act as natural antioxidants, and are becoming increasingly popular because of their potential role in contributing to human health. These compounds can also be used as indicators in studies into the floral and geographical origin of the honey and propolis themselves. We present here an overview of current analytical methods for measuring polyphenols in honey and propolis. The analytical procedure to determine individual phenolic compounds involves their extraction from the sample, analytical separation and quantification. The techniques reviewed are based on spectrophotometry as well as analytical separation techniques such as gas chromatography, high-pressure liquid chromatography and capillary electrophoresis.

Electrophoretic approaches to the analysis of complex polysaccharides

Complex polysaccharides, glycosaminoglycans (GAGs), are a class of ubiquitous macromolecules exhibiting a wide range of biological functions. They are widely distributed as sidechains of proteoglycans (PGs) in the extracellular matrix and at cellular level. The recent emergence of enhanced analytical tools for their study has triggered a virtual explosion in the field of glycomics. Analytical electrophoretic separation techniques, including agarose-gel, capillary electrophoresis (HPCE) and fluorophore-assisted carbohydrate electrophoresis (FACE), of GAGs and GAG-derived oligosaccharides have been employed for the structural analysis and quantification of hyaluronic acid (HA), chondroitin sulfate (CS), dermatan sulfate (DS), keratan sulfate (KS), heparan sulfate (HS), heparin (Hep) and acidic bacterial polysaccharides. Furthermore, recent developments in the electrophoretic separation and detection of unsaturated disaccharides and oligosaccharides derived from GAGs by enzymatic or chemical degradation have made it possible to examine alterations of GAGs with respect to their amounts and fine structural features in various pathological conditions, thus becoming applicable for diagnosis. In this paper, the electromigration procedures developed to analyze and characterize complex polysaccharides are reviewed. Moreover, a critical evaluation of the biological relevance of the results obtained by these electrophoresis approaches is presented.

Quadrupole magnetic field-flow fractionation for the analysis of magnetic nanoparticles

Field-flow fractionation (FFF) is an analytical scale separation and characterizatio technique for macromolecules and particles. A quadrupole magnetic FFF device has bee constructed for analyzing magnetic nanoparticles. It is shown to give reproducible results and be capable of distinguishing between different lots of a commercial magnetic nanoparticle material.

On‐line sample concentration techniques in capillary electrophoresis: Velocity gradient techniques and sample concentration techniques for biomolecules

Methods with a high sensitivity and high separation efficiency are goals in analytical separation techniques. On-line sample concentration techniques in capillary electrophoresis (CE) separations have rapidly grown in popularity over the past few years because they achieve this goal. This review describes the methodology and theory associated with a number of different techniques, including electrokinetic and chromatographic methods. For small molecules, several on-line concentration methods based on velocity gradient techniques are described, in which the electrophoretic velocities of the analyte molecules are manipulated by field amplification, sweeping, and isotachophoretic migration, resulting in the on-line concentration of the analyte zones. In addition, the on-line concentration methods for macromolecules are described, since the techniques used for macromolecules (DNAs and proteins), are different from those for small molecules, with respect to either mechanism or methodology. Recent studies relating to this topic are also discussed, including electrophoretic and chromatographic techniques on capillary or microchip.

Hyphenating liquid phase separation techniques with mass spectrometry: on-line or off-line

The advantages of hyphenating analytical separation techniques to mass spectrometers have been recognised since the 1970s. The first attempts to couple liquid phase separations to mass spectrometry(MS) were problematic, but the development of electrospray ionisation (ES) in 1984 facilitated the production of routinely used, commercial interfaces. This review considers the most recent major developments in hyphenating liquid chromatographic and electrophoretic separations to mass spectrometry, and considers the role the post genomic sciences have played in driving them. It also considers why, despite all of the advances in hyphenation, the future appears to hold a major role for off-line analysis.

Concept of Selectivity and Its Meaning in Analytical Separation Techniques

The ambiguity of the use of selectivity concept in currently available analytical separation techniques (including chromatographic techniques) is pointed out. In all cases when this term is applied, it should be specified for which analyte (or analytes) and with respect to which concomitant component (or components) selectivity is considered in order to eliminate uncertainties of this kind. It should also be specified whether the analysis as a whole, its individual stages, chromatographic systems, or their particular elements (such as columns or phases used in them) are characterized. Corresponding criteria should also be specified for derived terms such as selectivity control, selectivity improvement, etc.

Thin-Layer Chromatography: The "Eyes" of the Organic Chemist

Thin-layer chromatography (TLC) is a versatile, inexpensive, and rapid analytical separation technique available to the organic chemist. This inquiry-based experiment investigates the governing principles as to why and how the technique works. Students are led into expressing the position a sample travels in a systematic manner, the retention factor, Rf. It is found that compounds that are hydrogen bond acceptors (ethers and esters) are less strongly absorbed by the silica surface and possess larger Rf values. Compounds such as alcohols and carboxylic acids which are strong hydrogen bond donors and acceptors are more strongly absorbed by the surface and have smaller Rf values. A qualitative TLC study is also performed on compounds possessing different functional groups. The order of increasing functional group polarity is observed to be: ether < ester < ketone < aldehyde < amine < alcohol < carboxylic acid and directly corresponds to their observed Rf values. Knowledge gained in earlier sections is utiliz...

