High-performance liquid chromatography (HPLC) is the most widely used type of liquid chromatography. It employs high pressure to pass a liquid mobile phase and sample mixture through a column packed with a solid stationary phase. There are several types of HPLC based on the separation mechanism and stationary phase used, including partition, adsorption, ion exchange, size exclusion, and affinity chromatography. HPLC instrumentation includes pumps to pressurize the mobile phase, injection systems, columns of varying lengths and diameters, and detectors. Common applications involve the separation of pharmaceuticals, proteins, and other biological molecules.
This document provides an overview of refractometry, which is a technique used to measure the refractive index of substances. It discusses Snell's law, which describes how light refracts at the boundary between two materials. The refractive index is a measure of how much light bends when passing from one medium to another, and it can be used to identify substances or measure their purity. Factors like temperature, wavelength, and viscosity affect the refractive index. Common instruments for measuring refractive index include Abbe refractometers and immersion refractometers. Applications include quality control in industries like pharmaceuticals.
The document discusses high pressure liquid chromatography (HPLC). It begins with contact information for the author and provides a brief history of chromatography, including early techniques like paper chromatography. It then defines HPLC as using high pressure to push a liquid mobile phase through a packed column for separation. Key advantages of HPLC are its sensitivity, reproducibility, and suitability for separating nonvolatile compounds. The summary concludes by noting that HPLC systems consist of pumps to deliver the mobile phase at high pressure through an injector, column, and detectors.
HPTLC, or high performance thin layer chromatography, is an enhanced form of TLC that allows for more accurate quantitative measurements and higher resolution separation of mixtures compared to traditional TLC. Key features of HPTLC include use of pre-coated plates with smaller particle sizes, semi-automated or automatic sample application, shorter development times, and scanning densitometry for quantification and fingerprint analysis. The process involves selection of plates and mobile phase, sample preparation and application, chromatographic development and detection, followed by scanning and documentation.
Ion pair chromatography for pharmacy studentsabhishek rai
Ion-PairChromatography
A GENERALISED OVERVIEW
Chromatography
HPLC
Reverse Phase Chromatography
Ion Pair Chromatography
Ion Pair Reagent
Mechanism of Ion Pair Chromatography
Ion Pair Wash Procedure
Flame photometry is a technique that uses the characteristic colors emitted from flames to determine the presence of certain metal ions. When metal salts are aspirated into a flame, the metals are excited and emit light at characteristic wavelengths. The intensity of the emitted light is proportional to the concentration of the metal ion in solution. Common metal ions that can be analyzed using flame photometry include sodium, potassium, lithium, and calcium.
This document provides an overview of gas chromatography. It begins with an introduction to chromatography and lists some common chromatographic techniques. It then describes the basic components and working of gas chromatography, including the carrier gas, columns, temperature control, detectors, and how the chromatographic process separates components based on partitioning between a mobile and stationary phase. The principle of gas chromatography is described as partition, with examples of different types of columns and factors that influence chromatographic separation. The key components of a gas chromatography system and their functions are also summarized.
Ion exchange chromatography is a process that separates ions and polar molecules based on their charge using an ion exchange resin. There are two main types of ion exchange - cation exchange which uses a negatively charged resin to adsorb positively charged proteins, and anion exchange which uses a positively charged resin to adsorb negatively charged proteins. The process involves equilibrating the resin, applying the sample mixture, then eluting the bound molecules by altering conditions such as pH or ionic strength to cause differential elution. Ion exchange chromatography is useful for purifying proteins and other charged biomolecules.
This document provides an overview of refractometry, which is a technique used to measure the refractive index of substances. It discusses Snell's law, which describes how light refracts at the boundary between two materials. The refractive index is a measure of how much light bends when passing from one medium to another, and it can be used to identify substances or measure their purity. Factors like temperature, wavelength, and viscosity affect the refractive index. Common instruments for measuring refractive index include Abbe refractometers and immersion refractometers. Applications include quality control in industries like pharmaceuticals.
The document discusses high pressure liquid chromatography (HPLC). It begins with contact information for the author and provides a brief history of chromatography, including early techniques like paper chromatography. It then defines HPLC as using high pressure to push a liquid mobile phase through a packed column for separation. Key advantages of HPLC are its sensitivity, reproducibility, and suitability for separating nonvolatile compounds. The summary concludes by noting that HPLC systems consist of pumps to deliver the mobile phase at high pressure through an injector, column, and detectors.
HPTLC, or high performance thin layer chromatography, is an enhanced form of TLC that allows for more accurate quantitative measurements and higher resolution separation of mixtures compared to traditional TLC. Key features of HPTLC include use of pre-coated plates with smaller particle sizes, semi-automated or automatic sample application, shorter development times, and scanning densitometry for quantification and fingerprint analysis. The process involves selection of plates and mobile phase, sample preparation and application, chromatographic development and detection, followed by scanning and documentation.
Ion pair chromatography for pharmacy studentsabhishek rai
Ion-PairChromatography
A GENERALISED OVERVIEW
Chromatography
HPLC
Reverse Phase Chromatography
Ion Pair Chromatography
Ion Pair Reagent
Mechanism of Ion Pair Chromatography
Ion Pair Wash Procedure
Flame photometry is a technique that uses the characteristic colors emitted from flames to determine the presence of certain metal ions. When metal salts are aspirated into a flame, the metals are excited and emit light at characteristic wavelengths. The intensity of the emitted light is proportional to the concentration of the metal ion in solution. Common metal ions that can be analyzed using flame photometry include sodium, potassium, lithium, and calcium.
