bluuebunny/crossref_metadata_2025 huggingface dataset:
Starch Analysis in Food
Authors: Englyst Klaus N. , Hudson Geoffrey J. , Englyst Hans N.
Published: September 2006

Starch is quantitatively an important component of the human diet, being present in grains, tubers and legumes. Starch has for a long time been considered by many as being slowly but completely digested in the small intestine, resulting in modest glycemic responses and with no physiological role other than as an energy source. It is now understood that in fact the metabolic fate and physiological properties of starch can vary considerably, and both the botanical source and the effects of food processing are major determinants of starch digestibility. In addition to the nature of the starch itself, the site, rate and extent of digestion of starch in the human small intestine are influenced by a number of host factors. The rate at which starch is digested in the human small intestine results in a wide range of glycemic responses, and this physiological measurement has been used to rank foods by their glycemic index. In vitro studies have indicated that glycemic response and the rate of starch digestion are closely correlated. Rapidly digestible starch (RDS) and slowly digestible starch (SDS) fractions together represent the starch that is likely to be digested completely in the human small intestine, with any remaining starch defined as the resistant starch (RS) fraction that is available for fermentation in the large bowel. Measurements of RDS, SDS and RS can be obtained by one simple procedure. Values for the different starch fractions obtained by the in vitro method described here represent reproducible measurements that can be used to classify dietary starch according to its potential digestibility. In addition to these starch fractions, two terms, rapidly available glucose (RAG) and slowly available glucose (SAG), are introduced to reflect the rate at which glucose (from both sugars and starch) is likely to be absorbed in the small intestine.

The proportions of RAG, SAG, RDS, SDS and RS in foods can be controlled by food processing. The implications of altering the rate and extent of starch digestion are potentially of great importance to public health. A full understanding of the links between dietary carbohydrates and health and the underlying mechanisms will come only from the specific measurement of individual types of dietary carbohydrates.
Viscosity of Food: Measurement and Application
Authors: Rao M.A.
Published: September 2006

The measurement of viscosity and viscoelastic behavior of fluid foods are reviewed. The role of factors such as rate of shear, temperature, and concentration of soluble and insoluble solids on the viscosity of food is covered. Two important areas in food technology, sensory assessment of viscosity and role of viscosity in pipe flow, are discussed.
Vitamins Analysis in Food
Authors: Ye Lin , Eitenmiller Ronald R.
Published: September 2006

Vitamin assay of biological materials represents a large and rapidly expanding area. Methods now rely heavily on high‐performance liquid chromatography (HPLC). However, microbiological assay of the water‐soluble vitamins is still routinely used for food analysis owing to the sensitivity and specificity of the methods. An extensive table on physical properties of the vitamins is provided. Such information is dispersed widely in the literature and is often not at the analyst's fingertips when needed. This article concentrates on providing the reader with current HPLC approaches to vitamin analysis of food. Tables are provided that give method summaries referenced to the original work. Because of the scope of the area, recent text references are provided for readers requiring more in‐depth knowledge.
Ion Mobility Spectrometry in Forensic Science
Authors: Karpas Zeev
Published: September 2006

The ion mobility spectrometer (IMS) is a device used primarily for detection, identification, and monitoring of trace amounts of gases or vapors. Generally, sample introduction, ionization, ion separation, and detection all take place at ambient pressure, so that elaborate vacuum systems are not required. IMSs are typically small (portable, hand‐held or even pocket‐sized) and respond within seconds to part per billion (ppb), or even part per trillion (ppt) concentration levels. The IMS is not a universal detector and is not suitable for all compounds. It has a limited dynamic range due to its finite reservoir of charge, and may suffer from matrix effects which could interfere with the ionization or ion separation processes and cause spectral interferences. These drawbacks have limited the application of IMS technology to a few fields that require high sensitivity and rapid response, such as the detection of chemical warfare agents (CWA), explosives, and illicit drugs, and the identification and monitoring of toxic gases or vapors.

The array of field and laboratory instruments available today have become useful tools in the prevention of illicit use of contraband substances and in the rapid identification of these and other toxic substances. This article describes the principle of operation of IMSs and the basic gas‐phase ion chemistry underlying the technology. The proven experience, both in the field and in the laboratory, of IMSs for forensic science applications is also described, and the merits and shortcomings of present‐day IMS technology, and future trends, are discussed.
Nuclear Magnetic Resonance Spectroscopy for the Detection and Quantification of Abused Drugs
Authors: Dawson Brian
Published: September 2006

