Fast protein liquid chromatography (FPLC) is a type of liquid chromatography used to analyze or purify proteins. It introduces samples onto a column containing resin beads, then uses buffers to differentially elute bound protein. FPLC allows separation of heat-labile biomolecules like proteins under mild conditions like 4°C. It has advantages like simple reproducible separation, efficient resolution, and support for a wide range of columns and procedures under low pressure. Limitations include needing glass columns and inability to handle high pressures.
Fplc(fast protein liquid chromatography )Safina Kouser
This document describes Fast Protein Liquid Chromatography (FPLC), which is a chromatography system used to separate proteins and other biomolecules. FPLC uses stationary phases like resins and mobile phases like buffers. It works on the principles of size exclusion, ion exchange, and affinity chromatography. The key components of an FPLC system include pumps, mixers, injection valves, columns, fraction collectors, and detectors. Compared to HPLC, FPLC uses lower pressures, inert materials, and larger columns. Its advantages include reproducibility, simple operation, and suitability for analytical and preparative purposes. Common applications of FPLC include protein analysis, purification of venoms and plasma proteins.
Ultracentrifugation is a technique that uses high centrifugal forces generated by rotational speeds of up to 150,000 rpm to separate particles in solution based on differences in size, shape, density, and viscosity. It is an important tool in biochemical research used to isolate molecules like DNA, RNA, lipids, and separate organelles from cells. There are two main types - analytical ultracentrifugation which studies molecular interactions and properties, and preparative ultracentrifugation which isolates and purifies particles using techniques like density gradient centrifugation. Proper rotor selection and maintenance of the centrifuge are important for safe and effective use of this technique.
Centrifugation uses centrifugal force to separate particles in a solution based on properties like size, shape, density. There are several types of centrifugation including density gradient centrifugation, differential centrifugation, and ultracentrifugation. Density gradient centrifugation separates particles based on buoyant density into zones, while differential centrifugation separates organelles from cells. Ultracentrifugation uses very high speeds and forces particle separation. Centrifugation has many applications in areas like water treatment, biomedical research, and industries like sugar and oil production.
Precipitation techniques can be used to recover proteins from liquid and remove unwanted byproducts like nucleic acids. Precipitation is usually induced by neutral salts, organic solvents, changing the pH to the isoelectric point, ionic or non-ionic polymers, metal ions, or heat treatment. The most common precipitating agent is ammonium sulfate, which causes proteins to aggregate and precipitate out of solution by restricting water availability and increasing hydrophobic interactions between proteins.
2D-PAGE is a technique used to separate complex protein mixtures based on isoelectric point and molecular weight. It involves two sequential steps - isoelectric focusing and SDS-PAGE. In isoelectric focusing, proteins are separated based on their isoelectric point in an immobilized pH gradient. They are then separated by SDS-PAGE based on their molecular weight. The separated proteins can then be visualized through staining and identified through mass spectrometry. While useful for proteomic analysis, 2D-PAGE has limitations such as low reproducibility and dynamic range.
Sequence assembly refers to aligning and merging fragments from a longer DNA sequence in order to reconstruct the original sequence. This is needed as DNA sequencing technology cannot read whole genomes in one go, but rather reads small pieces of between 20 and 30,000 bases, depending on the technology used. Typically the short fragments, called reads, result from shotgun sequencing genomic DNA, or gene transcript (ESTs).
The problem of sequence assembly can be compared to taking many copies of a book, passing each of them through a shredder with a different cutter, and piecing the text of the book back together just by looking at the shredded pieces. Besides the obvious difficulty of this task, there are some extra practical issues: the original may have many repeated paragraphs, and some shreds may be modified during shredding to have typos. Excerpts from another book may also be added in, and some shreds may be completely unrecognizable.
Reversed phase chromatography is an adsorption technique used to separate nonpolar substances. It works by having a nonpolar stationary phase and a polar mobile phase, opposite of normal phase chromatography. Molecules like proteins, peptides, and nucleic acids can be separated using reversed phase chromatography. The separation depends on the hydrophobic binding of solutes from the mobile phase to the hydrophobic ligands attached to the stationary phase. Common stationary phases use silica beads with attached alkyl hydrocarbon chains of varying lengths. Gradient elution with mixtures of water and organic solvents like acetonitrile or methanol is typically used for separation. Reversed phase chromatography has applications in preparative purification of proteins, peptides, and other biomolecules.
Fast protein liquid chromatography (FPLC) is a type of liquid chromatography used to analyze or purify proteins. It introduces samples onto a column containing resin beads, then uses buffers to differentially elute bound protein. FPLC allows separation of heat-labile biomolecules like proteins under mild conditions like 4°C. It has advantages like simple reproducible separation, efficient resolution, and support for a wide range of columns and procedures under low pressure. Limitations include needing glass columns and inability to handle high pressures.
Fplc(fast protein liquid chromatography )Safina Kouser
This document describes Fast Protein Liquid Chromatography (FPLC), which is a chromatography system used to separate proteins and other biomolecules. FPLC uses stationary phases like resins and mobile phases like buffers. It works on the principles of size exclusion, ion exchange, and affinity chromatography. The key components of an FPLC system include pumps, mixers, injection valves, columns, fraction collectors, and detectors. Compared to HPLC, FPLC uses lower pressures, inert materials, and larger columns. Its advantages include reproducibility, simple operation, and suitability for analytical and preparative purposes. Common applications of FPLC include protein analysis, purification of venoms and plasma proteins.
