HPLC and FPLC are types of chromatography that separate compounds using high pressure to force a mobile liquid phase through a column packed with a stationary phase. The key principles are that sample components are carried by the mobile phase and interact differently with the stationary phase based on their properties, becoming separated as they move through the column at different speeds. HPLC uses smaller particle sizes (<10μm) and higher pressures (500-6000 psi) than column chromatography to achieve better separation over shorter time periods.
HPLC[ HIGH PERPROMANCE LIQUID CHROMATOGRAPHY OR HIGH PRESSURE LIQUID CHROMAT...Dr. Ravi Sankar
GASSCHROMATOGRAPHY[GC], ADVANCED STUDY OF THE FOLLOWING AND THEIR APPLICATIONS, INTRODUCTION, THEORY, COLUMN OPERATION,INSTRUMENTATION AND DETECTION,APPLICATIONS AND ADVANTAGES OF GC,PRINCIPLE OF SEPARATION IN GC, HOW GC MECHINE WORKS? COLUMN, DETECTORS.
BY P.RAVISANKAR, VIGNAN PHARMACY COLLEGE, VADLAMUDI, GUNTUR, A.P, INDIA.
HPLC- Introduction, Theory, Instrumentation, Advantage, Applications
High-performance liquid chromatography or commonly known as HPLC, is an analytical technique used to separate, identify or quantify each component in a mixture.
The mixture is separated using the basic principle of column chromatography and then identified and quantified by spectroscopy.
In the 1960s, the column chromatography LC with its low-pressure suitable glass columns was further developed to the HPLC with its high-pressure adapted metal columns.
HPLC is thus basically a highly improved form of column liquid chromatography. Instead of a solvent being allowed to drip through a column under gravity, Solvent is forced through under high pressures of up to 400 atmospheres.
Principle
The separation principle of HPLC is based on the distribution of the analyte (sample) between a mobile phase (eluent) and a stationary phase (packing material of the column). Depending on the chemical structure of the analyte, the molecules are retarded while passing the stationary phase.
Instrumentation
1.Solvent reservoir and degassing system
2. Pumping System (Screw- driven syringe pump, Reciprocating pump, Pneumatic or constant- pressure pump)
3. Sample injection system(Septum injectors, Stop flow septum- less injection, Micro- volume sampling valves)
4. Columns- (1. Guard columns 2.separating column)
5. Detectors( The commonly used detectors in HPLC are
Bulk property detectors- examples
1. Refractive-index detectors
2. Conductivity detectors
Solute property detectors- Examples
1. UV detectors
2. Fluorescence detectors
Multipurpose detectors- Example-
1. Perkin-Elmer 3D system (UV absorption+ fluorescence + conductometric detection altogethers)
Electrochemical Detectors- Examples
1.Amperometric, 2. Coulometric detectors)
6. Recorder( There are various types of data processors; from a simple system consisting of the in-built printer and word processor while those with software that are specifically designed for an LC system which not only data acquisition but features, like peak-fitting baseline correction, automatic concentration calculation, molecular weight determination, etc.) Type of HPLC- Normal phase, Reverse Phase, ion exchange, size exclusion, Applications-Stability study- eg Atropin
Bioassays- HPLC is commonly used for the bioassay and analysis of peptide harmones and some antibiotics- cotrimoxazole, penicillins, sulphates and chloramphenicol
In cosmetic industries- used for analyzing the quality of various cosmetic products such as lipsticks, gels, creams etc
Isolation of Natural pharmaceutically Active Compounds– use in the isolation of different type of Alkaloids and Glycosides ( analysis of cinchona, liquorice, ergot extracts and digitalis.)
Control of microbiological processes- HPLC is used in analyse antibiotics (eg. Tetracyclines, chloramphenicol, strptomycin and penicillins )
Assay of cephalosporins
Advantage, Limitation
This document provides information about Ahmad Aqel Ifseisi, an assistant professor of analytical chemistry at King Saud University in Saudi Arabia, including his contact information. It then provides an overview of high performance liquid chromatography (HPLC), including descriptions of its components like the column, pump, injector, detector, and various modes of separation. The document discusses advantages and disadvantages of HPLC compared to gas chromatography and provides details on optimization of the mobile phase and properties of common mobile phases.
This document provides information about High Performance Liquid Chromatography (HPLC) and Gas Chromatography. It discusses the basic principles, instrumentation, and applications of HPLC, including the types of columns, mobile phases, pumps, injectors, detectors, and data acquisition systems used. It also summarizes the basic principles of Gas Chromatography, where a gas mobile phase is used to separate components of a vaporized sample based on interactions with a stationary phase. Key applications of HPLC mentioned include pharmaceutical analysis, environmental monitoring, clinical analysis, and food and flavor analysis.
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.