Nuclear magnetic resonance (NMR) spectroscopy provides the forensic analyst with an extremely powerful tool for the detection and quantification of abused drugs. A whole range of one‐dimensional (1‐D) and two‐dimensional (2‐D) NMR techniques is available for performing the required analyses. These NMR methods may be used for routine purposes, such as to confirm the identity of a drug or quantify the amount of illicit substance present in a police exhibit. However, the area where NMR stands out as an analytical tool is in the identification of unknown compounds, such as “designer drugs”. NMR is also used in police intelligence work, as it can provide clues to the synthetic route used to prepare the drug. This is done by impurity profiling or by determining the drug's optical purity. Although NMR has been used for many years to analyze abused drugs, even the most modern spectrometers lack the sensitivity obtainable by other techniques such as mass spectrometry (MS) or high‐performance liquid chromatography (HPLC). However, NMR is a nondestructive technique which provides essential structural information which cannot be obtained from these other methods. NMR also has the distinct advantage of not requiring reference standards for the identification of unknowns.
Polymerase Chain Reaction in the Forensic Analysis ofDNA
Authors: Strom Charles M.
Published: September 2006

This article summarizes the theory behind the uses of DNA analysis for identification purposes. Various techniques are summarized and their strengths and weaknesses are contrasted. The theoretical basis of the polymerase chain reaction (PCR) and various types of PCR reactions are introduced. A table is provided with loci that can be used for the forensic analysis of DNA. The availability of kits is summarized. Finally, a discussion of mitochondrial DNA analysis is performed.
Pyrolysis Gas Chromatography in Forensic Science
Authors: Blackledge Robert D.
Published: September 2006

Pyrolysis (as the term is used by analytical chemists) is the breakdown of organic molecules by the application of heat in the absence of oxygen. Because oxygen is absent, combustion (which would produce primarily carbon dioxide and water vapor) does not occur, and the pyrolysis products are characteristic of their parent molecules. Pyrolysis is most often applied to characterize polymeric materials or other samples that cannot normally be vaporized without undergoing decomposition. In pyrolysis gas chromatography (PY/GC) the carrier gas (usually helium or nitrogen) sweeps the pyrolysis products onto a gas chromatography (GC) column.
X‐Ray Fluorescence in Forensic Science
Authors: Roux Claude , Lennard Chris
Published: September 2006

This article describes the applications of X‐ray fluorescence (XRF) as an analytical tool in forensic science, including limitations and practical aspects. The use of XRF is specifically discussed for the forensic analysis of materials of forensic interest such as metals, gunshot residues (GSR), paint, glass, soil, fibers, plastic and general polymers, and miscellaneous types of evidence. Recent developments such as micro XRF and total XRF are also described. These specialized techniques have highly desirable characteristics for the elemental profiling of a wide range of forensic samples, and hence it can be anticipated that they will find increasing use in forensic laboratories in the future.
Chromatographic Techniques in Industrial Hygiene
Authors: Hahne Rolf M.A.
Published: September 2006

Chromatographic techniques in industrial hygiene are defined as those separation and analysis techniques which rely on the separation of multiple chemical constituents by taking advantage of the differential adsorption, absorption, mobility, or affinity of the components in a system with a stationary phase (solid, liquid coated on solid, reactive substituent attached to solid surface, ion exchange resin, etc.) and a mobile phase (gas or liquid) which transports the separated constitutents to a detector for immediate identification and possible quantitation or to a collector for later identification and possible quantitation.

By industrial (occupational) hygiene is meant that science and art which is the anticipation, identification, assessment, and control of chemical and physical hazards in the workplace (meant to include offices, places of business, retail establishments, and schools) and home. Included among those methods that make possible the identification and assessment of such hazards are those methods which are used to determine the concentration of airborne and surface chemical contaminants, as well as those which assess levels of exposure to chemical contaminants by the analysis of exhaled breath, blood, urine, or other biological samples.
Parent and Progeny Compounds in Exhaled Breath, Determination of
Authors: Que Hee Shane S.
Published: September 2006

Breath analysis is the sampling and analysis of an analyte in the exhaled breath of living animals, and the subsequent interpretation of the results.
1
Breath sampling is noninvasive of the body as is urine sampling, unlike blood sampling. All three modes of biological monitoring can be very useful and often are necessary. Breath sampling is especially useful for chemically inert volatile organic compounds (VOCs).

The related branches of scientific endeavor include:

Pulmonary function testing is the measurement of pulmonary parameters to diagnose pulmonary disorders.
2

Pulmonary toxicology is the study of adverse effects of inspired toxicants on the respiratory system.

Inhalation toxicology is the study of adverse effects of inspired toxicants on body target organs and tissues. Pulmonary toxicology is thus a subset of inhalation toxicology. The health consequences of “sniffing” chemicals and “poppers” are included. Many scientists use inhalation toxicology as a term to describe animal experimentation.

Anesthesiology is the intentional exposure of anesthetics in nonadverse doses to humans to aid medical operations where pain would be excessive.

Taste and odor science is the investigation of taste and odor in inspired breath and oral ingestion from natural sources (decaying vegetation, decomposing bodies, and environmental odors) and synthetic sources (food, drink, drugs, cosmetics, perfumes, toiletries, wine tasting and bouquet, beer taste and odor, sewer headspaces and emissions, pesticide applications, garbage and waste sites, natural and potable waters), and environmental and indoor air quality situations. Often taste and odor panels are a part of the evaluation of such phenomena. When used for risk assessment purposes, taste, odor, and other aesthetic considerations are called organoleptic criteria.