Ultracentrifugation is a technique that uses high centrifugal forces generated by rotational speeds of up to 150,000 rpm to separate particles in solution based on differences in size, shape, density, and viscosity. It is an important tool in biochemical research used to isolate molecules like DNA, RNA, lipids, and separate organelles from cells. There are two main types - analytical ultracentrifugation which studies molecular interactions and properties, and preparative ultracentrifugation which isolates and purifies particles using techniques like density gradient centrifugation. Proper rotor selection and maintenance of the centrifuge are important for safe and effective use of this technique.
Centrifugation uses centrifugal force to separate particles in a solution based on properties like size, shape, density. There are several types of centrifugation including density gradient centrifugation, differential centrifugation, and ultracentrifugation. Density gradient centrifugation separates particles based on buoyant density into zones, while differential centrifugation separates organelles from cells. Ultracentrifugation uses very high speeds and forces particle separation. Centrifugation has many applications in areas like water treatment, biomedical research, and industries like sugar and oil production.
Precipitation techniques can be used to recover proteins from liquid and remove unwanted byproducts like nucleic acids. Precipitation is usually induced by neutral salts, organic solvents, changing the pH to the isoelectric point, ionic or non-ionic polymers, metal ions, or heat treatment. The most common precipitating agent is ammonium sulfate, which causes proteins to aggregate and precipitate out of solution by restricting water availability and increasing hydrophobic interactions between proteins.
2D-PAGE is a technique used to separate complex protein mixtures based on isoelectric point and molecular weight. It involves two sequential steps - isoelectric focusing and SDS-PAGE. In isoelectric focusing, proteins are separated based on their isoelectric point in an immobilized pH gradient. They are then separated by SDS-PAGE based on their molecular weight. The separated proteins can then be visualized through staining and identified through mass spectrometry. While useful for proteomic analysis, 2D-PAGE has limitations such as low reproducibility and dynamic range.
Sequence assembly refers to aligning and merging fragments from a longer DNA sequence in order to reconstruct the original sequence. This is needed as DNA sequencing technology cannot read whole genomes in one go, but rather reads small pieces of between 20 and 30,000 bases, depending on the technology used. Typically the short fragments, called reads, result from shotgun sequencing genomic DNA, or gene transcript (ESTs).
The problem of sequence assembly can be compared to taking many copies of a book, passing each of them through a shredder with a different cutter, and piecing the text of the book back together just by looking at the shredded pieces. Besides the obvious difficulty of this task, there are some extra practical issues: the original may have many repeated paragraphs, and some shreds may be modified during shredding to have typos. Excerpts from another book may also be added in, and some shreds may be completely unrecognizable.
Reversed phase chromatography is an adsorption technique used to separate nonpolar substances. It works by having a nonpolar stationary phase and a polar mobile phase, opposite of normal phase chromatography. Molecules like proteins, peptides, and nucleic acids can be separated using reversed phase chromatography. The separation depends on the hydrophobic binding of solutes from the mobile phase to the hydrophobic ligands attached to the stationary phase. Common stationary phases use silica beads with attached alkyl hydrocarbon chains of varying lengths. Gradient elution with mixtures of water and organic solvents like acetonitrile or methanol is typically used for separation. Reversed phase chromatography has applications in preparative purification of proteins, peptides, and other biomolecules.
This document provides an overview of flow cytometry. It discusses that flow cytometry allows for quantitative and qualitative analysis of cell properties as cells pass in single file in front of a laser. It describes the main components of flow cytometry as the fluidics system that transports cells, optics that illuminate cells and detect light scattering/fluorescence, and electronics that convert light signals to digital data. The document also outlines applications such as cell counting, sorting, and analysis of blood, bone marrow, and chromosomes.
Chromatofocusing is a type of ion exchange chromatography that uses an internally developed pH gradient to separate proteins based on their isoelectric point. It was developed by Sluyterman and colleagues. A pH gradient is established through the column as a polybuffer elution buffer passes through and titrates amines on the resin and proteins. Proteins are retained near the top of the column if their pH is above their pI and migrate down and bind when they reach a zone where the pH is above their pI, resulting in proteins with the highest pI eluting first and lowest pI eluting last.
Hydrophobic interaction chromatography (HIC) is used to purify biomolecules like proteins by exploiting differences in hydrophobicity. It works by separating substances based on their varying strength of interaction with hydrophobic groups attached to an uncharged gel matrix. HIC is useful for downstream purification as it can handle large sample volumes with high salt concentrations and is gentler than other methods, maintaining protein activity. Factors like ligand type, salt concentration, pH, and temperature affect HIC separation. The document evaluates HIC for monitoring plasmid DNA purification, finding it is a simple, rapid and reproducible method to assess purity.
Flow cytometry and fluorescence activated cell sorting (FACS)Abu Sufiyan Chhipa
Flow cytometry is a technology that analyzes physical and chemical characteristics of particles in fluid as they pass through a laser. It is used for cell counting, sorting, biomarker detection, and protein engineering. The basic principle is passing cells in single file past a laser for detection, counting, and sorting. It has applications in leukocyte analysis, DNA analysis, detecting enzymatic deficiencies, minimal residual disease, detecting autoantibodies, fetal-maternal hemorrhage quantification, and reticulocyte analysis.
Centrifugation is a process which involves the use of the centrifugal force for the sedimentation of heterogeneous mixtures to separate the two miscible substances ,and also to analyze the hydrodynamic properties of macro molecule with a centrifuge , used in industry and in laboratory setting.
1. Ultracentrifugation is a technique that uses very high speeds to separate particles in solution based on properties like size, shape, density.