HPLC is a popular and versatile technique for separating, identifying, and quantifying constituents of complex organic samples. It works by forcing a pressurized mobile phase through a column containing a stationary phase, which interacts differently with different compounds and allows them to be separated. Key components of an HPLC system include the pump, injector, column, detector, and data analysis software. HPLC can separate compounds based on differences in how strongly they interact with the mobile and stationary phases.
HPLC[ HIGH PERPROMANCE LIQUID CHROMATOGRAPHY OR HIGH PRESSURE LIQUID CHROMAT...Dr. Ravi Sankar
GASSCHROMATOGRAPHY[GC], ADVANCED STUDY OF THE FOLLOWING AND THEIR APPLICATIONS, INTRODUCTION, THEORY, COLUMN OPERATION,INSTRUMENTATION AND DETECTION,APPLICATIONS AND ADVANTAGES OF GC,PRINCIPLE OF SEPARATION IN GC, HOW GC MECHINE WORKS? COLUMN, DETECTORS.
BY P.RAVISANKAR, VIGNAN PHARMACY COLLEGE, VADLAMUDI, GUNTUR, A.P, INDIA.
HPLC- Introduction, Theory, Instrumentation, Advantage, Applications
High-performance liquid chromatography or commonly known as HPLC, is an analytical technique used to separate, identify or quantify each component in a mixture.
The mixture is separated using the basic principle of column chromatography and then identified and quantified by spectroscopy.
In the 1960s, the column chromatography LC with its low-pressure suitable glass columns was further developed to the HPLC with its high-pressure adapted metal columns.
HPLC is thus basically a highly improved form of column liquid chromatography. Instead of a solvent being allowed to drip through a column under gravity, Solvent is forced through under high pressures of up to 400 atmospheres.
Principle
The separation principle of HPLC is based on the distribution of the analyte (sample) between a mobile phase (eluent) and a stationary phase (packing material of the column). Depending on the chemical structure of the analyte, the molecules are retarded while passing the stationary phase.
Instrumentation
1.Solvent reservoir and degassing system
2. Pumping System (Screw- driven syringe pump, Reciprocating pump, Pneumatic or constant- pressure pump)
3. Sample injection system(Septum injectors, Stop flow septum- less injection, Micro- volume sampling valves)
4. Columns- (1. Guard columns 2.separating column)
5. Detectors( The commonly used detectors in HPLC are
Bulk property detectors- examples
1. Refractive-index detectors
2. Conductivity detectors
Solute property detectors- Examples
1. UV detectors
2. Fluorescence detectors
Multipurpose detectors- Example-
1. Perkin-Elmer 3D system (UV absorption+ fluorescence + conductometric detection altogethers)
Electrochemical Detectors- Examples
1.Amperometric, 2. Coulometric detectors)
6. Recorder( There are various types of data processors; from a simple system consisting of the in-built printer and word processor while those with software that are specifically designed for an LC system which not only data acquisition but features, like peak-fitting baseline correction, automatic concentration calculation, molecular weight determination, etc.) Type of HPLC- Normal phase, Reverse Phase, ion exchange, size exclusion, Applications-Stability study- eg Atropin
Bioassays- HPLC is commonly used for the bioassay and analysis of peptide harmones and some antibiotics- cotrimoxazole, penicillins, sulphates and chloramphenicol
In cosmetic industries- used for analyzing the quality of various cosmetic products such as lipsticks, gels, creams etc
Isolation of Natural pharmaceutically Active Compounds– use in the isolation of different type of Alkaloids and Glycosides ( analysis of cinchona, liquorice, ergot extracts and digitalis.)
Control of microbiological processes- HPLC is used in analyse antibiotics (eg. Tetracyclines, chloramphenicol, strptomycin and penicillins )
Assay of cephalosporins
Advantage, Limitation
This document provides information about Ahmad Aqel Ifseisi, an assistant professor of analytical chemistry at King Saud University in Saudi Arabia, including his contact information. It then provides an overview of high performance liquid chromatography (HPLC), including descriptions of its components like the column, pump, injector, detector, and various modes of separation. The document discusses advantages and disadvantages of HPLC compared to gas chromatography and provides details on optimization of the mobile phase and properties of common mobile phases.
This document provides information about High Performance Liquid Chromatography (HPLC) and Gas Chromatography. It discusses the basic principles, instrumentation, and applications of HPLC, including the types of columns, mobile phases, pumps, injectors, detectors, and data acquisition systems used. It also summarizes the basic principles of Gas Chromatography, where a gas mobile phase is used to separate components of a vaporized sample based on interactions with a stationary phase. Key applications of HPLC mentioned include pharmaceutical analysis, environmental monitoring, clinical analysis, and food and flavor analysis.
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.
HPLC is a popular and versatile technique for separating, identifying, and quantifying constituents of complex organic samples. It works by forcing a pressurized mobile phase through a column containing a stationary phase, which interacts differently with different compounds and allows them to be separated. Key components of an HPLC system include the pump, injector, column, detector, and data analysis software. HPLC can separate compounds based on differences in how strongly they interact with the mobile and stationary phases.
