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Methods in Molecular Biology (Clifton,... 2011Fast protein liquid chromatography (FPLC) is a form of high-performance chromatography that takes advantage of high resolution made possible by small-diameter stationary...
Fast protein liquid chromatography (FPLC) is a form of high-performance chromatography that takes advantage of high resolution made possible by small-diameter stationary phases. It was originally developed for proteins and features high loading capacity, biocompatible aqueous buffer systems, fast flow rates, and availability of stationary phases in most common chromatography modes (e.g., ion exchange, gel filtration, reversed phase, and affinity). The system makes reproducible separation possible by incorporating a high level of automation including autosamplers, gradient program control, and peak collection. In addition to proteins, the method is applicable to other kinds of biological samples including oligonucleotides and plasmids. The most common type of FPLC experiment is anion exchange of proteins. This chapter describes such an experiment carried out using an ÄKTA FPLC explorer system (Amersham Pharmacia Biotech, Sweden).
Topics: Analytic Sample Preparation Methods; Chromatography, Gel; Chromatography, High Pressure Liquid; Chromatography, Ion Exchange; Proteins
PubMed: 20978981
DOI: 10.1007/978-1-60761-913-0_25 -
British Journal of Hospital Medicine... Feb 2014
Review
Topics: Chromatography, High Pressure Liquid; Humans
PubMed: 24521830
DOI: 10.12968/hmed.2014.75.Sup2.C18 -
Annual Review of Chemical and... Jun 2021Since its first appearance in the 1960s, solid support-free liquid-liquid chromatography has played an ever-growing role in the field of natural products research. The... (Review)
Review
Since its first appearance in the 1960s, solid support-free liquid-liquid chromatography has played an ever-growing role in the field of natural products research. The use of the two phases of a liquid biphasic system, the mobile and stationary phases, renders the technique highly versatile and adaptable to a wide spectrum of target molecules, from hydrophobic to highly polar small molecules to proteins. Generally considered a niche technique used only for small-scale preparative separations, liquid-liquid chromatography currently lags far behind conventional liquid-solid chromatography and liquid-liquid extraction in process modeling and industrial acceptance. This review aims to expose a broader audience to this high-potential separation technique by presenting the wide variety of available operating modes and solvent systems as well as structured, model-based design approaches. Topics currently offering opportunities for further investigation are also addressed.
Topics: Chromatography, Liquid; Countercurrent Distribution; Hydrophobic and Hydrophilic Interactions; Solvents
PubMed: 33848424
DOI: 10.1146/annurev-chembioeng-101420-033548 -
Journal of Chromatography. A Aug 2020Modern liquid-liquid chromatography mainly refers to the following two kinds of chromatographic apparatuses: countercurrent chromatography based on hydrodynamic...
Modern liquid-liquid chromatography mainly refers to the following two kinds of chromatographic apparatuses: countercurrent chromatography based on hydrodynamic equilibrium systems and centrifugal partition chromatography based on hydrostatic equilibrium systems. In this paper, the recent advancements in enantioseparations by liquid-liquid chromatography, including the separation mechanism, chiral selector, two-phase solvent system, methods to improve the peak resolution and recent applications, are reviewed. The future outlook for liquid-liquid chromatography in enantioseparations is also proposed.
Topics: Algorithms; Chromatography, Liquid; Countercurrent Distribution; Hydrogen-Ion Concentration; Ligands; Solvents; Stereoisomerism
PubMed: 32797825
DOI: 10.1016/j.chroma.2020.461345 -
Journal of Separation Science Sep 2022Pavel Jandera was a world-leading analytical chemist who devoted his entire professional life to research in the field of high-performance liquid chromatography. During... (Review)
Review
Pavel Jandera was a world-leading analytical chemist who devoted his entire professional life to research in the field of high-performance liquid chromatography. During his scientific career, he worked at the Department of Analytical Chemistry at the University of Pardubice, Czech Republic. His greatest contribution to the field of liquid chromatography was the introduction of a comprehensive theory of liquid chromatography with programmed elution conditions. He was also involved in the research of gradient elution techniques in preparative chromatography, modeling of retention and selectivity in various phase systems, preparation of organic monolithic microcolumns, and, last but not least, in the development of theory and practical applications of two-dimensional liquid chromatography, mainly in the comprehensive form. In this review article, we have tried to capture the highlights of his scientific career and provide the readers with a detailed overview of Pavel Jandera's contribution to the evolution of separation sciences.
Topics: Chromatography, High Pressure Liquid; Chromatography, Liquid; Humans; Male
PubMed: 35932491
DOI: 10.1002/jssc.202200618 -
Journal of Separation Science Jan 2022The use of micro, capillary, and nano liquid chromatography systems for forensic analysis has excellent potential. In a field where sample size is often limited, several... (Review)
Review
The use of micro, capillary, and nano liquid chromatography systems for forensic analysis has excellent potential. In a field where sample size is often limited, several studies have presented the viability of capillary columns with microflow and nanoflow, and when using mass spectrometric analysis limits of detection can be improved. Reduction in flow rates result in significant reduction in operating costs. Recent advances in miniaturized liquid chromatography systems also aim at in-laboratory and on-site detection, which have already been applied to forensic drug cases. This critical review will discuss the advantages, disadvantages, and applicability of microflow and nano liquid chromatography. In this regard, included in this article is a discussion of some promising areas not yet applied to forensic research.
