This document discusses high performance liquid chromatography (HPLC). It begins by providing background on the founder of liquid chromatography, Mikhail Tsvet. It then describes the basic concepts of HPLC including qualitative and quantitative analysis using retention time and peak area/height comparisons. The document outlines the types of HPLC including partition, adsorption, ion exchange, size-exclusion, and affinity chromatography. It also describes the various components of an HPLC system including the solvent system, injection valve, column, and detector.
Supercritical fluid extraction and Supercritical fluid chromatography are techniques which use supercritical fluids as solvent for both extraction and separation respectively.
The properties such as density, viscosity and diffusion constant of the supercritical fluids are intermediate between those of a substance in gaseous and liquid state.
This helps in efficient extraction and chromatographic separation compared to other techniques.
This document outlines the five main steps for developing an analytical HPLC method: 1) selecting the initial HPLC method and conditions, 2) selecting the initial chromatographic conditions, 3) optimizing selectivity, 4) optimizing system parameters, and 5) validating the method. Key aspects of each step are discussed, including selecting the type of chromatography, column, detector, and mobile phase based on the analytes. The goal is to develop a validated method that provides adequate resolution and selectivity within a desired analysis time.
This document provides an overview of flash chromatography. It defines flash chromatography as a hybrid of medium and short column chromatography that uses slightly smaller silica gel particles and pressurized gas to drive solvents through the column more rapidly than gravity column chromatography. The key aspects of flash chromatography covered include the instrumentation, theory, selection of stationary and mobile phases, and procedures.
This document provides an overview of key concepts in chromatography. It defines terminology like stationary phase, mobile phase, retention time, and gradient vs isocratic elution. It also describes different types of chromatography like normal phase vs reverse phase, planar chromatography, column chromatography, and preparative chromatography. Quantitative analysis techniques and factors that influence column performance are also briefly covered.
The document discusses key concepts in gas chromatography including theoretical plates, resolution, retention time, retention volume, separation factor, height equivalent to a theoretical plate (HETP), peak asymmetry, stationary phases, and considerations for choosing stationary phases. It provides definitions and equations for these terms and concepts. Examples of common stationary phase materials and their applications are also presented.
HPLC Method Development & Method Validation (mr.s)22suresh
This document describes the development and validation of an HPLC method for estimating drugs. It discusses the principles of HPLC, steps in method development including selecting the method, column, mobile phase and detector. Method validation parameters like accuracy, precision, specificity, linearity and robustness are also summarized. The document provides details on the optimization process and validation procedures to ensure the method is suitable for its intended use.
This document discusses various detectors used in gas chromatography including the flame ionization detector, thermal conductivity detector, flame photometric detector, photoionization detector, atomic emission detector, sulfur chemiluminescence detector, nitrogen chemiluminescence detector, and others. For each detector, it provides information on the basic principle of operation, components, applications, and limitations. The document focuses on explaining how each detector is able to detect and measure compounds eluting from the gas chromatography column based on their specific characteristics.
HPLC is characterized by using high pressure to push a mobile phase through a stationary phase column, allowing for the separation of complex mixtures. Validation of analytical methods demonstrates that the methods are accurate, precise, specific, sensitive, linear, and robust for their intended purpose. Key aspects of validation include accuracy, precision, specificity, detection limit, quantitation limit, linearity, range, and robustness. Validation ensures methods provide reliable results.
Supercritical fluid extraction and Supercritical fluid chromatography are techniques which use supercritical fluids as solvent for both extraction and separation respectively.
The properties such as density, viscosity and diffusion constant of the supercritical fluids are intermediate between those of a substance in gaseous and liquid state.
This helps in efficient extraction and chromatographic separation compared to other techniques.
This document outlines the five main steps for developing an analytical HPLC method: 1) selecting the initial HPLC method and conditions, 2) selecting the initial chromatographic conditions, 3) optimizing selectivity, 4) optimizing system parameters, and 5) validating the method. Key aspects of each step are discussed, including selecting the type of chromatography, column, detector, and mobile phase based on the analytes. The goal is to develop a validated method that provides adequate resolution and selectivity within a desired analysis time.
This document provides an overview of flash chromatography. It defines flash chromatography as a hybrid of medium and short column chromatography that uses slightly smaller silica gel particles and pressurized gas to drive solvents through the column more rapidly than gravity column chromatography. The key aspects of flash chromatography covered include the instrumentation, theory, selection of stationary and mobile phases, and procedures.
This document provides an overview of key concepts in chromatography. It defines terminology like stationary phase, mobile phase, retention time, and gradient vs isocratic elution. It also describes different types of chromatography like normal phase vs reverse phase, planar chromatography, column chromatography, and preparative chromatography. Quantitative analysis techniques and factors that influence column performance are also briefly covered.
The document discusses key concepts in gas chromatography including theoretical plates, resolution, retention time, retention volume, separation factor, height equivalent to a theoretical plate (HETP), peak asymmetry, stationary phases, and considerations for choosing stationary phases. It provides definitions and equations for these terms and concepts. Examples of common stationary phase materials and their applications are also presented.
HPLC Method Development & Method Validation (mr.s)22suresh
This document describes the development and validation of an HPLC method for estimating drugs. It discusses the principles of HPLC, steps in method development including selecting the method, column, mobile phase and detector. Method validation parameters like accuracy, precision, specificity, linearity and robustness are also summarized. The document provides details on the optimization process and validation procedures to ensure the method is suitable for its intended use.
This document discusses various detectors used in gas chromatography including the flame ionization detector, thermal conductivity detector, flame photometric detector, photoionization detector, atomic emission detector, sulfur chemiluminescence detector, nitrogen chemiluminescence detector, and others. For each detector, it provides information on the basic principle of operation, components, applications, and limitations. The document focuses on explaining how each detector is able to detect and measure compounds eluting from the gas chromatography column based on their specific characteristics.
HPLC is characterized by using high pressure to push a mobile phase through a stationary phase column, allowing for the separation of complex mixtures. Validation of analytical methods demonstrates that the methods are accurate, precise, specific, sensitive, linear, and robust for their intended purpose. Key aspects of validation include accuracy, precision, specificity, detection limit, quantitation limit, linearity, range, and robustness. Validation ensures methods provide reliable results.