This document provides an overview of gas chromatography. It begins with an introduction to chromatography and lists some common chromatographic techniques. It then describes the basic components and working of gas chromatography, including the carrier gas, columns, temperature control, detectors, and how the chromatographic process separates components based on partitioning between a mobile and stationary phase. The principle of gas chromatography is described as partition, with examples of different types of columns and factors that influence chromatographic separation. The key components of a gas chromatography system and their functions are also summarized.
Ion exchange chromatography is a process that separates ions and polar molecules based on their charge using an ion exchange resin. There are two main types of ion exchange - cation exchange which uses a negatively charged resin to adsorb positively charged proteins, and anion exchange which uses a positively charged resin to adsorb negatively charged proteins. The process involves equilibrating the resin, applying the sample mixture, then eluting the bound molecules by altering conditions such as pH or ionic strength to cause differential elution. Ion exchange chromatography is useful for purifying proteins and other charged biomolecules.
Ion exchange chromatography uses ion exchange resins to separate ionic compounds based on competition for binding sites on the resin. Cation exchange resins contain negatively charged functional groups like sulfonic acid that bind positively charged cations from solution. Anion exchange resins contain positively charged functional groups like quaternary ammonium that bind negatively charged anions. Eluting solutions with varying salt concentrations are used to displace bound ions from the resin selectively. Ion exchange chromatography finds application in water softening and analysis of ions in environmental and biological samples.
High Performance Liquid Chromatography (HPLC) is described. HPLC uses high pressure to force a mobile phase through a column at a fast rate, increasing resolution. It discusses the types of chromatography used in HPLC, including normal phase, reverse phase, ion-exchange, and size-exclusion. The instrumentation of HPLC is also summarized, including components like the pump, mixing unit, degasser, injector, column, and detector.
Ion-Pair chromatography is an alternative to ion exchange chromatography.
ion pair reagent.
Mechanism of ion pair chromatography.
Factors influencing retention.
experimental conditions.
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, chromatography column, detectors, and data collection system. It provides details on how each component works and its purpose. Overall, the document serves as a comprehensive introduction to HPLC principles, methodology, and instrumentation.
Column chromatography is a separation technique that uses a column packed with a stationary phase. When a mixture is passed through the column via a mobile phase, the components separate as they interact differently with the stationary phase. The document discusses the principles, common terminology, types including adsorption and partition chromatography, practical steps like sample application and detection, and applications in separating mixtures like dyes, alkaloids, and purifying compounds. Advantages are the ability to separate many types of mixtures while disadvantages include being time-consuming and using large amounts of solvents.
chromatography, principle, adsorbent of TLC, mobile phase of TLC, techniques in TLC, preparation of TLC plate, standards for TLC, advantages, disadvantages of TLC, Application of TLC.
This document discusses the principles and instrumentation of high performance thin layer chromatography coupled with mass spectrometry (HPTLC-MS). HPTLC-MS combines the separation capabilities of HPTLC with the identification abilities of mass spectrometry. It works by separating compounds via HPTLC and then using an interface to extract zones of interest from the HPTLC plate and transfer them to a mass spectrometer for analysis. This allows for structural confirmation and elucidation of targeted analytes. Some advantages of HPTLC-MS are that it is cost-effective, allows identification of unknown substances, and is useful for applications like drug analysis, metabolism studies, and characterization of impurities.
Sample introduction techniques in gas chromatographyVrushali Tambe
This document discusses various techniques used for sample introduction in gas chromatography. It describes 12 different techniques: direct injection, flash vaporizer, split and splitless injection, rotary valve injector, pyrolysis, purge and trap, Grob injection, head space sampling, direct thermal extraction, solid phase microextraction, and autosampler. For each technique, it provides details on the methodology and applications.
This document discusses supercritical fluid chromatography (SFC). SFC uses supercritical fluids like carbon dioxide as the mobile phase. Carbon dioxide is most widely used as it is non-toxic, inexpensive, and has a critical temperature and pressure that are easily reached. SFC works on the principles of adsorption and partition chromatography. It can be used to analyze and purify low to moderate weight compounds, including chiral separations. SFC instrumentation includes pumps to deliver the mobile phase, an oven for temperature control, various injectors, columns, a backpressure regulator, and detectors. SFC finds applications in fields like pharmaceuticals and has advantages over HPLC like using less toxic solvents.
Analytical chemistry involves separating, identifying, and quantifying components of matter. Hyphenated techniques combine two analytical methods, such as gas chromatography coupled with mass spectrometry (GC-MS). GC-MS separates chemical mixtures using gas chromatography and then identifies components using mass spectrometry. Other common hyphenated techniques include liquid chromatography-mass spectrometry (LC-MS) and gas chromatography-infrared spectroscopy (GC-IR). These coupled techniques provide enhanced sensitivity and accuracy for analyzing organic compounds, pollutants, drugs, proteins, and more.
