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Scanning Tunneling Microscopy/Spectroscopy in Analysis of Surfaces
Authors: Dharmadhikari C.V.
Published: September 2006

Scanning tunneling microscopy (STM), scanning tunneling spectroscopy (STS) and related techniques with their unique capability to resolve topological and electronic structures at atomic level have revolutionized the power of experimental techniques in high resolution imaging of surfaces. In STM, a sharp conducting tip is mechanically scanned over a conducting or semi‐conducting sample at the distance of few angstroms from the surface. A voltage bias applied between the tip and the sample leads to flow of tunnel current, which decreases exponentially as the gap increases. By keeping the tunnel current constant with an electronic feedback controller the tip is maintained at a fixed distance during the scanning by means of a 3‐D scanner made of a piezoelectric transducer. The trajectory of the tip then traces out a profile of the surface, including the bumps due to individual atoms. In the context of atomic resolution imaging capability, there are only a few techniques such as scanning transmission electron microscopy (STEM), transmission electron microscopy (TEM) and field ion microscopy (FIM) which perform as well as STM, but they do so under extremely special circumstances. The traditional spectroscopic techniques that can be compared with STS are those of conventional (elastic) electron tunneling spectroscopy (ETS) and inelastic electron tunneling spectroscopy (IETS), X‐ray photoelectron spectroscopy (XPS), ultraviolet photoemission spectroscopy (UPS) and inverse photoemission spectroscopy (IPS). But all these techniques detect and average the data from a relatively large area, a few microns to a few millimeters across. The STM/STS, in contrast, can take spectra localized to as small an area as an individual atom. The most important characteristic that distinguishes STM/STS from other techniques is ability to operate in a variety of different environments such as air, reactive gases, liquids, electrolytes and biological fluids, leading to its applications not only in basic research in surface science but also in such diverse fields as lithography, electrochemistry, biology and medicine. An inherent limitation of STM is that it always operates at high resolution and, therefore, works better for atomically flat surfaces.
SoftX‐Ray Photoelectron Spectroscopy in Analysis of Surfaces
Authors: Yarmoff Jory A.
Published: September 2006

X‐ray photoelectron spectroscopy (XPS) is widely used to measure the composition and chemical states of species in the near‐surface region of a solid material in ultrahigh vacuum (UHV). In XPS, the core‐level, or inner‐shell, electrons that are emitted via the photoelectric effect are measured. The spectra provide information related to the electronic structure and chemical composition of the near‐surface region and to the bonding of adsorbates onto the surface. Because the mean free path for electrons traveling in solids is small, the spectra reflect the composition of the outermost few atomic layers. When synchrotron light is used as the excitation source, the technique is known as soft X‐ray photoelectron spectroscopy (SXPS). There are many advantages of synchrotron radiation over conventional XPS. For example, the photon energy is tunable over a wide range, which allows for the enhancement of particular spectral features and for unique types of spectroscopy in which the photon energy is continuously varied. The spectral resolution is also greatly enhanced, which allows for the observation of features not otherwise discernible. Also, the brightness of the beam is much greater than that of conventional X‐ray sources, thereby allowing for the detection of very small photoelectron signals. SXPS has been applied to a wide variety of materials, including metals, semiconductors, adsorbates on surfaces and nanoparticles. The information obtained from SXPS includes determining the core‐level binding energies of individual surface atoms, the position of the Fermi level within the gap of a semiconductor, and the surface work function. Shifts of the core‐level binding energy are observed for the outermost atoms on clean surfaces because they are in a different chemical environment than the atoms in the bulk. When an adsorbate attaches to a surface atom, there is a shift in binding energy that is related to the degree of charge transfer during bonding, i.e. the oxidation state. Analysis of the intensity of the shifted components provides the coverage and chemical state distribution of each adsorbate. Novel uses of SXPS include ultrahigh‐resolution spectroscopy, SXPS microscopy, photoelectron diffraction and spin‐polarized photoemission. The equipment utilized for SXPS includes an electron storage ring, an X‐ray monochromator, and an electron spectrometer.
Flow Injection Analysis Techniques in Atomic Spectroscopy
Authors: Pereiro Rosario , Sanz‐Medel Alfredo
Published: September 2006

Flow injection analysis (FIA), developed originally for the automation of serial assays, has become a powerful tool most adequate for performing on‐line any sample preparations before final measurement (e.g. sample dissolution, dilutions, matrix removal, preconcentration, etc.). It is not surprising that the combination of FIA with atomic spectrometric techniques has enlarged the analytical potential of atomic methods and expanded their wide field of applications. The collection of sample manipulation processes which can be covered today by flow operation procedures is amazing and therefore the general instrumentation required is reviewed in this contribution.

