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Size-Exclusion Chromatography
Abstract
Size-exclusion chromatography is also called gel-filtration or gel-permeation chromatography. This method uses porous particles to separate molecules of different sizes. It is generally used to separate biological molecules and to determine molecular weights and molecular weight distributions of polymers, and the separation of molecules is called fractionation. It is usually applied to large molecules or macromolecular complexes such as proteins and industrial polymers. The size of the pores in the beads determines the exclusion limit (what goes through the beads and what goes around the beads). There are different types of methods to separate the molecules based on different conditions, such as temperature variation. The usage of size-exclusion chromatography for different applications is also presented.
... Another type of partition chromatography that uses porous particles to differentiate molecules of various sizes is called size-exclusion chromatography. This method is also commonly known as gel filtration or gel permeation chromatography (Giridhar et al. 2017). The main idea behind this technique is that the molecules are separated according to size into mobile and stationary phases (comprising a porous matrix) with a specific porosity. ...
Chromatography, as a powerful analytical tool, plays a pivotal role in diverse fields such as chemistry, biology, pharmaceuticals, and environmental sciences. The current chapter provides a comprehensive overview of traditional chromatographic techniques, such as gas chromatography (GC), liquid chromatography (LC), and thin-layer chromatography (TLC), while also spotlighting recent innovations and emerging trends. The discussion encompasses the fundamental principles underlying chromatography, elucidating the mechanisms that enable the precise separation of complex mixtures. The latest advancements in ultra-high-performance liquid chromatography (UHPLC) and high-performance liquid chromatography (HPLC) are given particular attention, shedding light on their enhanced efficiency, sensitivity, and speed. Additionally, the chapter explores the integration of chromatography with other analytical methods, which are mass spectrometry (MS) and nuclear magnetic resonance (NMR), amplifying the capabilities for compound recognition and structural elucidation.
Furthermore, the applications of chromatography in diverse research domains are explored, ranging from pharmaceutical analysis to environmental monitoring and food safety. The chapter highlights case studies that exemplify the practical implementation of chromatography in addressing complex analytical challenges and underscores its role in advancing scientific knowledge. As chromatography continues to evolve, the chapter concludes with a forward-looking perspective on emerging trends, including the rise of miniaturized and portable chromatographic systems, developments in stationary phase technology, and the integration of artificial intelligence in data analysis. This investigation is an invaluable tool for students, scholars, learners, and experts aiming for a thorough comprehension of the latest advancements in chromatography and their implications for advancing analytical methodologies across various scientific disciplines.
... Additionally, these methods may be incompatible with a wide range of nanoparticle sizes, polydispersity, and nanoparticles that adhere to the column material. However, they do offer the advantage of investigating individual protein-nanoparticle association/dissociation rates and attraction, as well as collecting diverse portions of an experiment with minimal disruptive effects on particle-protein complexes [121]. ...
Polymeric nanomaterials, nanogels, and solid nanoparticles can be fabricated using single or double emulsion methods. These materials hold great promise for various biomedical applications due to their biocompatibility, biodegradability, and their ability to control interactions with body fluids and cells. Despite the increasing use of nanoparticles in biomedicine and the plethora of publications on the topic, the biological behavior and efficacy of polymeric nanoparticles (PNPs) have not been as extensively studied as those of other nanoparticles. The gap between the potential of PNPs and their applications can mainly be attributed to the incomplete understanding of their biological identity. Under physiological conditions, such as specific temperatures and adequate protein concentrations, PNPs become coated with a “protein corona” (PC), rendering them potent tools for proteomics studies. In this review, we initially investigate the synthesis routes and chemical composition of conventional PNPs to better comprehend how they interact with proteins. Subsequently, we comprehensively explore the effects of material and biological parameters on the interactions between nanoparticles and proteins, encompassing reactions such as hydrophobic bonding and electrostatic interactions. Moreover, we delve into recent advances in PNP-based models that can be applied to nanoproteomics, discussing the new opportunities they offer for the clinical translation of nanoparticles and early prediction of diseases. By addressing these essential aspects, we aim to shed light on the potential of polymeric nanoparticles for biomedical applications and foster further research in this critical area.
