This document provides an introduction and overview of high performance liquid chromatography (HPLC). It discusses the history and development of HPLC from its origins in 1903 to present day applications. The document covers various HPLC modes including normal phase, reversed phase, ion exchange, and size exclusion chromatography. It explains the separation principles, stationary phases, and applications of each mode. The document also discusses HPLC method development, validation, and the expanding role of HPLC in areas like drug discovery and analysis.
Group presentation on HPLC (High-performance liquid chromatography). The group members are listed. HPLC can separate mixtures and works by forcing a mobile phase through a column packed with porous particles. Components interact differently with the stationary phase based on properties like affinity, allowing separation. There are various types of HPLC based on the separation mode or principle, including reverse phase, ion exchange, and size exclusion. The basic instrumentation includes a pump, injector, column, detector, and recorder/computer. Common detectors are UV-Vis, fluorescence, and mass spectrometry. Applications of HPLC include pharmaceutical analysis, forensics, clinical analysis, and more.
High performance liquid chromatography (HPLC) is a technique used to separate components in a mixture. It works by pumping a sample mixture through a column containing chromatographic packing material at high pressure. The sample components interact differently with the stationary phase in the column, causing them to elute out at different rates and allowing separation. HPLC has many applications in fields like pharmaceuticals, environmental analysis, food and flavors testing, clinical testing, and forensics. It provides a powerful analytical tool for identifying and quantifying compounds in samples.
HPLC is a form of column chromatography that separates compounds based on their polarity and interaction with the stationary phase. It utilizes a pump to push the mobile phase and analytes through a column under high pressure. Various detectors can then provide the retention time of analytes as they exit the column. Key aspects of HPLC include the types of columns, mobile phases, and detectors used, which are selected based on the compounds being analyzed. HPLC is commonly used to analyze biological, pharmaceutical, environmental, and forensic samples.
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.
High Performance Liquid Chromatography (HPLC)VaibhaviPatil21
HPLC stands for High Performance Liquid Chromatography. previously HPLC was known as High pressure Liquid Chromatography as high pressure is required to pump the liquid,due to its efficiency ,later it came to know as High Performance liquid chromatography.It is an analytical technique which is used to analyze compunds in samples in pharma industry,chemical industry as well as in Bioanalytical Labs for studying BABE studies.
It is the form of Liquid column chromatography.In HPLC,the mobile phase is liquid and the stationary phase is solid.
HPLC can be divided into two types-
1) Normal phase HPLC
2) Reverse phase HPLC
HPLC can further be combined with Mass Spectrometry (LC-MS) which allows to seperate the samples with the analysis of mass.
HPLC, or high performance liquid chromatography, is a separation technique that uses the differential partitioning of analytes between a stationary and mobile liquid phase. The document discusses various HPLC methods used for compounds like pesticides, explosives, and PAHs. It describes the basic components of an HPLC system including the pump, injector, column, detector and how separation is based on a compound's relative solubility in the mobile and stationary phases. Common reverse phase columns and solvents are also outlined.
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.
This document provides an overview of high performance liquid chromatography (HPLC). It discusses the basic principles of chromatographic separation and defines key terms like retention time and resolution. It also describes different HPLC techniques including normal phase, reversed phase, ion exchange, size exclusion, and ion-pair chromatography. The document outlines the typical instrumentation used in HPLC including the pump, injector, columns, detectors, and data collection systems. It provides details on how each component works and its role in the chromatography process.
Group presentation on HPLC (High-performance liquid chromatography). The group members are listed. HPLC can separate mixtures and works by forcing a mobile phase through a column packed with porous particles. Components interact differently with the stationary phase based on properties like affinity, allowing separation. There are various types of HPLC based on the separation mode or principle, including reverse phase, ion exchange, and size exclusion. The basic instrumentation includes a pump, injector, column, detector, and recorder/computer. Common detectors are UV-Vis, fluorescence, and mass spectrometry. Applications of HPLC include pharmaceutical analysis, forensics, clinical analysis, and more.
High performance liquid chromatography (HPLC) is a technique used to separate components in a mixture. It works by pumping a sample mixture through a column containing chromatographic packing material at high pressure. The sample components interact differently with the stationary phase in the column, causing them to elute out at different rates and allowing separation. HPLC has many applications in fields like pharmaceuticals, environmental analysis, food and flavors testing, clinical testing, and forensics. It provides a powerful analytical tool for identifying and quantifying compounds in samples.
HPLC is a form of column chromatography that separates compounds based on their polarity and interaction with the stationary phase. It utilizes a pump to push the mobile phase and analytes through a column under high pressure. Various detectors can then provide the retention time of analytes as they exit the column. Key aspects of HPLC include the types of columns, mobile phases, and detectors used, which are selected based on the compounds being analyzed. HPLC is commonly used to analyze biological, pharmaceutical, environmental, and forensic samples.
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.