This article will focus on human exhaled breath analysis, except where specified otherwise.
Sensors in the Measurement of Toxic Gases in the Air
Authors: Groves William A.
Published: September 2006

The use of sensors to measure toxic gases and vapors in air has increased rapidly due to several factors, including a growing public awareness of the hazards associated with many airborne chemicals, increasing regulatory requirements for industries using hazardous chemicals, and continuous advancements in the technology and instrumentation available to measure these compounds. The principal advantage of sensor systems for toxic gases and vapors relative to traditional sampling and analytical methods, is the ability to measure chemicals over short time periods, that is, in a matter of seconds or minutes, thus providing the information necessary to protect workers from acute health effects. Modern sensor‐based instrumentation provides many options for processing and storing measurements, thereby allowing extreme flexibility in evaluating exposures to toxic gases and vapors relative to ceiling limits, short‐term exposure limits (STELs), or longer term average concentrations based on user selectable time periods. This extreme range of options for processing data makes sensors an attractive, and in some cases, the only alternative for measuring toxic gases and vapors in air. Most of these devices can be broadly categorized as: (1) electrochemical sensors which constitute the vast majority of devices currently available for detection of toxic gases and vapors; (2) mass sensors that are based on piezoelectric materials including surface acoustic wave (SAW) devices; or (3) optical sensors that rely on the interaction of electromagnetic radiation with an analyte, resulting in some alteration of the properties of the radiation. The most common applications of sensors for measuring toxic gases and vapors in air include industrial hygiene monitoring, confined space monitoring, process emission monitoring, and leak detection.
Surface and Dermal Monitoring
Authors: Ness Shirley A.
Published: September 2006

Skin contamination can produce the same types of toxic effects as inhalation if a chemical can significantly penetrate the skin. Surface contamination can be ingested orally, create airborne exposures when surface buildup is disturbed, and be transferred into other areas, including from the workplace into the home. When used to determine the potential for dermal exposure, methods are often categorized as indirect or direct. Indirect methods involve the use of filters, gauze pads, or swabs to wipe surfaces; the use of instruments to “sniff” surfaces for volatile compounds; and the use of vacuum methods and adhesive tape to lift dust off surfaces. Direct methods measure exposure to the body and include the use of gauze, cellulose, or charcoal pads for dosimetry; analysis of clothing, skin wipes; washing the skin with solvent; and monitoring for skin color change and fluorescence.

Current sampling strategies emphasize air exposures and overlook the contribution of dermal exposures and surface contamination. Integration of surface and dermal monitoring techniques into sampling strategies is important so that a better estimation of exposures and therefore controls to protect against them are developed.
Aptamers
Authors: James William
Published: September 2006

Aptamers are artificial nucleic acid ligands that can be generated against amino acids, drugs, proteins and other molecules. They are isolated from complex libraries of synthetic nucleic acid by an iterative process of adsorption, recovery and reamplification. They have potential applications in analytical devices, including biosensors, and as therapeutic agents.
Genome Physical Mapping UsingBACs
Authors: Altmann Thomas
Published: September 2006

A physical map of a region or the entirety of the genome of an organism is a representation of the genome providing information on the relative order and the arrangement of physically defined entities. For most physical maps, these entities are cloned genomic DNA fragments propagated in cosmids, bacteriophage P1, bacterial artificial chromosomes (BACs), P1‐derived artificial chromosomes (PACs), or yeast artificial chromosomes (YACs). The various cloning systems differ in the sizes of the cloned DNA fragments and the fidelity of DNA structure maintenance. Owing to its excellent properties, the BAC system is the premier system for this purpose. Several different, highly efficient procedures have been developed and applied successfully to identify overlaps between the DNA fragments represented in different clones. The procedures are based on different experimental techniques, including nucleic acid hybridization, DNA amplification through polymerase chain reaction (PCR), DNA restriction fragment analysis, and DNA sequencing. Applied separately or in combination, these procedures have been used successfully to generate extensive physical maps of human, animal, and plant genomes.
Capillary Electrophoresis of Nucleic Acids
Authors: Kawamura Kunio
Published: September 2006