Infrared spectroscopy is a technique that analyzes infrared light absorbed by a molecule to determine its structure. There are several types of molecular vibrations that can be observed, including stretching and bending vibrations. Samples can be analyzed in solid, liquid, or gas form using different sample handling methods. The main components of an IR spectrometer are the radiation source, monochromator, sample cell, detector, and recorder. Dispersive and Fourier transform IR spectrometers are two common instrument types, with Fourier transform having advantages like faster scanning. Functional groups can be identified by their characteristic absorption bands. Factors like coupling, hydrogen bonding, and electronic effects can influence vibrational frequencies.
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.
Gas chromatography-mass spectrometry (GC-MS) is a hyphenated technique that combines gas chromatography and mass spectrometry. GC is used to separate compounds in a mixture, while MS identifies the compounds based on their mass-to-charge ratios. The document discusses the basic principles, instrumentation, and applications of GC-MS. It explains how the gas chromatograph separates compounds and the mass spectrometer ionizes and detects them, providing both separation and identification capabilities in a single technique.
This document provides an overview of gas chromatography (GC). It describes the basic components and principles of how GC works, including the carrier gas, injection port, separation column in an oven, and detector. It explains that GC separates volatile organic compounds based on how they partition between the mobile gas phase and stationary liquid phase. The document also outlines different types of columns, sample preparation procedures, common detectors like the flame ionization detector, and applications of GC in fields like environmental analysis and refineries.
Phosphorescence involves the emission of light from electronically excited triplet states in a material. It is a spin-forbidden process that results in longer-lived emission compared to fluorescence. Phosphorescence occurs when electrons in the excited triplet state relax to the ground singlet state. Instrumentation for measuring phosphorescence requires cryogenic temperatures to reduce thermal quenching, as well as a phosphoroscope to separate the longer-lived phosphorescence from short-lived fluorescence. Applications of phosphorescence include security markers, toys, watches, and switches that glow in the dark.
This document discusses factors that affect fluorimetry and quenching. It lists several factors that can influence fluorescence, including the nature of molecules, substituents, concentration, adsorption, light, oxygen, pH, temperature, and viscosity. It also describes different types of quenching such as self-quenching, chemical quenching, static quenching, and collisional quenching. Chemical quenching can occur due to changes in pH, presence of oxygen, or heavy metals. Static quenching involves complex formation between the fluorophore and quencher. Collisional quenching occurs through interactions between an excited fluorophore and quencher molecule.
INSTRUMENTAL METHODS OF ANALYSIS, B.PHARM 7TH SEM. AND FOR BSC,MSC CHEMISTRY. This is Geeta prasad kashyap (Asst. Professor), SVITS, Bilaspur (C.G) 495001
Polarography is an electroanalytical technique invented in 1922 by Jaroslav Heyrovsky for which he won the Nobel Prize. It involves measuring the current in a solution under an applied potential using a dropping mercury electrode and a reference electrode such as SCE. Mercury is used as the working electrode due to its wide negative potential range and ability to regenerate its surface. A polarogram is generated by plotting current versus applied potential, showing residual, diffusion, and limiting currents. Polarography can be used for qualitative and quantitative analysis of metals, drugs, and other compounds.
High performance liquid chromatography (HPLC) is a technique that forces a solvent through a column under high pressure to separate samples into their constituent parts. HPLC uses a pump to force a mobile phase through a column containing a stationary phase, and a detector measures the analytes as they elute from the column. There are several types of HPLC that separate samples based on polarity (normal phase), hydrophobic interactions (reverse phase), molecular size (size-exclusion), or ionic charge (ion-exchange). HPLC has many applications in fields like pharmaceuticals, environmental analysis, forensics, food and flavors, and clinical testing.
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) is presented. HPLC is a chromatographic technique used to separate mixtures by using high pressure to force a liquid mobile phase and sample through a column packed with solid stationary phase. Key aspects summarized include:
1. HPLC provides simultaneous analysis, high resolution, sensitivity, repeatability for qualitative and quantitative analysis.
2. It works on principles of adsorption and partition chromatography depending on the stationary phase.
3. Instrumentation includes pumps, injector, analytical column, detector, and recorder/integrator.
4. Parameters like retention time, capacity factor, separation factor, and plate height provide information about sample separation and column efficiency.
Ion exchange chromatography uses ion exchange resins to separate ionic compounds based on competition for binding sites on the resin. Cation exchange resins contain negatively charged functional groups like sulfonic acid that bind positively charged cations from solution. Anion exchange resins contain positively charged functional groups like quaternary ammonium that bind negatively charged anions. Eluting solutions with varying salt concentrations are used to displace bound ions from the resin selectively. Ion exchange chromatography finds application in water softening and analysis of ions in environmental and biological samples.
High Performance Liquid Chromatography (HPLC) is described. HPLC uses high pressure to force a mobile phase through a column at a fast rate, increasing resolution. It discusses the types of chromatography used in HPLC, including normal phase, reverse phase, ion-exchange, and size-exclusion. The instrumentation of HPLC is also summarized, including components like the pump, mixing unit, degasser, injector, column, and detector.
Ion-Pair chromatography is an alternative to ion exchange chromatography.
ion pair reagent.
Mechanism of ion pair chromatography.
Factors influencing retention.
experimental conditions.
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, chromatography column, detectors, and data collection system. It provides details on how each component works and its purpose. Overall, the document serves as a comprehensive introduction to HPLC principles, methodology, and instrumentation.