... Larger molecules are eluted earlier than small compounds, as the beads have cross-linked polyacrylamide, agarose, and dextran, where smaller compounds enter the sieves in the matrix of the stationary phase (Duong-Ly and Gabelli 2014a). According to Giridhar et al. (2017), porosity, i.e., pore size, is an important parameter. Because SEC separates molecules according to their size in solution, the process occurs wholly within the pore volume, which should be as large as possible. ...
α-Amylases (E.C 3.2.1.1) hydrolyse starch into smaller moieties such as maltose and glucose by breaking α-1,4-glycosidic linkages. The application of α-amylases in various industries has made the large-scale productions of these enzymes crucial. Thermostable α-amylase that catalyses starch degradation at the temperatures higher than 50 °C is favourable in harsh industrial applications. Due to ease in genetic manipulation and bulk production, this enzyme is most preferably produced by microorganisms. Bacillus sp. and Escherichia coli are commonly used microbial expression hosts for α-amylases (30 to 205 kDa in molecular weight). These amylases can be purified using ultrafiltration, salt precipitation, dialysis, and column chromatography. Recently, affinity column chromatography has shown the most promising result where the recovery rate was 38 to 60% and purification up to 13.2-fold. Microbial thermostable α-amylases have the optimum temperature and pH ranging from 50 °C to 100 °C and 5.0 to 10.5, respectively. These enzymes have high specificity towards potato starch, wheat starch, amylose, and amylopectin. EDTA (1 mM) gave the highest inhibitory effect (79%), but Ca2+ (5 mM) was the most effective co-factor with 155%. This review provides insight regarding thermostable α-amylases obtained from microbial sources for industrial applications.
... In order to assess the complex between CisPt and NaHA, we decided to exploit their great difference in molecular weight [32]. An HPLC method was developed using a Size Exclusion Chromatography (SEC) column. ...
Pleural mesothelioma is a lung diffuse tumor, whose complete resection is unlikely. Consequently, metastases reappear where the primary tumor was removed. This paper illustrates the orphan medicine designation procedure of an intracavitary cisplatin film and related pharmaceutical development aspects requested by the European Medicines Agency (EMA) in its Scientific Advice. Since cisplatin pharmacokinetics from the implanted film in sheep resulted substantially modified compared to intravenous administration, the formation of a cisplatin/hyaluronan complex had been hypothesized. Here, the interaction between sodium hyaluronate (NaHA) and cisplatin (CisPt) was demonstrated. Size exclusion chromatography qualitatively evidenced the complex in the film-forming mixture, only showing the NaHA peak. Atomic absorption spectroscopy of the corresponding fraction revealed platinum, confirming the interaction. Reverse phase HPLC quantified about 5% free cisplatin in the film-forming mixture, indirectly meaning that 95% was complexed. Finally, a study of CisPt release from the film assessed how CisPt/NaHA complex affected drug availability. In water, a medium without chloride ions, there was no release and the film remained intact for 48 h and longer, whereas the placebo film dissolved in 15 min. In 0.9% NaCl medium, the film became more soluble, dissolving within 3–4 h. However, cisplatin release was still controlled by the existing complex in solution until chloride ions displaced it. While the film modified its dissolution with aging, CisPt release remained unaffected (90% released in 48 h).
... Composed of monosaccharide unit long chains linked by glycosidic bonds, polysaccharides are polymeric carbohydrate molecules [14]. Gel permeation chromatography (GPC) was widely applied for characterization of polysaccharides with identification of molecular weight averages and molecular weight distributions offering numerous advantages [15,16]. In this study, polysaccharides were extracted from G. lucidum using UAEE method. ...
Red Ganoderma lucidum (G. lucidum) is a popular medicinal herb commonly used in Vietnamese traditional remedies due to its potential value for health. In this study, polysaccharides were extracted from G. lucidum using ultrasound-assisted enzymatic extraction method. The response surface methodology and Box–Behnken design were employed to investigate the effects of pH, extraction temperature, extraction time, and ultrasonic power on the content of polysaccharides. Based on ultraviolet-visible spectroscopy analysis, the highest content of polysaccharides in the extract was 32.08 mg/g under optimum experimental parameters including enzyme concentration of 3%, pH of 5.5, extraction temperature of 45°C, extraction time of 30 min, and ultrasonic power of 480 W. The Fourier-transform infrared spectroscopy was also used to identify the functional groups in the extracts. The molecular weights of polysaccharides were determined by gel permeation chromatography. The obtained extract was then evaluated for anticancer activities by using (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay, showing the anticancer activities with the half-maximal inhibitory concentration value of more than 512 μg/mL. This result suggested that UAEE could be considered as an appropriate and effective extraction method for bioactive crude polysaccharides from G. lucidum.