Capillary electrophoresis (CE) is a powerful and relatively new technique for nucleic acid mapping, and can be used along with conventional slab electrophoresis and high‐performance liquid chromatography (HPLC). There are several types of separation techniques of CE, for instance capillary zone electrophoresis (CZE), micellar electrokinetic chromatography (MEKC), capillary gel electrophoresis (CGE), and capillary affinity gel electrophoresis (CAGE), all of which are capable of being used for quantification and qualification of nucleic acids. CZE and MEKC are suitable for the separation of bases, nucleoside and nucleotide monomers, and short nucleic acids. Although the sensitivity of ultraviolet (UV) detection is not as high as HPLC, since the light path length of CE is more than 100 times smaller than that of HPLC, a laser‐excited fluorescence detection system enables highly sensitive detection of nucleic acids. CGE and CAGE, in which electrophoresis is performed in molecular sieving materials of polymer gels, are used rather than HPLC and slab gel electrophoresis (SGE) for sequencing of large nucleic acids for several reasons: high‐resolution power, high‐speed sequencing, ease of automation, and small sample volume. Combining the advantages of CGE and laser fluorescence detection with a four‐ or two‐color labeling method enables sequencing of more than 1000 bases within 1 h. Moreover, recent improvements in capillary array electrophoresis (CAE) with high‐throughput and high‐speed sequencing systems contribute to an expansion of the sequence analysis of DNA fragments in order to achieve the goal of the Human Genome Project (HGP).
Comparative Genomics: Differential Display and Subtractive Hybridization
Authors: Smith Cassandra L. , Bouchard Joseph , Surdi Gregg , Nguyen Giang , Pimpis K. , Tolstoi Linda G.
Published: September 2006

An understanding of genome function within the context of human cells requires new methodology. This article describes comparative approaches that are used for genomes and genes in situ. However, the major focus of the article is on differential display (DD) and subtractive hybridization (SH) because these two methods are most accessible to a large number of researchers. The basic principles of these methods are described along with some fundamental reminders about the important factors that must be kept in mind for successful experiments.
DNAStructures of Biological Relevance, Studies of Unusual Sequences
Authors: Wang Yuh‐Hwa
Published: September 2006

DNA molecules with unusual structures can have significant consequences in many DNA metabolic processes. This article summarizes structural studies of DNA molecules containing bulged and mismatched bases and trinucleotide repeating DNAs. Extra base bulges and mismatches in double‐stranded DNA can arise from replication errors or imperfect recombination. If these lesions escape the repair system, they would subsequently cause mutagenesis. Using a combination of gel electrophoresis, electron microscopy (EM), chemical modification, and probing with RecA protein, the structure of bulged and mismatched DNAs was examined and factors such as base composition and flanking base sequence have been shown to influence the overall structure of these molecules.

Expansion of trinucleotide repeats is a major cause of several inherited neurodegenerative diseases. Described here are findings that show, by in vitro nucleosome assembly assay, that the expanded CTG triplet sequences derived from myotonic dystrophy (DM) patients form the most stable naturally occurring nucleosomes that are known. In contrast, long tracts of repeating CCG triplets (expanded in fragile X syndrome (FraX) patients) resist nucleosome assembly. This information could provide critical clues about how these unusual DNAs cause harmful biological effects in living cells.
Mass Spectrometry of Nucleic Acids
Authors: Gross Julia , Hillenkamp Franz
Published: September 2006

Nucleic acids are prone to fragmentation in the ionization process of mass spectrometry (MS). The cause can be found in the polar nature of this analyte class. Following the introduction of the so‐called soft‐ionization techniques of electrospray ionization (ESI) and matrix‐assisted laser desorption ionization (MALDI) at the end of the 1980s, mass spectrometric analysis of nucleic acids has gained broader applicability. In combination with further developments of the associated instrumentation and optimized preparation and purification methods, a dramatic enhancement of the mass range, detection sensitivity, and resolution became possible – in routine analysis oligonucleotides up to a length of 50 nucleotides are accessible. At present the necessary quantity of sample is in the subfemtomole range.

This article describes the fundamentals of mass spectrometric analyses of nucleic acids and common applications, such as the analysis of noncovalent complexes, mixture analysis, different sequencing strategies and clinical diagnostics. The focus is on MALDI/MS (matrix‐assisted laser desorption ionization/mass spectrometry).
Nuclear Magnetic Resonance and Nucleic Acid Structures
Authors: Eriksson Magdalena
Published: September 2006

Nuclear magnetic resonance (NMR) is one of the most important and widely used techniques for studying nucleic acid structure. With modern techniques, including isotope labeling and multidimensional spectroscopy, structures of large molecules – nucleic acids alone or in complexes, for example with proteins or ligands – can be determined. Here is given an overview of experimental NMR methods for nucleic acids studies, complemented by illustrative examples of DNA, RNA and PNA structures.
Nucleic Acid Structural Energetics
Authors: Hardin Charles C. , Moon Andrea F.
Published: September 2006