Column chromatography is a separation technique that uses a column packed with a stationary phase. When a mixture is passed through the column via a mobile phase, the components separate as they interact differently with the stationary phase. The document discusses the principles, common terminology, types including adsorption and partition chromatography, practical steps like sample application and detection, and applications in separating mixtures like dyes, alkaloids, and purifying compounds. Advantages are the ability to separate many types of mixtures while disadvantages include being time-consuming and using large amounts of solvents.
chromatography, principle, adsorbent of TLC, mobile phase of TLC, techniques in TLC, preparation of TLC plate, standards for TLC, advantages, disadvantages of TLC, Application of TLC.
This document discusses the principles and instrumentation of high performance thin layer chromatography coupled with mass spectrometry (HPTLC-MS). HPTLC-MS combines the separation capabilities of HPTLC with the identification abilities of mass spectrometry. It works by separating compounds via HPTLC and then using an interface to extract zones of interest from the HPTLC plate and transfer them to a mass spectrometer for analysis. This allows for structural confirmation and elucidation of targeted analytes. Some advantages of HPTLC-MS are that it is cost-effective, allows identification of unknown substances, and is useful for applications like drug analysis, metabolism studies, and characterization of impurities.
Sample introduction techniques in gas chromatographyVrushali Tambe
This document discusses various techniques used for sample introduction in gas chromatography. It describes 12 different techniques: direct injection, flash vaporizer, split and splitless injection, rotary valve injector, pyrolysis, purge and trap, Grob injection, head space sampling, direct thermal extraction, solid phase microextraction, and autosampler. For each technique, it provides details on the methodology and applications.
This document discusses supercritical fluid chromatography (SFC). SFC uses supercritical fluids like carbon dioxide as the mobile phase. Carbon dioxide is most widely used as it is non-toxic, inexpensive, and has a critical temperature and pressure that are easily reached. SFC works on the principles of adsorption and partition chromatography. It can be used to analyze and purify low to moderate weight compounds, including chiral separations. SFC instrumentation includes pumps to deliver the mobile phase, an oven for temperature control, various injectors, columns, a backpressure regulator, and detectors. SFC finds applications in fields like pharmaceuticals and has advantages over HPLC like using less toxic solvents.
Analytical chemistry involves separating, identifying, and quantifying components of matter. Hyphenated techniques combine two analytical methods, such as gas chromatography coupled with mass spectrometry (GC-MS). GC-MS separates chemical mixtures using gas chromatography and then identifies components using mass spectrometry. Other common hyphenated techniques include liquid chromatography-mass spectrometry (LC-MS) and gas chromatography-infrared spectroscopy (GC-IR). These coupled techniques provide enhanced sensitivity and accuracy for analyzing organic compounds, pollutants, drugs, proteins, and more.
Infrared spectroscopy is a technique that analyzes infrared light absorbed by a molecule to determine its structure. There are several types of molecular vibrations that can be observed, including stretching and bending vibrations. Samples can be analyzed in solid, liquid, or gas form using different sample handling methods. The main components of an IR spectrometer are the radiation source, monochromator, sample cell, detector, and recorder. Dispersive and Fourier transform IR spectrometers are two common instrument types, with Fourier transform having advantages like faster scanning. Functional groups can be identified by their characteristic absorption bands. Factors like coupling, hydrogen bonding, and electronic effects can influence vibrational frequencies.
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.
Gas chromatography-mass spectrometry (GC-MS) is a hyphenated technique that combines gas chromatography and mass spectrometry. GC is used to separate compounds in a mixture, while MS identifies the compounds based on their mass-to-charge ratios. The document discusses the basic principles, instrumentation, and applications of GC-MS. It explains how the gas chromatograph separates compounds and the mass spectrometer ionizes and detects them, providing both separation and identification capabilities in a single technique.
This document provides an overview of gas chromatography (GC). It describes the basic components and principles of how GC works, including the carrier gas, injection port, separation column in an oven, and detector. It explains that GC separates volatile organic compounds based on how they partition between the mobile gas phase and stationary liquid phase. The document also outlines different types of columns, sample preparation procedures, common detectors like the flame ionization detector, and applications of GC in fields like environmental analysis and refineries.
Phosphorescence involves the emission of light from electronically excited triplet states in a material. It is a spin-forbidden process that results in longer-lived emission compared to fluorescence. Phosphorescence occurs when electrons in the excited triplet state relax to the ground singlet state. Instrumentation for measuring phosphorescence requires cryogenic temperatures to reduce thermal quenching, as well as a phosphoroscope to separate the longer-lived phosphorescence from short-lived fluorescence. Applications of phosphorescence include security markers, toys, watches, and switches that glow in the dark.
This document discusses factors that affect fluorimetry and quenching. It lists several factors that can influence fluorescence, including the nature of molecules, substituents, concentration, adsorption, light, oxygen, pH, temperature, and viscosity. It also describes different types of quenching such as self-quenching, chemical quenching, static quenching, and collisional quenching. Chemical quenching can occur due to changes in pH, presence of oxygen, or heavy metals. Static quenching involves complex formation between the fluorophore and quencher. Collisional quenching occurs through interactions between an excited fluorophore and quencher molecule.