... Larger molecules are eluted earlier than small compounds, as the beads have cross-linked polyacrylamide, agarose, and dextran, where smaller compounds enter the sieves in the matrix of the stationary phase (Duong-Ly and Gabelli 2014a). According to Giridhar et al. (2017), porosity, i.e., pore size, is an important parameter. Because SEC separates molecules according to their size in solution, the process occurs wholly within the pore volume, which should be as large as possible. ...
Amylases (E.C 3.2.1.1) hydrolyse starch into smaller moieties such as maltose and glucose by breaking α-1,4-glycosidic linkages. The application of α-amylases in various industries has made the large-scale productions of these enzymes crucial. Thermostable α-amylase that catalyses starch degradation at the temperatures higher than 50 °C is favourable in harsh industrial applications. Due to ease in genetic manipulation and bulk production, this enzyme is most preferably produced by microorganisms. Bacillus sp. and Escherichia coli are commonly used microbial expression hosts for α-amylases (30 to 205 kDa in molecular weight). These amylases can be purified using ultrafiltration, salt precipitation, dialysis, and column chromatography. Recently, affinity column chromatography has shown the most promising result where the recovery rate was 38 to 60% and purification up to 13.2-fold. Microbial thermostable α-amylases have the optimum temperature and pH ranging from 50 °C to 100 °C and 5.0 to 10.5, respectively. These enzymes have high specificity towards potato starch, wheat starch, amylose, and amylopectin. EDTA (1 mM) gave the highest inhibitory effect (79%), but Ca 2+ (5 mM) was the most effective co-factor with 155%. This review provides insight regarding thermostable α-amylases obtained from microbial sources for industrial applications.
... AUC, whether by sedimentation velocity or Analytical SEC is a quantitative and relatively robust, simple method that separates macromolecules during flow through a packed column. The principles and applications of SEC are well presented in several reviews 18,19,20 and in the handbook "Size Exclusion Chromatography: Principles and Methods" 21 . The differences in retention are due to different amounts of time spent diffusing into and out of the pores in the stationary phase before eluting from the end of the column. ...
Analytical size-exclusion chromatography (SEC), commonly used for the determination of the molecular weight of proteins and protein-protein complexes in solution, is a relative technique that relies on the elution volume of the analyte to estimate molecular weight. When the protein is not globular or undergoes non-ideal column interactions, the calibration curve based on protein standards is invalid, and the molecular weight determined from elution volume is incorrect. Multi-angle light scattering (MALS) is an absolute technique that determines the molecular weight of an analyte in solution from basic physical equations. The combination of SEC for separation with MALS for analysis constitutes a versatile, reliable means for characterizing solutions of one or more protein species including monomers, native oligomers or aggregates, and heterocomplexes. Since the measurement is performed at each elution volume, SEC-MALS can determine if an eluting peak is homogeneous or heterogeneous and distinguish between a fixed molecular weight distribution versus dynamic equilibrium. Analysis of modified proteins such as glycoproteins or lipoproteins, or conjugates such as detergent-solubilized membrane proteins, is also possible. Hence, SEC-MALS is a critical tool for the protein chemist who must confirm the biophysical properties and solution behavior of molecules produced for biological or biotechnological research. This protocol for SEC-MALS analyzes the molecular weight and size of pure protein monomers and aggregates. The data acquired serve as a foundation for further SEC-MALS analyses including those of complexes, glycoproteins and surfactant-bound membrane proteins.
... The prerequisite for a direct correlation between elution time and molecular size of the analyte is an assumption of no interactions between stationary phase and analyte. Otherwise, nonexclusion effects such as ionic exclusion and sorption will also govern constituent separation as shown in Figure 13.3 (Belenkii, 1998 ;Dubin, 1988 ). ...