The general relations between structure and energetics of nucleic acids are described. A detailed, multileveled definition of the structures adopted by one, two, three and four strands is presented, with emphasis on the chemical functionalities of the structural subcomponents. Descriptions include results from studies of the thermodynamics of transformations and structures (e.g. double helix formation, superhelical coiling). Different aspects of this large array of problems have been determined using X‐ray diffraction and spectroscopic techniques, such as ultraviolet (UV) and infrared (IR) absorption, circular dichroism (CD), Raman scattering and nuclear magnetic resonance (NMR). Interactions, within one strand or between two or more strands, with cations, buffers, aromatic chemicals and proteins are all described. Methods include spectroscopy, calorimetry and computational approaches (statistical mechanics, molecular mechanics, dynamics, quantum mechanics). A Further Reading section is included to provide an idea of the vast scope of this area.
X‐Ray Structures of Nucleic Acids
Authors: Berman Helen M.
Published: September 2006

This article first summarizes the procedures used in the X‐ray crystallographic determination of nucleic acids. The methods used to analyze the results of these determinations are presented, followed by a survey of the highlights of the more than 500 structures determined by these methods since the 1970s.
Sequencing and Fingerprinting
DNA
by Hybridization with Oligonucleotide Probes
Authors: Drmanac Radoje , Drmanac Snezana , Little Deane
Published: September 2006

Sequencing by hybridization (SBH) determines the sequences of DNA or RNA targets using hybridization data obtained from sets of overlapping oligonucleotide probes of known sequence. SBH shares interesting parallels with computer‐based keyword searches of text files. In SBH, chemically synthesized short oligonucleotides (usually 4–25 bases in length) are hybridized to the target under conditions that predominantly allow formation of fully complementary probe/target hybrids. An oligonucleotide hybridization search is effectively a highly parallel molecular computation process with fully random access to the polynucleotide chain “input data”. The target sequence is assembled by aligning sequences of the subset of positively hybridizing oligonucleotides. Probe sequence overlap allows indirect assignment of which of four bases corresponds to each position in the analyzed DNA chain, without performing any actual positional measurements on the sample. The fundamental characteristics of the SBH process confer unique opportunities for miniaturization and parallel analyses (currently in the form of high‐density arrays), leading to rapid, cost‐effective DNA sequencing. A unique advantage of SBH is its ability to analyze in a single reaction complex DNA or RNA samples that may be thousands of bases long. Hybridization of smaller selected subsets of probes provides a very efficient way of partially sequencing and fingerprinting samples. A very important advantage of SBH is that in a single reaction each base is “read” by multiple overlapping probes. To achieve these advantages, hybridization reactions are miniaturized and coupled with automated data acquisition and efficient computational analyses that can process data sets containing both false‐positive and false‐negative probe scores. Successful assembly of long target sequences involves synthesis and hybridization of a large number of long probes, due to the higher probability that given short oligonucleotide sequences will be repeated within the target sequence. Overall, SBH is a rapid and cost‐effective method of obtaining large amounts of accurate DNA sequence information.
Polymerase Chain Reaction and Other Amplification Systems
Authors: Christopoulos Theodore K.
Published: September 2006

The discovery of restriction endonucleases is perhaps the most significant technical milestone in the development of recombinant DNA technology. For, together with ligation and transformation procedures, they enabled the insertion of genes of any organism in suitable vectors (e.g. plasmids), introduction of the vector in bacteria and subsequent replication of the DNA of interest during cell division (cloning). Other important technical milestones include the development of methods for DNA sequencing and the chemical synthesis of oligodeoxynucleotides.

The polymerase chain reaction (PCR) is another technique which had a major impact in every area of research that involves nucleic acid analysis. PCR is a method of exponentially increasing the concentration of a specific nucleic acid sequence in a reaction mixture. A great advantage of PCR is that the target nucleic acid does not need to be particularly pure. It can be a minute part of an extremely complex mixture of biological material. Crude cell lysates provide a very good starting material. The ability to propagate, in a few hours, minute amounts of DNA that are too small for standard amplification (i.e. cloning) techniques gives PCR an extraordinary power and sensitivity.

The advent of PCR stimulated the research activity in the development of alternative exponential amplification systems such as the ligase chain reaction (LCR), the self‐sustained sequence replication (3SR), the nucleic acid sequence‐based amplification (NASBA) and the strand displacement amplification system (SDA). An essential feature of target amplification techniques is that the amplification is an iterative process consisting of successive cycles. The products synthesized in one cycle act as templates for the next cycle.

Amplification techniques can be used to amplify trace amounts of nucleic acid sequences in blood, cells, water, food and other clinical and environmental samples. Fixed tissue specimens, buccal cells from mouth washes, human hair, dried blood at the scene of a crime, single lymphoid or sperm cells are examples of starting material that may be used for nucleic acid analysis.
Radiation Hybrid Mapping
Authors: Newell William R.
Published: September 2006