2. There are two main types - preparative ultracentrifugation which handles large volumes to separate molecules, and analytical ultracentrifugation which uses small volumes and optical detection to study purified molecules.
3. Preparative ultracentrifugation techniques include differential centrifugation, density gradient centrifugation, and zonal centrifugation to separate organelles, proteins, and other molecules. Analytical ultracentrifugation determines molecular weight and detects conformational changes.
This document discusses nucleic acid probes and their use in hybridization experiments. It notes that probes are short sequences of nucleotides that bind to specific target sequences. The degree of homology between the probe and target determines how stable the hybridization is. Probes can range in size from 10 to over 10,000 nucleotide bases, with most common probes being 14 to 40 bases. Short probes hybridize quickly but have less specificity, while longer probes hybridize more stably. The document then describes different methods for labeling probes, including nick translation, primer extension, RNA polymerase transcription, end-labeling, and direct labeling. It also discusses factors that affect probe specificity and hybridization conditions.
The document discusses 2D gel electrophoresis and the limitations of conventional 2D gels. It introduces Difference Gel Electrophoresis (DIGE), which uses spectrally distinct fluorescent dyes to label protein samples before running multiple samples on the same 2D gel. This allows direct comparison of protein abundance levels between samples and eliminates gel-to-gel variation. The document outlines the experimental design, statistical analysis software, and advantages of DIGE over conventional 2D gels such as increased accuracy, reduced variation, and ability to detect small protein differences.
Ultracentrifugation is a technique that uses very high rotational speeds, up to 80,000 rpm, to separate particles via centrifugal force up to 600,000g. There are two main types: analytical ultracentrifugation monitors particles in real-time to study molecular interactions and properties, while preparative ultracentrifugation isolates and purifies particles like organelles. Common techniques include differential centrifugation to separate organelles and density gradient centrifugation to separate mixtures based on density.
Differential centrifugation is a technique used to separate cell organelles based on their densities. It involves homogenizing tissue to break open cells and mix organelle contents. The homogenate is then centrifuged at increasing speeds, causing organelles like mitochondria and lysosomes to pellet out after centrifuging at 1000g for 15 minutes. Repeating this process with the supernatant at higher speeds allows separation of organelles into fractions based on their sedimentation rates in a centrifugal field. While convenient and economical, differential centrifugation yields impure preparations and poor recovery of organelles.
Isoelectric focusing is a technique that separates molecules like proteins based on their isoelectric point, which is the pH at which the molecule has no net charge. It was developed in the 1960s and allows for much better resolution than older techniques. The process involves creating an immobilized pH gradient using carrier ampholytes, loading protein samples, and applying an electric field to cause proteins to migrate to the point in the gradient matching their isoelectric point. The separated proteins can then be visualized through staining. Isoelectric focusing is useful for applications like identifying serum proteins and aiding in proteomics research.
This is technique used widely for protein separation from a mixture and is very easy and less costly method. Slides cover all essential points about EMSA and it is quite interesting to know that how it detect and separate different proteins and their mobility shift assay.
Nucleic Acid Quantification Methods - DNA / RNA Quantificationajithnandanam
Nucleic acids are quantified to check the concentration and purity of DNA/RNA present in the solution mixture.it is important to know the concentration and purity of the nucleic acid for the use in further applications like PCR, restriction digestion etc. Spectrophotometric analysis is the most commonly used method of quantifying DNA, agarose gel electrophoresis can also be used to analyse the DNA sample for purity.
Protein purification involves a series of steps to isolate a single protein from a complex mixture. These steps may separate proteins based on size, charge, or binding affinity. Common techniques include precipitation with ammonium sulfate, chromatography methods like ion exchange, affinity, size exclusion, and reversed-phase chromatography, and electrophoresis. The goal is to free the protein of interest from other materials, separate it from other proteins, and finally isolate it in a pure form for characterization and use.
This document summarizes the steps of a ChIP-Seq (Chromatin Immunoprecipitation Sequencing) assay. The key steps are: 1) cross-linking proteins to DNA, 2) fragmenting chromatin, 3) immunoprecipitating the DNA-protein complex using antibodies specific to the protein of interest, 4) purifying and analyzing the DNA. ChIP-Seq allows researchers to identify the genomic binding sites of transcription factors and histone modifications genome-wide.
HIGH PERFORMANCE LIQUID CHROMATOGRAPHY (HPLC)Suneal Saini
This document provides an introduction to high performance liquid chromatography (HPLC). It discusses the basic components and principles of HPLC, including the stationary and mobile phases, pumps to move the mobile phase through the column at high pressure, and various detectors used to analyze the separated components as they elute from the column. It also describes the different types of HPLC based on the mode of separation, elution technique, scale of operation, and type of analysis performed.
This document provides an overview of flow cytometry. It discusses that flow cytometry allows for quantitative and qualitative analysis of cell properties as cells pass in single file in front of a laser. It describes the main components of flow cytometry as the fluidics system that transports cells, optics that illuminate cells and detect light scattering/fluorescence, and electronics that convert light signals to digital data. The document also outlines applications such as cell counting, sorting, and analysis of blood, bone marrow, and chromosomes.
Chromatofocusing is a type of ion exchange chromatography that uses an internally developed pH gradient to separate proteins based on their isoelectric point. It was developed by Sluyterman and colleagues. A pH gradient is established through the column as a polybuffer elution buffer passes through and titrates amines on the resin and proteins. Proteins are retained near the top of the column if their pH is above their pI and migrate down and bind when they reach a zone where the pH is above their pI, resulting in proteins with the highest pI eluting first and lowest pI eluting last.