... . , , ------_ . _ ------_ .. _-------::---24 Kelen-Tudos plot for copolymerisation of p-AA TBTB-MA (estimated by' H NMR spectroscopy) ...
Thesis (Ph. D.)--Loughborough University of Technology, 1996.
Antidiabetic biologicals (ADBs) have revolutionized the treatment of diabetes mellitus, once considered incurable through conventional medicine. These biological products, derived from natural sources via extraction, semi-synthesis, or recombinant DNA technology, include insulin and its analogs, GLP-1 receptor agonists, amylin analogs, and the recently approved monoclonal antibody teplizumab. Regulatory authorities worldwide have established QC parameters outlined in pharmacopoeias, alongside analytical techniques to ensure their safety and efficacy. This review focuses on the analytical techniques used to assess QC parameters of ADBs, including chromatographic methods, spectroscopic techniques, capillary electrophoresis, immunoassays, and endotoxin testing. Key parameters such as identification, potency, purity, and impurity profiling are thoroughly examined. The paper provides a comprehensive and up-to-date compilation of QC requirements and methodologies, along with a detailed comparison of analytical techniques. In doing so, it highlights their advantages and limitations, offering valuable insights for researchers and regulatory professionals involved in selecting suitable methods for QC assessment and understanding the complexities of ADBs evaluation. Furthermore, the article discusses the paramount importance of QC and future perspectives, emphasizing the transition to advanced versions of current techniques driven by the need for efficiency and reliability in quality testing.
The promising field of regenerative medicine is thrilling as it can repair and restore organs for various debilitating diseases. Mesenchymal stem cells are one of the main components in regenerative medicine that work through the release of secretomes. By adopting the use of the secretome in cell-free-based therapy, we may be able to address the challenges faced in cell-based therapy. As one of the components of cell-free-based therapy, secretome has the advantage of a better safety and efficacy profile than mesenchymal stem cells. However, secretome has its challenges that need to be addressed, such as its bioprocessing methods that may impact the secretome content and its mechanisms of action in clinical settings. Effective and standardization of bioprocessing protocols are important to ensure the supply and sustainability of secretomes for clinical applications. This may eventually impact its commercialization and marketability. In this review, the bioprocessing methods and their impacts on the secretome profile and treatment are discussed. This improves understanding of its fundamental aspects leading to potential clinical applications.
Prostate cancer is the second leading cause of men's death worldwide. Although early diagnosis and therapy for localized prostate cancer have improved, the majority of men with metastatic disease die from prostate cancer annually. Therefore, identification of the cellular‐molecular mechanisms underlying the progression of prostate cancer is essential for overcoming controlled proliferation, invasion, and metastasis. Exosomes are small extracellular vesicles that mediate most cells' interactions and contain membrane proteins, cytosolic and nuclear proteins, extracellular matrix proteins, lipids, metabolites, and nucleic acids. Exosomes play an essential role in paracrine pathways, potentially influencing Prostate cancer progression through a wide variety of mechanisms. In the present review, we outline and discuss recent progress in our understanding of the role of exosomes in the Prostate cancer microenvironment, like their involvement in prostate cancer occurrence, progression, angiogenesis, epithelial–mesenchymal transition, metastasis, and drug resistance. We also present the latest findings regarding the function of exosomes as biomarkers, direct therapeutic targets in prostate cancer, and the challenges and advantages associated with using exosomes as natural carriers and in exosome‐based immunotherapy. These findings are a promising avenue for the expansion of potential clinical approaches.
Background
The development of nanostructures for food applications is growing exponentially, due to their potential to improve foods' nutritional and health value as well as foods' shelf-life. However, there is still a lack of information regarding the nanostructures' effects on the complex food matrices and vice versa, leading to some concerns about nanostructures’ safety and food quality. One of the reasons is the difficulty to identify and characterize organic nanostructures within food matrices.
Scope and approach
This article reports a critical review of some of the most recent applications of organic nanostructures in food products, and focus on the methodologies that can be used to evaluate the food matrix/nanostructures interactions. The most used techniques for organic nanostructures' identification and characterization in food matrices are presented, and other potential techniques/methodologies are suggested. Furthermore, some suitable techniques to evaluate the effects of nanostructures’ incorporation in food products were also described and discussed.