Application of Conducting Polymers in Chemical Analysis

AbstractFor Abstract see ChemInform Abstract in Full Text.

Application of Conducting Polymers in Chemical Analysis

This paper presents recent achievements in the field of analytical application of conducting polymers, most of which were published in the last five years. Based on 171 references mainly to original papers in international scientific journals, the applications of conducting polymers in the design of electrochemical and optical sensors, enzymatic biosensors and immunosensors are reviewed together with applications in analytical preconcentration procedures and high-performance separation techniques.

Pharmaceutical and biomedical applications of chiral capillary electrophoresis and capillary electrochromatography: an update.

Capillary electrophoresis is often considered an ideal method for the chiral analysis of compounds due to the high separation power of the technique and has therefore found widespread acceptance for the analysis of drugs and pharmaceuticals. In contrast, capillary electrochromatography is still more or less in an infancy state searching for its place among the analytical separation techniques although interesting applications have been published. The present review summarizes recent developments and applications of chiral pharmaceutical analysis by electromigration techniques published in 2002 and early 2003.

Analytical separation techniques for the determination of chemical warfare agents.

Today, the determination of chemical warfare agents (CWAs) is an important area of application in analytical chemistry. Chromatographic, capillary electrophoretic and mass spectrometric techniques are primarily used for the identification and quantification of a broad field of classical CWAs in environmental samples and neutralization masses, obtained after destruction of CWAs. This overview is illustrative for the state of the art and mainly focuses on the literature published since 1996.

Development of comparative methods using gas chromatography–mass spectrometry and capillary electrophoresis for determination of endocrine disrupting chemicals in bio-solids

Two analytical separation techniques are being investigated for their potential in determining a wide range of endocrine disrupting chemicals (EDCs) in the environment. Capillary electrophoresis (CE) in the micellar mode in conjunction with a cyclodextrin (CD) modifier is shown to have potential for determination of alkylphenol breakdown products. Gas chromatography with mass spectrometric (GC–MS) detection is being utilised for validation of the CE method development and in addition as a separation technique to optimise preconcentration using solid-phase extraction. GC has demonstrated potential for the separation of 26 priority chemicals suspected as being endocrine disrupting compounds. The challenge of the method development process lies in the fact that these compounds are of differing polarities, size and charge and therefore are difficult to separate in a single run. Capillary electrophoresis in the CD–MEKC (micellar electrokinetic chromatography) mode is showing potential in this regard. Limits of determination are in the low mg/l range for CE and GC, however, using preconcentration it is possible to improve detection sensitivity with >80% recovery for some analytes and up to 100% recovery for most target species.

Capillary Electrophoresis of Water-Soluble Vitamins: An Undergraduate Experiment

Capillary electrophoresis (CE) is a relatively new analytical separation technique that is not usually introduced in the undergraduate analytical chemistry curriculum. The technique’s growing popularity in research, industrial, and commercial laboratories, however, should be a reason to consider its introduction at this level. Here, we describe an exercise utilizing capillary zone electrophoresis and micellar electrokinetic chromatography. This exercise provides a suitable introduction to capillary electrophoresis and illustrates the mechanism for the separation of ionized and nonionized water-soluble vitamins (B1, B2 phosphate, B3 niacinamide, and B12). Joule heating can also be easily introduced as part of the exercise.

Evaluation of micro-electrospray ionization with ion mobility spectrometry/mass spectrometry

In recent years, the resolving power of ion mobility instruments has been increased significantly, enabling ion mobility spectrometry (IMS) to be utilized as an analytical separation technique for complex mixtures. In theory, decreasing the drift tube temperature results in increased resolution due to decreased ion diffusion. However, the heat requirements for complete ion desolvation with electrospray ionization (ESI) have limited the reduction of temperatures in atmospheric pressure ion mobility instruments. Micro-electrospray conditions were investigated in this study to enable more efficient droplet formation and ionization with the objective of reducing drift tube temperatures and increasing IMS resolution. For small molecules (peptides), the drift tube temperature was reduced to ambient temperature with good resolution by employing reduced capillary diameters and flow rates. By employing micro-spray conditions, experimental resolution values approaching theoretically predicted resolution were achieved over a wide temperature range (30 to 250 °C). The historical heat requirements of atmospheric pressure IMS due to ESI desolvation were eliminated due to the use of micro-spray conditions and the high-resolution IMS spectra of GLY-HIS-LYS was obtained at ambient temperature. The desolvation of proteins (cytochrome c) was found to achieve optimal resolution at temperatures greater than 125 °C. This is significantly improved from earlier IMS studies that required drift tube temperatures of 250°C for protein desolvation.