Hydrophobic interaction chromatography (HIC) is used to purify biomolecules like proteins by exploiting differences in hydrophobicity. It works by separating substances based on their varying strength of interaction with hydrophobic groups attached to an uncharged gel matrix. HIC is useful for downstream purification as it can handle large sample volumes with high salt concentrations and is gentler than other methods, maintaining protein activity. Factors like ligand type, salt concentration, pH, and temperature affect HIC separation. The document evaluates HIC for monitoring plasmid DNA purification, finding it is a simple, rapid and reproducible method to assess purity.
Flow cytometry and fluorescence activated cell sorting (FACS)Abu Sufiyan Chhipa
Flow cytometry is a technology that analyzes physical and chemical characteristics of particles in fluid as they pass through a laser. It is used for cell counting, sorting, biomarker detection, and protein engineering. The basic principle is passing cells in single file past a laser for detection, counting, and sorting. It has applications in leukocyte analysis, DNA analysis, detecting enzymatic deficiencies, minimal residual disease, detecting autoantibodies, fetal-maternal hemorrhage quantification, and reticulocyte analysis.
Centrifugation is a process which involves the use of the centrifugal force for the sedimentation of heterogeneous mixtures to separate the two miscible substances ,and also to analyze the hydrodynamic properties of macro molecule with a centrifuge , used in industry and in laboratory setting.
1. Ultracentrifugation is a technique that uses very high speeds to separate particles in solution based on properties like size, shape, density.
2. There are two main types - preparative ultracentrifugation which handles large volumes to separate molecules, and analytical ultracentrifugation which uses small volumes and optical detection to study purified molecules.
3. Preparative ultracentrifugation techniques include differential centrifugation, density gradient centrifugation, and zonal centrifugation to separate organelles, proteins, and other molecules. Analytical ultracentrifugation determines molecular weight and detects conformational changes.
This document discusses nucleic acid probes and their use in hybridization experiments. It notes that probes are short sequences of nucleotides that bind to specific target sequences. The degree of homology between the probe and target determines how stable the hybridization is. Probes can range in size from 10 to over 10,000 nucleotide bases, with most common probes being 14 to 40 bases. Short probes hybridize quickly but have less specificity, while longer probes hybridize more stably. The document then describes different methods for labeling probes, including nick translation, primer extension, RNA polymerase transcription, end-labeling, and direct labeling. It also discusses factors that affect probe specificity and hybridization conditions.
The document discusses 2D gel electrophoresis and the limitations of conventional 2D gels. It introduces Difference Gel Electrophoresis (DIGE), which uses spectrally distinct fluorescent dyes to label protein samples before running multiple samples on the same 2D gel. This allows direct comparison of protein abundance levels between samples and eliminates gel-to-gel variation. The document outlines the experimental design, statistical analysis software, and advantages of DIGE over conventional 2D gels such as increased accuracy, reduced variation, and ability to detect small protein differences.
Ultracentrifugation is a technique that uses very high rotational speeds, up to 80,000 rpm, to separate particles via centrifugal force up to 600,000g. There are two main types: analytical ultracentrifugation monitors particles in real-time to study molecular interactions and properties, while preparative ultracentrifugation isolates and purifies particles like organelles. Common techniques include differential centrifugation to separate organelles and density gradient centrifugation to separate mixtures based on density.
Differential centrifugation is a technique used to separate cell organelles based on their densities. It involves homogenizing tissue to break open cells and mix organelle contents. The homogenate is then centrifuged at increasing speeds, causing organelles like mitochondria and lysosomes to pellet out after centrifuging at 1000g for 15 minutes. Repeating this process with the supernatant at higher speeds allows separation of organelles into fractions based on their sedimentation rates in a centrifugal field. While convenient and economical, differential centrifugation yields impure preparations and poor recovery of organelles.
Isoelectric focusing is a technique that separates molecules like proteins based on their isoelectric point, which is the pH at which the molecule has no net charge. It was developed in the 1960s and allows for much better resolution than older techniques. The process involves creating an immobilized pH gradient using carrier ampholytes, loading protein samples, and applying an electric field to cause proteins to migrate to the point in the gradient matching their isoelectric point. The separated proteins can then be visualized through staining. Isoelectric focusing is useful for applications like identifying serum proteins and aiding in proteomics research.
This is technique used widely for protein separation from a mixture and is very easy and less costly method. Slides cover all essential points about EMSA and it is quite interesting to know that how it detect and separate different proteins and their mobility shift assay.
Nucleic Acid Quantification Methods - DNA / RNA Quantificationajithnandanam
Nucleic acids are quantified to check the concentration and purity of DNA/RNA present in the solution mixture.it is important to know the concentration and purity of the nucleic acid for the use in further applications like PCR, restriction digestion etc. Spectrophotometric analysis is the most commonly used method of quantifying DNA, agarose gel electrophoresis can also be used to analyse the DNA sample for purity.
Protein purification involves a series of steps to isolate a single protein from a complex mixture. These steps may separate proteins based on size, charge, or binding affinity. Common techniques include precipitation with ammonium sulfate, chromatography methods like ion exchange, affinity, size exclusion, and reversed-phase chromatography, and electrophoresis. The goal is to free the protein of interest from other materials, separate it from other proteins, and finally isolate it in a pure form for characterization and use.
This document summarizes the steps of a ChIP-Seq (Chromatin Immunoprecipitation Sequencing) assay. The key steps are: 1) cross-linking proteins to DNA, 2) fragmenting chromatin, 3) immunoprecipitating the DNA-protein complex using antibodies specific to the protein of interest, 4) purifying and analyzing the DNA. ChIP-Seq allows researchers to identify the genomic binding sites of transcription factors and histone modifications genome-wide.