The description of the different flow strategies is carried out according to a hierarchy going from simple dilutions (including isotopic dilution of particular interest when using mass spectrometric detection), reagent mixing or standard additions, to more sophisticated flow manifolds such as those based on the use of two phases (e.g. gas–liquid, liquid–liquid or solid–liquid) for separation/preconcentration purposes. Modern approaches allowing for on‐line decomposition/dissolution of solid samples (e.g. on‐line chemical oxidation, photo‐oxidation and microwave heating) are also described. Finally, manifolds for the in situ uptake of the sample and its complete on‐line pretreatment are discussed.

The coupling of the above flow methodologies to a variety of atomization/excitation/ionization sources (flames, quartz tubes, graphite furnaces, inductively coupled plasmas (ICPs), microwave coupled plasmas, glow discharges (GDs), etc.) is detailed, in order to understand the usefulness of this combination both in atomic techniques based on photon measurements (absorption, emission and fluorescence spectrometry techniques) and on ions measurement mass spectrometry (MS).
Graphite Furnace Atomic Absorption Spectrometry
Authors: Holcombe James A.
Published: September 2006

Graphite furnace atomic absorption spectrometry (GFAAS) is an atomic spectroscopic technique in which a small sample is placed inside a graphite tube that is then resistively heated to accomplish sample desolvation (for liquid samples), ashing or charring (to decompose the sample and volatilize some of the matrix) and finally atomization. The light from a line source characteristic of the element being determined is passed longitudinally through the tube and the absorbance resulting from the presence of free analyte atoms in the gas phase is measured. The signal is transient in character, lasting approximately 1–5 s, and the area under this transient peak is generally used in the construction of a calibration curve. Modern instrumentation provides high levels of automation with capabilities of background correction as well as routine methods of sample analysis, for example quality assurance/quality control (QA/QC), standard additions, matrix modification, etc. Since the technique was first introduced in 1969, a lot of progress has been made in understanding the processes occurring within the graphite tube atomizer that ultimately produces the absorbance signal. An understanding of the free atoms formation process has facilitated the application of the technique to the analysis of a variety of complex samples.

Graphite furnace (also known as an electrothermal atomizer or ETA) atomic absorption (AA) is generally considered an ultratrace and microtrace analytical technique with limits of detection (LODs) in the low picogram range, precision of a few percent (relative standard deviation) and a dynamic range of about three orders of magnitude. In addition to its excellent sensitivity, it is unique in its ability to handle microsamples including aqueous solutions, viscous liquids, slurries and even solids. In general, there is considerable literature detailing methods and procedures for the determination of a variety of analytes in complex matrices that can be used by the analyst to apply the approach to new, complex analytical needs. When used correctly, this analytical tool can provide precise, accurate analysis for a wide range of sample types.
Laser‐Induced Breakdown Spectroscopy
Authors: Cremers David A. , Knight Andrew K.
Published: September 2006

Laser‐induced breakdown spectroscopy (LIBS) is a novel method of elemental analysis based on a laser‐generated plasma. Pulses from a laser are focused on a sample to atomize a small amount of material resulting in the formation of a microplasma. Because of the high plasma temperature, the resulting atoms are electronically excited to emit light. The plasma light is spectrallyresolved and detected to determine the elemental composition of the sample based on the unique emission spectrum of each element. Because of the simplicity of the method, it is suited for analyses that cannot be carried out using conventional methods of atomic emission spectroscopy (AES). This is particularly true for measurements that must be conducted outside of an analytical laboratory. A particular advantage of LIBS is the ability to analyze most types of samples without any preparation. This means that samples can be interrogated in situ, providing rapid measurement capability and permitting the method to be used in the analysis of gases, liquids, and solids in a variety of different sampling configurations. Although LIBS provides sensitive detection for many elements, it is not an ultrasensitive detection technique. In addition, under field conditions, the method typically does not provide the high accuracy and precision offered by laboratory‐based methods of AES.
Microwave‐Induced Plasma Systems in Atomic Spectroscopy
Authors: Broekaert José A.C. , Engel Ulrich
Published: September 2006

The use of microwave plasmas as radiation sources for optical atomic emission (AES), absorption (AAS) and fluorescence (AFS) spectroscopy and for laser ionization spectroscopy is treated and reference is also made to the use of microwave‐induced plasmas (MIPs) as ion sources for mass spectrometry (MS). Devices for producing both single‐electrode and electrodeless microwave plasmas are treated, in addition to methods for their diagnostics, and results for the analytically relevant plasma parameters are presented. Methods for sample introduction are discussed. They include dry aerosol generation techniques (hydride generation (HG), electrothermal evaporation, spark and laser ablation (LA)) and possibilities for the uptake of wet aerosols. Special reference is made to coupling with gas chromatography (GC) and also to the potential for coupling with high‐performance liquid chromatography (HPLC) Further, the use of microwave plasmas for cross‐excitation in the case of glow discharges (GDs) is treated. The analytical figures of merit in the case of AES with low‐power microwave plasmas, single‐electrode microwave plasmas, plasma torch sources and stabilized capacitively coupled plasmas are given also in the case of atomic absorption, fluorescence and laser ionization with these sources. The developments in MS in the case of both low‐power and high‐power microwave plasmas and in the case of various types of sample introduction are discussed.