Radiation hybrid (RH) mapping is a convenient and widely used method for rapidly locating new DNA sequences on genomes in relation to other DNA markers. The aim of the technique is to determine the correct order and relative distances between a set of markers, or to locate a new marker to its most likely location on a genome. We review the history of the technique, its current status, and the main analytical techniques used to generate maps from raw data. The physical processes and assumptions underlying the technique are examined. These processes are used to derive models from which the required information, location of markers with respect to each other on a linear map of DNA, is obtained by a variety of computational means. The RH technique has proved indispensable in mapping the human genome, since, unlike genetic mapping, the conditions under which panels are made is completely under experimental control, so mapping at any degree of resolution is possible. The other major advantage of RH mapping over genetic mapping is that markers need not be polymorphic; it is presence or absence of a marker that is detected, not type. It has therefore been used to map the large numbers of expressed sequence tags that have been generated as part of the human genome project (HGP). This has given rise to the first human gene map and subsequent updates, so that currently 40 000 genes have been assigned locations on the human genome.
Structural Analysis of Ribozymes
Authors: Ananvoranich Sirinart , Perreault Jean‐Pierre
Published: September 2006

We use delta ribozymes as a model in illustrating the various steps involved in the structural analysis of ribozymes. Delta ribozymes were originally identified as a self‐cleaving motif located on the single‐stranded RNA genome of hepatitis delta virus (HDV). The methodologies are presented in the order of their requirement for determination of the structure/function relationship of a ribozyme, beginning with the preparation of the RNA molecules required for the studies. Primary structural information is needed in order to identify potential ribozymes, then secondary and tertiary structural information is required for ribozyme characterization. The manuscript was written to guide an investigator from the initial observation of RNA catalytic activity (a ribozyme discovery) to the deduction of a structural model of the ribozyme.
Centrifugation in Particle Size Analysis
Authors: Mächtle Walter
Published: September 2006

Millions of tons per year of submicron particles are produced in industrial processes around the world. They are also referred to as nanoparticles, colloids, latices, polymer dispersions, mini‐emulsions or micro‐emulsions. These solid or liquid particles, which are mostly dispersed in an aqueous medium, have a diameter (D) in the 10 < D < 3000 nm range. They are used as dyes and pigments, in paints, adhesives and coatings applied to paper, as impact modifiers in plastics, as catalysts, in cosmetic and photographic emulsions, metal sols and electrodeposition coatings applied to automobiles, as excipients for pharmaceuticals, etc.

Most of the valuable practical properties of these nanoparticle dispersions/emulsions result from their extremely small size and their high specific surface area. This implies that it is essential to be able to control and measure the particle size in order to produce nanoparticle systems of consistent quality. The particle size distribution is just as important as the average particle diameter. The practical consequences of changing from a very narrow to a very broad particle size distribution with the same average diameter can be quite dramatic in many applications. For example, the viscosity of a high‐solid polymer latex will decrease, the impact strength of plastic material will increase, surfaces coated with emulsion paints will become more level, etc.

Many methods, such as light scattering, electron microscopy, field flow fractionation (FFF), capillary hydrodynamic chromatography and ultracentrifugation, are used to measure the average particle diameters and overall particle size distribution of nanoparticles. Analytical ultracentrifugation (AUC) is the most versatile method with the broadest diameter range (1–5000 nm) and the highest resolution. The nanoparticles are forced to move through a predominantly aqueous medium inside an AUC measuring cell under a centrifugal force of 500–500 000 × g (rotor speed 100–60 000 rpm). Sedimentation causes fractionation to take place, because the larger particles move faster. The sedimentation velocities s
i
of the different fractions i are measured with refractive index, ultraviolet (UV)‐absorption or light scattering/turbidity detectors. The diameters D
i
of the different fractions can be calculated from the difference in s
i
, i.e. the complete particle size distribution, according to Stokes' law. The resolution of the AUC method is so high because, according to Stokes, s
i
is proportional to the square of D
i
. The AUC particle size distribution method can give spurious results if D is too high, i.e. if the particles form a sediment too quickly or if they display a broad chemical heterogeneity (i.e. no uniform particle density).
Optical Particle Counting
Authors: Lieberman Alvin
Published: September 2006

This discussion of optical particle counting (OPC) points out application areas where the procedure is used to collect data that would not be available otherwise. These areas include determination of particle size distributions from the size data of individual particles, identification of particles that do not fit within a specified size distribution function, and characterizing the particles present in a process of product fluid. The designs, performance, and operation of OPCs for both gasborne and liquidborne particles are discussed. Capabilities and limitations in terms of particle size ranges, counting accuracy, concentration, and so on are pointed out. The primary limitation is related to the fact that OPCs respond not only to the size of the particles, but also to the optical nature of the particles and of the fluid. Data are also affected by the counter design. All respond to a physical and/or chemical property of the particles, as well as to their physical size and morphology. The limitations of OPCs in relation to those factors are considered.