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 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
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 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.
Content include basic introduction to chromatography. Brief view of Liquid Chromatography. HPLC introduction, other names, types of HPLC, detailed instrumentation with image of each part, and applications. Sources of content described in 'References' entitled slide. This presentation was prepared for the partial fulfillment of Master of Pharmacy.
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.
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.
a brief introduction to countercurrent chromatography with its principle. working and modes of operation along with little bit of history, the types of CCC and its applications
Supercritical fluid chromatography uses a supercritical fluid as the mobile phase to carry samples through a chromatographic column. As analyte bands exit the column, their identities and quantities are determined by a detector. Common supercritical fluids used are gases like carbon dioxide above their critical temperature and pressure, giving them properties between liquids and gases. Separation is based on interaction between analytes and the stationary phase. SFC offers advantages over HPLC like rapid separations without organic solvents and over GC through analysis of thermally labile compounds at lower temperatures. It finds applications in areas like pharmaceuticals, polymers, foods, and enantiomer separations.
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.
High performance liquid chromatography (hplc)Pharm Ajahson
HPLC is a type of column chromatography that uses high pressure to pass a sample mixture in a mobile liquid phase through a column containing a stationary solid phase, allowing the components to separate. It provides high resolution and sensitivity for analyzing chemicals and biological molecules. Key components include a solvent reservoir, pump, injector, column, detector, and recorder. Separation occurs as each component interacts differently with the stationary phase based on properties like polarity and solubility. The detector measures the components as they elute from the column, generating a chromatogram to identify the components. HPLC has many applications in fields like pharmaceuticals, chemicals, foods, biosciences, and more.
HPLC is a liquid chromatography technique used to separate mixtures of compounds. It involves injecting a sample into a column packed with porous particles, through which a liquid mobile phase is pumped under high pressure. The separated components interact differently with the stationary phase and elute from the column at different retention times, producing a chromatogram. HPLC is used for qualitative and quantitative analysis of compounds, identification of unknown mixtures, and preparation of pure samples. It is well-suited for separation of non-volatile and thermally-fragile compounds across various application areas.
High- performance Liquid Chromatography”/
(High- pressure Liquid Chromatography) is a powerful tool in analysis, it yields High Performance and high speed compared to traditional columns chromatography
Chromatography introduction ppt by Akshay patelakshay patel
This document provides a basic introduction to chromatography, including definitions of key terms and descriptions of basic chromatography instrumentation and concepts. It discusses the basic components and functions of a liquid chromatograph, including the eluent delivery system, pumps, injection systems, columns, detectors, and common types of chromatography like adsorption and partition chromatography. It also provides explanations of fundamental chromatography concepts like retention, theoretical plates, rate theory, the van Deemter equation, and resolution.
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.
Chromatography is a technique that separates components of a mixture based on how quickly they travel through a stationary and mobile phase. It was invented in 1903 by Russian botanist Mikhail Tswett who used it to separate plant pigments on a calcium carbonate column. The components form colored bands corresponding to different pigments. Chromatography has many applications and separates components based on differences in how they partition between phases. Common techniques include thin layer chromatography, gas chromatography, high performance liquid chromatography, and paper chromatography.
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.
it describes the bony anatomy including the femoral head , acetabulum, labrum . also discusses the capsule , ligaments . muscle that act on the hip joint and the range of motion are outlined. factors affecting hip joint stability and weight transmission through the joint are summarized.
Physiology and chemistry of skin and pigmentation, hairs, scalp, lips and nail, Cleansing cream, Lotions, Face powders, Face packs, Lipsticks, Bath products, soaps and baby product,
Preparation and standardization of the following : Tonic, Bleaches, Dentifrices and Mouth washes & Tooth Pastes, Cosmetics for Nails.
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 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
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 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.
Content include basic introduction to chromatography. Brief view of Liquid Chromatography. HPLC introduction, other names, types of HPLC, detailed instrumentation with image of each part, and applications. Sources of content described in 'References' entitled slide. This presentation was prepared for the partial fulfillment of Master of Pharmacy.
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.
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.
a brief introduction to countercurrent chromatography with its principle. working and modes of operation along with little bit of history, the types of CCC and its applications
Supercritical fluid chromatography uses a supercritical fluid as the mobile phase to carry samples through a chromatographic column. As analyte bands exit the column, their identities and quantities are determined by a detector. Common supercritical fluids used are gases like carbon dioxide above their critical temperature and pressure, giving them properties between liquids and gases. Separation is based on interaction between analytes and the stationary phase. SFC offers advantages over HPLC like rapid separations without organic solvents and over GC through analysis of thermally labile compounds at lower temperatures. It finds applications in areas like pharmaceuticals, polymers, foods, and enantiomer separations.
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.