Applications of MIP atomic spectroscopy are in the fields of biological samples with special reference to microanalysis, and of environmental samples with special emphasis on metal speciation, on‐line monitoring and direct solids analysis. A critical comparison of the methodology with other methods for the determination of the elements and their species is given.
Classical and Nonclassical Optimization Methods
Authors: Wehrens Ron , Buydens Lutgarde M.C.
Published: September 2006

Optimization problems are abundant in analytical chemistry, examples being the determination of optimal conditions for experiments or optimal settings for instruments. In general, all the required information should be obtained from as few experiments as possible. Classical techniques such as response surface models or simplex optimization are often used. These techniques, which can be very efficient in cases where the underlying assumptions are fulfilled, are called “strong” methods.

With the advent of the computer in the laboratory, a new class of optimization problems arose which could not be tackled with the standard methodologies. For these search‐type problems, new strategies such as simulated annealing (SA) and genetic algorithms (GA) are applied. Although these are not guaranteed to give the optimal result, in almost all cases they are able to find very good solutions where other techniques fail completely. These methods find themselves in an intermediate position between the strong methods, mentioned above, and weak methods, where hardly any assumptions are made.

This article provides an overview of classical and nonclassical optimization techniques, and stresses the differences in their areas of application. The key ideas are highlighted and references to important publications are given.
Signal Processing in Analytical Chemistry
Authors: Wentzell Peter D. , Brown Christopher D.
Published: September 2006

Signal processing refers to a variety of operations that can be carried out on a continuous (analog) or discrete (digital) sequence of measurements in order to enhance the quality of information it is intended to convey. In the analog domain, electronic signal processing can encompass such operations as amplification, filtering, integration, differentiation, modulation/demodulation, peakdetection, and analog‐to‐digital (A/D) conversion. Digital signal processing can include a variety of filtering methods (e.g. polynomial least‐squares smoothing, differentiation, median smoothing, matched filtering, boxcar averaging, interpolation, decimation, and Kalman filtering) and domain transformations (e.g. Fourier transform (FT), Hadamard transform (HT), and wavelet transform (WT)). Generally the objective is to separate the useful part of the signal from the part that contains no useful information (the noise) using either explicit or implicit models that distinguish these two components. Signal processing at various stages has become an integral part of most modern analytical measurement systems and plays a critical role in ensuring the quality of those measurements.
Infrared Spectro‐Electrochemistry
Authors: Anderson Mark R. , Taylor C. Douglas
Published: September 2006

The combination of infrared (IR) spectroscopy and electrochemistry in a single measurement has been an area of active research interest since the late 1960s. IR spectroelectrochemical experiments provide insight about interfacial electrochemical phenomena and/or oxidation–reduction reaction mechanisms that cannot be obtained using either technique individually. The power of the measurement is in the complementary nature of the spectroscopic and electrochemical information available when obtained simultaneously. The combination of measurements, however, requires compromise between the IR spectroscopy and the electrochemistry because of their different sampling requirements. The need for compromise illustrates the unique strengths and weaknesses of the combined spectroelectrochemical measurement. Three spectral sampling methods are commonly used: transmission, internal reflection, and specular reflection sampling of the electrochemical experiment. Additional experimental perturbations, such as polarization and electrode potential modulation, are frequently applied to increase the spectral sensitivity toward changes confined to the electrode interfacial region. This article presents the instrumental requirements for, and a theoretical background of, different types of IR spectroelectrochemical measurements.
Liquid/Liquid Interfaces, Electrochemistry at
Authors: Reymond Frédéric , Girault Hubert H.
Published: September 2006

This article outlines the electrochemical methodology at the interface between two electrolyte solutions (ITIES). The fundamental concepts of the thermodynamics in biphasic systems are presented in order to show how ions are distributed between the two adjacent phases, and hence how a Galvani potential difference is established at an ITIES. Polarizable and nonpolarizable ITIES are then characterized, and it is further evidenced that the classical electroanalytical methodology at a solid electrode can be directly transposed to the ITIES, thereby allowing reversible charge‐transfer reactions to be easily monitored and interpreted.