Operational requirements for OPC are pointed out. These include good sample acquisition and handling procedures, consideration of the effects of differences between particle counter designs on reported data, requirements for careful calibration before use and the effects of differences between calibration particles and sampled particles. A brief summary of present national and international standard documents for calibration and operation of these instruments is included. Performance capabilities of current OPCs are summarized and possible future developments are discussed. Performance is considered in comparison with that of other particle measuring procedures.
Photon Correlation Spectroscopy in Particle Sizing
Authors: Tscharnuter Walther
Published: September 2006

Photon correlation spectroscopy (PCS) has become a powerful light‐scattering technique for studying the properties of suspensions and solutions of colloids, macromolecules and polymers, that is absolute, non‐invasive and non‐destructive. This text explains the principles of the technique and describes the required instrumentation. It also discusses new developments in instrumentation, which may substantially impact on future applications of this technique. Proper sample preparation is of major importance for the accuracy and precision of the mean size and size distribution results and thus one section is dedicated to sample handling.
Sedimentation in Particle Size Analysis
Authors: Bernhardt Claus
Published: September 2006

Particle size analysis by sedimentation is one of the most important techniques of analysis. The principle is based on the fact that it is possible to draw conclusions about the geometrical dimensions (size) of the particles from the speed of their movement in a fluid. In the past many different apparatuses were developed. Nowadays, methods that allow quick, automatic measurement are most popular.

This article consists of two general sections valid for all methods: the first concerns the undisturbed and disturbed movement of particles in a fluid at rest, and the second concerns the dispersion of solids in liquids.

The methods described are divided in gravitational methods and methods used in a centrifugal field, respectively. Finally, the appendix gives a list of liquids and dispersing agents valid for measurement.
Surface Area and Pore Size Distributions
Authors: Takei Takashi
Published: September 2006

The determination methods of the surface area and pore size distribution (PSD) of a solid have been extensively described. The surface area and PSD are important as the basic properties for a powder and porous material. Nowadays the surface area and PSD are easily obtained by an automatic measuring instrument. However, there is no method which covers the complete range of a specific surface area and a pore size. All of the methods have their limits of measurement. In many cases the definitions of the surface area and pore size in each method differ from one another. The features and principles of the methods have been discussed mainly in order to select an appropriate method and to analyze data exactly.
Capillary Electrophoresis of Proteins and Glycoproteins
Authors: Trout Amy , Muza Michelle M. , Landers James P.
Published: September 2006

As a result of their complex nature, the separation of proteins and glycoproteins by capillary electrophoresis (CE) is a challenge. Fortunately, a number of approaches allow for effective analysis of these biomolecules. Of particular concern is the proclivity of proteins and their glycoconjugates to adsorb to the capillary wall. Various covalent and dynamic capillary coatings are described which minimize this effect. We also address general concerns including electrode polarity, capillary conditioning and method development that are important for achieving optimal separation. Finally, each separation mode, including capillary zone electrophoresis (CZE), capillary gel electrophoresis (CGE), and capillary isoelectric focusing (CIEF), is discussed with particular attention to its niche in the realm of protein and glycoprotein analysis.
Fluorescence Spectroscopy in Peptide and Protein Analysis
Authors: Ladokhin Alexey S.
Published: September 2006

Fluorescence spectroscopy and its multiple applications to the life sciences have undergone rapid development. This is due to numerous technical advances in both instrumentation and methods of data analysis as well as to a vast proliferation of basic techniques. Applications of fluorescence spectroscopy to protein and peptide analysis are governed by three principal factors: the dynamic nature of the signal, its localized nature, and its redundancy. Although these features can complicate interpretation of the experimental result, they also can be exploited to obtain unique structural and dynamic information. The availability and simplicity of basic data acquisition and analysis are important practical features behind the popularity of fluorescence as compared to other spectroscopic techniques. Yet this simplicity does not appear to compromise its advantages. This article is intended first to provide an overview of the fluorescence phenomenon in proteins, and second to illustrate applications of fluorescence spectroscopy in advanced (but not necessarily high‐tech) studies. Three areas of protein studies, namely protein–ligand interactions, protein folding and studies of membrane proteins, have been chosen to demonstrate key advantages of fluorescence spectroscopy: it is sensitive, versatile, and it lends itself readily to fast data acquisition.
Protein Purification: Theoretical and Methodological Considerations
Authors: Takács Béla János
Published: September 2006

With the rapid advances in recombinant DNA technologies, it became possible to express large amounts of virtually any protein in different host cells, including bacteria, yeast, insect, and mammalian cells. Efficient purification methods are becoming more and more important as the need for protein drugs continues to expand. In this article we cover some basic procedures for the purification of soluble recombinant proteins expressed in Escherichia coli and methods to obtain soluble and active protein from insoluble and inactive inclusion bodies. Finally, methods for the purification of native intrinsic membrane proteins from human lymphoblastoid cell lines, using immobilized monoclonal antibodies (mAbs), are presented.

As a first step, the protein of interest has to be released into an aqueous extract. Following extraction, residual debris and intact cells are removed by centrifugation. Nucleic acids are either hydrolyzed or precipitated to reduce the viscosity of the extract. A crude enrichment for the protein of interest is made based on its differential solubility in ammonium sulfate. This is then followed by high‐resolution chromatographic methods. The type of chromatographic matrices and the order of their use are evaluated in pilot experiments for column selection. By combining the specificity and discriminatory powers of various chromatographic techniques, it is possible to isolate rapidly a single protein species from a heterogeneous mixture.