HIGH PERFORMANCE LIQUID CHROMATOGRAPHY (HPLC)Suneal Saini
This document provides an introduction to high performance liquid chromatography (HPLC). It discusses the basic components and principles of HPLC, including the stationary and mobile phases, pumps to move the mobile phase through the column at high pressure, and various detectors used to analyze the separated components as they elute from the column. It also describes the different types of HPLC based on the mode of separation, elution technique, scale of operation, and type of analysis performed.
HPLC- introduction, principle, types, working, instrumentation and operations of HPLC has been included with appropriate gifs and images for better understanding. What are all the things need to be known by a science student about HPLC (basics and working) is clearly given in this presentation.
Chromatography separates mixtures by exploiting differences in how components interact with stationary and mobile phases. High performance liquid chromatography (HPLC) uses a liquid mobile phase and high pressure to achieve high resolution separation over short periods. HPLC is useful for analyzing compounds that are non-volatile, thermally labile, or high molecular weight. It works by injecting samples onto a column where components separate as they pass through the column at different rates depending on their partitioning between the stationary and mobile phases.
Chromatography separates components in a mixture using a stationary and mobile phase. High performance liquid chromatography (HPLC) is a type of chromatography that uses high pressure to force a liquid mobile phase through a column packed with solid particles. The document discusses various aspects of HPLC including separation modes, selecting stationary and mobile phases, HPLC system components, and applications.
Chromatography techniques such as high performance liquid chromatography (HPLC), fast protein liquid chromatography (FPLC), and gas chromatography (GC) can be used to separate mixtures. HPLC uses high pressure to push a mobile phase through a column containing a stationary phase to separate complex mixtures. FPLC is a modified HPLC system designed for separating proteins more gently. GC vaporizes samples and uses an inert gas mobile phase to separate components in a sample based on differences in how they partition between the gas and a liquid or solid stationary phase.
HPLC stands for high-performance liquid chromatography. It is a separation technique used to separate mixtures of compounds. HPLC works by forcing a pressurized liquid mobile phase through a column packed with porous particles. As the different compounds in the sample interact differently with the stationary phase particles, they elute from the column at different rates, allowing separation. Key components of an HPLC system include the pump, injector, column, detector, and data processing unit. HPLC has many applications in fields like pharmaceutical analysis and natural products analysis.
HPLC is a type of column chromatography that uses high pressure to force a mobile liquid phase through a column packed with solid particles. This allows for faster separation of mixtures compared to standard column chromatography. HPLC instruments consist of a pump that forces the mobile phase through the column at high pressure, an injector for samples, a column for separation, a detector, and a computer system. There are different types of HPLC columns that separate mixtures based on polarity, ionic interactions, size, or other properties. HPLC is used in various applications to separate, identify, and quantify components in mixtures.
HPLC (High Performance Liquid Chromatography) is a separation technique used to separate, identify, and quantify compounds in mixtures. It works by injecting samples into a column with a stationary phase and passing a liquid mobile phase through under high pressure. Compounds are separated based on how they partition between the mobile and stationary phases. HPLC is useful for pharmaceutical analysis, clinical applications, chemical separations, and purification of compounds due to its high resolution, sensitivity, repeatability, and ability to separate both volatile and non-volatile compounds.
This document provides an introduction to high performance liquid chromatography (HPLC). It begins with definitions of HPLC and basic chromatographic terms. It then discusses the instrumentation of HPLC including common components like the solvent delivery pump, injector, column, detector, and data system. Different types of chromatography are also outlined, including reverse phase, normal phase, ion exchange, and size exclusion. Key terms used in HPLC like retention time, peak width, and tailing factor are defined. The principles of HPLC separation and factors that influence separation are described.
The document discusses chromatography and high performance liquid chromatography (HPLC). It defines chromatography as a method used to separate components in a complex mixture using two phases, a stationary phase and a mobile phase. It then discusses various types of chromatography, including normal phase and reversed phase chromatography, based on different factors like separation principle, elution technique, scale of operation, and type of analysis. The document also discusses key components of HPLC like pumps, injectors, columns, detectors and provides details on their functioning. It highlights advantages of HPLC like high resolution, sensitivity, reproducibility and its importance in qualitative and quantitative analysis.
Column chromatography is a method used to separate chemical mixtures by using a column filled with a stationary phase and pumping a mobile phase through it. Key steps include preparing the column with a dry or wet method, loading the sample, and collecting fractions as compounds elute from the column at different rates depending on their interactions with the stationary phase. Resolution, a measure of separation, can be calculated from chromatograms by measuring retention times and curve widths. Automated systems now streamline column chromatography but typically have lower resolution than HPLC.
This document provides an overview of high performance liquid chromatography (HPLC). It begins by defining chromatography and its basic components. It then discusses the development of HPLC and how it works, including the instrumentation used. The document outlines different types of HPLC based on the mode of chromatography, principle of separation, elution technique, and type of analysis. It provides examples of each type. Finally, the document lists several applications of HPLC in fields like chemistry, biochemistry, quality control, and pharmacokinetics.
HPLC is a form of liquid chromatography that uses pumps to pass a pressurized mobile liquid phase through a column packed with solid particles. This allows the components of a dissolved sample to be separated as they are transported through the column at different rates depending on their interactions with the stationary and mobile phases. HPLC instruments consist of a pump, injector, column, and detector. Separation is based on the partitioning of compounds between the mobile and stationary phases, and detectors are used to measure separated components as they exit the column. HPLC provides efficient, sensitive, and high-pressure separations of sample mixtures.