High Performance Liquid Chromatography (HPLC)VaibhaviPatil21
HPLC stands for High Performance Liquid Chromatography. previously HPLC was known as High pressure Liquid Chromatography as high pressure is required to pump the liquid,due to its efficiency ,later it came to know as High Performance liquid chromatography.It is an analytical technique which is used to analyze compunds in samples in pharma industry,chemical industry as well as in Bioanalytical Labs for studying BABE studies.
It is the form of Liquid column chromatography.In HPLC,the mobile phase is liquid and the stationary phase is solid.
HPLC can be divided into two types-
1) Normal phase HPLC
2) Reverse phase HPLC
HPLC can further be combined with Mass Spectrometry (LC-MS) which allows to seperate the samples with the analysis of mass.
HPLC, or high performance liquid chromatography, is a separation technique that uses the differential partitioning of analytes between a stationary and mobile liquid phase. The document discusses various HPLC methods used for compounds like pesticides, explosives, and PAHs. It describes the basic components of an HPLC system including the pump, injector, column, detector and how separation is based on a compound's relative solubility in the mobile and stationary phases. Common reverse phase columns and solvents are also outlined.
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.
This document provides an overview of high performance liquid chromatography (HPLC). It discusses the basic principles of chromatographic separation and defines key terms like retention time and resolution. It also describes different HPLC techniques including normal phase, reversed phase, ion exchange, size exclusion, and ion-pair chromatography. The document outlines the typical instrumentation used in HPLC including the pump, injector, columns, detectors, and data collection systems. It provides details on how each component works and its role in the chromatography process.
HPLC: Principle and Maintenance with Applicationijtsrd
High performance liquid chromatography (HPLC) is a significant qualitative and quantitative technique, usually used for the estimation of pharmaceutical and biological samples. The chromatography term is derived from the Greek words namely chroma (colour) and graphein (to write). The chromatography is very accepted technique and it is mostly used analytically. It is the most resourceful, safest, reliable and fastest chromatographic technique for the quality control of drug components. This technique involves 2 phases"™ stationary and mobile phases. There are different types of chromatographic techniques. The separation of constituents is based on the variation between the partition coefficients of the two phases. This article is primed with an aim to review different aspects of HPLC, such as principle, types, instrumentation and application with maintenance. Yogesh Kumar | Sayed Md Mumtaz | Mustaq Ahmad"HPLC: Principle and Maintenance with Application" Published in International Journal of Trend in Scientific Research and Development (ijtsrd), ISSN: 2456-6470, Volume-2 | Issue-5 , August 2018, URL: http://www.ijtsrd.com/papers/ijtsrd17134.pdf http://www.ijtsrd.com/pharmacy/pharmacology-/17134/hplc-principle-and-maintenance-with-application/yogesh-kumar
The document discusses High Performance Liquid Chromatography (HPLC), including its principles, types of separations, instrumentation, applications in fields like food and pharmaceuticals, and advantages like speed and accuracy over traditional chromatography techniques. HPLC uses high pressure to pass samples in liquid mobile phases through stationary phases in columns to separate mixtures based on properties like polarity, size, or ionic charge.
High Performance Liquid Chromatography (HPLC) is a separation technique that uses pumps to force a liquid mobile phase through a column packed with solid particles. Sample components interact differently with the stationary and mobile phases allowing separation. HPLC instruments consist of pumps, injectors, columns, detectors and computers. Samples are injected and the separated components are detected and data is analyzed to identify and quantify the components. HPLC is used in various fields to analyze complex mixtures like pharmaceuticals, chemicals, and biological samples.
HPLC Principle,Instrumentation and ApplicationAlakesh Pradhan
HPLC Chromatography and its principle
Liquid chromatography
High Performance Liquid Chromatography ( HPLC )
The components of the high performance liquid chromatograph (HPLC).
The separation process.
The chromatogram
This document discusses various concepts related to high performance liquid chromatography (HPLC) peak analysis including:
1. It describes factors that influence peak shape such as column packing, mobile phase composition, pH, and buffers which can improve peak symmetry and resolution.
2. Key parameters for characterizing chromatographic performance are discussed including retention factor (k), selectivity factor (α), plate number (N), and height equivalent of a theoretical plate (HETP).
3. Optimizing these parameters through adjusting mobile phase or column properties can enhance separation and analysis of chromatographic runs.
This document provides an overview of high-performance liquid chromatography (HPLC). It describes HPLC as a chromatographic technique used to separate components of a mixture for identifying, quantifying, or purifying individual components. The document outlines the history, instrumentation, operation, types, advantages, and disadvantages of HPLC. It explains that HPLC involves a mobile phase, column, pump, injector, detector, and computer to separate sample components based on their interactions with the stationary phase.
HPLC, or high-performance liquid chromatography, is an analytical technique used to separate, identify, and quantify components in a mixture. It works by pumping a pressurized mobile liquid phase through a column containing a stationary phase, which causes the components in a sample to separate as they migrate through the column at different rates. Common components of an HPLC system include the pump, injector, column, detector, and recorder or computer system. HPLC has advantages of speed, efficiency, accuracy, and versatility in chemical analysis. It is used in various applications such as drug analysis, environmental analysis, and industrial quality control.