INSTRUMENTAL METHODS OF ANALYSIS, B.PHARM 7TH SEM. AND FOR BSC,MSC CHEMISTRY. This is Geeta prasad kashyap (Asst. Professor), SVITS, Bilaspur (C.G) 495001
Polarography is an electroanalytical technique invented in 1922 by Jaroslav Heyrovsky for which he won the Nobel Prize. It involves measuring the current in a solution under an applied potential using a dropping mercury electrode and a reference electrode such as SCE. Mercury is used as the working electrode due to its wide negative potential range and ability to regenerate its surface. A polarogram is generated by plotting current versus applied potential, showing residual, diffusion, and limiting currents. Polarography can be used for qualitative and quantitative analysis of metals, drugs, and other compounds.
High performance liquid chromatography (HPLC) is a technique that forces a solvent through a column under high pressure to separate samples into their constituent parts. HPLC uses a pump to force a mobile phase through a column containing a stationary phase, and a detector measures the analytes as they elute from the column. There are several types of HPLC that separate samples based on polarity (normal phase), hydrophobic interactions (reverse phase), molecular size (size-exclusion), or ionic charge (ion-exchange). HPLC has many applications in fields like pharmaceuticals, environmental analysis, forensics, food and flavors, and clinical testing.
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) is presented. HPLC is a chromatographic technique used to separate mixtures by using high pressure to force a liquid mobile phase and sample through a column packed with solid stationary phase. Key aspects summarized include:
1. HPLC provides simultaneous analysis, high resolution, sensitivity, repeatability for qualitative and quantitative analysis.
2. It works on principles of adsorption and partition chromatography depending on the stationary phase.
3. Instrumentation includes pumps, injector, analytical column, detector, and recorder/integrator.
4. Parameters like retention time, capacity factor, separation factor, and plate height provide information about sample separation and column efficiency.
This document provides an overview of high performance liquid chromatography (HPLC). It discusses how HPLC refined traditional liquid chromatography by using smaller particle sizes, smaller column diameters, and high fluid pressures to provide enhanced separations over shorter periods of time. Key aspects of HPLC systems and processes are summarized, including the use of pumps to deliver mobile phases at high pressure through columns containing small stationary phase particles. Separation is achieved based on how sample components partition between the mobile and stationary phases. Various detectors are also outlined.
This document provides an overview of high-performance liquid chromatography (HPLC). It discusses the different types of stationary and mobile phases used in HPLC, including normal phase and reversed phase. It also describes the basic instrumentation components of an HPLC system, such as pumps, injectors, columns, detectors, and data systems. Various detector types are explained. The document then covers different modes of chromatography used in HPLC, including partition chromatography, gel permeation chromatography, ion exchange chromatography, affinity chromatography, and chiral chromatography.
High performance liquid chromatography (HPLC)Htet Wai Moe
High performance liquid chromatography (HPLC) is a separation technique used to separate mixtures. It uses columns packed with small particle sizes under high pressure, allowing better separation than traditional liquid chromatography. HPLC involves pumping a mobile phase through a column containing a stationary phase, separating components as they flow through at different rates based on interactions with the phases. Components are then detected and quantified as they exit the column. HPLC provides rapid, sensitive, and precise separation of mixtures and is widely used in fields like pharmaceuticals, chemistry, and environmental analysis.
High Performance Liquid Chromatography (HPLC) is a separation technique that involves injecting a small volume of liquid sample into a column packed with tiny particles. Individual components of the sample are then transported through the column by a mobile phase and separated based on interactions with the stationary phase. These separated components exit the column and are detected, providing a chromatogram. HPLC uses small particle sizes, high column pressures up to 6000-9000 psi, and flow rates of 1-3 mL/min to achieve fast, efficient, and high resolution separations of both volatile and non-volatile compounds.
Column chromatography is a method used to separate chemical mixtures by using a column filled with a stationary phase and pumping a mobile phase through it. Key steps include preparing the column with a dry or wet method, loading the sample, and collecting fractions as compounds elute from the column at different rates depending on their interactions with the stationary phase. Resolution, a measure of separation, can be calculated from chromatograms by measuring retention times and curve widths. Automated systems now streamline column chromatography but typically have lower resolution than HPLC.
Chromatography techniques such as high performance liquid chromatography (HPLC), fast protein liquid chromatography (FPLC), and gas chromatography (GC) can be used to separate mixtures. HPLC uses high pressure to push a mobile phase through a column containing a stationary phase to separate complex mixtures. FPLC is a modified HPLC system designed for separating proteins more gently. GC vaporizes samples and uses an inert gas mobile phase to separate components in a sample based on differences in how they partition between the gas and a liquid or solid stationary phase.
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.
Types Of Chromatography - liquid & Gas Chromatography(Mobile Phase).pptxPriyaDixit46
Liquid chromatography (LC) and gas chromatography (GC) are two common types of chromatography. In LC, the mobile phase is a liquid and separation is based on interactions between solutes and the mobile and stationary phases. GC uses an inert gas as the mobile phase, with separation dependent on solute boiling points. Both techniques can separate mixtures and are used in various applications like pharmaceutical analysis, environmental testing, and food and chemical quality control. However, GC is generally faster and provides better resolution than LC.
The document discusses high-performance liquid chromatography (HPLC). It defines HPLC and describes its basic principles, which involve separating mixtures by distributing components between a stationary and mobile phase under high pressure. The key components of an HPLC system are described, including pumps, injectors, columns, detectors, and data systems. Various modes, columns, and detectors are discussed. The document provides an overview of the technique of HPLC.