Key findings and conclusions
Despite the high number of recent works reporting the application of organic nanostructures in food products and the existence of techniques that can be used for their identification and characterization within food matrices, the standardization of existing methodologies and/or the development of new reliable techniques and methodologies for identification and characterization of organic nanostructures are still needed. Also, it is essential to conduct more studies to assess food matrix/nanostructures interactions, in order to minimize the existing knowledge gap between development of new organic nanostructures and their application in food products.
Complete polymer compositional information of polyolefins is critically valuable in understanding the polymer structure‐processing‐property relationship, thus helping tailor‐design polymers with desired end‐use properties. In this paper, a robust on‐line 2D analytical TREF combined with high‐throughput SEC technique (2D aTREF‐hSEC) is described that can rapidly determine the bivariate distribution of chemical composition and molecular weight of polyethylene resins. Unlike Nakano and Goto's pioneering cross fractionation apparatus and the commercial CFC instrument, this 2D aTREF‐hSEC system has the following unique characteristics: First of all, the GPC injection volume is not limited by the dead volume of the full TREF column. It can be set at any desired values to ensure optimized separation in SEC. Second, the flow rates for the TREF and SEC unit are independently controlled, which add operational flexibility and help optimize the separation efficiency in both the TREF and the SEC dimension. Third, the 2D aTREF‐hSEC system runs continuously instead of stepwise. Once started, the TREF column elutes continuously without stopping to wait for the completion of downstream SEC elution before the next SEC injection, increasing the resolution in TREF dimension. Fourth, experimental time is significantly reduced thanks to the employment of high throughput SEC column. And finally, this 2D aTREF‐hSEC technique in principle can be applied equally well to characterize other semi‐crystalline polymers and potentially any polymers whose solubility is a function of temperature.
There is a large and increasing variety of polymers currently in use both for domestic and industrial applications. The properties of polymers are deter mined not only by their chemical type, but also by their molecular mass and molecular mass distributions. However, while the chemical type of polymers can be determined relatively easily, the average molecular masses and molecular mass distributions are more difficult to measure. The molecular mass averages of a polymer are measured by specialized and complex techniques such as light scattering (for weight average) and osmometry (for number average). Thus, complete characterization of the molecular mass distribution of a polymer by such means requires separating the sample into many fractions which can then be examined individually. Since size exclusion chromatography was introduced as a rapid and straightforward technique for the characterization of polymer molecular mass distributions, there have been tremendous increases in development and applications, and it was felt appropriate to bring together into a single volume the information required by scientists from many disciplines who wish to use the technique. This book should be useful to existing users, those who are new to the technique, and those who may be familiar with the basic technique and now wish to extend their capabilities to more complex applications (or to consider the potential of a number of related techniques). The book will also be of general interest to the experienced liquid chromatographer.
SEC measurements can be carried out using an HPLC setup which consists of a solvent delivery system, a separation system, and a detection system. A solvent delivery system is an assembly of a solvent reservoir, an on-line degasser, and a solvent delivery pump. A pulse damper may be connected after the pump. A separation system consists of a sample injector and SEC columns with or without a thermostated column oven. A detection system is composed of a detector, a strip-chart recorder and a waste reservoir. A data handling system to analyze SEC chromatograms and to calculate molecular weight (MW) averages of samples can be used in place of a strip-chart recorder. An autosampler (an autoinjector) which is used to inject a sample solution into the separation system at a specified interval can also be used with the HPLC apparatus. A schematic diagram of a typical SEC assembly is shown in Fig. 3.1.
A wide variety of biological and medical analyses are based on the use of optical signals to report specific molecular events. Thanks to advances in nanotechnology, various nanostructures have been extensively used as optical reporters in bio- and chemical assays. This review describes recent progress in chemical sensing using noble metal nanoparticles (gold and silver), quantum dots and upconverting nanoparticles. It provides insights into various nanoparticle-based sensing strategies including fluorescence/luminescence resonance energy transfer nanoprobes as well as activatable probes sensitive to specific changes in the biological environment. Finally we list some research challenges to be overcome in order to accelerate the development of applications of nanoparticle bio- and chemical sensors.