This theoretical approach is completed by a review of the analytical methods used at ITIES, namely cyclic voltammetry, dropping electrolyte electrodes, optical techniques and microinterfaces (which are the biphasic analogs of microelectrodes). The last part deals with the practical applications that electrochemistry at ITIES has attracted during the last decade in the development of amperometric ion sensors and detectors, in the extraction of metal ions by interfacial formation of a complex and in the assessment of the lipophilicity of ionizable drugs.
Scanning Tunneling Microscopy, In Situ, Electrochemical
Authors: Itaya Kingo
Published: September 2006

This article describes the in situ scanning tunneling microscopy (STM) operated at electrode–electrolyte interfaces. It is demonstrated that in situ STM makes it possible to monitor with atomic resolution a wide variety of electrode processes such as the adsorption of inorganic and organic species and the dissolution and deposition of metals and semiconductors. Owing to limitations on space, the focus is on selected topics and mainly on our own experimental results.
Self‐Assembled Monolayers on Electrodes
Authors: Finklea Harry O.
Published: September 2006

Alkanethiols and related molecules spontaneously adsorb from solution or vapor phase onto oxide‐free metals, especially gold, to form close‐packed oriented monolayers. The ease and flexibility of the self‐assembly process provides a convenient method for altering the properties of the metal as an electrode. The close‐packed hydrocarbon layer blocks access of most solution species to the electrode surface. Consequently, interfacial capacitances are markedly reduced and most electron‐transfer reactions are strongly inhibited. The monolayers also provide a foundation for attachment of additional molecules or molecular layers to electrodes. Self‐assembled monolayers (SAMs) on electrodes have been used in voltammetric methods to improve the analytical sensitivity and to impart greater selectivity towards specific analytes. Redox molecules have been anchored at fixed and controllable distances from the electrode surface, thereby permitting the study of electron‐transfer kinetics over long distances and at large driving forces. The ability of SAMs to inhibit metal corrosion and to promote better adhesion of electroactive polymers has been demonstrated.
X‐Ray Methods for the Study of Electrode Interaction
Authors: Herrero Enrique
Published: September 2006

X‐ray methods provide an excellent tool for the determination of the structure and composition of the electrode/solution interphase. The possibility of carrying out in situ experiments, because of the low absorbability of the X‐rays by the solution, allows direct correlation to be made between the structural changes observed in the sample and the potential applied. Depending on the interaction of the X‐rays with matter, these techniques can be classified in two main groups: X‐ray absorption techniques and X‐ray scattering techniques. Within the absorption techniques, extended X‐ray absorption fine structure (EXAFS) and X‐ray absorption near edge structure (XANES) will be considered. EXAFS allows determination of the short‐range order of the sample, i.e. it provides information about the nature and distances of the closest neighbors to the absorbing atom. On the other hand, identification of the oxidation state of the absorbing atom can be done with XANES. Grazing incident X‐ray diffraction (GIXD) and crystal truncation rod (CTR) measurements belong to the scattering techniques. The information obtained from these measurements can be considered complementary since GIXD allows precise determination of the long‐range order structure of the interphase whereas CTR analysis gives information about the out‐of‐plane structure of the interphase. The last technique covered, X‐ray standing waves (XSW), is not a pure scattering technique, but rather one that combines X‐ray scattering and X‐ray interference. With this last technique, the exact position of a foreign atom in the interphase can be determined.
Circular Dichroism and Linear Dichroism
Authors: Rodger Alison
Published: September 2006

Circular dichroism (CD) is the difference in absorption,A, of left and right circularly polarized light, Equation (1):For randomly oriented systems such as solutions of molecules, only chiral molecules will show any CD intensity corresponding to their absorption bands. Chiral molecules are those molecules that cannot be superposed on their mirror images.1Chiral is derived from the Greek word χɛιρ meaning hand, hence the alternate term for chirality, “handedness”. Two molecules that are mirror images of each other are often referred to as enantiomers and equimolar mixtures of two enatiomers form a racemic mixture which has no net CD intensity in solution.

Linear dichroism (LD) is the difference in absorption of light linearly polarized parallel and perpendicular to an orientation axis, Equation (68):
Indirect Detection Methods in Capillary Electrophoresis
Authors: Shamsi Shahab A.
Published: September 2006

An overview is given of existing indirect detection methods of inorganic and organic anions in capillary zone electrophoresis (CZE). Kohlrausch's regulating function (KRF), used by several researchers to predict the detection sensitivity of ultraviolet (UV)‐inactive analytes in CZE, is reviewed. Sensitive indirect photometric detection (IPD) of ionic compounds in CZE is largely governed by the value of the change in absorbance in a solute zone. This in turn depends on a transfer ratio (TR), ratio of the charge, and ratio of the electrophoretic mobility of the analyte and IPD reagent. The influence of other experimental parameters, such as pH, type and concentration of the absorbing electrolyte, detection wavelength, and absorption pathlength, on detection sensitivity are also critically discussed. An extensive part of this review deals with the separation of various classes ofionic analytes using capillary zone electrophoresis/indirect photometric detection (CZE/IPD). Different approaches for simultaneous separation and indirect detection of nonabsorbing anions and cations are evaluated. From this evaluation, some rules of thumb as to when these approaches will be most effective are drawn. In addition, indirect fluorescence detection (IFD) in CZE and micellar electrokinetic chromatography (MEKC) are reviewed and possible application areas are highlighted. Some recent developments in other indirect detection modes, such as indirect chemiluminescence detection (ICLD) and indirect conductivity detection (ICD), are outlined.
Sample Preparation for Gas Chromatography
Authors: Torkos Kornél
Published: September 2006