This document provides an overview of high performance liquid chromatography (HPLC). It discusses how HPLC refined traditional liquid chromatography by using smaller particle sizes, smaller column diameters, and high fluid pressures to provide enhanced separations over shorter periods of time. Key aspects of HPLC systems and processes are summarized, including the use of pumps to deliver mobile phases at high pressure through columns containing small stationary phase particles. Separation is achieved based on how sample components partition between the mobile and stationary phases. Various detectors are also outlined.
This document provides information about High Performance Liquid Chromatography (HPLC). It defines HPLC as a technique that uses pumps to pass a pressurized liquid mobile phase through a column packed with adsorbent particles. This allows the separation of a sample mixture as its components interact differently with the stationary phase. The document outlines the basic components of an HPLC system including the sample injector, column, detector, and data analysis devices. It also describes various parameters that affect the separation like retention time and factors, temperature control, and types of columns and detectors commonly used.
High Performance Liquid Chromatography (HPLC) is presented. HPLC is a chromatographic technique used to separate mixtures by using high pressure to force a liquid mobile phase and sample through a column packed with solid stationary phase. Key aspects summarized include:
1. HPLC provides simultaneous analysis, high resolution, sensitivity, repeatability for qualitative and quantitative analysis.
2. It works on principles of adsorption and partition chromatography depending on the stationary phase.
3. Instrumentation includes pumps, injector, analytical column, detector, and recorder/integrator.
4. Parameters like retention time, capacity factor, separation factor, and plate height provide information about sample separation and column efficiency.
The document provides an overview of high performance liquid chromatography (HPLC). It begins with defining HPLC and explaining the basic principles of chromatography. It then describes the different types of HPLC based on the mode and principle of separation. The document also discusses HPLC instrumentation, including the solvent reservoir, pump, injector, column, and various detectors. It concludes by outlining some common applications of HPLC and discussing quantitative and qualitative analysis.
This document discusses high-performance liquid chromatography (HPLC), which is a widely used technique for separating and analyzing components in mixtures. It describes the basic components and principles of HPLC, including pumps to pass a pressurized liquid and sample mixture through a column containing stationary phase particles. The components interact differently with the stationary phase and are separated into bands that are then detected and analyzed. Common detectors described are UV-visible, fluorescence, and electrochemical detectors. The document also discusses various modes of operation like isocratic and gradient elution and types of columns and stationary phases used.
Chromatography separates mixtures into components based on molecular structure and composition. High performance liquid chromatography (HPLC) is a highly improved form of liquid chromatography that forces solvents through columns under high pressure, making it faster. HPLC instruments include pumps to force mobile phases, injectors for samples, columns for separation, detectors to analyze eluents, and data collection systems. HPLC is used in pharmaceutical quality control, environmental monitoring, forensics, food and flavor analysis, and clinical testing.
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Chapter wise All Notes of First year Basic Civil Engineering.pptxDenish Jangid
Chapter wise All Notes of First year Basic Civil Engineering
Syllabus
Chapter-1
Introduction to objective, scope and outcome the subject
Chapter 2
Introduction: Scope and Specialization of Civil Engineering, Role of civil Engineer in Society, Impact of infrastructural development on economy of country.
Chapter 3
Surveying: Object Principles & Types of Surveying; Site Plans, Plans & Maps; Scales & Unit of different Measurements.
Linear Measurements: Instruments used. Linear Measurement by Tape, Ranging out Survey Lines and overcoming Obstructions; Measurements on sloping ground; Tape corrections, conventional symbols. Angular Measurements: Instruments used; Introduction to Compass Surveying, Bearings and Longitude & Latitude of a Line, Introduction to total station.
Levelling: Instrument used Object of levelling, Methods of levelling in brief, and Contour maps.
Chapter 4
Buildings: Selection of site for Buildings, Layout of Building Plan, Types of buildings, Plinth area, carpet area, floor space index, Introduction to building byelaws, concept of sun light & ventilation. Components of Buildings & their functions, Basic concept of R.C.C., Introduction to types of foundation
Chapter 5
Transportation: Introduction to Transportation Engineering; Traffic and Road Safety: Types and Characteristics of Various Modes of Transportation; Various Road Traffic Signs, Causes of Accidents and Road Safety Measures.
Chapter 6
Environmental Engineering: Environmental Pollution, Environmental Acts and Regulations, Functional Concepts of Ecology, Basics of Species, Biodiversity, Ecosystem, Hydrological Cycle; Chemical Cycles: Carbon, Nitrogen & Phosphorus; Energy Flow in Ecosystems.
Water Pollution: Water Quality standards, Introduction to Treatment & Disposal of Waste Water. Reuse and Saving of Water, Rain Water Harvesting. Solid Waste Management: Classification of Solid Waste, Collection, Transportation and Disposal of Solid. Recycling of Solid Waste: Energy Recovery, Sanitary Landfill, On-Site Sanitation. Air & Noise Pollution: Primary and Secondary air pollutants, Harmful effects of Air Pollution, Control of Air Pollution. . Noise Pollution Harmful Effects of noise pollution, control of noise pollution, Global warming & Climate Change, Ozone depletion, Greenhouse effect
Text Books:
1. Palancharmy, Basic Civil Engineering, McGraw Hill publishers.
2. Satheesh Gopi, Basic Civil Engineering, Pearson Publishers.
3. Ketki Rangwala Dalal, Essentials of Civil Engineering, Charotar Publishing House.
4. BCP, Surveying volume 1
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This document provides an overview of wound healing, its functions, stages, mechanisms, factors affecting it, and complications.