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.
This document provides an overview of high performance liquid chromatography (HPLC) presented by Ravi Pratap Pulla. It introduces HPLC and its history. Key topics covered include HPLC components like columns and systems. Applications to pharmaceutical analysis are discussed. The document also reviews some basic HPLC terminology and concepts.
HPLC is a technique used to separate components in a mixture using pumps to pass a pressurized liquid solvent containing the sample through a column filled with an adsorbent material. Each component interacts slightly differently with the material, causing different flow rates and separation. HPLC has applications in manufacturing like pharmaceutical production, legal purposes like drug testing, research like separating biological samples, and medical uses like detecting vitamin levels. It provides superior resolving power over traditional chromatography to distinguish between compounds.
This document provides an overview of high performance liquid chromatography (HPLC). It describes the key components of an HPLC system, including the pump, injector, separation column, and detector. It explains how the different components of a mixture are separated through their interaction with the mobile and stationary phases in the column. The document also discusses various types of columns, detectors, and applications of HPLC in chemistry, biochemistry, and quality control.
High-performance liquid chromatography (HPLC) is an analytical technique used to separate, identify, and quantify components in a mixture. It works by pumping a sample mixture through a column at high pressure using a solvent. Components are separated into bands as they interact differently with the stationary and mobile phases of the column on their way through. HPLC can separate and identify compounds present in trace concentrations and is widely used in applications like analyzing food products, environmental samples, medical products, and more due to its accuracy, affordability, and ability to quickly obtain high resolution results.
Introduction to High Performance Liquid Chromatography (HPLC)Saurabh Arora
This presentation provides a brief introduction to HPLC and its parts. The technique has found immense scope of applications in both academic and industrial laboratories requiring identification and quantification of mixtures of organic compounds.It is essential for scientists working in any field to understand and know how to use a HPLC.
High-performance liquid chromatography (HPLC) is a popular analytical technique used to separate, identify, and quantify mixtures into their individual components. HPLC uses high pressure pumps to push a pressurized mobile phase through a column packed with adsorbent particles. Sample components interact differently with the stationary phase based on their properties and are eluted from the column at different retention times, allowing for separation. The separated components are then detected and quantified to analyze the sample. HPLC provides high-speed, efficient, and sensitive separation compared to traditional liquid chromatography.
High Performance Liquid chromatography (HPLC)Unnati Garg
The document provides an overview of high performance liquid chromatography (HPLC). It describes key components of HPLC systems including pumps to deliver solvent at stable flow rates, columns for molecular separation, and detectors for recognizing analytes. The separation principle is based on the distribution of analytes between a mobile liquid phase and stationary column packing material. Different constituents are eluted at different times, achieving separation. HPLC is widely used in pharmaceutical applications such as drug development, production quality control, and stability testing.
This is a PPT for HPLC which I made for presenting my assigned topic for Practice School during 7 sem of my graduation . Hope it is useful for you guys :)
This document provides an overview of high performance liquid chromatography (HPLC). It discusses the key components of an HPLC system including the solvent rack, pump, injector, separation column, and detector. It also describes different chromatography techniques such as normal phase chromatography, reversed phase chromatography, and ion exchange chromatography which separate compounds based on polarity, electrical charge, and molecular size. Finally, it discusses factors that influence separation such as stationary phase particle size and eluent composition.
Stationary Phase and Mobile Phase Selection for Liquid Chromatography
The presentation focuses on how to choose the appropriate mode of separation, the correct column and highlights the importance of the correct mobile phase. This approach will be applied to a wide selection of compound types ranging from proteins, peptides, glycans to small pharmaceutical molecules and their metabolites. It will also look at specific application areas for monoclonal antibody analysis, namely: titer, aggregation, charge and oxidation variant. Platform methods for biologics characterization are also discussed.
HPLC: Principle and Maintenance with Applicationijtsrd
High performance liquid chromatography (HPLC) is a significant qualitative and quantitative technique, usually used for the estimation of pharmaceutical and biological samples. The chromatography term is derived from the Greek words namely chroma (colour) and graphein (to write). The chromatography is very accepted technique and it is mostly used analytically. It is the most resourceful, safest, reliable and fastest chromatographic technique for the quality control of drug components. This technique involves 2 phases"™ stationary and mobile phases. There are different types of chromatographic techniques. The separation of constituents is based on the variation between the partition coefficients of the two phases. This article is primed with an aim to review different aspects of HPLC, such as principle, types, instrumentation and application with maintenance. Yogesh Kumar | Sayed Md Mumtaz | Mustaq Ahmad"HPLC: Principle and Maintenance with Application" Published in International Journal of Trend in Scientific Research and Development (ijtsrd), ISSN: 2456-6470, Volume-2 | Issue-5 , August 2018, URL: http://www.ijtsrd.com/papers/ijtsrd17134.pdf http://www.ijtsrd.com/pharmacy/pharmacology-/17134/hplc-principle-and-maintenance-with-application/yogesh-kumar
The document discusses High Performance Liquid Chromatography (HPLC), including its principles, types of separations, instrumentation, applications in fields like food and pharmaceuticals, and advantages like speed and accuracy over traditional chromatography techniques. HPLC uses high pressure to pass samples in liquid mobile phases through stationary phases in columns to separate mixtures based on properties like polarity, size, or ionic charge.