The success of a gas chromatographic analysis mostly depends on the sample preparation procedure. Biological and environmental samples are not suitable for direct analysis. Organic analytes can be separated from matrixes by extraction methods such as gas–solid, liquid–liquid, solid–liquid, purge‐and‐trap, headspace, solid‐phase (SPE), solid‐phase microextraction (SPME), supercritical fluid (SFE) and Soxhlet extractions. These extraction methods are called phase separation methods. Matrix solid‐phase dispersion (MSPD) is a special highly efficient phase separation procedure for biological matrixes such as meat, liver, muscle, vegetables, etc. The aim of the extraction procedure is not only separation of organic analytes from matrixes but also concentration in another phase.

If the organic analytes contain protic (active) hydrogen (alcohols, carboxylic acids, amines, oxo compounds, etc.) some derivatization reactions, such as acylation, alkylation and silylation, need to be carried out first. The aim of the derivatization methods is to increase the sensitivity of detection and thermal stability of the organic analytes. The other aspect of derivatization is to reduce the polarity of the compounds.
Theory of Infrared Spectroscopy
Authors: Turrell George
Published: September 2006

This article is devoted to the fundamentals of infrared spectroscopy. While it is well known that infrared spectroscopy is an extremely important analytical technique, its theoretical basis is not easily assimilated by most analytical chemists. It must be admitted that a certain understanding of abstract concepts, such as those encountered in quantum mechanics, is required. However, with all apologies to the reader, it is the objective here to present the essentials of these ideas, as needed in analytical applications of infrared spectroscopy. It should be noted that most of these principles apply equally to Raman spectroscopy, a subject which is described in other articles.

It is the Born–Oppenheimer approximation that provides the theoretical basis for the analysis of infrared spectra. The rotational and vibrational degrees of freedom of a molecule are specified to allow the spectra of molecules in the gas phase to be interpreted. It is shown that the logical extension of these arguments can be employed in the analysis of the spectra of liquids and crystalline solids.

In later sections, an effort has been made to explain how the basic ideas developed earlier can be applied to the interpretation of the infrared spectra of hydrogen‐bonded molecules, polymers and amorphous systems. A brief description of the notion of characteristic group frequencies, as routinely employed in organic chemistry, has been included to allow the practicing chemist to appreciate the possibilities – as well as the limitations – of this most important approach to the subject. To advance his appreciation of the advantages of infrared spectroscopy in analytical chemistry, the reader should refer to the various applications, as listed at the end of this article.
Microspectroscopy
Authors: Lang Patricia L.
Published: September 2006

The essential features of an infrared (IR) microscope accessory include

reflective optics for focusing, collecting, and imaging transmitted or reflected IR radiation from the sample onto a detector,

one or more variable apertures located in an image plane of the sample which serve to define the area to be recorded, and

a visible light path which is parfocal and collinear with the IR light path so that the sample can be viewed and positioned.

The accessory is coupled to a Fourier transform IR (FTIR) spectrometer by a set of transfer optics from the interferometer into the microscope.

IR microspectroscopy provides the analyst with the capability to perform IR transmission, absorption‐reflection, specular reflection, and diffuse reflection experiments on microscopic samples. Using special microscope objectives or accessories, attenuated total reflection (ATR) and grazing angle reflection experiments can be performed as well. IR microspectroscopy can also be used to map a sample's heterogeneity using a motorized stage in conjunction with a standard mercury cadmium telluride (MCT) detector. Alternatively, spatial and spectral information can be collected simultaneously using a step‐scan interferometer coupled with an IR microscope and a focal plane array (FPA) detector. In either case, the superior contrast attained by functional group imaging and the selectivity found in the mid‐IR region has been shown to be a powerful analytical tool.

However, diffraction ultimately limits the quality of the spectral information obtained using the IR microscope, and its effects may include poor spatial resolution, a reduction in photometric accuracy, and unreliable band intensities. Furthermore, unwanted optical effects from microscopic samples can also affect the quality of the spectrum, and sample preparation techniques must often take into account a sample's thickness, diameter, refractive index, and shape.
Kinetic Determinations: Introduction
Authors: Mottola Horacio A.
Published: September 2006

This introduction opens by discussing the distinction between kinetic and thermodynamic determinations. After defining equilibrium, pseudo‐equilibrium, and dynamic measurements, a historical perspective is given that covers early tests and publications to the present overall scope of kinetic determinations. In closing, a listing of selected bibliography is presented; this follows a short narrative emphasizing that kinetics goes beyond determinations in analytical chemistry.
Data Treatment and Error Analysis in Kinetics
Authors: Baeza‐Baeza Juan J. , Pérez‐Pla Francisco F.
Published: September 2006

Methods of data treatment in kinetics are reviewed from a practical viewpoint. The equations to describe the kinetics of chemical reactions and the methodology to obtain the models are presented. Moreover, the integration methods that are needed for the simulation of complex reactions are explained, from the simple first‐order reaction to the complex general mechanism.