Analytical method development and validation for simultaneous estimationProfessor Beubenz
The document discusses analytical method development and validation for the simultaneous estimation of active pharmaceutical ingredients (APIs). It covers topics such as the need for method development, the steps involved, parameters for validation as outlined in ICH guidelines, and the use of high performance liquid chromatography (HPLC). The key objectives of validation are to demonstrate that the developed method is suitable for its intended purpose and can reliably quantify the APIs.
High Performance Liquid Chromatography (HPLC) is described. HPLC uses high pressure to force a mobile phase through a column at a fast rate, increasing resolution. It discusses the types of chromatography used in HPLC, including normal phase, reverse phase, ion-exchange, and size-exclusion. The instrumentation of HPLC is also summarized, including components like the pump, mixing unit, degasser, injector, column, and detector.
The document discusses the process and history of paper chromatography. It begins with an introduction to chromatography and its use in separating mixtures. It then covers the history from early experiments in the 1860s to modern developments. The main types and techniques of paper chromatography are explained, including the use of a stationary phase, mobile phase, and capillary action to separate components by affinity and travel distance. Key steps like sample application and developing the paper strip are outlined.
HPLC is a type of liquid chromatography that uses high pressure to force a sample through a column packed with porous particles. This allows for faster separations compared to traditional chromatography. Key parameters in HPLC include retention time, which measures how long components spend in the column; capacity factor k', which is a ratio of time spent in the stationary vs mobile phase; selectivity factor α, which is the ratio of k' values and describes separation of adjacent peaks; and theoretical plates N, which estimates column efficiency based on peak widths and retention times. Optimizing these parameters can improve resolution of components in the mixture.
Chromatography is a technique used to separate mixtures into individual components. Paper chromatography is a type of chromatography that uses paper as the stationary phase. The mixture is applied to the paper and then placed in a developing chamber with the mobile phase solvent. As the solvent travels up the paper, the different components of the mixture separate based on how strongly they interact with the stationary and mobile phases. This creates discrete spots that can be analyzed to identify the components in the original mixture. Paper chromatography is a simple, inexpensive, and effective technique for separating and analyzing mixtures.
HPLC is a type of chromatography that uses high pressure to force a liquid mobile phase through a column packed with solid particles. This allows for faster analysis times and better separation of components compared to traditional liquid chromatography. HPLC systems include a pump to deliver the mobile phase, an injector for samples, a column inside an oven, a detector, and a data processor. The interaction of sample components with the stationary and mobile phases causes separation as components move through the column at different speeds.
Super Critical Fluid Chromatography was first proposed in 1958 and involves using fluids above their critical temperature and pressure to separate mixtures. Carbon dioxide is commonly used as the mobile phase due to its stability and ability to dissolve large molecules. SFC provides faster analysis than HPLC and can analyze non-volatile compounds without derivatization like GC. It finds applications in pharmaceuticals, natural products, lipids, pesticides and more due to its mild conditions and speed.
This document discusses HPLC columns, including:
1. Silica is commonly used as the surface for HPLC columns, with silanols bonding to the surface. Pore size and surface area impact analyte retention and loading capacity.
2. Column particle sizes have decreased over time from 100 μm to below 2 μm, increasing theoretical plate counts. Column dimensions and particle sizes are selected based on the application.
3. Pore size should be larger than analyte molecules to allow entry without hindrance. Pore sizes of 60-80Å or 95-300Å are recommended for small molecules or proteins, respectively.
general description for the high performance liquid chromatography,the types,the equipment, principles, and HPLC uses for quantitative and qualitative analysis.
This document presents information on HPLC method development and validation. It begins with an introduction to analytical chemistry and chromatography. It then discusses the principles, types, and modes of HPLC, as well as factors to consider in method development such as column selection and mobile phase selection. The document concludes with a discussion of method validation parameters such as system suitability, specificity, linearity, precision, accuracy, limit of detection, limit of quantification, and robustness. References on the topic are also provided.
This document provides information about gas chromatography. It defines chromatography and gas chromatography, describes the basic components of a gas chromatography instrument including the carrier gas, injector, column, oven, and common detectors. It explains the principles of gas chromatography and separation, and provides details about different types of columns, injection techniques, and detectors such as FID, TCD, ECD, and GC-MS.
This document provides information about different types of columns used in high performance liquid chromatography (HPLC). It discusses normal phase and reverse phase chromatography columns. It describes various column packing materials, particle sizes, dimensions, costs and specifications. It provides details on columns from several major manufacturers like Waters, Phenomenex, Agilent, GE Healthcare and others. Preparative chromatography is also briefly mentioned. Resources for further information are listed at the end.
This document provides an overview of high performance liquid chromatography (HPLC). It describes the key components of an HPLC system including the stationary phase, mobile phase, injector, chromatographic column, pumping system, and detectors. It explains the separation process, noting that differences in how compounds partition between the mobile and stationary phases allows for separation. It also discusses normal phase and reverse phase chromatography, and provides examples of applications such as pharmaceutical analysis, food and flavor testing, and environmental and clinical analysis.
Thin layer chromatography (TLC) is a technique used to separate mixtures of compounds and identify their components. It involves spotting a sample onto a thin layer of adsorbent material and using a mobile phase solvent to migrate the components at different rates based on their interactions with the stationary and mobile phases. TLC is useful for identifying unknown compounds, analyzing purity, and separating mixtures. It has advantages over column chromatography like being faster, using less solvent, and allowing detection of both colored and non-colored compounds.
Chromatographic techniques such as thin layer chromatography (TLC), high performance thin layer chromatography (HPTLC), column chromatography, and high performance liquid chromatography (HPLC) separate mixtures by distributing components between a stationary and mobile phase. HPLC uses high pressure to force a mobile liquid or gas phase through a column packed with solid particles. Components elute from the column at different rates and are detected and analyzed. Chromatographic techniques have applications in pharmaceutical analysis, environmental monitoring, food and flavor analysis, and forensics.