High performance liquid chromatography (hplc)Pharm Ajahson
HPLC is a type of column chromatography that uses high pressure to pass a sample mixture in a mobile liquid phase through a column containing a stationary solid phase, allowing the components to separate. It provides high resolution and sensitivity for analyzing chemicals and biological molecules. Key components include a solvent reservoir, pump, injector, column, detector, and recorder. Separation occurs as each component interacts differently with the stationary phase based on properties like polarity and solubility. The detector measures the components as they elute from the column, generating a chromatogram to identify the components. HPLC has many applications in fields like pharmaceuticals, chemicals, foods, biosciences, and more.
HPLC is a liquid chromatography technique used to separate mixtures of compounds. It involves injecting a sample into a column packed with porous particles, through which a liquid mobile phase is pumped under high pressure. The separated components interact differently with the stationary phase and elute from the column at different retention times, producing a chromatogram. HPLC is used for qualitative and quantitative analysis of compounds, identification of unknown mixtures, and preparation of pure samples. It is well-suited for separation of non-volatile and thermally-fragile compounds across various application areas.
High- performance Liquid Chromatography”/
(High- pressure Liquid Chromatography) is a powerful tool in analysis, it yields High Performance and high speed compared to traditional columns chromatography
Chromatography introduction ppt by Akshay patelakshay patel
This document provides a basic introduction to chromatography, including definitions of key terms and descriptions of basic chromatography instrumentation and concepts. It discusses the basic components and functions of a liquid chromatograph, including the eluent delivery system, pumps, injection systems, columns, detectors, and common types of chromatography like adsorption and partition chromatography. It also provides explanations of fundamental chromatography concepts like retention, theoretical plates, rate theory, the van Deemter equation, and resolution.
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.
Chromatography is a technique that separates components of a mixture based on how quickly they travel through a stationary and mobile phase. It was invented in 1903 by Russian botanist Mikhail Tswett who used it to separate plant pigments on a calcium carbonate column. The components form colored bands corresponding to different pigments. Chromatography has many applications and separates components based on differences in how they partition between phases. Common techniques include thin layer chromatography, gas chromatography, high performance liquid chromatography, and paper chromatography.
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.
it describes the bony anatomy including the femoral head , acetabulum, labrum . also discusses the capsule , ligaments . muscle that act on the hip joint and the range of motion are outlined. factors affecting hip joint stability and weight transmission through the joint are summarized.
Physiology and chemistry of skin and pigmentation, hairs, scalp, lips and nail, Cleansing cream, Lotions, Face powders, Face packs, Lipsticks, Bath products, soaps and baby product,
Preparation and standardization of the following : Tonic, Bleaches, Dentifrices and Mouth washes & Tooth Pastes, Cosmetics for Nails.
Strategies for Effective Upskilling is a presentation by Chinwendu Peace in a Your Skill Boost Masterclass organisation by the Excellence Foundation for South Sudan on 08th and 09th June 2024 from 1 PM to 3 PM on each day.
This presentation includes basic of PCOS their pathology and treatment and also Ayurveda correlation of PCOS and Ayurvedic line of treatment mentioned in classics.
A workshop hosted by the South African Journal of Science aimed at postgraduate students and early career researchers with little or no experience in writing and publishing journal articles.
The simplified electron and muon model, Oscillating Spacetime: The Foundation...RitikBhardwaj56
Discover the Simplified Electron and Muon Model: A New Wave-Based Approach to Understanding Particles delves into a groundbreaking theory that presents electrons and muons as rotating soliton waves within oscillating spacetime. Geared towards students, researchers, and science buffs, this book breaks down complex ideas into simple explanations. It covers topics such as electron waves, temporal dynamics, and the implications of this model on particle physics. With clear illustrations and easy-to-follow explanations, readers will gain a new outlook on the universe's fundamental nature.
Main Java[All of the Base Concepts}.docxadhitya5119
This is part 1 of my Java Learning Journey. This Contains Custom methods, classes, constructors, packages, multithreading , try- catch block, finally block and more.
Macroeconomics- Movie Location
This will be used as part of your Personal Professional Portfolio once graded.
Objective:
Prepare a presentation or a paper using research, basic comparative analysis, data organization and application of economic information. You will make an informed assessment of an economic climate outside of the United States to accomplish an entertainment industry objective.
This presentation was provided by Steph Pollock of The American Psychological Association’s Journals Program, and Damita Snow, of The American Society of Civil Engineers (ASCE), for the initial session of NISO's 2024 Training Series "DEIA in the Scholarly Landscape." Session One: 'Setting Expectations: a DEIA Primer,' was held June 6, 2024.
Introduction to AI for Nonprofits with Tapp NetworkTechSoup
Dive into the world of AI! Experts Jon Hill and Tareq Monaur will guide you through AI's role in enhancing nonprofit websites and basic marketing strategies, making it easy to understand and apply.