However, it is essential to know the main sources of error in data treatment in order to reduce their effects on the results. Stress is put on the analysis of the errors due to the simplification of complex models. The different factors that influence the kinetics of the reaction are reviewed. How to take advantage of the kinetic information contained in the experimental data with an analytical aim is described. Finally, the propagation of errors in the analytical results is studied, reviewing both the simple univariate methods, used to determine one species, as well as the complex multivariate methods devoted to the determination of multicomponent mixtures.
Differential Rate Determinations
Authors: Pérez‐Bendito M. Dolores
Published: September 2006

Kinetic differential rate determinations are a measurement approach in kinetic methods of analysis where two or more components are determinated based on the different rates at which they interact with a common reagent.
Enzymatic Kinetic Determinations
Authors: Hüttl Regina
Published: September 2006

Kinetic methods for analytical applications can be divided into noncatalytic and catalytic methods. Enzyme kinetic determinations are a special type of catalytic methods. Enzymatic reactions are used analytically to determine enzyme activities – for instance in the diagnosis of diseases, substrate concentrations in food industry or medicine and concentrations of effectors – for example in trace analysis. Due to the high selectivity of enzymatic analysis the importance of this method in various fields is growing rapidly. The widest use is observed in clinical chemistry and in food chemistry.

This article can only give a general survey of the great potential of enzymatic analysis, with references to more detailed literature.

Enzymatic reactions are characterized by some special features, explained after the introduction. The theoretical background for kinetic determination is the comprehension of the principles on enzymatic kinetics. In section 3 the simplest mechanism, outlined by Michaelis and Menten and several transformations of this equation will be described. In this section two kinetic parameters, enzyme activity and the Michaelis constant, will be introduced. Section 4 deals with substances, so called effectors, influencing the rate of an enzymatic reaction. Simple mechanisms of these effectors will be shown. The different methods for determination of substrates, enzymes and effectors, illustrated by many examples, are represented in section 5. While substrate concentrations can be determined using both ways, equilibrium or kinetic methods, the determination of enzymes and effectors can only be performed by kinetic approaches. In the last two sections the application of immobilized enzymes in flow systems and biosensor development will be shown.
Luminescence‐based Kinetic Determinations
Authors: Gómez‐Hens A.
Published: September 2006

Luminescence‐based kinetic determinations are of great analytical interest and flexibility as they combine the high selectivity of kinetic methodology with the low detection limits afforded by the different luminescence techniques.
Uncatalyzed Kinetic Determinations
Authors: Koupparis Michael A.
Published: September 2006

Analytical procedures in which the measurement step (chemical, physical or physicochemical) is influenced by a transient (kinetic) process, can strictly be classified as kinetic method. Thus, the majority of modern analytical methods (continuous‐flow, time‐resolved fluorimetric and chromatographic methods, among others) are kinetic in nature. Nevertheless, the term “kinetic method”, for historical reasons, is incorrectly limited to methods based on direct or indirect measurements of the rate of a chemical reaction, which should be classified as “reaction‐rate methods”. Analytical methods based on signal measurements made under dynamic conditions compete in efficiency with static and equilibrium measurements. The growing development of kinetic methods is due to the need for quantification of microamounts of substances, increasing knowledge of reaction mechanisms, and breakthroughs in instrumentation and data handling and interpretation. The most popular classification of kinetic methods of analysis is based on the chemistry of reactions employed and distinguishes between catalytic (nonenzymatic, enzymatic and electrochemical), and noncatalytic methods. The latter are further categorized into those used to determine a single species or several components in mixtures (differential reaction‐rate methods). This article deals with kinetic methods for the determination of single noncatalytic species. Although the majority of kinetic‐based determinations depend on catalytic systems, some analytical applications using the rate of uncatalyzed reactions are of analytical interest, mainly those involving organic chemical species. Advances in instrumentation: (1) improved signal detection, (2) resolving of problems arising from time measurements especially in fast processes, and (3) availability of automated mixing devices and data processing (error‐compensated methods, curve‐fitting and predictive approaches) establish noncatalytic methods as powerful alternatives to equilibrium methods. The main field of application of noncatalytic reactions is organic analysis, unlike catalytic reactions where a metal ion usually acts as the catalyst.
Affinity Chromatography
Authors: Hage David S.
Published: September 2006