HPLC
Chromatography
Mobile Phase & Stationary Phase
CLASSIFICATION OF CHROMATOGRAPHY
Characteristics of HPLC
Purpose
Superiority of HPLC
TYPES OF HPLC TECHNIQYES
Principle
PHASING SYSTEM & (normal vs reversed phase)
INSTRUMENTATION
Flow diagram of HPLC instrument
Advantages of HPLC
Sample preparation is an essential part of HPLC analysis to provide a reproducible and homogenous solution suitable for injection onto the column. The goal of sample preparation is to remove interferences and ensure the sample is compatible with the HPLC method without damaging the column. Sample matrices can be organic or inorganic solids, semisolids, liquids or gases, with liquids being easiest to prepare. Solid and semisolid samples require reducing particle size through processes like blending or grinding. Filtration is also important to remove particles that could damage the column. Common pretreatment methods for liquid samples include liquid-liquid extraction and solid phase extraction, while newer techniques are used for solid samples like supercritical fluid extraction. Derivatization can improve
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.
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.
Analytical method development and validation for simultaneous estimationProfessor Beubenz
The document discusses analytical method development and validation for the simultaneous estimation of active pharmaceutical ingredients (APIs). It covers topics such as the need for method development, the steps involved, parameters for validation as outlined in ICH guidelines, and the use of high performance liquid chromatography (HPLC). The key objectives of validation are to demonstrate that the developed method is suitable for its intended purpose and can reliably quantify the APIs.
High Performance Liquid Chromatography (HPLC) is described. HPLC uses high pressure to force a mobile phase through a column at a fast rate, increasing resolution. It discusses the types of chromatography used in HPLC, including normal phase, reverse phase, ion-exchange, and size-exclusion. The instrumentation of HPLC is also summarized, including components like the pump, mixing unit, degasser, injector, column, and detector.
The document discusses the process and history of paper chromatography. It begins with an introduction to chromatography and its use in separating mixtures. It then covers the history from early experiments in the 1860s to modern developments. The main types and techniques of paper chromatography are explained, including the use of a stationary phase, mobile phase, and capillary action to separate components by affinity and travel distance. Key steps like sample application and developing the paper strip are outlined.
HPLC is a type of liquid chromatography that uses high pressure to force a sample through a column packed with porous particles. This allows for faster separations compared to traditional chromatography. Key parameters in HPLC include retention time, which measures how long components spend in the column; capacity factor k', which is a ratio of time spent in the stationary vs mobile phase; selectivity factor α, which is the ratio of k' values and describes separation of adjacent peaks; and theoretical plates N, which estimates column efficiency based on peak widths and retention times. Optimizing these parameters can improve resolution of components in the mixture.
Chromatography is a technique used to separate mixtures into individual components. Paper chromatography is a type of chromatography that uses paper as the stationary phase. The mixture is applied to the paper and then placed in a developing chamber with the mobile phase solvent. As the solvent travels up the paper, the different components of the mixture separate based on how strongly they interact with the stationary and mobile phases. This creates discrete spots that can be analyzed to identify the components in the original mixture. Paper chromatography is a simple, inexpensive, and effective technique for separating and analyzing mixtures.
HPLC is a type of chromatography that uses high pressure to force a liquid mobile phase through a column packed with solid particles. This allows for faster analysis times and better separation of components compared to traditional liquid chromatography. HPLC systems include a pump to deliver the mobile phase, an injector for samples, a column inside an oven, a detector, and a data processor. The interaction of sample components with the stationary and mobile phases causes separation as components move through the column at different speeds.
Super Critical Fluid Chromatography was first proposed in 1958 and involves using fluids above their critical temperature and pressure to separate mixtures. Carbon dioxide is commonly used as the mobile phase due to its stability and ability to dissolve large molecules. SFC provides faster analysis than HPLC and can analyze non-volatile compounds without derivatization like GC. It finds applications in pharmaceuticals, natural products, lipids, pesticides and more due to its mild conditions and speed.
This document discusses HPLC columns, including:
1. Silica is commonly used as the surface for HPLC columns, with silanols bonding to the surface. Pore size and surface area impact analyte retention and loading capacity.
2. Column particle sizes have decreased over time from 100 μm to below 2 μm, increasing theoretical plate counts. Column dimensions and particle sizes are selected based on the application.
3. Pore size should be larger than analyte molecules to allow entry without hindrance. Pore sizes of 60-80Å or 95-300Å are recommended for small molecules or proteins, respectively.
general description for the high performance liquid chromatography,the types,the equipment, principles, and HPLC uses for quantitative and qualitative analysis.
This document presents information on HPLC method development and validation. It begins with an introduction to analytical chemistry and chromatography. It then discusses the principles, types, and modes of HPLC, as well as factors to consider in method development such as column selection and mobile phase selection. The document concludes with a discussion of method validation parameters such as system suitability, specificity, linearity, precision, accuracy, limit of detection, limit of quantification, and robustness. References on the topic are also provided.
This document provides information about gas chromatography. It defines chromatography and gas chromatography, describes the basic components of a gas chromatography instrument including the carrier gas, injector, column, oven, and common detectors. It explains the principles of gas chromatography and separation, and provides details about different types of columns, injection techniques, and detectors such as FID, TCD, ECD, and GC-MS.
This document provides information about different types of columns used in high performance liquid chromatography (HPLC). It discusses normal phase and reverse phase chromatography columns. It describes various column packing materials, particle sizes, dimensions, costs and specifications. It provides details on columns from several major manufacturers like Waters, Phenomenex, Agilent, GE Healthcare and others. Preparative chromatography is also briefly mentioned. Resources for further information are listed at the end.
This document provides an overview of high performance liquid chromatography (HPLC). It describes the key components of an HPLC system including the stationary phase, mobile phase, injector, chromatographic column, pumping system, and detectors. It explains the separation process, noting that differences in how compounds partition between the mobile and stationary phases allows for separation. It also discusses normal phase and reverse phase chromatography, and provides examples of applications such as pharmaceutical analysis, food and flavor testing, and environmental and clinical analysis.
Thin layer chromatography (TLC) is a technique used to separate mixtures of compounds and identify their components. It involves spotting a sample onto a thin layer of adsorbent material and using a mobile phase solvent to migrate the components at different rates based on their interactions with the stationary and mobile phases. TLC is useful for identifying unknown compounds, analyzing purity, and separating mixtures. It has advantages over column chromatography like being faster, using less solvent, and allowing detection of both colored and non-colored compounds.