Multi-shell Cd-free nanocrystals were designed such that (1) the outer-most shell was free of reactive sulfide to prevent corrosion (2) the position of the band-edge emission remained unchanged during multi-shell coating. The photoluminescence and photostability were markedly enhanced and allowed the fabrication of light-emitting diode devices with high color-rendering abilities and high photostability.
As newly developed inorganic materials, semiconductor nanocrystals (NCs), or quantum dots (QDs), have received considerable attention because of their unique nano-related properties including high quantum yield, simultaneous excitation with multiple fluorescence colors, and electrochemical properties. This review presents a general description of the electrochemical properties of QDs with their electrochemical applications including indirect and direct effects. The fields of inorganic substance analysis, organic analysis, immunoanalysis, DNA analysis and aptamer analysis are discussed in detail.
Wide-bore hydrodynamic chromatography in a polyether ether ketone (PEEK) capillary (I.D. 0.7 mm, length 20 m) was used to determine the weight average diameter dw of colloidal particles. The method was applied to cadmium sulphide and gold sols in the diameter range between 3 nm and 27 nm. The method is based on the radial distribution of the analyte in the capillary due to the hydrodynamic flow profile in the capillary and due to the diffusion coefficient of the particles, which is dependent on their diameter. The diameter was calculated from the ratio of the heights of convection peak and diffusion peak. The size-quantization effect of small semiconductor particles made it possible to visualise the separation inside of the capillary. One important advantage of the applied method is the very much reduced adsorption, which often causes serious problems in the HPLC especially of inorganic colloids. The results of wide-bore hydrodynamic chromatography, size exclusion chromatography and transmission electron microscopy were compared.
Colloidal cadmium sulfide with diameters of ca. 17 nm was used as a model substance to study the effects of sample concentration on elution time and peak area in size exclusion chromatography (SEC) of colloidal inorganic particles in the low nm-size regime. A clear distinction had to be made between the pure particle concentration and that of the accompanying electrolytes. The effects were astonishingly high taking into account that particles and small electrolytes are separated immediately in SEC. The reasons for these phenomena are discussed. The electrical double layer was found to play an important role. The results obtained for solid particles were compared with those for organic polymers in SEC. A memory effect of the column was observed due to temporarily adsorbed particles.
In the reported experiments, high-speed gel-permeation chromatography (GPC) of water-soluble polymers has been investigated by using TSK-GEL, type-PW columns packed with small porous particles. Being semirigid, the column packing could be operated under high pressure and, therefore, it was possible to achieve high-speed GPC. A resolution higher than that of ordinary GPC in organic solvent system was attained when the measurement of one sample was completed within 40 min and a 6-ft column set. Samples with a wide range of molecular weights, from oligomers to polymers having a molecular weight greater than 10**6, could be fractionated. Dextran, polyethylene glycol, polyacrylamide, poly(vinyl alcohol), and poly(vinyl pyrrolidone) were separated according to molecular size with no evidence of adsorption. Investigation of other water soluble polymers is now in progress
Part I. Separation Mechanisms. Size exclusion parameters (M.E. Himmel, P.G. Squire). Partitioning: Hydrophobic interactions (M. Janado). Electrostatic effects (P.L. Dubin). Exclusion chromatography of inorganic compounds (M. Shibukawa, N. Ohta). II. Characterization of Stationary Phases. Pore size distributions (L. Hagel). Structural analysis of porous materials by measurement of size exclusion (S. Kuga). Column efficiency (S. Mori). III. New Packings. Native and bonded silicas in aqueous SEC (K.K. Unger, J.N. Kinkel). Rigid polymer gels for SEC and their application to biopolymers (K. Makino, H. Hatano). IV. Biopolymers. Biopolymers. I. Protein chromatography in denaturing and non-denaturing solvents (R.C. Montelaro). Biopolymers. II. Serum lipoproteins (M. Okazaki, I. Hara). Application of SEC/LALLS to biopolymer assemblies (K. Konishi). V. Associating Systems. Measurement of equilibrium constants by exclusion methods (T.K. Korpela, J.-P. Himanen). 14. Frontal boundary analysis in size exclusion chromatography of self-associating proteins (G.W. Becker). 15. Exclusion chromatography of micelles (K.S. Birdi). Subject Index.