HPLC involves injecting a liquid sample into a column packed with tiny adsorbent particles. Components are separated as they interact differently with the stationary phase and are eluted by the mobile phase. The separated components are then detected and analyzed. Key components of HPLC include the solvent reservoir, pump, injector, column, and various detectors. There are different modes of separation including reversed phase, normal phase, ion exchange, and size exclusion chromatography. Parameters like retention time, theoretical plate number, and resolution are used to characterize chromatographic separations.
High performance liquid chromatography (HPLC) is a technique used to separate compounds in a mixture. It works by forcing a pressurized liquid mobile phase through a column containing a stationary phase, which causes the compounds in a sample to separate as they interact differently with the phases. HPLC provides faster, more efficient separations than traditional column chromatography due to the use of high pressure. It has various applications including drug analysis, polymer analysis, and purification of biopolymers.
Chromatography; history and its types.Zeeshan Awan
This document provides an overview of chromatography techniques. It discusses the history of chromatography, developed in 1900 by Mikhail Tsvet to separate plant pigments. It explains the basic principle of chromatography, which works by differential distribution of components between a stationary and mobile phase. Several types of chromatography are described, including liquid chromatography, gas chromatography, ion exchange chromatography, thin layer chromatography, paper chromatography, column chromatography, and affinity chromatography. Each technique is explained briefly in terms of its components, principle, and uses. The document concludes that chromatography has many applications in biochemistry and organic chemistry for separation of mixtures.
This document discusses high-performance liquid chromatography (HPLC), which is a widely used technique for separating and analyzing components in mixtures. It describes the basic components and principles of HPLC, including pumps to pass a pressurized liquid and sample mixture through a column containing stationary phase particles. The components interact differently with the stationary phase and are separated into bands that are then detected and analyzed. Common detectors described are UV-visible, fluorescence, and electrochemical detectors. The document also discusses various modes of operation like isocratic and gradient elution and types of columns and stationary phases used.
High performance liquid chromatography (HPLC) is an improved form of liquid chromatography that forces solvent through a column at high pressure. It separates mixtures by interacting differently with stationary and mobile phases in the column based on molecular structure. HPLC uses pumps to push solvent through an injector, column, detector, and recorder/computer. The column contains porous particles that substances differentially bind to. Detectors identify separated substances and recorders display chromatograms showing separation and quantification. HPLC has many applications like pharmaceutical quality control and forensic drug analysis due to its accuracy, precision, and versatility.
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) and its use in pathology. It describes the basic components and separation mechanisms of chromatography, including mobile phase, stationary phase, and how separation occurs. It then discusses various types of chromatography techniques like ion exchange, partition, size exclusion, and affinity chromatography. The document focuses on HPLC, describing its instrumentation including reservoirs, pumps, injectors, columns, and detectors. It explains how HPLC provides improved resolution, speed, and reproducibility over other chromatography methods.
Partition chromatography & partition paper chromatographyArtina Aquitania
Partition chromatography separates mixtures based on differences in how components partition between two immiscible liquid phases. The stationary phase is a liquid coated on a solid support, and separation occurs as the mobile phase passes through based on each component's partition coefficient. Paper partition chromatography uses the moisture in filter paper as the stationary phase, with an organic solvent or buffer as the mobile phase. Components separate based on their relative solubilities in the paper versus solvent, with their positions after development quantified using Rf values. Factors like solvent choice, paper quality, and development conditions influence separations and Rf values.
HPLC- introduction, principle, types, working, instrumentation and operations of HPLC has been included with appropriate gifs and images for better understanding. What are all the things need to be known by a science student about HPLC (basics and working) is clearly given in this presentation.
Have you ever been confused by the myriad of choices offered by AWS for hosting a website or an API?
Lambda, Elastic Beanstalk, Lightsail, Amplify, S3 (and more!) can each host websites + APIs. But which one should we choose?
Which one is cheapest? Which one is fastest? Which one will scale to meet our needs?
Join me in this session as we dive into each AWS hosting service to determine which one is best for your scenario and explain why!
Dive into the realm of operating systems (OS) with Pravash Chandra Das, a seasoned Digital Forensic Analyst, as your guide. 🚀 This comprehensive presentation illuminates the core concepts, types, and evolution of OS, essential for understanding modern computing landscapes.
Beginning with the foundational definition, Das clarifies the pivotal role of OS as system software orchestrating hardware resources, software applications, and user interactions. Through succinct descriptions, he delineates the diverse types of OS, from single-user, single-task environments like early MS-DOS iterations, to multi-user, multi-tasking systems exemplified by modern Linux distributions.
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The narrative then shifts to a captivating exploration of prominent desktop OSs, Windows, macOS, and Linux. Windows, with its globally ubiquitous presence and user-friendly interface, emerges as a cornerstone in personal computing history. macOS, lauded for its sleek design and seamless integration with Apple's ecosystem, stands as a beacon of stability and creativity. Linux, an open-source marvel, offers unparalleled flexibility and security, revolutionizing the computing landscape. 🖥️
Moving to the realm of mobile devices, Das unravels the dominance of Android and iOS. Android's open-source ethos fosters a vibrant ecosystem of customization and innovation, while iOS boasts a seamless user experience and robust security infrastructure. Meanwhile, discontinued platforms like Symbian and Palm OS evoke nostalgia for their pioneering roles in the smartphone revolution.