Chromatographic techniques such as thin layer chromatography (TLC), high performance thin layer chromatography (HPTLC), column chromatography, and high performance liquid chromatography (HPLC) separate mixtures by distributing components between a stationary and mobile phase. HPLC uses high pressure to force a mobile liquid or gas phase through a column packed with solid particles. Components elute from the column at different rates and are detected and analyzed. Chromatographic techniques have applications in pharmaceutical analysis, environmental monitoring, food and flavor analysis, and forensics.
HPLC
Chromatography
Mobile Phase & Stationary Phase
CLASSIFICATION OF CHROMATOGRAPHY
Characteristics of HPLC
Purpose
Superiority of HPLC
TYPES OF HPLC TECHNIQYES
Principle
PHASING SYSTEM & (normal vs reversed phase)
INSTRUMENTATION
Flow diagram of HPLC instrument
Advantages of HPLC
Sample preparation is an essential part of HPLC analysis to provide a reproducible and homogenous solution suitable for injection onto the column. The goal of sample preparation is to remove interferences and ensure the sample is compatible with the HPLC method without damaging the column. Sample matrices can be organic or inorganic solids, semisolids, liquids or gases, with liquids being easiest to prepare. Solid and semisolid samples require reducing particle size through processes like blending or grinding. Filtration is also important to remove particles that could damage the column. Common pretreatment methods for liquid samples include liquid-liquid extraction and solid phase extraction, while newer techniques are used for solid samples like supercritical fluid extraction. Derivatization can improve
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.
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.
1. Chromatography is a technique used to separate components of a mixture using a stationary and mobile phase. Molecules spend varying amounts of time in each phase, becoming separated as they move through the column at different rates.
2. HPLC uses a liquid mobile phase pumped under pressure through a column packed with solid particles. Components separate based on interactions with the stationary and mobile phases, and are detected as they exit the column.
3. In reverse phase HPLC using a methanol/water mobile phase, prednisolone would elute just before betamethasone due to its slightly greater polarity from lacking an additional methyl group. Betamethasone dipropionate would elute last due to
Chromatography is a technique used to separate mixtures by distributing compounds between a stationary and mobile phase. High-performance liquid chromatography (HPLC) is commonly used and separates compounds using a column with a stationary phase and liquid mobile phase. HPLC can identify, detect, quantify, and purify individual components in a mixture using an apparatus including a pump, injector, column, detector, and recorder. The separation occurs as the compounds interact differently with the stationary phase in the column.
powerpoint presentation on high performance liquid chromatography which include its definition, classification, principles of seperation, instrumentation and application.
This document discusses several chromatography techniques used in forensic science analysis, including high performance liquid chromatography (HPLC), gas chromatography (GC), and inductively coupled plasma mass spectrometry (ICP-MS). It describes the basic principles, instrumentation components, and applications of each technique. HPLC uses high pressure to separate mixtures based on interactions with a stationary and mobile liquid phase. GC separates volatile compounds using an inert gas mobile phase and liquid stationary phase. ICP-MS uses plasma to ionize elements and masses to identify unknown samples at very low concentrations.
HPLC is a liquid chromatography technique used to separate compounds in a solution. It works by exploiting differences in how compounds partition between a stationary phase and mobile phase. There are four main types: partition, ion exchange, size exclusion, and affinity chromatography. HPLC systems consist of solvent reservoirs, pumps, injectors, columns, detectors, and data acquisition components. HPLC is used for research, quality control, environmental monitoring, and regulatory purposes to analyze complex mixtures and isolate compounds.
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.
HPLC is a form of liquid chromatography that uses high pressure to generate flow through a column packed with small particles. It allows for efficient separation of compounds based on differences in how they interact with the stationary and mobile phases. Key aspects of HPLC include pumps to deliver mobile phases at high pressure, injectors for sample introduction, columns packed with particles or beads, detectors to identify eluting compounds, and data systems to analyze results. Common modes are reverse phase, normal phase, size exclusion, and ion exchange chromatography. HPLC finds wide application in fields like pharmaceuticals, biochemistry, and environmental analysis.
High Performance Liquid Chromatography (HPLC) is a form of column chromatography that pumps a sample mixture or analyte in a solvent (known as the mobile phase) at high pressure through a column with chromatographic packing material (stationary phase).
1. Chromatography involves separating mixtures based on differences in how components distribute themselves between a stationary and mobile phase.
2. Common stationary phases include silica gel, alumina, and ion exchange resins. The interaction between the stationary phase and components leads to separation as they travel through the column at different rates.
3. Types of chromatography covered include liquid column chromatography, high performance liquid chromatography (HPLC), and ion exchange chromatography. Key applications are separating pharmaceuticals, proteins, food/industrial chemicals, and pollutants.
Chromatography is a technique used to separate mixtures by distributing components between a stationary and mobile phase. High-performance liquid chromatography (HPLC) uses high pressure to pass a solvent or solvent mixture through a column containing a stationary phase to separate components in a mixture. HPLC consists of several major components including a pump, injector, column, column compartment, detector, and degasser. The injector introduces the sample into the mobile phase which passes through the column, allowing separation based on interactions between components and the stationary phase. A detector then measures and records separated components as they elute from the column.
This document provides information about handling and operating high performance liquid chromatography (HPLC). It discusses the basic components and schematic of an HPLC system. It also summarizes key differences between thin layer chromatography (TLC) and HPLC. The document then covers HPLC theory, proposed reverse phase mechanisms, column selection guidelines, buffers, ion pair reagents, common stationary phase properties, detectors, and system suitability parameters.
This document provides information about handling and operating high performance liquid chromatography (HPLC). It discusses the basic components and setup of an HPLC instrument. It also compares HPLC to thin layer chromatography. The document explains reverse phase and normal phase HPLC, proposed mechanisms, column selection, mobile phase preparation including buffers, ion pair reagents, column properties, and common detectors. It provides guidelines for handling HPLC including preparation of samples, standards and mobile phases, and assessing system suitability.
1. Quantitative analysis using chromatography relies on measuring either the height or area of analyte peaks. Peak area provides a better measure of concentration since it is unaffected by variations in column efficiency.
2. The concentration of an analyte is determined by comparing its peak area to that of a standard of known concentration. Internal standards are used to control for variable experimental conditions between runs.
3. Calibration curves relate the detector response of external standards to their known concentrations. As long as injection volumes are identical, they provide accurate and precise results. Internal standards use the ratio of analyte to internal standard peak areas to normalize for variable conditions.