Topics: Animals; Chromatography, Liquid; Forensic Sciences; Humans
PubMed: 34626162
DOI: 10.1002/jssc.202100631 -
Journal of Chromatography. A Aug 2002The status of the theory and the main methods of implementation of preparative liquid chromatography are reviewed. On the theory front, the focus has recently shifted.... (Review)
Review
The status of the theory and the main methods of implementation of preparative liquid chromatography are reviewed. On the theory front, the focus has recently shifted. The theory of non-linear, non-ideal chromatography has given rise to numerous models whose advantages, disadvantages and ranges of application are now well understood. Interest now resides in investigating the equilibrium thermodynamics of complex new systems, in the study of the kinetics of mass transfers in conventional chromatographic systems, and in the application of the various models of chromatography to optimize the experimental conditions. Progress in computer technology allows the use of sophisticated models, provided their parameters can be measured. This allows the detailed investigation of separations for which the mass transfer kinetics is slow such as chiral separations, the purification of basic compounds, and the extraction of recombinant proteins. On the applied front, in addition to numerous incremental improvements in reliability and economic performance, a few essential new features should be noted, i.e. the availability of instruments for simulated moving bed separations at the scale needed for preparative chiral separations, the use of expanded beds for the extraction of recombinant proteins from fermentation broths, and the attention given to improvements in the performance of packed beds. A survey of the literature dealing with practical applications and recent meetings shows that preparative chromatography is becoming a well established separation and purification method in the pharmaceutical industry.
Topics: Chromatography, Liquid; Models, Theoretical
PubMed: 12236522
DOI: 10.1016/s0021-9673(01)01471-6 -
Current possibilities of liquid chromatography for the characterization of antibody-drug conjugates.Journal of Pharmaceutical and... Jan 2018Antibody Drug Conjugates (ADCs) are innovative biopharmaceuticals gaining increasing attention over the last two decades. The concept of ADCs lead to new therapy... (Review)
Review
Antibody Drug Conjugates (ADCs) are innovative biopharmaceuticals gaining increasing attention over the last two decades. The concept of ADCs lead to new therapy approaches in numerous oncological indications as well in infectious diseases. Currently, around 60 CECs are in clinical trials indicating the expanding importance of this class of protein therapeutics. ADCs show unprecedented intrinsic heterogeneity and address new quality attributes which have to be assessed. Liquid chromatography is one of the most frequently used analytical method for the characterization of ADCs. This review summarizes recent results in the chromatographic characterization of ADCs and supposed to provide a general overview on the possibilities and limitations of current approaches for the evaluation of drug load distribution, determination of average drug to antibody ratio (DAR), and for the analysis of process/storage related impurities. Hydrophobic interaction chromatography (HIC), reversed phase liquid chromatography (RPLC), size exclusion chromatography (SEC) and multidimensional separations are discussed focusing on the analysis of marketed ADCs. Fundamentals and aspects of method development are illustrated with applications for each technique. Future perspectives in hydrophilic interaction chromatography (HILIC), HIC, SEC and ion exchange chromatography (IEX) are also discussed.
Topics: Animals; Antibodies, Monoclonal; Biopharmaceutics; Chromatography, High Pressure Liquid; Chromatography, Liquid; Chromatography, Reverse-Phase; Humans; Immunoconjugates
PubMed: 28688616
DOI: 10.1016/j.jpba.2017.06.022 -
Analytical Chemistry Apr 1986
Review
Topics: Chemical Phenomena; Chemistry, Physical; Chromatography, Gel; Chromatography, Ion Exchange; Chromatography, Liquid; Electrochemistry; Mass Spectrometry; Micelles; Photometry; Spectrometry, Fluorescence; Spectrophotometry
PubMed: 3521387
DOI: 10.1021/ac00296a016 -
Journal of Separation Science Aug 2023Humic acid was the main compound in soil and reduced the availability of some organic compounds in soils. In this work, humic acid was immobilized for the first time on... (Review)
Review
Humic acid was the main compound in soil and reduced the availability of some organic compounds in soils. In this work, humic acid was immobilized for the first time on a homemade neutravidin poly(glycidyl methacrylate-co-ethylene dimethacrylate) capillary column with a 20 μm i.d. for the screening of potential ligands to humic acid and the evaluation of their molecular recognition mechanism. This homemade humic acid column enabling it to work at very low backpressure (0.60 MPa at 20 nl/min flow rate), had a long lifetime, excellent repeatability, and negligible non-specific binding sites. The performance of this affinity humic acid column was demonstrated by the evaluation of recognition assay for a series of known ligands of humic acid (a series of rodenticide molecules) which is the heart of the fragment-based drug design. In addition, this column was used successfully for highlighting the binding mechanism to humic acid of the severe acute respiratory syndrome coronavirus-2-spike protein. As well this new humic acid miniaturized liquid chromatography column developed in this work could be used in the feature for another solute molecule-humic acid binding studies or for a separative mode.
Topics: Chromatography, Liquid; Ligands; Humic Substances
PubMed: 37254734
DOI: 10.1002/jssc.202300203