The journey concludes with a reflection on the ever-evolving landscape of OS, underscored by the emergence of real-time operating systems (RTOS) and the persistent quest for innovation and efficiency. As technology continues to shape our world, understanding the foundations and evolution of operating systems remains paramount. Join Pravash Chandra Das on this illuminating journey through the heart of computing. 🌟
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2. • High Performance Liquid Chromatography (HPLC) is the most
widely used type of elution chromatography
• In Liquid Chromatography, the columns were packed with 50 to
500 cm lengths of solid particles coated with an adsorbed liquid
that formed the stationary phase and the mobile phase is a liquid
solvent containing the sample as a mixture of solutes
• The types of high – performance liquid chromatography are often
classified by separation mechanism or by the type of the
stationary phase
(a) Partition, or liquid – liquid chromatography
(b) Adsorption, or liquid – solid chromatography
(c) Ion – exchange, or ion chromatography
(d) Size exclusion chromatography
(e) Affinity chromatography
(f) Chiral chromatography
3. • High–Performance Liquid Chromatography (HPLC) is a type of
chromatography that employs a liquid mobile phase and a finely
divided stationary phase
• To obtain satisfactory flow rates, the liquid must be pressurized
to several hundreds or more pounds per square inch
Applications of Liquid Chromatography
• Molecular masses greater than 10,000:
Size exclusion: gel permeation for non-
polar compounds & gel filtration for
polar compounds
• Ionic compounds of lower masses:
Ion exchange chromatography
• Smaller but non-ionic: Partition
methods
4. High – Performance Partition Chromatography
• Partition Chromatography
(a) Mobile Phase: Liquid
(b) Stationary Phase: A second liquid that is immiscible with the
liquid mobile phase
• Partition Chromatography is sub-divided into
(a) Liquid–Liquid Chromatography
The stationary phase is a solvent that is held in place by
adsorption on the surface of packing particles
(b) Liquid bonded–phase chromatography
The stationary phase is an organic species that is attached to
the surface of the packing materials by chemical bonds
6. • Pumping pressures of several hundred atmospheres are required
to achieve reasonable rates with packings in the 3- to 10-mm size
range
1. Mobile phase reservoirs and solvent treatment systems
2. Pumping systems
3. Sample injection system
4. Columns
5. Detectors
Important Components of HPLC
Instrumentation Contd…..
7. 1. Mobile phase reservoirs and solvent treatment systems
• A modern HPLC apparatus is equipped with one or more glass
reservoirs which contains 500 mL or more of a solvent
• Gases and dust are present in the solvents and they are to be
removed. Gases produce bubbles in the column and thereby cause
band spreading
• Degassers may consist of vacuum pumping system, a distillation
system, a device for heating and stirring – mainly causes sparging
Sparging: It is a process in which dissolved gases are swept out of
a solvent by fine bubbles of an inert gas that is not soluble in the
mobile phase
8. • A isocratic elution in HPLC is one in which the solvent
composition remains constant
• A gradient elution in HPLC is one in which the composition of the
solvent in changed continuously or in a series of steps
9. Pumping Systems
• Requirements for liquid chromatographic pumps include
(a) ability to generate of up to 6000 psi (lb/in2)
(b) pulse-free output
(c) flow rates ranging from 0.1 to 10 mL/min
(d) flow reproducibilities of 0.5% relative or better
(e) resistance to corrosion by a variety of solvents
There are three major types of pumps:
(a) The screw driven syringe pump
(b) The reciprocating pump
(c) The pneumatic or constant pressure pump
• The most widely used pump is reciprocating pump
10. Pumping Systems
• Consists of small cylindrical
chamber that is filled and
then emptied by the back-
and-forth motion of a piston
• The pumping motion
produces a pulsed flow that
must be subsequently
damped
Advantages
• Small internal volume
• High output pressure
• Gradient elution is possible
• Constant flow rates
A reciprocating pump for HPLC
12. Columns for HPLC
• Most columns range in length from 10 to 30 cm and have inside
diameters of 2 to 5 mm
• The most common packing for liquid chromatography is prepared
from silica particles, which are synthesized by agglomerating
submicron silica particles under conditions that lead to larger
particles with highly uniform
• The resulting particles are often coated with thin organic films,
which are chemically or physically bonded to the surface
Detectors
• The detector should have low dead volume to minimize extra
column band broadening
• Should be small and compatible with liquid flow
13. Bonded – Phase Packings
• Most bonded – packings are prepared by the reaction of an
organochlorosilane with the –OH groups on the surface of silica
particles
where R = straight chain octyl or octyldecyl group
• Other organic functional groups include: aliphatic amines, ethers,
and nitriles, as well as aromatic hydrocarbons
• Bonded – phase packings have the advantage of markedly greater
stability than the physically held stationary phases. With the
later, periodic recoating of the solid surfaces is required because
the stationary phase is gradually dissolved away in the mobile
phase.
14. Partition Chromatography:
Normal – and Reversed – Phase Packings
• Two types of partition chromatography are distinguishable based
on the relative polarities of the mobile and stationary phases
(a) Normal Phase Chromatography
The stationary phase is highly polar such as triethylene glycol
or water and relatively nonpolar solvent such as hexane or i-propyl
ether the served as mobile phase.