High performance liquid chromatography (HPLC) is described, including the basic principles and components of HPLC systems. HPLC uses high pressure to pass a liquid mobile phase through a column packed with solid adsorbent particles or porous beads. This allows for separation of mixtures based on differences in how components partition between the stationary and mobile phases. Key components reviewed are the solvent reservoirs, pump, injector, column, and detectors. Common applications of HPLC mentioned are qualitative and quantitative analysis of both volatile and non-volatile compounds.
High performance liquid chromatography (HPLC) is summarized as follows:
HPLC is a technique used to separate mixtures by distributing the components between a stationary and mobile phase. It can be used for both qualitative and quantitative analysis. HPLC utilizes high pressure pumps to pass a mobile phase through a column packed with adsorbent particles, allowing separation of components based on differences in their partitioning behavior between the mobile and stationary phases. Common detectors used in HPLC include UV/Vis, refractive index, fluorescence, and mass spectrometry.
High performance liquid chromatography (HPLC) is described, including the basic principles and components of HPLC systems. HPLC uses high pressure to pass a liquid mobile phase through a column packed with solid adsorbent particles or porous beads. This allows for separation of mixtures based on differences in how components partition between the stationary and mobile phases. Key components reviewed are the solvent reservoirs, pump, injector, column, and detectors. Common applications of HPLC mentioned are qualitative and quantitative analysis of both volatile and non-volatile compounds.
This document provides an introduction to spectroscopic analysis methods. It describes how electromagnetic radiation can be modeled as waves with properties like wavelength and frequency. It explains the basic components of instruments used for optical analysis, including a radiant energy source, wavelength selector, sample containers, radiation detector, and signal processor. The document also discusses the electromagnetic spectrum, photon energy, and atomic and molecular energy levels relevant to spectroscopic techniques.
1. NMR spectroscopy uses strong magnetic fields and radio waves to analyze atomic nuclei and their magnetic properties. Nuclei with spin can align parallel or anti-parallel to an external magnetic field, with the parallel alignment having lower energy.
2. The frequency at which nuclei absorb radio waves depends on the magnetic field strength, but reporting frequencies as chemical shifts relative to a standard allows comparisons between instruments. Chemical shifts distinguish different types of protons in a molecule.
3. NMR spectra provide information about chemical shifts, peak integrals, and spin-spin splitting between neighboring protons, which must all be considered to interpret spectra and determine molecular structures.
This document discusses molecular absorption spectroscopy, including definitions of key terms, Beer's law, instrumentation, and applications. It defines terms like absorbance, transmittance, molar absorptivity, and path length. Beer's law states that absorbance is directly proportional to concentration and path length. Limitations to Beer's law are discussed. Instrumentation covered includes UV-Vis and infrared spectrophotometers, highlighting features of single beam, double beam, and Fourier transform instruments.
This document discusses molecular absorption spectroscopy, including definitions of key terms, Beer's law, instrumentation, and applications. It defines terms like absorbance, transmittance, molar absorptivity, and path length. Beer's law states that absorbance is directly proportional to concentration and path length. Limitations to Beer's law are discussed. Instrumentation covered includes UV-Vis and infrared spectrophotometers, describing single beam, double beam, and Fourier transform designs.
The document discusses alkanes and cycloalkanes. It describes how alkanes are found naturally in petroleum and natural gas. Petroleum is separated through distillation into fractions like gasoline and kerosene. Alkanes can be refined and processed through technologies like cracking, isomerization, and reforming to produce smaller alkanes, branched alkanes, and aromatics for use in fuels and petrochemicals. The physical properties of alkanes are also covered, including combustion, heats of combustion, and octane ratings. Naming conventions for alkanes like alkyl groups and IUPAC nomenclature are outlined.
- Precision refers to how closely repeated measurements are clustered together, while accuracy describes how close measurements are to the true value. There are various ways to express accuracy and precision numerically.
- Accuracy can be expressed as absolute error or relative error compared to the true value. Precision can be expressed using values like standard deviation, deviation from the mean/median, and range.
- Errors can be determinate (systematic) or indeterminate (random). Determinate errors are consistent and can be avoided, while indeterminate errors follow a normal distribution and cannot be eliminated. Statistical analysis is needed to understand random error.
Gravimetric analysis involves determining the amount of analyte by measuring the mass of a pure substance containing the analyte. It has two main types - precipitation and volatilization. In precipitation, the analyte is converted to a solid precipitate using a reagent, then the precipitate is filtered, washed, purified and weighed. In volatilization, the analyte or its products are vaporized and collected or the mass loss is measured. Key steps in gravimetric analysis include preparation of the analyte solution, precipitation, digestion, filtration, washing, drying/ignition, and weighing to calculate the analyte amount. It is potentially more accurate and precise than volumetric analysis but requires proper technique.
Introduction to plant design economicsSunita Jobli
The document discusses different approaches to conceptual process design for a chemical process. It describes the onion model heuristic approach, which designs the process sequentially from the inner layers outward - starting with the reactor design and building outward by adding separation/recycle systems, heat exchanger networks, and utilities. This approach keeps the process structure "irreducible" by only adding units if economically justified. In contrast, the hierarchical approach classifies design decisions into 5 levels and generates/assesses alternatives at each level. The mathematical programming approach formulates the entire design problem into equations to optimize the "superstructure" and reduce it to an optimal feasible solution.
This document discusses gas chromatography apparatus and applications. It describes the key components of a GC system including the carrier gas supply, sample injection system, column, column thermostating, and detectors such as thermal conductivity and flame ionization. It also discusses stationary phases, qualitative and quantitative analysis applications, calibration with standards, and internal standard methods. Quantitative GC analysis is based on comparing analyte peak heights or areas to calibration standards to obtain concentration.
KuberTENes Birthday Bash Guadalajara - K8sGPT first impressionsVictor Morales
K8sGPT is a tool that analyzes and diagnoses Kubernetes clusters. This presentation was used to share the requirements and dependencies to deploy K8sGPT in a local environment.