A Strategic Approach: GenAI in EducationPeter Windle
Artificial Intelligence (AI) technologies such as Generative AI, Image Generators and Large Language Models have had a dramatic impact on teaching, learning and assessment over the past 18 months. The most immediate threat AI posed was to Academic Integrity with Higher Education Institutes (HEIs) focusing their efforts on combating the use of GenAI in assessment. Guidelines were developed for staff and students, policies put in place too. Innovative educators have forged paths in the use of Generative AI for teaching, learning and assessments leading to pockets of transformation springing up across HEIs, often with little or no top-down guidance, support or direction.
This Gasta posits a strategic approach to integrating AI into HEIs to prepare staff, students and the curriculum for an evolving world and workplace. We will highlight the advantages of working with these technologies beyond the realm of teaching, learning and assessment by considering prompt engineering skills, industry impact, curriculum changes, and the need for staff upskilling. In contrast, not engaging strategically with Generative AI poses risks, including falling behind peers, missed opportunities and failing to ensure our graduates remain employable. The rapid evolution of AI technologies necessitates a proactive and strategic approach if we are to remain relevant.
2. Several chromatographic techniques
Even though each method utilizes different
techniques to separate compounds, the
principles are the same.
Common to all:
Stationary phase-
a solid or a liquid supported on a solid
Mobile phase-
A liquid or gas
Chromatography Theory Review
3. As the mobile phases passes through the
stationary phase, it carries the components of
the sample mixture with it.
The components of the sample will be attracted to
the stationary phase, but there will also be a
competing attraction for the mobile phase.
Each component will have its own characteristic
balance of attraction to the mobile/stationary
phase.
So the components will not move at the same
speed and are separated.
Chromatography Theory Review
4. Column Chromatography
Similar to thin layer
chromatography
Stationary phase =
silica gel on support
Mobile phase = liquid solvent
In column chromatography,
this stationary phase is
packed into a vertical glass
column.
Mobile phase moves down
the column as a result of
gravity.
5. Column Chromatography
Blue compound = more polar
Adsorb more to the
silica gel
Elutes slower
Yellow compound =
less polar
Spends much of its
time in the mobile
phase
Elutes faster
Example of column chromatography separation:
6. HPLC Introduction:
HPLC = improved form of column chromatography
Instead of the mobile phase moving through the
column as a result of gravity, it is forced through
the column under high pressure.
Typical operating pressures: 500-6000psi
To get improved separation – smaller sized
packing material is required (<10µm).
Smaller packing = greater resistance to flow
Low flow rate = solute diffusion
Higher pressures needed to generate the needed solvent flow
Gravity is too slow- high pressure greatly speeds up the
procedure.
7. 1903: Russian botanist Mikhail Tswett
Separated plant pigments through
column adsorption chromatography
Packed open glass column with particles
Calcium carbonate and alumina
Poured sample into column, along with
pure solvent
As the sample moved down the vertical
column, different colored bands could
be seen.
Bands correlated to the sample
components.
Coined the term chromatography from
the Latin word meaning “color writing”.
HPLC History
8. Early 1950s: First appearance of GC
Almost immediately became popular.
Work began on improving LC
1964: J. Calvin Giddings
Published a paper entitled “Comparison of the
Theoretical Limit of Separating Ability in Gas and
Liquid Chromatography” in the journal Analytical
Chemistry.
Outlined ways to improve LC: smaller packing size,
increased pressure
In theory, he demonstrated how LC could actually be more
efficient than GC.
Increased number of theoretical plates
HPLC History
9. HPLC History
1966: Horváth
Built the first HPLC instrument and gave it its name
HPLC = High Pressure Liquid Chromatography.
1970’s: HPLC became popular with an increase in
technology
Improved columns and detectors
Production of small silica packing material
By 1972 particle sizes less than 10µm were introduced
This allowed for more precise and rapid separations.
As new technology continued to develop, HPLC
became more efficient.
HPLC = High Performance Liquid Chromatography
10. Overview of the HPLC Process
Mobile phase pumped through column at high pressure.
Sample is injected into the system.
Separation occurs as the mobile phase and sample are
pumped through the column.
Each sample component will elute from the column, one at a
time, and will be detected by one of several possible
detector types.
The response of the detector to each component eluted will
be displayed on a chart or computer screen.
Known as a chromatogram.
Each compound eluted will show up as a peak on this chromatogram.
Data processing equipment are used to analyze the data
generated.
http://www.waters.com/WatersDivision/flash/hplc_primer/hplcsys_primer.html
13. Diagram of HPLC Apparatus:
1.
http://www.cem.msu.edu/~cem333/Week16.pdf
14. Design & Operation of an HPLC
Instrument
1) Mobile phase degassing:
Dissolved gases in the mobile phase can come out of
solution and form bubbles as the pressure changes
from the column entrance to the exit.
May block flow through the system
Sparging is used to remove any dissolved gas from
the mobile phase.
An inert and virtually insoluble gas, such as helium, is forced
into the mobile phase solution and drives out any dissolved
gas.
Degassing may also be achieved by filtering the
mobile phase under a vacuum.