A wound is a break in the integrity of the skin or tissues, which may be associated with disruption of the structure and function.
Healing is the body’s response to injury in an attempt to restore normal structure and functions.
Healing can occur in two ways: Regeneration and Repair
There are 4 phases of wound healing: hemostasis, inflammation, proliferation, and remodeling. This document also describes the mechanism of wound healing. Factors that affect healing include infection, uncontrolled diabetes, poor nutrition, age, anemia, the presence of foreign bodies, etc.
Complications of wound healing like infection, hyperpigmentation of scar, contractures, and keloid formation.
Level 3 NCEA - NZ: A Nation In the Making 1872 - 1900 SML.pptHenry Hollis
The History of NZ 1870-1900.
Making of a Nation.
From the NZ Wars to Liberals,
Richard Seddon, George Grey,
Social Laboratory, New Zealand,
Confiscations, Kotahitanga, Kingitanga, Parliament, Suffrage, Repudiation, Economic Change, Agriculture, Gold Mining, Timber, Flax, Sheep, Dairying,
2. INTRODUCTION
• Chromatography is a method of separating mixture of
components into individual components through equilibrium
distribution between two phases.
• Each chromatographic method essentially consists of 2 phases
a stationary phase and mobile phase.
Stationary phase :- solid or liquid
Mobile phase :- liquid or gas
3. INTRODUCTION
• Chromatography is based on differential migration
(differences in the rate at which components move through
stationary phase under the influence of mobile phase.)
• Solutes with a greater affinity for the mobile phase will spend
more time in mobile phase than the solutes that have greater
affinity for stationary phase.
• As the solutes move through the stationary phase they
separate. This is called chromatographic development.
https://www.sciencedirect.com/topics/biochemistry-genetics-and-molecular-biology/column-chromatography
4. DistributionCoefficient(Equilibrium
Distribution)
• DEFINITION :-
Concentration of component in stationary phase
Concentration of component in mobile phase
Due difference in distribution Coefficient of various component of mixture,
there mobility in two phases differ hence, separation
Measure of this mobility is called RF value i.e Relative Fraction value
RF = Distance travelled by substance
Distance travelled by solvent
6. FPLC -
• FPLC is basically a “ protein friendly
“ HPLC system.
• FPLC is an intermediate between
classical column chromatography
and HPLC.
• FPLC was introduced by Pharmacia
in 1982.
• It is a complete system for
chromatographic separations of
proteins and other biomolecules
such as nucleic acids.
Fig:-AKTA pure 25 FPLC
7. FPLC -
• Fast protein liquid chromatography
(FPLC), is a form of liquid
chromatography that is often used to
analyze or purify mixtures of proteins
• In FPLC the mobile phase is an
aqueous solution, or “ Buffer".
• The stationary phase is a resin
composed of beads, usually of cross-
linked agarose, packed into a
cylindrical glass or plastic column.
• FPLC resins are available in a wide
range of bead sizes and surface ligands
depending on the application
https://www.slideshare.net/PratheebaSubramani/fplc-fast-protein-liquid-chromatography
8. Difference between HPLC and
FPLC
FPLC HPLC
Goal Purification Analytics purification
Pressure Max.40bar (4MPa) 1500 bar (150MPa)
Sample Volume From 100µl to several
millilitres
<100µl also it depends on
loop vol
Column type Plastic or glass columns
are used
Columns is made up of
steel
Column Materials agarose/polymer, only low
pressure, big particles
sizes, high flow rates
Silica beads, pressure
stable, small particles
sizes, low flow rates
Methods Size-exclusion, ion-
exchange, affinity, or
hydrophobic interaction
Normal phase, Reversed
phase
Highest advantage Ease of operation and
versatility
High resolution
11. 1. Affinity Chromatography
• Separate protein based on a highly specific interaction such as
that between antigen and antibody, enzyme and substrate or
receptor and ligand.
12. Affinity Chromatography
• Types of column can be used for affinity chromatography :-
HisTrap HP HisTrap Streptoavidin
HiTrap Talon
13. 2. Size (size exclusion chromatography)
• Separate proteins according to their size. Also termed as ‘’Gel
Filtration Chromatography ‘’
17. Instrumentation :-
• Pump:- The majority of systems utilize two piston
pumps which delivers buffer or sample in
purification runs
• Mixer:- Mixes the buffers delivered from the
system pumps to a homogeneous buffer
composition.
• Injection valve:- The injection valve has a sample
loading port through which the sample can be
loaded into the injection loop.
• UV monitor:- Measures the UV absorbance at a
fixed wavelength of 280 nm.
18. Instrumentation :-
• UV monitor:- Measures the UV/Vis absorbance at
up to three wavelengths simultaneously in the
range 190-700 nm
• Conductivity monitor:- Measures the conductivity
of buffers and eluted proteins.
• Versatile valve:- which can be used when adding
extra features to the flow path.
• Fraction collector:- The fraction collector is
typically a rotating rack that can be filled with test
tubes or similar containers.it allows sample to be
collected in fixed volume.
32. Work Flow
Protein mixture dissolve in 100% buffer A pumped
through the column
The proteins of interest bind to the resin other
components are carried out in the buffer.
The proportion of buffer B (the "elution" buffer) is
gradually increased from 0% to 100% according to a
programmed change in concentration (the
"gradient").
At some point during this process each of the bound
proteins dissociates and appears in the eluant
The eluant passes through two detectors which
measure salt concentration and protein
concentration.