High Performance Liquid Chromatography (HPLC) is a separation technique that uses pumps to force a liquid mobile phase through a column packed with solid particles. Sample components interact differently with the stationary and mobile phases allowing separation. HPLC instruments consist of pumps, injectors, columns, detectors and computers. Samples are injected and the separated components are detected and data is analyzed to identify and quantify the components. HPLC is used in various fields to analyze complex mixtures like pharmaceuticals, chemicals, and biological samples.
HPLC Principle,Instrumentation and ApplicationAlakesh Pradhan
HPLC Chromatography and its principle
Liquid chromatography
High Performance Liquid Chromatography ( HPLC )
The components of the high performance liquid chromatograph (HPLC).
The separation process.
The chromatogram
This document discusses various concepts related to high performance liquid chromatography (HPLC) peak analysis including:
1. It describes factors that influence peak shape such as column packing, mobile phase composition, pH, and buffers which can improve peak symmetry and resolution.
2. Key parameters for characterizing chromatographic performance are discussed including retention factor (k), selectivity factor (α), plate number (N), and height equivalent of a theoretical plate (HETP).
3. Optimizing these parameters through adjusting mobile phase or column properties can enhance separation and analysis of chromatographic runs.
This document provides an overview of high-performance liquid chromatography (HPLC). It describes HPLC as a chromatographic technique used to separate components of a mixture for identifying, quantifying, or purifying individual components. The document outlines the history, instrumentation, operation, types, advantages, and disadvantages of HPLC. It explains that HPLC involves a mobile phase, column, pump, injector, detector, and computer to separate sample components based on their interactions with the stationary phase.
HPLC, or high-performance liquid chromatography, is an analytical technique used to separate, identify, and quantify components in a mixture. It works by pumping a pressurized mobile liquid phase through a column containing a stationary phase, which causes the components in a sample to separate as they migrate through the column at different rates. Common components of an HPLC system include the pump, injector, column, detector, and recorder or computer system. HPLC has advantages of speed, efficiency, accuracy, and versatility in chemical analysis. It is used in various applications such as drug analysis, environmental analysis, and industrial quality control.
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.
This document provides an overview of high performance liquid chromatography (HPLC) presented by Ravi Pratap Pulla. It introduces HPLC and its history. Key topics covered include HPLC components like columns and systems. Applications to pharmaceutical analysis are discussed. The document also reviews some basic HPLC terminology and concepts.
HPLC is a technique used to separate components in a mixture using pumps to pass a pressurized liquid solvent containing the sample through a column filled with an adsorbent material. Each component interacts slightly differently with the material, causing different flow rates and separation. HPLC has applications in manufacturing like pharmaceutical production, legal purposes like drug testing, research like separating biological samples, and medical uses like detecting vitamin levels. It provides superior resolving power over traditional chromatography to distinguish between compounds.
This document provides an overview of high performance liquid chromatography (HPLC). It describes the key components of an HPLC system, including the pump, injector, separation column, and detector. It explains how the different components of a mixture are separated through their interaction with the mobile and stationary phases in the column. The document also discusses various types of columns, detectors, and applications of HPLC in chemistry, biochemistry, and quality control.
High-performance liquid chromatography (HPLC) is an analytical technique used to separate, identify, and quantify components in a mixture. It works by pumping a sample mixture through a column at high pressure using a solvent. Components are separated into bands as they interact differently with the stationary and mobile phases of the column on their way through. HPLC can separate and identify compounds present in trace concentrations and is widely used in applications like analyzing food products, environmental samples, medical products, and more due to its accuracy, affordability, and ability to quickly obtain high resolution results.
Introduction to High Performance Liquid Chromatography (HPLC)Saurabh Arora
This presentation provides a brief introduction to HPLC and its parts. The technique has found immense scope of applications in both academic and industrial laboratories requiring identification and quantification of mixtures of organic compounds.It is essential for scientists working in any field to understand and know how to use a HPLC.
High-performance liquid chromatography (HPLC) is a popular analytical technique used to separate, identify, and quantify mixtures into their individual components. HPLC uses high pressure pumps to push a pressurized mobile phase through a column packed with adsorbent particles. Sample components interact differently with the stationary phase based on their properties and are eluted from the column at different retention times, allowing for separation. The separated components are then detected and quantified to analyze the sample. HPLC provides high-speed, efficient, and sensitive separation compared to traditional liquid chromatography.