15. Design & Operation of an HPLC
Instrument
2) Solvent reservoirs:
Individual reservoirs store the mobile phase
components until
they are mixed
and used.
May also manually
prepare the mobile
phase mixture and
store in a single
reservoir.
2.
16. 3) Mobile phase mixing:
Solvent proportioning valve can be programmed
to mix specific
amounts of solvent
from the various
reservoirs to
produce the
desired mobile
phase composition.
Design & Operation of an HPLC
Instrument
3.
17. 3) Mobile phase mixing:
Isocratic elution:
operate at a single, constant mobile phase composition
Gradient elution:
Vary the mobile phase composition with time
If there is a wide polarity range of components to be eluted.
Allows for faster runs.
Ex: mobile phase composition can be programmed to vary
from 75% water: 25% acetonitrile at time zero to
25% water: 75% acetonitrile at the end of the run.
More polar components will tend to elute first.
More non-polar components will elute later in the gradient.
Design & Operation of an HPLC
Instrument
18. Design & Operation of an HPLC
Instrument
4) HPLC pump:
Fill stroke: mobile phase is pulled from the
solvent side
Exhaust stroke:
the mobile phase
is pushed from the
injector to the
column head.
This is where the
high pressure is
generated
4.
19. 4) HPLC pump:
Most common =
reciprocating piston type
Flow rates change during
pumping cycle
Want to minimize flow surges
Pulse dampener
Dual pistons
While one piston fills, the other delivers
Design & Operation of an HPLC
Instrument
http://www.lcresources.com/resources/getstart/2b01.htm
20. 5) Injector:
Introduces the sample into the mobile phase stream to
be carried into the
column.
Syringe = impractical
for use in highly
pressurized systems.
Rotary injection
valve is used.
For more
information visit:
http://www.lcresources.com/resources/getstart/2c01.htm
Design & Operation of an HPLC
Instrument
5.
21. 6) Column:
Usually constructed of stainless steel
glass or Tygon may
be used for lower
pressure applications
(<600 psi).
Length: 5-100cm
10 to 20cm common
Diameter:
Typical: 2.1, 3.2, or
4.5mm
Up to 30mm for preparative applications
Design & Operation of an HPLC
Instrument
6.
22. Column packing:
Usually spherical silica particles of
uniform diameter (2-10µm)
The smaller particles yield higher
separation efficiencies.
The silica particles are very porous
Allows for greater surface area for
interactions between the stationary
phase and the analytes.
Other packing materials may also be
used:
Zirconia (ZrO2)
http://www.lcresources.com/resources/getstart/3a01.htm
Design & Operation of an HPLC
Instrument
http://hplc.chem.shu.edu/NEW/HP
LC_Book/Adsorbents/ads_part.html
23. 6) Column:
Guard column: Protects the analytical column
Particles
Interferences
Prolongs the life of the analytical column
Analytical column: Performs the separation
Design & Operation of an HPLC
Instrument
24. 7) Detector:
The component that
emits a response due
to the eluting sample
compound and
subsequently signals
a peak on the
chromatogram.
A wide variety of
detectors exist.
Must have high sensitivity- small sample sizes are used with
most HPLC columns
Design & Operation of an HPLC
Instrument
7.
25. Detection in HPLC
*There are six major HPLC detectors:
Refractive Index (RI) Detector
Evaporative Light Scattering Detector (ELSD)
UV/VIS Absorption Detectors
The Fluorescence Detector
Electrochemical Detectors (ECDs)
Conductivity Detector
* The type of detector
utilized depends on
the characteristics
of the analyte of
interest. http://www.waters.com/WatersDivision/Contentd.asp?watersit=JDRS-6UXGZ4
26. Refractive Index Detector
Based on the principle that every transparent
substance will slow the speed of light passing through
it.
Results in the bending of light
as it passes to another material
of different density.
Refractive index = how much
the light is bent
The presence of analyte molecules
in the mobile phase will generally
change its RI by an amount
almost linearly proportional to its concentrations.
http://farside.ph.utexas.edu/teaching/302l/
lectures/img1154.png
27. Refractive Index Detector
Affected by slight changes in mobile phase
composition and temperature.
Universal-based on a property of the mobile phase
It is used for analytes which give no response with
other more sensitive and selective detectors.
RI = general
responds to the presence of all solutes in the mobile
phase.
Reference= mobile phase
Sample= column effluent
Detector measures the
differences between the RI of
the reference and the sample. http://hplc.chem.shu.edu/HPLC/index.html
28. Analyte particles don’t scatter light
when dissolved in a liquid mobile
phase.
Three steps:
1) Nebulize the mobile phase effuent into
droplets.
Passes through a needle and mixes
with hydrogen gas.
2) Evaporate each of these droplets.
Leaves behind a small particle of
nonvolatile analyte
3) Light scattering
Sample particles pass through a cell
and scatter light from a laser beam
which is detected and generates a
signal.