Semiconductor quantum dots (QDs) are light-emitting particles on the nanometer scale that have emerged as a new class of fluorescent labels for chemical analysis, molecular imaging, and biomedical diagnostics. Compared with traditional fluorescent probes, QDs have unique optical and electronic properties such as size-tunable light emission, narrow and symmetric emission spectra, and broad absorption spectra that enable the simultaneous excitation of multiple fluorescence colors. QDs are also considerably brighter and more resistant to photobleaching than are organic dyes and fluorescent proteins. These properties are well suited for dynamic imaging at the single-molecule level and for multiplexed biomedical diagnostics at ultrahigh sensitivity. Here, we discuss the fundamental properties of QDs; the development of next-generation QDs; and their applications in bioanalytical chemistry, dynamic cellular imaging, and medical diagnostics. For in vivo and clinical imaging, the potential toxicity of QDs remains a major concern. However, the toxic nature of cadmium-containing QDs is no longer a factor for in vitro diagnostics, so the use of multicolor QDs for molecular diagnostics and pathology is probably the most important and clinically relevant application for semiconductor QDs in the immediate future. Expected final online publication date for the Annual Review of Analytical Chemistry Volume 6 is June 15, 2013. Please see http://www.annualreviews.org/catalog/pubdates.aspx for revised estimates.
HPLC is used to determine the size distribution of CdS colloids. These distributions agree with those obtained from electron microscopy. The method is applied to recognize 'magic' agglomeration numbers and to follow thermal particle growth and photodissolution. In the thermal growth a gradual increase in size is ascribed to Ostwald ripening. However, in the beginning a rather abrupt increase in size takes place that is ascribed to particle combination. In the photodissolution of a sample containing two size distributions, the larger particles disappear more rapidly than the smaller ones, this effect being particularly pronounced when light of longer wavelengths is used where the small particles have little absorption.
A fast flow technique is described in which two solutions containing Cd2+ and SH- ions are mixed, and the absorption spectrum of the resulting CdS sol is rapidly recorded using a diode array detector. Experiments were also performed in which the sol passed through a high-performance liquid chromatography column between the mixing chamber and diode array. The colloidal particles formed with and without the column had practically the same absorption spectrum (very small particles with a diameter down to 1.3 nm, strongly quantized). However, the particles formed using the column grew thermally much more slowly. The effect is attributed to the existence of "active" particles after the precipitation which promote the thermal growth. The active particles are filtered out by the column. With increasing Cd2+/SH- concentration ratio in the mixing chamber, smaller CdS particles are formed. In the most extreme cases studied, new species with a strong absorption maximum at 211 nm were obtained and their size (1.5 nm) was determined by size exclusion chromatography, the calibration of which was carried out by transmission electron microscopy. The flow technique is useful for rapid optimization of the precipitation conditions as well as for easy preparation of very small particles with extremely narrow size distribution.
It is shown that steric exclusion chromatography can be used to measure the size distribution of colloidal gold sols. Good separation can be achieved for particles ranging in size from 3.0 to 20 nm using columns containing 25-mum silica particles (pore size 50-100 nm) and with aqueous trisodium citrate (0.001 M) as the eluent.
Palladium clusters protected by a series of n-alkanethiolates, Pd:SCn (SCn = n-CnH2n+1S, n = 10, 12, 14, 16, and 18), were prepared by a ligand exchange approach: Pd clusters protected by poly (N-vinyl-2-pyrrolidone) (PVP) were transferred from the aqueous phase to the toluene phase containing the thiols. The structures and stabilities of the thiolate monolayers of the Pd:SCn clusters were investigated by gel permeation chromatography (GPC) together with TEM, XPS, and FT-IR. The thicknesses of the thiolate layers formed on the Pd clusters were evaluated from the differences between the hydrodynamic diameters and core diameters of the Pd:SCn clusters, determined by GPC and TEM, respectively. The thicknesses thus obtained are in good agreement with the lengths of the corresponding thiolates in the all-trans conformations, illustrating that the alkanethiolates in nearly straight configurations are aligned almost perpendicularly to the core surfaces. Fractionation of the Pd:SC18 clusters by GPC yielded a series of the purified samples: the clusters in each fraction are different in their core sizes. The GPC measurements on the Pd:SCn clusters with small n revealed the decomposition of the monolayers through spontaneous etching and their reconstruction by heat treatment in the presence of the free thiols. The mechanism of these processes is discussed. The present study demonstrates that the GPC provides an elemental and versatile means to characterize and purify the monolayer-protected clusters.