Literature Review Basics and Understanding Reference Management.pptxDr Ramhari Poudyal
Three-day training on academic research focuses on analytical tools at United Technical College, supported by the University Grant Commission, Nepal. 24-26 May 2024
Advanced control scheme of doubly fed induction generator for wind turbine us...IJECEIAES
This paper describes a speed control device for generating electrical energy on an electricity network based on the doubly fed induction generator (DFIG) used for wind power conversion systems. At first, a double-fed induction generator model was constructed. A control law is formulated to govern the flow of energy between the stator of a DFIG and the energy network using three types of controllers: proportional integral (PI), sliding mode controller (SMC) and second order sliding mode controller (SOSMC). Their different results in terms of power reference tracking, reaction to unexpected speed fluctuations, sensitivity to perturbations, and resilience against machine parameter alterations are compared. MATLAB/Simulink was used to conduct the simulations for the preceding study. Multiple simulations have shown very satisfying results, and the investigations demonstrate the efficacy and power-enhancing capabilities of the suggested control system.
CHINA’S GEO-ECONOMIC OUTREACH IN CENTRAL ASIAN COUNTRIES AND FUTURE PROSPECTjpsjournal1
The rivalry between prominent international actors for dominance over Central Asia's hydrocarbon
reserves and the ancient silk trade route, along with China's diplomatic endeavours in the area, has been
referred to as the "New Great Game." This research centres on the power struggle, considering
geopolitical, geostrategic, and geoeconomic variables. Topics including trade, political hegemony, oil
politics, and conventional and nontraditional security are all explored and explained by the researcher.
Using Mackinder's Heartland, Spykman Rimland, and Hegemonic Stability theories, examines China's role
in Central Asia. This study adheres to the empirical epistemological method and has taken care of
objectivity. This study analyze primary and secondary research documents critically to elaborate role of
china’s geo economic outreach in central Asian countries and its future prospect. China is thriving in trade,
pipeline politics, and winning states, according to this study, thanks to important instruments like the
Shanghai Cooperation Organisation and the Belt and Road Economic Initiative. According to this study,
China is seeing significant success in commerce, pipeline politics, and gaining influence on other
governments. This success may be attributed to the effective utilisation of key tools such as the Shanghai
Cooperation Organisation and the Belt and Road Economic Initiative.
A SYSTEMATIC RISK ASSESSMENT APPROACH FOR SECURING THE SMART IRRIGATION SYSTEMSIJNSA Journal
The smart irrigation system represents an innovative approach to optimize water usage in agricultural and landscaping practices. The integration of cutting-edge technologies, including sensors, actuators, and data analysis, empowers this system to provide accurate monitoring and control of irrigation processes by leveraging real-time environmental conditions. The main objective of a smart irrigation system is to optimize water efficiency, minimize expenses, and foster the adoption of sustainable water management methods. This paper conducts a systematic risk assessment by exploring the key components/assets and their functionalities in the smart irrigation system. The crucial role of sensors in gathering data on soil moisture, weather patterns, and plant well-being is emphasized in this system. These sensors enable intelligent decision-making in irrigation scheduling and water distribution, leading to enhanced water efficiency and sustainable water management practices. Actuators enable automated control of irrigation devices, ensuring precise and targeted water delivery to plants. Additionally, the paper addresses the potential threat and vulnerabilities associated with smart irrigation systems. It discusses limitations of the system, such as power constraints and computational capabilities, and calculates the potential security risks. The paper suggests possible risk treatment methods for effective secure system operation. In conclusion, the paper emphasizes the significant benefits of implementing smart irrigation systems, including improved water conservation, increased crop yield, and reduced environmental impact. Additionally, based on the security analysis conducted, the paper recommends the implementation of countermeasures and security approaches to address vulnerabilities and ensure the integrity and reliability of the system. By incorporating these measures, smart irrigation technology can revolutionize water management practices in agriculture, promoting sustainability, resource efficiency, and safeguarding against potential security threats.
TIME DIVISION MULTIPLEXING TECHNIQUE FOR COMMUNICATION SYSTEMHODECEDSIET
Time Division Multiplexing (TDM) is a method of transmitting multiple signals over a single communication channel by dividing the signal into many segments, each having a very short duration of time. These time slots are then allocated to different data streams, allowing multiple signals to share the same transmission medium efficiently. TDM is widely used in telecommunications and data communication systems.
### How TDM Works
1. **Time Slots Allocation**: The core principle of TDM is to assign distinct time slots to each signal. During each time slot, the respective signal is transmitted, and then the process repeats cyclically. For example, if there are four signals to be transmitted, the TDM cycle will divide time into four slots, each assigned to one signal.
2. **Synchronization**: Synchronization is crucial in TDM systems to ensure that the signals are correctly aligned with their respective time slots. Both the transmitter and receiver must be synchronized to avoid any overlap or loss of data. This synchronization is typically maintained by a clock signal that ensures time slots are accurately aligned.
3. **Frame Structure**: TDM data is organized into frames, where each frame consists of a set of time slots. Each frame is repeated at regular intervals, ensuring continuous transmission of data streams. The frame structure helps in managing the data streams and maintaining the synchronization between the transmitter and receiver.
4. **Multiplexer and Demultiplexer**: At the transmitting end, a multiplexer combines multiple input signals into a single composite signal by assigning each signal to a specific time slot. At the receiving end, a demultiplexer separates the composite signal back into individual signals based on their respective time slots.
### Types of TDM
1. **Synchronous TDM**: In synchronous TDM, time slots are pre-assigned to each signal, regardless of whether the signal has data to transmit or not. This can lead to inefficiencies if some time slots remain empty due to the absence of data.
2. **Asynchronous TDM (or Statistical TDM)**: Asynchronous TDM addresses the inefficiencies of synchronous TDM by allocating time slots dynamically based on the presence of data. Time slots are assigned only when there is data to transmit, which optimizes the use of the communication channel.
### Applications of TDM
- **Telecommunications**: TDM is extensively used in telecommunication systems, such as in T1 and E1 lines, where multiple telephone calls are transmitted over a single line by assigning each call to a specific time slot.
- **Digital Audio and Video Broadcasting**: TDM is used in broadcasting systems to transmit multiple audio or video streams over a single channel, ensuring efficient use of bandwidth.
- **Computer Networks**: TDM is used in network protocols and systems to manage the transmission of data from multiple sources over a single network medium.