Affinity chromatography is a type of liquid chromatography that makes use of biological‐like interactions for the separation and specific analysis of sample components. This article discusses the basic principles behind the theory and use of this method. Various factors that are important in the development of an affinity chromatographic system are considered, such as the choice of affinity ligand, support material, immobilization method, and elution conditions. A survey of the different types of affinity chromatography is also presented; this includes bioaffinity chromatography, immunoaffinity chromatography, dye–ligand affinity chromatography, immobilized metal affinity chromatography, and analytical affinity chromatography. In addition, some areas of expected future growth are considered, such as the use of molecular imprints or aptamers as ligands in affinity columns, and the coupling of affinity chromatography with other analytical techniques.
Gradient Elution Chromatography
Authors: Dolan J.W. , Snyder L.R.
Published: September 2006

Elution chromatography can be carried out in either isocratic or gradient modes. In isocratic elution, the mobile‐phase composition is held constant during separation of the sample, e.g. 60% v acetonitrile–water. In gradient elution, the mobile‐phase composition will be varied during sample separation, e.g. changing from 0 to 100% v acetonitrile–water. Gradient elution requires special chromatographic equipment, as well as somewhat greater care on the part of the operator, but it has important advantages for many separations. Thus, in isocratic elution (Figure 1a), sample peaks tend to “bunch up” at the beginning of the chromatogram (often with decreased resolution) and to broaden at the end of the chromatogram (with reduced detection sensitivity). Gradient elution (Figure 1b), on the other hand, provides a more even spacing of peaks, similar widths throughout the chromatogram, and often a shorter run time. For these and other reasons, gradient elution is preferred for the separation of many samples.
Micellar Electrokinetic Chromatography
Authors: Pyell Ute
Published: September 2006

Micellar electrokinetic chromatography (MEKC) is a highly efficient miniaturized separation technique that is performed with the same experimental set‐up that is used with capillary electrophoresis (CE). While the separation principle of CE is based on differences in the electrophoretic mobility of the solutes to be separated (only charged species can be separated), in MEKC separation is also based on differences in the distribution constants between two phases migrating at different velocity due to electrokinetic effects. The second phase is introduced into the separation electrolyte by adding an ionic surfactant (anionic or cationic) at a concentration above the critical micelle concentration (CMC). Aggregates of surfactant monomers (micelles) are present in the separation electrolyte. These aggregates have an electrophoretic mobility and are able to incorporate solutes or interact strongly with species dissolved in the surrounding medium.

MEKC extends the applicability of CE to neutral solutes. In addition, MEKC has been proven to be superior to CE in the separation of very complex mixtures of solutes with similar electrophoretic mobility, because of the various factors that are available for the manipulation of separation selectivity. Currently, MEKC can be regarded as a separation technique with a similar scope to that of reversed‐phase high‐performance liquid chromatography (RPHPLC), having advantages over the latter concerning the efficiency of the separation system, separation speed, cost and tolerance to matrix constituents, as in MEKC no stationary phase is present that can be fouled by irreversible sorption of matrix constituents of the sample. Owing to the small injection volume (a few nanoliters) and small detection volume that is necessary to avoid intolerable instrumental band broadening, current MEKC instrumentation restricts the applicability of MEKC to the determination of those compounds in complex mixtures that are present in concentrations sufficiently high with respect to the limits of detection that can be obtained with such instrumentation.
Reversed Phase Liquid Chromatography
Authors: Lough W. John
Published: September 2006

Reversed‐phase liquid chromatography (RPLC) is an elution procedure used in liquid chromatography (LC) in which the mobile phase is significantly more polar than the stationary phase, e.g. a microporous silica‐based material with chemically bonded alkyl chains. Typically, the mobile phase is a mixture of an aqueous component, often containing a buffer to control pH, and a water‐miscible polar organic solvent. This most frequently used mode of LC is suitable for the analysis of compounds with a very wide range of polarities, using experimental variables such as mobile‐phase pH and ratio of organic to aqueous mobile phase components to manipulate selectivity and retention to separate sample components from one another.
Thin‐Layer Chromatography
Authors: Sherma Joseph
Published: September 2006