High Performance Liquid chromatography (HPLC)Unnati Garg
The document provides an overview of high performance liquid chromatography (HPLC). It describes key components of HPLC systems including pumps to deliver solvent at stable flow rates, columns for molecular separation, and detectors for recognizing analytes. The separation principle is based on the distribution of analytes between a mobile liquid phase and stationary column packing material. Different constituents are eluted at different times, achieving separation. HPLC is widely used in pharmaceutical applications such as drug development, production quality control, and stability testing.
This is a PPT for HPLC which I made for presenting my assigned topic for Practice School during 7 sem of my graduation . Hope it is useful for you guys :)
This document provides an overview of high performance liquid chromatography (HPLC). It discusses the key components of an HPLC system including the solvent rack, pump, injector, separation column, and detector. It also describes different chromatography techniques such as normal phase chromatography, reversed phase chromatography, and ion exchange chromatography which separate compounds based on polarity, electrical charge, and molecular size. Finally, it discusses factors that influence separation such as stationary phase particle size and eluent composition.
Stationary Phase and Mobile Phase Selection for Liquid Chromatography
The presentation focuses on how to choose the appropriate mode of separation, the correct column and highlights the importance of the correct mobile phase. This approach will be applied to a wide selection of compound types ranging from proteins, peptides, glycans to small pharmaceutical molecules and their metabolites. It will also look at specific application areas for monoclonal antibody analysis, namely: titer, aggregation, charge and oxidation variant. Platform methods for biologics characterization are also discussed.
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.
This document provides an overview of high performance liquid chromatography (HPLC). It discusses the history and development of chromatography. The major components of an HPLC system are described, including pumps, detectors, columns, and recorders. The main separation modes are explained, including reversed-phase, normal-phase, ion exchange, and size exclusion chromatography. Parameters for method development and validation are outlined. Applications of HPLC in qualitative and quantitative analysis are also summarized.
The document discusses various aspects of high performance liquid chromatography (HPLC), including the types of separations, columns and stationary phases, mobile phases, injection, and detection methods. It provides details on normal phase, reversed phase, adsorption, and size exclusion chromatography. The roles of the stationary phase, mobile phase, column, and detector are explained. Common detection techniques like UV-VIS, diode array detection, refractive index, and evaporative light scattering are outlined.
High performance liquid chromatography (HPLC) is a technique used to separate compounds based on differences in their interactions with a stationary phase. There are various modes of separation including ion exchange, size exclusion, hydrophobic interaction, and affinity chromatography. The choice of technique depends on the physicochemical properties of the compounds being separated. Reversed-phase HPLC, which uses a non-polar stationary phase and polar mobile phase, is the most commonly used mode, allowing separation based on a compound's relative polarity.
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.
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.
High-performance liquid chromatography (HPLC) is an analytical chemistry technique used to separate, identify, and quantify components in mixtures. It works by forcing a pressurized liquid solvent through a column packed with adsorbent particles under high pressure. This allows for better separation than traditional column chromatography due to smaller particle sizes and detection methods. HPLC has applications in manufacturing, legal, research, and medical fields such as drug analysis, food testing, and pharmaceutical development.
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).
This document discusses liquid chromatography (LC) techniques. It begins with an introduction to LC, noting its advantages over gas chromatography including its ability to separate thermally labile compounds and greater flexibility in optimizing separations. The document then discusses different types of LC, including low-performance LC using large particle columns, and high-performance LC using smaller particle columns. It also discusses isocratic and gradient elution techniques. Finally, it discusses different retention mechanisms for LC including adsorption, partition, ion-exchange, affinity, and size-exclusion chromatography.
HPLC (HIGH PERFOMANCE LIQUID CHROMATOGRAPHY)RAJA K
The document discusses high performance liquid chromatography (HPLC). It introduces HPLC and provides details on the types of HPLC techniques, operating principle, instrumentation including pumps, columns, detectors, and parameters used in HPLC like retention time. The advantages of HPLC are that it allows for fast, efficient separations of complex mixtures and accurate quantitative analysis using a variety of column types and mobile phases.
This document provides an overview of high performance liquid chromatography (HPLC). It discusses the basic principles of chromatography and how HPLC works to separate compounds. HPLC uses a stationary phase and mobile phase to separate samples based on properties like polarity. Different separation modes are used like normal phase, reversed phase, ion exchange, size exclusion, and affinity chromatography. Instrumentation includes the column, detector, pump, injection port, and auto-injector. Various detectors can be used like UV/Vis detectors and photo diode array detectors. HPLC provides high resolution, sensitivity, repeatability and is useful for analyzing small samples and purifying compounds.
Liquid chromatography is a technique used to separate mixtures based on differential interactions between components and a stationary phase and mobile phase. It works by pumping a mobile liquid or gas phase through a column containing a stationary solid or liquid phase. The document discusses the history and basic principles of chromatography, different modes (liquid, gas), parameters used to characterize separations, and factors that influence separation efficiency and selectivity.