### Advantages of TDM
- **Efficient Use of Bandwidth**: TDM all
Optimizing Gradle Builds - Gradle DPE Tour Berlin 2024Sinan KOZAK
Sinan from the Delivery Hero mobile infrastructure engineering team shares a deep dive into performance acceleration with Gradle build cache optimizations. Sinan shares their journey into solving complex build-cache problems that affect Gradle builds. By understanding the challenges and solutions found in our journey, we aim to demonstrate the possibilities for faster builds. The case study reveals how overlapping outputs and cache misconfigurations led to significant increases in build times, especially as the project scaled up with numerous modules using Paparazzi tests. The journey from diagnosing to defeating cache issues offers invaluable lessons on maintaining cache integrity without sacrificing functionality.
Using recycled concrete aggregates (RCA) for pavements is crucial to achieving sustainability. Implementing RCA for new pavement can minimize carbon footprint, conserve natural resources, reduce harmful emissions, and lower life cycle costs. Compared to natural aggregate (NA), RCA pavement has fewer comprehensive studies and sustainability assessments.
2. 6.1 HPLC INTRODUCTION
6.1.1 Founder of Liquid Chromatography
Mikhail Tsvet
Born on May 14, 1872
Asti, Italy
Invented chromatography in 1901 while
performing research on plant pigments
Column adsorption liquid
chromatography
3. 6.1.2 HPLC Analysis
Qualitative Analysis
Identification of compound identity
Require a known standard
Identified by comparing retention time
Quantitative Analysis
Identify the amount/concentration of the compound
Require a standard with known amount of concentrations
Identified by interpolating the area of unknown into a set of
standards with known concentration
5. 6.1.3 Basic Concept of HPLC
Sample are injected into a HPLC system.
Interactions happen between the samples with the mobile phase
and stationary phase (column) which results in separation of
samples which is detected through the detector and converted into
a chromatogram.
7. 6.1.4 HPLC Peaks
A separation by changing the relative speed of each analyte band
(Competition between the mobile phase and stationary phase).
8. As analyte “Bands” pass
through Detector Flow Cell, an
electrical signal is sent to the
Computer Data Station
(recorder) to draw the “Peak”.
Blue band is now the broadest as it
exits the column since it was
moving so slowly in the column, it
takes a lot of mobile phase to
finally sweep it all out – this is why
the latest eluting peaks are the
broadest.
How Peaks Are Created?
ISOCRATIC
CONDITIONS
9. Few, Earliest
Arriving
Analyte
Molecules
Front
Apex
Back
Chromatogram
Analyte “Band” flowing
Into Detector Cell
Most Analyte
Molecules
Arrive
(Highest
Concentration)
Few, Latest
Arriving
Analyte
Molecules
Computer
Data Station
Signal from the
Analyte “Band” in the
Detector
Flow Cell
is Translated into a
“Peak”
The higher the
analyte
concentration, the
higher the peak
height –
better Sensitivity
Most Concentrated
Point in the
Analyte “Band”
Time
D
E
T
E
C
T
O
R
Mobile
Phase
Mobile
Phase
Mobile Phase
Base Line
Mobile Phase
Base Line0
How an Analyte Becomes a Peak?
10. 6.1.5 Band Spreading
Narrow Band –
Narrow Peak
More Concentrated –
Increased Peak
Height/Sensitivity
More Resolution
Capability
Broad Band – Broad Peak
Less Concentration –
Less Sensitivity
Less Resolution Capability
Broader “Band”
More “Band Spreading”
Broader Peak
Narrower “Band”
Less “Band Spreading”
Narrower, Taller Peak
Mobile
Phase
Mobile
Phase
Mobile
Phase
Less
Concentrated
0 0
Mobile
Phase
11. Better separation
More concentrated “Bands”
Higher Sensitivity
In this region, both analytes (blue and red)
are not separated [a partial co-elution –
shown as a “purple” band]
System with
MORE
Band Spreading
System with
LESS
Band Spreading
Narrower, Sharper Bands create Narrower Sharper Peaks, which
provide a better separation.
This results in better Resolution between the 2 peaks and
greater peak heights for better Sensitivity
12. 6.2 TYPES OF HPLC
HPLC : is a type of chromatography that employs a liquid mobile
phase and a very finely divided stationary phase.
The types of HPLC are often classified by separation mechanism or
by the type of stationary phase :
Partition / Liquid-liquid chromatography
Adsorption / liquid solid chromatography
Ion exchange
Size-exclusion
Affinity chromatography
Chiral chromatography
15. 6.3 APPARATUS
In HPLC solvent at high pressure is forced through a column to
obtain a separation
The instrument is made of a solvent system, an injection valve, a
column, and a detector.
18. 6.3.1 (a) Mobile Phase Reservoir and Solvent Treatment
Systems
Solvent reservoir is made from glass and it contains 500 mL or
more of a solvent volume.
However, dissolved gas and dust from liquid can form bubbles in
column » band spreading. Thus, Degassers is needed for a
sparging process.
Sparging : the removal of an unwanted dissolved gas by aeration
with an inert gas.
Elution can be performed isocratically or with a gradient :
Isocratic elution is performed with a constant solvent (pure or
constant mixture).
Gradient elution is the solvent mixture is altered during the run
if one solvent is not satisfactory.
19. Isocratic elution
Mixture of acetonitrile and aqueous
phase buffer solution (KH2PO4)
Separation of aromatic compounds,
including alcohols and ketones.
22. 6.3.1 (b) Pumping System
The requirement of pump :
Ability to generate pressures up to 6000 psi
Pulse-free output
From rate ranging 0.1 to 10 mL/min
Flow reproducibilities of 0.5% relative or better
Resistance to corrosion by a
variety of solvent
There are 3 types of pumps
Syringe-type pumps
Reciprocating pump
Pnuematic or constant pressure pump
23. A reciprocating pump for HPLC
6.3.1 (c) Sample Injection System
The most widely used method of sample introduction in liquid
chromatography is based on a sampling loop.
These devices are an integral part of some liquid chromatography
equipment.
24. Interchangeable loops are available to provide
a choice of sample sizes ranging from 5 to 500 μL.
Many HPLC instruments have autosampler
with an automatic injector.
25.