(b) Reversed Phase Chromatography
The stationary phase is non-polar, often a hydrocarbon, and
the mobile phase is a relatively polar solvent (such as water,
methanol, acetonitrile, or tetrahydrofuran)
15. In normal – phase chromatography, the least polar component is eluted
first; increasing the polarity of the mobile phase decreases the elution
time.
In reverse – phase chromatography, the most polar component elutes
first, and increasing the mobile phase polarity increases the elution time
• It has been estimated that more than three quarters of all HPLC
separations are currently performed with reversed-phase, bonded,
octyl- or octyldecyl-siloxane packings.
• With such preparations, the long chain hydrocarbon groups are aligned
parallel to one another and perpendicular to the surface of the
particle, giving a brush like non-polar, hydro carbon surface
• Mobile phase is water with various conc. of methanol, acetonitrile,
THF
16. Choice of Mobile and Stationary Phases
• Successful partition chromatography requires a proper balance
of intermolecular forces among the three participants in the
separation process – the analyte, the mobile phase, and the
stationary phase
• These intermolecular forces are described qualitatively in terms
of the relative polarity possessed by each of the three
components
• Most chromatographic separations are achieved by matching the
polarity of the analyte to that of the stationary phase; a mobile
phase of considerably different polarity is then used
Increasing polarity
17. Applications of Partition Chromatography
Soft-drink additives
Column is packed with polar (nitrile)
bonded-phase packing
Organophosphate Insecticides
Column is packed with C8 bonded-
phase packing
19. Ion-Exchange Chromatography
• Stationary phase is a resin and mobile phase is a polar solvent
• In most cases, conductivity measurements are used to measure
the eluents
• Two types of Ion-Chromatography are currently in use:
(a) Suppressor-based and
(b) Single-Column
They differ in the method to prevent the conductivity of the eluting
electrolyte from interfering with the measurement of analyte
concentrations
20. Conductivity Detectors – Detectors for Ion–Exchange Columns
• Conductivity detectors are highly sensitive, universal for charged
species
• They respond to concentration changes
• Disadvantage
High electrolyte concentrations are required to elute most
analyte ions in a reasonable time. As a consequence, the
conductivity of the mobile phase components tend to swamp
that from the analyte ions, thus greatly reducing the detector
sensitivity
• So Eluent Suppressor Column is used
21. • The suppressor column is packed with an second ion-exchange
resin that effectively converts the ions of the eluting solvent to a
molecular species of limited ionization without effecting the
conductivity due to analyte ions
Eg: (a) When cations are being separated and determined,
hydrochloric acid is chosen as the eluting reagent, and the
suppressor column is an anion-exchange resin in the hydroxide
form. The product of the reaction in the suppressor is water
The analyte cations are not retained by this second column
Suppressor based Ion-Exchange Column
22. Eg: (b) For anion separations, the suppressor packing is the acid
form of a cation-exchange resin, and sodium bicarbonate or
carbonate is the eluting agent. The reaction in the suppressor is
The largely undissociated carbonic acid does not contribute
significantly to the conductivity
23. Single-Column Ion Chromatography
• No suppressor column is needed
• Analyte ions are separated on a low capacity ion exchanger by
means of a low-ionic strength eluent that does not interfere with
the conductometric detection of analyte ions
24. • Fractionation is based on molecular size
• Powerful technique that is particularly applicable to high-
molecular weight species
Column Packings
• Packings consist of small (~10 mm) silica or polymer particles
containing a network of uniform pores into which solute and
solvent molecules can diffuse
• In the pores, the molecules are effectively trapped and removed
from the flow of the mobile phase
• The average residence time of the analyte molecules depends on
their effective size
Size-Exclusion Chromatography
25. • Molecules that are significantly larger than the average pore size
of the packing are excluded and thus suffer no retention i.e.,
they travel through the column at the rate of the mobile phase
• Molecules that are appreciably smaller than the pores can
penetrate through the pore maze and thus entrapped for the
greatest time; they are last to elute
• Intermediate size molecules whose average penetration into the
pores of the packing depends on their diameters – undergo
fractionation dependent on molecular size and molecular shape
• No physical and chemical interactions with the stationary phase
Column packings in Size-Exclusion Chromatography
26. • Gel filtration:
Type of size-exclusion chromatography in which the packing
is hydrophilic. It is used to separate polar species
• Gel Permeation:
Type of size-exclusion chromatography in which the packing
is hydrophobic. It is used to separate nonpolar species
27. Affinity Chromatography
• Affinity Chromatography involves covalently bonding a reagent,
called an affinity ligand, to a solid support.
• Typical affinity ligands are
Antibodies, Enzyme inhibitors, or other molecules that
reversibly and selectively bind to analyte molecules in the sample
• Procedure
(1) When sample passes through the column, only the molecules
that selectively bind to the affinity ligand are retained
(2) Molecules that do not bind pass through the column with the
mobile phase
(3) After the desired molecules are removed, the retained analytes
can be eluted by changing the mobile phase conditions
28. Stationary Phase
• Solid such as a agarose or a porous glass bead to which affinity
ligand is immobilized
Mobile phase has two important roles
(a) First it should support the binding of the analyte molecules to
the ligand
(b) Second, once the undesired species are removed, the mobile
phase must weaken or eliminate the analyte-ligand interaction
so that the analyte can be eluted
Applications
(1) Extraordinary specificity
(2) Rapid isolation of biomolecules during preparative work