1) Ion pair chromatography is a type of column chromatography that uses ion pairing agents to neutralize charged analytes and allow their separation on a reversed-phase column.
2) By adding counter ions with the opposite charge to the mobile phase, ion pairs form between the counter ions and analytes, neutralizing their charge and increasing their hydrophobicity.
3) The use of ion-pairing reagents as mobile phase additives allows the separation of ionic and highly polar substances that cannot otherwise be separated by reversed-phase chromatography.
High Performance Liquid Chromatography HPLC is a process of separating components in a liquid mixture. A liquid sample is injected into a stream of solvent mobile phase flowing through a column packed with a separation medium stationary phase . Sample components separate from one another by a process of differential migration as they flow through the column.As bands emerge from the column, flow carries them to one or more detectors which deliver a voltage response as a function of time. This is called a chromatogram. For each peak, the time at which it emerges identifies the sample constituent with respect to a standard. The peak’s area represents the quantity .HPLC provides a highly specific, reasonably precise, and fairly rapid analytical method for a plethora of complicated samples.This is difficult in detecting compounds. Low sensitivity of some compounds towards the stationary phase in the columns is difficult. Mohd Ali | Panjak Chasta | Dr. Kausal Kishore Chandrul "High Performance Liquid Chromatography (HPLC)" Published in International Journal of Trend in Scientific Research and Development (ijtsrd), ISSN: 2456-6470, Volume-5 | Issue-5 , August 2021, URL: https://www.ijtsrd.com/papers/ijtsrd45146.pdf Paper URL: https://www.ijtsrd.com/pharmacy/other/45146/high-performance-liquid-chromatography-hplc/mohd-ali
HPLC_A practical guide for the beginner users.pdfSherif Taha
This lecture presents an introduction to the beginner user on the usage of high-performance liquid chromatography. The main topics are; selecting a buffer solution, and the stationary & mobile phases.
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.
2. DISCOVERY
M.S. Tswett, 1903, Warsaw
Polytechnic Institute (Separation
of the chlorophylls of green leaves
extract)
Calcium carbonate and non polar eluent
3. HPLC Introduction
G S C G L C
G A S S F C
N P R P I E C
G P C G F C
S E C
C o lu m n
T L C P a p e r
P la n a r
L I Q U I D
C H R O M A T O G R A P H Y
4. HPLC Retention
Adsorption Chromatography (NP, RP, IEX)
− Interactions of the analyte with the adsorbent surface
causing its slower movement compared to the eluent
molecules
Size-Exclusion Chromatography
− Exclusion of the analyte molecules from the
adsorbent pore volume due to their size
− No interactions with the adsorbent surface
5. NORMAL PHASE
1980 2004
30% 5% Silica Gel
47% 44% Silica based bonded phases
• Diol
• Amino
• Cyano
3% 1% Alumina
20% 50% Chiral Bonded Phases
Principle: Adsorption of analytes on the polar, weakly acidic surface of
silica gel.
Stationary Phase.: Silica (pH 2-8), Alumina (pH 2 - 12), Bonded Diol,
and NH2, or CN
Mobile Phase: Non-polar solvents (Hexane, CHCl3)
Applications: Non-polar and semi-polar samples; hexane soluble;
positional isomers.
6. NP: SEPARATION PRINCIPLE
Polar (specific but nonionic) interactions of analyte with polar
adsorption sites (SiOH, -NH2, -CN, Diol) cause its retention
Different sorption affinities between analytes result in their
separation
− More polar analytes retained longer
− Analytes with larger number of polar functional group are
retained longer
− Structural isomers are often separated
7. Reversed-Phase HPLC
Principle: Partition of analytes between mobile phase and stagnant phase inside the
pore space + adsorption on the surface of bonded phase.
Stationary Phase: Hydrophobic surfaces of moieties bonded on silica (C18, C8,
C5, Phenyl, CN)
Mobile phase: Methanol or Acetonitrile and Water.
Applications: ~80% of all separations performed with RP HPLC.