26. 6.3.1 (d) Analytical Columns
HPLC column :
made from stainless steel
10 – 30 cm long
2 – 5 mm internal diameter
Particles size for column packing is 3 – 10 μm and provide
40000 – 60000 plates/m
Microcolumn :
3 – 7.5 cm long
1 – 4.6 mm internal diameter
Particles size for column packing is 3 – 5 μm and provide
100000 plates/m
Have disadvantage of speed and minimal solvent consumption.
27. The most packing for liquid chromatography is prepared from silica
particles which are synthesized by agglomerating submicron silica
particles under conditions that lead to larger particles with highly
uniform diameters.
The resulting particles are coated with thin organic films, which are
chemically or physically bonded to the surface.
28. Guard Column
Positioned ahead of the analytical column to increase the life of
the analytical column by removing particulate matter and
contaminants from solvents.
It also serves to saturate the mobile phase with the stationary
phase so that losses of stationary phase from the analytical
column are minimized.
Composition is similar to analytical column
except particle size
is larger to minimize pressure drop.
29. Column Thermostats
Better chromatograms are obtained by maintaining column
temperatures constant to a few tenths of a degree celcius.
Equipped with heaters to control column temperatures.
Columns may be fitted with water jackets from a constant-
temperature bath to give precise temperature control.
30. 6.3.1 (e) Detectors
Detectors for HPLC must have low dead volume to minimize extra-
column band broadening.
The detector :
small and compatible with liquid flow
not highly sensitive
universal detector system
There are 2 types of detector :
Bulk property detector : measure property of mobile phase
(refractive index, dielectric constant, density).
31. Solute property detector : measure property of solute
present in mobile phase (UV absorbance, fluorescence, IR
absorbance).
The most widely used detectors for liquid chromatography are
based on absorption of ultraviolet or visible radiation.
Both photometer and spectrophotometer specifically designed for
use with chromatographic column are commercially available.
Source :
single line (arc or hollow cathode lamp)
continuum (Xe, D2 lamp)
Detector :
photodiode/ photomultiplier tube
photodiode array
34. 6.4 HIGH PERFORMANCE PARTITION
CHROMATOGRAPHY
The most widely used and popular type of HPLC → the stationary
phase is a second liquid that is immiscible with the liquid mobile
phase.
Sample molecules equilibrate (PARTITION) between liquid
stationary phase and mobile phase.
Retention depends on a sample molecule's escaping tendency into
the mobile phase versus its solubility in the stationary phase.
35. It can be divided by 2 types, liquid-liquid and liquid-phase-bounded
chromatography.
i. Liquid-liquid partition chromatography » the stationary phase is a
solvent that is held in place by absorption on the surface of
packing material.
ii. Liquid-bounded-phase chromatography » the stationary phase is
an organic species that is attached to the surface of the packing
particles by chemical bonds.
36. 6.4.1 Bonded-Phase Packings
Most of the bonded-phase packings are prepared by rxn of an
organochlororsilane with the ―OH groups formed on the surface of
silica particles (3, 5 or 10 μm) by hydrolysis in hot, dilute HCL to
produce organosiloxane.
Advantage : not required periodic recoating of stationary phase
due to thee phase will be dissolved away by mobile phase and very
practical for gradient elution.
37. 6.4.2 Normal and Reversed-Phase Packings
Base on the relative polarities of the mobile and stationary phase :
Normal-Phase HPLC → nonpolar (solvent)/ polar (column)
Reversed-Phase HPLC → polar (solvent)/ nonpolar (column)
38. In Normal-Phase Chromatography → the least polar component is
eluted first » ↑ the polarity of the mobile phase, ↓ the elution time.
In Reversed-Phase Chromatography → the most polar components
elutes first, ↑ the polarity of the mobile phase, ↑ the elution time.
6.4.3 Choice of Mobile and Stationary Phases
The partition chromatography requires a proper balance of
intermolecular force among analyte, mobile phase and stationary
phase.
This intermolecular force are describes qualitatively in terms of the
relative polarity posses by each components.
The order of polarities of common mobile phase solvents » water>
acetonitrile> methanol> ethanol > tetrahydrofuron > propanol >
cyclohexane > hexane.
39. For stationary phase choice : choose column with similar polarity to
analyte for maximum interaction.
Reversed-Phase » column (nonpolar)
Normal-Phase » column (polar)
For mobile phase choice : Polar strong solvent interacts most with
polar analyte (solute) – elute faster but less resolution.
41. 6.5 HIGH PERFORMANCE ION-EXCHANGE
CHROMATOGRAPHY
The interactions between the chromatographic medium and the
proteins in the mixture are based primarily on ionic charge.
Ion exchangers are resins often coupled on cross-linked
polysaccharides that can exchange ions with water solutions.
42. Most common types of ion-exchangers:
DEAE (diethylaminoethyl)-cellulose; an anion exchange resin
used primarily for neutral and acidic proteins
CM (carboxymethyl)-cellulose; a cation exchanger used
primarily for the separation of neutral and basic proteins.
Elution from the column:
by altering the pH of the elution buffer
by increasing the ionic strength of the elution buffer
Two types of ion chromatography in use :
Suppressor based
Single Column
43. 6.5.1 Ion Chromatography Based on Suppression
In suppression-based ion chromatography, the ion exchange
column is followed by a suppressor column or by a suppressor
membranes that converts an ionic eluent into a non-ionic species
that does not interfere with the conductometric detection of analyte
ions.
6.5.2 Single Column Ion Chromatography
In single-column ion-exchange chromatography, analyte ions are
separated on a low-capacity ion exchanger by means of low-ionic
strength eluent that does not interfere with the conductometric
detection of analyte ions
44. 6.6 HIGH PERFORMANCE SIZE-EXCLUSION
CHROMATOGRAPHY
It is based on molecular size.
Used for large MW compounds protein and polymers.
Separation mechanism is sieving not portioning.
Stationary phase » porous silica or polymer particles (Polystyrene,
polyacrylamide)(5 – 10 μm)
45. 6.6.1 Column Packing
Well-defined pore sizes (40 – 2500 Å)
Large molecules excluded from pores – not retained, first
eluted (exclusion limit – term of MW).
Intermediate molecules – retained, intermediate elution times.
Small molecules permeate into pores – strongly retained last
eluted (permeation limit – term of MW).
6.6.2 Applications
Separation of sugars in cane
Separation of proteins/ peptide
Determination of molecular mass of large polymers or natural
products