An HPLC technique for the size determination of colloidal cadmium sulphide and zinc sulphide in a diameter range from 20 down to 2 nm using silica with pore sizes from 30 to 100 nm is described. The growth of the particles during the run was suppressed by the addition of stabilizers to the eluent and by the use of reversed-phase silica as the stationary phase for inorganic stabilizers. The calibration of the column sets by electron microscopy resulted in a linear relationship between the logarithm of the particle diameter and the elution time. The analysis was carried out within 4–10 min. The lateral resolution lay between 1.3% for larger particles and 1.9% for smaller particles. Below a diameter of 13 nm these values were better than those found from electron microscopy. From the comparison of the calibration lines for various colloidal materials, the differences in their electrical double layers could be estimated. The limitations of the method are discussed and the size-exclusion chromatographic and electron microscopic methods are compared.
The shape separations of suspended gold nanoparticles were investigated using size-exclusion chromatography. The separations in shapes were identified by examining the 3-D chromatograms obtained by employing a diode-array detection system and were further confirmed by analyzing TEM images of fractional collection of particles. This shape separation was achieved by adding a mixed-surfactant system containing sodium dodecyl sulfate and poluoxyethylene (23) dodecanol (Brij-35) into the eluent, which apparently affects the adsorption behaviors of both rodlike and spherical Au nanoparticles onto the column packing materials. While the overall particle gross sizes of these two shapes were similar, the baseline resolution was unfortunately not obtainable. However, the absorption spectra from the diode-array detector could be utilized to interpret the shapes of Au nanoparticles. The potential capability for the size separation of Au nanoparticles by size-exclusion chromatography with diode-array detection was also demonstrated.
In a liquid containing ions an electrical double layer is formed close to any charged solid surface. Thus a charged dispersed species wears a “coat” of ions. In size-exclusion chromatography (SEC) of charged colloidal particles the effective size including this electrical double layer has to be considered. For the investigation of the effective size crystalline colloids with rigid core are advantageous, as their core diameters can be determined by electron microscopy. According to the Gouy–Chapman model the thickness of the double layer is inverse proportional to the square root of the ionic strength of the liquid. Thus the particles investigated (CdS colloids) were chromatographed on the same SEC column with eluents with different ionic strength. It was found that the retention times increased with increasing concentration and valency of the electrolyte present in the eluent. It could be shown that adsorption or repulsion did not play a significant role for this retention time shift as long as the electrolyte content is not too high. The layer thickness and its ionic strength dependence, derived from the SEC experiments with CdS particles in the low nm range were found in good agreement with theory. The sum of diameter of the particle core and the Debye length 1/κ (as predicted from theory) agreed with effective particle size measured chromatographically.
We have developed a facile means for the refolding of milligram quantities of purified proteins that employs gel filtration chromatography. We demonstrate by electrophoretic mobility shift and NMR spectroscopy that human ETS-1 protein, bovine ribonuclease A and E. coli integration host factor can be refolded into the native conformation using this technique. We have extended this strategy to the preparation of milligram quantities of macromolecular complexes suitable for structural analysis by NMR spectroscopy or X-ray crystallography. The diverse challenges to overcome in refolding these proteins illustrates the potential of this technique as a general approach for recovery of recombinant proteins produced as insoluble inclusion bodies.
Size exclusion chromatography and centrifugation separation protocols were developed and compared for isolating enriched fractions of phenylethynyl-bridged metal nanoparticle dimers and trimers from the monomeric particle starting material. Both methods enabled the isolation of enriched fractions of a desired array without causing significant sample aggregation or replacement of the phenylethynyl bridge. Solutions containing ca. 70% bridged gold dimers were obtained using either method. The further development of methods for separating discrete arrays of covalently bridged nanoparticle homo and hetero structures is expected to help advance our understanding of collective metal particle electronic structure-function relationships.
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