In thin‐layer chromatography (TLC), the sample solution is applied as a spot or band on the origin of the layer spread on a support (the plate). After evaporation of the sample solvent, the plate is placed in a sealed chamber containing a solvent chosen as the mobile phase. Development occurs as the mobile phase moves through the layer, and the components of the sample move at different rates to create the separation. The plate is removed from the chamber, and the separated zones are detected by physical or chemical methods, identified by comparison of their migration to standard zones on the same plate, and quantified by visual or instrumental methods based on measurement of zone sizes and intensities. A great variety of compounds can be separated, including organic, inorganic, biological, and chiral. Environmental, pharmaceutical, biomedical, and food samples are among the sample types commonly analyzed by TLC. Advantages of TLC include rapid analysis time because many samples can be analyzed simultaneously, low solvent usage on a per‐sample basis, a high degree of accuracy and precision for instrumental TLC, and sensitivity in the nanogram or picogram range. Coupling of TLC with other analytical methods such as high‐performance liquid chromatography (HPLC), mass spectrometry (MS), and Fourier transform infrared spectrometry (FTIR) provides enhanced opportunities for sample analysis. Disadvantages of TLC include application to only nonvolatile compounds, limited resolution capability (separation numbers or peak capacities of 10–50), and the absence of fully automated systems, although the individual steps of the technique can be automated.
Atmospheric Pressure Ionization Mass Spectrometry
Authors: Tarr Matthew A. , Zhu Junhua , Cole Richard B.
Published: September 2006

The atmospheric pressure ionization (API) techniques serve to transform neutral analytes into ionic species that may then be analyzed by mass spectrometry (MS). Ionization takes place under ambient pressure (∼1 atm), and formed ions are subsequently directed into the low‐pressure regions of the mass spectrometer for mass analysis. The API techniques have offered notable successes in enabling ionization of nonvolatile compounds contained in solution that are not amenable to “classical” mass spectrometric ionization techniques such as electron ionization and conventional chemical ionization. Under favorable API conditions, neutral analytes may be converted to ionic forms in extremely high efficiencies approaching 100%. Fundamental aspects and applications of the two most widely used API techniques, atmospheric pressure chemical ionization (APCI) and electrospray ionization (ESI), are discussed at length in this article.
Discrete Energy Electron Capture Negative Ion Mass Spectrometry
Authors: Laramée James A. , Cody Robert B. , Deinzer Max L.
Published: September 2006

Standard electron capture negative ion mass spectrometry (ECNIMS) has long been known as a sensitive and selective method for the analysis of molecules with positive electron affinities. Applications of this technique for the analysis of environmental contaminants, particularly of halogenated compounds such as polychlorobiphenyls (PCBs), are common. However, the requirement for a moderating gas in the ion source to lower the energy of the electrons into the range required for resonance electron capture generates new experimental variables that are difficult to control. Differences in the moderating gas pressure cause changes in the electron energy distribution and in the resulting ion abundances, whereas small fluctuations in temperature often cause major differences in ion abundance ratios. Even more serious are the uncontrollable reactions between ions, molecules, and radicals within the high‐pressure ion source region that give unpredictable spurious product ions, making spectral interpretation difficult and leading to wide variations of negative ion spectra from different instruments. Because of these difficulties, the advantages in sensitivity and specificity of ECNIMS for analysis of electronegative compounds often have been unrealized.

Better results can be obtained by having direct control over the electron energies. Control over electron energies can be achieved using a trochoidal electron monochromator, which allows the operator to tune to or scan a range of electron energies necessary for resonance electron capture. With this device some of the inherent difficulties encountered with ECNIMS can be avoided and reliable spectra for a broader range of compounds becomes possible. The ability to tune to a desired electron energy or to scan over a range of electron energies makes another dimension of analytical information available for identifying compounds. Quadrupole and sector instruments have been equipped with electron monochromators and the results are very promising. A clear advantage of these units is the reproducibility of the data they produce, the potential for three‐dimensional spectra for easier interpretation of the results, and the availability of a new dimension of analytical information. These instruments have not shown the sensitivity inherent in the ECNIMS method. Improvements of about two or three orders of magnitude in sensitivity will be required before these instruments can compete with existing technology for trace analysis of environmental residues or other electron‐capturing compounds. An improvement in the sensitivity of this magnitude is well within reasonable expectations and it should not be long before gas chromatography/electron monochromator mass spectrometry (GC/EMMS) becomes a standard technique for detecting environmental compounds, explosives and substances of abuse.
Electron Ionization Mass Spectrometry
Authors: Watson J. Throck
Published: September 2006

Electron ionization (EI) is the basis for one of the most effective mass spectrometric means for identification of a given organic compound. The identification is based on an indication of the molecular weight (MW) coupled with a fragmentation pattern that serves as a chemical fingerprint for the compound. Of the many ionization techniques available in mass spectrometry, EI is one of the most harsh in terms of promoting fragmentation of the molecular ion. The analyst can take advantage of this extensive fragmentation by using some of the fundamental principles of organic mechanistic chemistry and physical chemistry for the purposes of rationalizing the fragmentation pathways that characterize various structural features in an organic molecule. Four basic mechanisms of fragmentation are considered in this chapter; most of them operate on an odd‐electron ion, although some pathways will be applicable to even‐electron ions as well. The multiplicity of peaks that represent a given ion allows one to estimate the elemental composition of an ion. This information on composition, coupled with a rationalization of a given fragmentation pathway, often enables the structure of a given organic compound to be deduced.
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