Evaporative Light Scattering Detector
(ELSD)
http://www.sedere.com/WLD/whatis.html
29. UV/VIS Absorption Detectors
Different compounds will absorb different amounts of
light in the UV and visible regions.
A beam of UV light is shined through the analyte after
it is eluted from the column.
A detector is positioned on the opposite side which
can measure how much light is absorbed and
transmitted.
The amount of light
absorbed will depend on
the amount of the
compound that is passing
through the beam. http://www.chemguide.co.uk/analysis/chromatography/hplc.html
30. UV/VIS Absorption Detectors
Beer-Lambert law: A=εbc
absorbance is proportional to the compound
concentration.
Fixed Wavelength: measures at one wavelength,
usually 254 nm
Variable Wavelength: measures at one wavelength
at a time, but can detect over a wide range of
wavelengths
Diode Array Detector (DAD): measures a spectrum
of wavelengths simultaneously
31. The Fluorescence Detector
Measure the ability of a compound to absorb then
re-emit light at given wavelengths
Some compounds will absorb
specific wavelengths of light which,
raising it to a higher energy state.
When the compound returns to its
ground state, it will release a
specific wavelength of light which
can be detected.
Not all compounds can fluoresce /
more selective than UV/VIS detection.
http://mekentosj.com/science/fret/images/
fluorescence.jpg
32. Electrochemical Detectors (ECDs):
Electrochemical Detectors (ECDs):
Used for compounds that undergo
oxidation/reduction reactions.
Detector measures the current resulting
from an oxidation/reduction reaction of
the analyte at a suitable electrode.
Current level is directly proportional to
the concentration of analyte present.
Conductivity Detector:
Records how the mobile phase
conductivity changes as different sample
components are eluted from the column.
http://hplc.chem.shu.edu/HPLC/index.html
http://hplc.chem.shu.edu/HPLC/index.html
33. Interfacing HPLC to Mass Spectrometry
Mass Spectrometry = an analytical tool used
to measure the molecular mass of a sample.
Measures the mass to
charge ratio
Allows for the definitive
identification of each
sample component.
Most selective HPLC
detector, but also the
most expensive.
http://www.chem.queensu.ca/FACILI
TIES/NMR/nmr/mass-spec/index.htm
34. Picture of a Typical HPLC System
http://www.waters.com/WatersDivision/ContentD.asp?watersit=JDRS-6UXGYA&WT.svl=1
35. Retention Time- tR
The elapsed time between the time of analyte
injection and the time which the maximum peak
height for that compound is detected.
Different compounds will have different retention
times.
Each compound will have its own characteristic balance
of attraction to the mobile/stationary phase.
So they will not move at the same speed.
Running conditions can also effect tR:
Pressure used, nature of the stationary phase, mobile
phase composition, temperature of the column
36. Retention Time- tR
If you are careful to keep the conditions
constant, you may use tR to help you identify
compounds present.
Must have measured tR for the pure compounds
under identical conditions.
37. Determining Concentration
In most cases, sample peaks on the chromatogram
can be used to
estimate the
amount of a
compound
present.
The more
concentrated,
the stronger
the signal, the
larger the
peak. http://www.waters.com/WatersDivision/Contentd.asp?watersit=JDRS-6UXGZ4
38. tR: Retention time
t’R: Adjusted retention time = (tR- Tm)
Tm: Dead time
W0,5: Peak width at half height
h: Height of signal
39. Types of HPLC
There are numerous types of HPLC which
vary in their separation chemistry.
All chromatographic modes are possible:
Ion-exchange
Size exclusion
Also can vary the stationary & mobile
phases:
Normal phase HPLC
Reverse phase HPLC
40. Chromatographic Modes of HPLC
Ion exchange:
Used with ionic or ionizable samples.
Stationary phase has a charged surface.
opposite charge to the sample ions
The mobile phase = aqueous buffer
The stronger the charge on the analyte, the more it will be
attracted to the stationary phase, the slower it will elute.
Size exclusion:
Sample separated based on size.
Stationary phase has specific pore sizes.
Larger molecules elute quickly.
Smaller molecules penetrate inside the pores of the
stationary phase and elute later.
41. Normal Phase HPLC
Stationary phase: polar, silica particles
Mobile phase: non-polar solvent or mixture of
solvents
Polar compounds:
Will have a higher affinity for the polar,
stationary phase
Will elute slower
Non-polar compounds:
Will have a higher affinity for the non-polar,
mobile phase
Will elute faster
42. Reverse Phase HPLC
Stationary phase: non-polar
Non-polar organic groups are covalently attached to the
silica stationary particles.
Most common attachment is a long-chain
n-C18 hydrocarbon
Octadecyl silyl group, ODS
Mobile phase: polar liquid or
mixture of liquids
Polar analytes will spend more time
in the polar mobile phase.
Will elute quicker than non-polar analytes
Most common type of HPLC used today.
http://www.lcresources.com/
resources/getstart/3a01.htm
43. What is Ultra Performance Liquid
Chromatography?