80% Octadecylsilica (ODS,
C18)
10% Octylsilica (C8)
5% Butylsilica (C4)
3% Phenyl
2% Cyano (CN)
8. REVERSED PHASE
SEPARATION PRINCIPLE
Nonpolar (nonspecific) interactions of analyte with hydrophobic
adsorbent surface (-C18, C8, Phenyl, C4)
Different sorption affinities between analytes results in their
separation
− More polar analytes retained less
− Analytes with larger hydrophobic part are retained
longer
− structural isomers maybe more challenging in this mode
9. Reversed-Phase vs. Normal Phase
Nonspecific (hydrophobic) interactions are at least ten times weaker than
polar
− small differences in component molecular structure could have a
significant effect in their retention
0
5
10
15
20
25
0 1 2 3 4 5 6 7 8 9 10
0
1
2
3
4
5
6
7
8
0 1 2 3 4 5 6 7 8 9 10
0
5
10
15
20
25
0 1 2 3 4 5 6 7 8 9 10
0
1
2
3
4
5
6
7
8
9
10
0 1 2 3 4 5 6 7 8 9 10
Separation of 2-Me-Phenol and 4-Me-Phenol in RP and NP
Reversed-Phase
MeOH/Water, Luna-C18
Normal Phase
Hexane/IPA, Luna-Si
90/10
60/40
95/5
98/2
10. R1
CH3
R2
OH
O
CH3
CH3
CH3CH3CH3CH3
CH3
Tocopherols
O CH3
CH3
CH3CH3
CH3
OH
CH3
R1
R2
Tocopherols Tocotrienols R1
R
α-Tocopherol (α-T) α-Tocotrienol (α-3)_ Me Me
β-Tocopherol (β-T) β-Tocotrienol (β-3) Me H
-Tocopherol (γ-T) γ-Tocotrienol (γ-3) H Me
-Tocopherol (δ-T) δ-Tocotrienol (δ-3) H H
Tocotrienols
Genesis silica column (250 x 4.6 mm, 4 µm).
Mobile phase: Hexane-1,4-dioxane (96:4).
. J. Chromatogr. A, 881 (2000) 217-227
Separation of synthetic tocopherols by reversed phase
HPLC (280 nm) :1) d-tocopherol, 2) g-tocopherol,
3) b-tocopherol, 4) a-tocopherol, 5)a-tocopheryl acetate
Food Chemistry, 76 (2002) 357 – 362.
Experimental Comparison of NP and RP HPLC
11. Ion Exchange
Principle: Reversible adsorption of ions on S.P. with oppositely charged functional
groups.
Anionic polymers are known as cation exchange resins and these
resins can be strong or weak cation exchange resins which are
strongly dependent upon the anionic group that is bonded to
the polymer.
Cationic polymers on the other hand are known as anion exchange
resins and these resins can also be weak or strong anion
exchange.
Stationary Phase:
• For cations (cation exchange) - SO3
- (strong)
, CO2
- (weak)
• For anions (anion exchange)- NR4
+ (strong)
, NH3
+ (weak)
Mobile Phase: Aqueous buffer with pH and buffer strength carefully controlled.
Applications: All ionic compounds common anions, cations, sugars, amines, etc.
Anionic polymer
Cationic polymer
12. Size Exclusion
Principle: Internal pores of stationary phase exclude analytes molecules based on their
hydrodynamic volume. Vr is correlated to M.W. by calibration curve.
Stationary Phase: Porous polymeric particles (SDVB) with pore diameters of 80, 100, 150,
300, 500 or 1000 Å.
Mobile Phase: Good solvent for polymer. Solvent must suppress all possible interactions with
the stationary phase surface.
Applications: Organic polymers, biopolymers.
13. Step 2: Determine the optimal w
wpH range of the aqueous portion of the mobile phase
by running one linear gradient with 6 different w
wpH mobile phases (2 – 10.8) using an
automated pH screening approach. (AutoChrom Wave 1)
Step 2a Multiple columns can be screened with a pH value within the optimal w
wpH range using
same linear gradient. Select column that gives the best selectivity, check for co-eluting components.
(AutoChrom Wave 2). Do Stopped Flow study with best column/pH.
Step 3*: Gradient scouting studies (shallow/steep x 2 temps) with the
optimal w
wpH (suitable buffer) of the aqueous phase.
(Autochrom Wave 3) Use crude and forced degraded samples.
Check for coeluting components
Step 1: Analyze the molecule:
Physicochemical properties:
solubility, pKa, UV spectra, log P, log D.
α Acceptable
Step 5: Verification run.
Check peak purity (MS and DAD)
α Unacceptable
α Acceptable
Step 4b: Screen additional
columns/ mobile phases with the
optimal w
wpH of the aqueous
phase; use column switcher
Step 4: Use Drylab or ACD (LC-Sim) to
determine the desired resolution/selectivity.
Finished
Optimize Speed:
Scale flow rate/gradient time
Not spectrally
homogeneous
Spectrally
homogeneous
*Choosing the buffer: Wavelength
considerations
*Choosing the Diluent: Solution
stability/solubility
Considerations-
15. PART I HPLC THEORY AND PRACTICE.
1 Introduction (Yuri Kazakevich and Rosario LoBrutto).
2 HPLC Theory (Yuri Kazakevich).
3 Stationary Phases (Yuri Kazakevich and Rosario LoBrutto).
4 Reversed-Phase HPLC (Rosario LoBrutto and Yuri Kazakevich).
5 Normal-Phase HPLC (Yong Liu and Anant Vailaya).
6 Size-Exclusion Chromatography (Yuri Kazakevich and Rosario LoBrutto).
7 LC/MS: Theory, Instrumentation, and Applications to Small Molecules
(Guodong Chen, Li-Kang Zhang, and Birendra N. Pramanik).