For the purification of native intrinsic membrane proteins, the use of immunoaffinity methods is emphasized, since specific mAbs may be obtained even against low‐copy‐number membrane proteins by reducing a multispecific antibody response to a series of monospecific responses by cloning. The use of immobilized, highly specific mAbs is demonstrated in the purification of human leukocyte antigen D‐related (HLA‐DR) antigens.

Rapid advances in complete genome sequences provide us with a continuously expanding number of new potential therapeutic targets. These proteins will have to be purified and characterized. The existence of a functional protein blueprint will be invaluable not only for the diagnosis but possibly also for the treatment of many diseases.
Proteolytic Mapping
Authors: Nadler Tim
Published: September 2006

Proteolytic mapping is a technique used to verify the primary structure of a protein. It involves the cleavage of the protein at specific amino acids to generate a unique set of peptides which is then separated and analyzed. The chromatogram generated during the separation is known as the proteolytic map of the protein, which serves as a unique “fingerprint” for that protein. A proteolytic map can be used to verify the identity of a protein and to detect mutations in a protein. This technique is faster than two‐dimensional (2D) nuclear magnetic resonance (NMR), X‐ray crystallography, or complete amino acid sequencing for verifying the primary structure of a protein. However, it does not allow determination of the sequence of a protein de novo as can be accomplished with sequencing, nor does it yield the higher order structural information that may be obtained with 2D NMR or X‐ray crystallography.

The process of proteolytic mapping involves sample preparation, enzyme digestion, and map development. Each of these steps should be optimized for each new protein to obtain a good map; alternatively, generic protocols can be used to produce satisfactory results for some proteins. A typical protocol requires about a day to complete owing to the time required to digest a protein with an enzyme. However, more recent automated techniques are capable of completing the process in a few hours because they employ a highly concentrated immobilized enzyme which speeds digestion. Proteolytic maps are used in both quality control (QC) and research environments, with the most recent application in the field of proteomics.
Reversed‐Phase High‐Performance Liquid Chromatography in Peptide and Protein Analysis
Authors: Aguilar Marie‐Isabel
Published: September 2006

Reversed‐phase high‐performance liquid chromatography (RPHPLC) involves the separation of molecules on the basis of hydrophobicity. The separation depends on the hydrophobic binding of the solute molecule from the mobile phase to the immobilized hydrophobic ligands attached to the stationary phase, i.e. the sorbent. The solute mixture is initially applied to the sorbent in the presence of aqueous buffers, and the solutes are eluted by the addition of organic solvent to the mobile phase. Elution can proceed either under isocratic conditions, where the concentration of organic solvent is constant, or by gradient elution, whereby the amount of organic solvent is increased over a period of time. The solutes are therefore eluted in order of increasing molecular hydrophobicity. RPHPLC is a very powerful technique for the analysis of peptides and proteins because of a number of factors which include (1) the excellent resolution that can be achieved under a wide range of chromatographic conditions for very closely related molecules in addition to structurally distinct molecules, (2) the experimental ease with which chromatographic selectivity can be manipulated through changes in mobile phase characteristics, (3) the generally high recoveries and hence high productivity and (4) the excellent reproducibility of repetitive separations carried out over a long period of time, which is due partly to the stability of the sorbent materials under a wide range of mobile phase conditions. However, RPHPLC can cause the irreversible denaturation of protein samples, thereby reducing the potential recovery of material in a biologically active form.
X
‐Ray Crystallography of Biological Macromolecules
Authors: Messerschmidt Albrecht , Huber Robert
Published: September 2006

X‐ray crystallography of biological macromolecules comprises the determination of the three‐dimensional structures of proteins, nucleic acids and other biological macromolecules at atomic resolution by diffraction of X‐rays on crystals of such macromolecules. The atomic structure is obtained from the electron density distribution of the macromolecular crystal, which is the Fourier transform of the waves diffracted by the crystal. The amplitudes of the diffracted waves are determined directly from the diffraction experiment. The diffraction phases are revealed (i) from additional diffraction data of isomorphous heavy atom derivatives [multiple isomorphous replacement (MIR) technique], (ii) from multiple anomalous diffraction (MAD), if suitable anomalous scatterers are in the crystal and using tunable synchrotron radiation, and (iii) by Patterson search techniques (molecular replacement), if structural information on the biological macromolecule under investigation is available. X‐ray crystallography of biological macromolecules is the unique method for the elucidation of the spatial structures at atomic resolution of complex biomolecules with molecular masses greater than 30 kDa. The structures represent a time average and spatial average of molecules packed in a crystal. The preparation of suitable crystals may be a limiting factor.