2004: Further advances in column technology
and chromatography instrumentation
Utilized even smaller packing particle sizes (1.7µm)
Higher pressures (15000psi)
Allowed for significant increases in LC speed,
reproducibility, and sensitivity.
New research utilizing particle sizes as small as
1µm and pressures up to 100,000psi!
WHO KNOWS WHAT THE FUTURE MAY BRING!
44. HPLC Applications
Can be used to isolated and purify compounds
for further use.
Can be used to identify the presence of specific
compounds in a sample.
Can be used to determine the concentration of
a specific compound in a sample.
Can be used to perform chemical separations
Enantiomers
Biomolecules
45. HPLC Applications
*HPLC has an vast amount of current & future applications*
Some uses include:
Forensics: analysis of explosives, drugs, fibers, etc.
Proteomics: can be used to separate and purify protein
samples
Can separate & purify other biomolecules such as: carbohydrates,
lipids, nucleic acids, pigments, proteins, steroids
Study of disease: can be used to measure the presence &
abundance of specific biomolecules correlating to disease
manifestation.
Pharmaceutical Research: all areas including early
identification of clinically relevant molecules to large-scale
processing and purification.
46. FPLC- A Modification of HPLC
In 1982 Pharmacia introduced a new
chromatographic method called FPLC.
FPLC = Fast Protein Liquid Chromatography
FPLC is basically a “protein friendly” HPLC system.
Stainless steel components replaced with glass and
plastic.
Stainless steel was thought to denature proteins
Also many ion-exchange separations of proteins involve salt
gradients; thought that these conditions could results in
attack of stainless steel systems.
FPLC can also be used to separate other biologically
active molecules, such as nucleic acid.
47. FPLC- A Modification of HPLC
FPLC is an intermediate between classical
column chromatography and HPLC.
FPLC pump delivers a solvent flow rate in the
range of 1-499ml/hr
HPLC pump= 0.010-10ml/min
FPLC operating pressure: 0-40 bar
HPLC= 1-400bar
classic chromatography= atmospheric pressure
Since lower pressures are used in FPLC than in
HPLC, a wider range of column supports are
possible.
48. Applications of FPLC/HPLC to Proteomics
Protein characterization and measurement is
essential to understanding life at the
molecular level.
The first step in protein analysis is isolation
and purification.
Proteins can be separated by FPLC/HPLC in
various forms.
Reversed phase
Ion exchange
Size exclusion
Affinity
Used to isolate/purify without loss of
biological activity
49. Applications of FPLC/HPLC to Proteomics
Not only can FPLC/HPLC systems be used to
isolate and purify proteins, but they can be
coupled to other instruments for further
analysis.
UV/VIS
Mass Spectrometer
This instrument interfacing can allow for the
determination of:
Protein amino acid sequence
Structural information
Functional information
51. The Impact of HPLC/FPLC
HPLC/FPLC has such widespread application it
is impossible to convey its extensive impact.
Has many advantages in situations were a
nonvolatile or thermally unstable sample must be
separated.
As with many biochemical samples
Great speed and resolution
Resolution = how well solutes are separated
Columns don’t have to be repacked
Adaptable to large-scale, preparative procedures.
52. References
Ashcroft, A.E. An Introduction to Mass Spectrometry. URL:
http://www.astbury.leeds.ac.uk/facil/MStut/mstutorial.htm
. Accessed: July 1, 2007.
Detectors and Detection Limits. URL: http://kerouac.pharm
.uky.edu/ASRG/HPLC/detectors.html. Accessed: July 2,
2007.
Filmore, David; Lesney, Mark S. Performing Under
Pressure: The Rise of HPLC. URL: http://pubs.acs.org/jour
nals/chromatography/chap4.html. Accessed: July 1, 2007.
FPLC??. URL: http://www.lcresources.com/discus/messag
es/5133/2395.html?TuesdayAugust520031156am. Accesed
July2, 2007.
Getting Started in HPLC. URL: http://www.lcresources.co
m/resources/get start/. Accessed: June 22, 2007.
53. References:
High Performance Liquid Chromatography- HPLC. URL:
http://www.chemguide.co.uk/analysis/chromatography/hplc.
html#top. Accessed: June 15, 2007.
High Performance Liquid Chromatography (HPLC)
Primer.URL: http://www.waters.com/WatersDivision/Conten
td.asp?Watersit =JDRS-6UXGZ4. Accessed: June 22, 2007.
Kazakevich, Y; McNair, H.M. Basic Liquid Chromatography.
URL: http://hplc.chem.shu.edu/HPLC/index.html. Accessed:
June 22, 2007.
Boyer, Rodney. Modern Experimental Biochemistry.3rd Ed.
Addison Wesley Longman. San Frisco, CA. 2000. 87-100.
Robertson, James W.; Frame, Eileen M.; Frame, George M.
Undergraduate Instrumental Analysis. Marcel Dekker: New
York, NY. 2005.797-836.