8 Method Development (Rosario LoBrutto). .
9 Method Validation (Rosario LoBrutto and Tarun Patel). .
10 Computer-Assisted HPLC and Knowledge Management
(Yuri Kazakevich, Michael McBrien, and Rosario LoBrutto).
PART II HPLC IN THE PHARMACEUTICAL INDUSTRY.
11 The Expanding Role of HPLC in Drug Discovery (Daniel B. Kassel).
12 Role of HPLC in Preformulation (Irina Kazakevich).
13 The Role of Liquid Chromatography–Mass Spectrometry in Pharmacokinetics and
Drug Metabolism
(Ray Bakhtiar, Tapan K. Majumdar, and Francis L. S. Tse).
14 Role of HPLC in Process Development
(Richard Thompson and Rosario LoBrutto). .
15 Role of HPLC During Formulation Development
(Tarun S. Patel and Rosario LoBrutto).
16 The Role of HPLC in Technical Transfer and Manufacturing (Joseph Etse).
PART III HYPHENATED TECHNIQUES AND SPECIALIZED HPLC SEPARATIONS.
17 Development of Fast HPLC Methods (Anton D. Jerkovich and Richard V. Vivilecchia).
18 Temperature as a Variable in Pharmaceutical Applications (Roger M. Smith).
19 LC/MS Analysis of Proteins and Peptides in Drug Discovery
(Guodong Chen, Yan-Hui Liu, and Birendra N. Pramanik).
20 LC-NMR Overview and Pharmaceutical Applications (Maria Victoria Silva Elipe).
21 Trends in Preparative HPLC (Ernst Kuesters).
22 Chiral Separations (Nelu Grinberg, Thomas Burakowski, and Apryll M. Stalcup).
16. •LoBrutto,R.*, Kazakevich, Y.V.* “Chaotropic effects in RP-HPLC” (Invited Review) for volume 44 of “The
Advances in Chromatography” series, editors, Professor Eli Grushka and Nelu Grinberg (September 2005).
•LoBrutto, R. Normal Phase Stationary Phases (Encyclopedia chapter), Cazes, J., Editor, "Encyclopedia of
Chromatography", New York, Marcel Dekker, 553-556 (2001).
•Grinberg, N., LoBrutto, R. Efficiency in Chromatography, (Encyclopedia chapter), Cazes, J., Editor,
"Encyclopedia of Chromatography", New York, Marcel Dekker, 274-276 (2001).
•LoBrutto, R., Kazakevich, Y.V. Retention of Ionizable Components in Reversed Phase HPLC (Book Chapter),
Practical Problem Solving in HPLC, Wiley-VCH, 122-158 (2000).
•Jerkovich, A.D*, LoBrutto,R., and Vivilecchia, R.V., The Use of Acquity UPLC™ in Pharmaceutical
Development, published in LC-GC,( 2005 )
•Chan, F, Yeung, L.S, LoBrutto,R*, Kazakevich,Y*. Characterization of phenyl-type HPLC adsorbents • Journal
of Chromatography A, 1069, Issue 2,April 2005, 217-224.
•Kazakevich, Y*., LoBrutto,R* and Vivilecchia, R. Reversed-Phase HPLC Behavior of Chaotropic
Counteranions, Journal of Chromatography, 1064, 9-18, (2005)
•Pan, L, LoBrutto, R.*, Kazakevich, Y.*, and Thompson, R. Influence of inorganic mobile phase additives on the
retention, efficiency, and peak symmetry of protonated basic compounds in reversed phase liquid chromatography,
Journal of Chromatography A, 1049, 63 -73 (2004).
•Jones, A., LoBrutto, R*., Kazakevich, Y.V.* Effect of the counter-anion type and concentration on the HPLC
retention of beta-blockers, Journal of Chromatography A, 964, 179-187 (2002).
•Rustamov, I., Farcas, T., Ahmed, F., Chan, F., LoBrutto, R., McNair, H.M,. Kazakevich, Y.V. Geometry of
Chemically Modified Silica, Journal of Chromatography A, 913, 49 – 63 (2001).
•Kazakevich, Y.V., LoBrutto, R., Chan, F., Patel, T. Interpretation of the excess adsorption isotherms of organic
eluent components on the surface of reversed phase adsorbents: Effect on the analyte retention, Journal of
Chromatography A, 913, 75-87 (2001).
•LoBrutto, R., Jones, A., Kazakevich, Y.V. Effect of counter-anion concentration on HPLC retention of
protonated basic analytes, Journal of Chromatography A, 913, 189 – 196 (2001).
•LoBrutto, R., Jones, A., Kazakevich, Y.V., McNair, H.M. Effect of the Eluent pH and Acidic Modifiers on the
HPLC Retention of Basic Analytes, Journal of Chromatography A, 913, 173-187 (2001).
HPLC References