As each protein is eluted, it appears in the eluant as a
"peak" in protein concentration
Sheehan D, O'Sullivan S (2003). "Fast protein liquid chromatography". In Cutler P (ed.). Protein Purification Protocols. Methods in Molecular Biology.
Vol. 244. pp. 253–8.
It was originally called fast performance liquid chromatography to contrast it with HPLC or High performance liquid chromatography
FPLC is generally applied only to proteins; however, because of the wide choice of resins and buffers it has broad applications
Flow rate is 5ml/min fplc
Flow rate is 1ml/min HPLC
FPLC purifies large biomolecules like proteins or DNA
The chromatography of biomolecules is very demanding and sensitive because they cannot stand high temperatures, high pressures, or the solvents usually used in HPLC.
It utilizes a combination of these principles to separate and analyze proteins based on their affinity, size, and charge.
Affinity chromatography is a separation method based on a specific binding interaction between an immobilized ligand and its binding partner
HisTrap HP prepacked columns preparative purification of histidine-tagged (His-tag) recombinant proteins.
Columns prepacked with cobalt-based chromatography media designed for purification of histidine-tagged recombinant proteins by immobilized metal affinity chromatography
HiTrap Streptavidin HP is a column prepacked with Streptavidin Sepharose High Performance for purification of biotinylated proteins and biomolecules
SEC is a type of chromatography used to separate proteins based on their size. It is based on the principles that protein with larger molecular weights will be excluded from the pores of the stationary phases, while proteins with smaller mol wt will pass through the pores.
In FPLC , SEC used to separate proteins based on their size. This help to identify and isolate proteins that share similar properties
Superdex prep grade (pg) is produced by covalent bonding of dextran to highly cross-linked agarose.The separation properties of these media are determined by the dextran component.
columns prepacked with Sephadex G-25 Superfine resin for fast and convenient desalting and buffer exchange.
In FPLC , ion exchange chromatography used to separate proteins based on their surface charges. This help to identify and isolate proteins that share similar properties such as those having positive or negative surface charges
Quaternary ammonium (Q) strong anion exchanger for high resolution ion exchange chromatography
HiTrap CM FF is prepacked with CM Sepharose Fast Flow, and is a weak cation exchanger for small-scale protein purifications
:- The majority of systems utilize two piston pumps which delivers buffer or sample in purification runs. The flow rate is varied based on scale of preparation i.e; analytical or preparative chromatography.
It is Pressure monitor . It Reads the system pressure after System pump A and System pump B.
Mixer which is Power and controlled by the pump. Especially important when forming gradients between buffer sources. The mixer ensures that the buffer used are in the correct proportion during the the FPLC run
The inlet valves are used to select which buffers or samples to use in a run
The outlet valve is used to direct the flow to the fraction collector, to an outlet port, or to waste.
The injection valve is a motorized valve which links the mixer and sample loop to the column. The injection valve has a sample loading port through which the sample can be loaded into the injection loop.
Inject sample into column. Loop volume can range from a few microliters to 50 ml or more.
UV monitor:- Measures the UV/Vis absorbance at up to three wavelengths simultaneously in the range 190-700 nm
Measures the conductivity of buffers and eluted proteins.
Enables the pH electrode to be included in the flow path or bypassed during a run.
The colums is a glass or plastic cylinder packed with stationary phase-beads of resin.thess are mounted vertically with the buffer flowing downward from top to bottom
The fraction collector is typically a rotating rack that can be filled with test tubes or similar containers.it allows sample to be collected in fixed volume.
. A mixture containing one or more proteins of interest is dissolved in 100% buffer A and pumped into the column. The proteins of interest bind to the resin while other components are carried out in the buffer.[2] The total flow rate of the buffer is kept constant; however, the proportion of buffer B (the "elution" buffer) is gradually increased from 0% to 100% according to a programmed change in concentration (the "gradient"). At some point during this process each of the bound proteins dissociates and appears in the eluant. The eluant passes through two detectors which measure salt concentration (by conductivity) and protein concentration (by absorption of ultraviolet light at a wavelength of 280nm). As each protein is eluted, it appears in the eluant as a "peak" in protein concentration, and can be collected for further use
. A mixture containing one or more proteins of interest is dissolved in 100% buffer A and pumped into the column. The proteins of interest bind to the resin while other components are carried out in the buffer.[2] The total flow rate of the buffer is kept constant; however, the proportion of buffer B (the "elution" buffer) is gradually increased from 0% to 100% according to a programmed change in concentration (the "gradient"). Because different protein may elute at different gradient .At some point during this process each of the bound proteins dissociates and appears in the eluant. The eluant passes through two detectors which measure salt concentration (by conductivity) and protein concentration (by absorption of ultraviolet light at a wavelength of 280nm). As each protein is eluted, it appears in the eluant as a "peak" in protein concentration, and can be collected for further use
So With every columns there come a standard chromatogram so if am using superdex 75 increase which is a size exclusion chromatography they give known protein with know mol wt. they also mention the sample volume and flow rates and buffer. Wen iam passing these sample through the column iam getting a peak bet 1ml and 2ml
So any protein eluting before 1ml is considered to be unresolved as because of higher mol mass or may be due to aggregartion
Measures the conductivity of buffers and eluted proteins.
Addition of elution buffer could separate slightly negatively charges molecules that give us a small peak so addition of elution buffer with increased concentration to separate highly negatively charged molecules
Equilibrate the columns with buffer that is compatible with the protein of interest,
Binding of protein
Columns Wash so that unbound proteins will remove
Elution buffer