This document provides an overview of high-performance liquid chromatography (HPLC). It discusses how HPLC uses a liquid mobile phase and finely divided stationary phase to separate components. Key aspects covered include the principles of HPLC, typical instrumentation such as solvent delivery systems and columns, and considerations for the mobile phase like composition, polarity, and isocratic versus gradient elution methods. The document also examines stationary phase materials like modified silica and how they impact selectivity.
Chromatography is a technique used to separate mixtures by distributing components between a stationary and mobile phase. High-performance liquid chromatography (HPLC) uses high pressure to pass a solvent or solvent mixture through a column containing a stationary phase to separate components in a mixture. HPLC consists of several major components including a pump, injector, column, column compartment, detector, and degasser. The injector introduces the sample into the mobile phase which passes through the column, allowing separation based on interactions between components and the stationary phase. A detector then measures and records separated components as they elute from the column.
1. Chromatography is a technique used to separate components of a mixture using a stationary and mobile phase. Molecules spend varying amounts of time in each phase, becoming separated as they move through the column at different rates.
2. HPLC uses a liquid mobile phase pumped under pressure through a column packed with solid particles. Components separate based on interactions with the stationary and mobile phases, and are detected as they exit the column.
3. In reverse phase HPLC using a methanol/water mobile phase, prednisolone would elute just before betamethasone due to its slightly greater polarity from lacking an additional methyl group. Betamethasone dipropionate would elute last due to
High performance liquid chromatography (HPLC) is a technique used to separate compounds based on differences in their interactions with a stationary phase. There are various modes of separation including ion exchange, size exclusion, hydrophobic interaction, and affinity chromatography. The choice of technique depends on the physicochemical properties of the compounds being separated. Reversed-phase HPLC, which uses a non-polar stationary phase and polar mobile phase, is the most commonly used mode, allowing separation based on a compound's relative polarity.
HPLC (High Performance Liquid Chromatography) is a separation technique used to separate, identify, and quantify compounds in mixtures. It works by injecting samples into a column with a stationary phase and passing a liquid mobile phase through under high pressure. Compounds are separated based on how they partition between the mobile and stationary phases. HPLC is useful for pharmaceutical analysis, clinical applications, chemical separations, and purification of compounds due to its high resolution, sensitivity, repeatability, and ability to separate both volatile and non-volatile compounds.
HPLC is a technique used to separate components in a mixture using pumps to pass a pressurized liquid solvent containing the sample through a column filled with an adsorbent material. Each component interacts slightly differently with the material, causing different flow rates and separation. HPLC has applications in manufacturing like pharmaceutical production, legal purposes like drug testing, research like separating biological samples, and medical uses like detecting vitamin levels. It provides superior resolving power over traditional chromatography to distinguish between compounds.
HPLC is a form of liquid chromatography that uses pumps to pass a pressurized mobile liquid phase through a column packed with solid particles. This allows the components of a dissolved sample to be separated as they are transported through the column at different rates depending on their interactions with the stationary and mobile phases. HPLC instruments consist of a pump, injector, column, and detector. Separation is based on the partitioning of compounds between the mobile and stationary phases, and detectors are used to measure separated components as they exit the column. HPLC provides efficient, sensitive, and high-pressure separations of sample mixtures.
This document discusses high performance liquid chromatography (HPLC). It begins by providing background on the founder of liquid chromatography, Mikhail Tsvet. It then describes the basic concepts of HPLC including qualitative and quantitative analysis using retention time and peak area/height comparisons. The document outlines the types of HPLC including partition, adsorption, ion exchange, size-exclusion, and affinity chromatography. It also describes the various components of an HPLC system including the solvent system, injection valve, column, and detector.
Chromatography is a laboratory technique used to separate components of a mixture through differential partitioning between a stationary and mobile phase. The key aspects are:
- Mixtures are separated based on how components partition between a mobile liquid or gas phase and a stationary solid or liquid phase.
- There are various types including adsorption chromatography which uses interactions between components and a solid stationary phase, partition chromatography which relies on differing solubilities in mobile and stationary liquid phases, and ion-exchange chromatography which separates based on charge.
- Factors like pH, salt concentration, temperature and column properties influence the separation in chromatography. It has many applications in analyzing compounds like drugs, proteins, sugars and more.
Chromatography is a technique used to separate mixtures by distributing components between a stationary and mobile phase. High-performance liquid chromatography (HPLC) uses high pressure to pass a solvent or solvent mixture through a column containing a stationary phase to separate components in a mixture. HPLC consists of several major components including a pump, injector, column, column compartment, detector, and degasser. The injector introduces the sample into the mobile phase which passes through the column, allowing separation based on interactions between components and the stationary phase. A detector then measures and records separated components as they elute from the column.
1. Chromatography is a technique used to separate components of a mixture using a stationary and mobile phase. Molecules spend varying amounts of time in each phase, becoming separated as they move through the column at different rates.
2. HPLC uses a liquid mobile phase pumped under pressure through a column packed with solid particles. Components separate based on interactions with the stationary and mobile phases, and are detected as they exit the column.
3. In reverse phase HPLC using a methanol/water mobile phase, prednisolone would elute just before betamethasone due to its slightly greater polarity from lacking an additional methyl group. Betamethasone dipropionate would elute last due to
High performance liquid chromatography (HPLC) is a technique used to separate compounds based on differences in their interactions with a stationary phase. There are various modes of separation including ion exchange, size exclusion, hydrophobic interaction, and affinity chromatography. The choice of technique depends on the physicochemical properties of the compounds being separated. Reversed-phase HPLC, which uses a non-polar stationary phase and polar mobile phase, is the most commonly used mode, allowing separation based on a compound's relative polarity.
HPLC (High Performance Liquid Chromatography) is a separation technique used to separate, identify, and quantify compounds in mixtures. It works by injecting samples into a column with a stationary phase and passing a liquid mobile phase through under high pressure. Compounds are separated based on how they partition between the mobile and stationary phases. HPLC is useful for pharmaceutical analysis, clinical applications, chemical separations, and purification of compounds due to its high resolution, sensitivity, repeatability, and ability to separate both volatile and non-volatile compounds.
HPLC is a technique used to separate components in a mixture using pumps to pass a pressurized liquid solvent containing the sample through a column filled with an adsorbent material. Each component interacts slightly differently with the material, causing different flow rates and separation. HPLC has applications in manufacturing like pharmaceutical production, legal purposes like drug testing, research like separating biological samples, and medical uses like detecting vitamin levels. It provides superior resolving power over traditional chromatography to distinguish between compounds.
HPLC is a form of liquid chromatography that uses pumps to pass a pressurized mobile liquid phase through a column packed with solid particles. This allows the components of a dissolved sample to be separated as they are transported through the column at different rates depending on their interactions with the stationary and mobile phases. HPLC instruments consist of a pump, injector, column, and detector. Separation is based on the partitioning of compounds between the mobile and stationary phases, and detectors are used to measure separated components as they exit the column. HPLC provides efficient, sensitive, and high-pressure separations of sample mixtures.
This document discusses high performance liquid chromatography (HPLC). It begins by providing background on the founder of liquid chromatography, Mikhail Tsvet. It then describes the basic concepts of HPLC including qualitative and quantitative analysis using retention time and peak area/height comparisons. The document outlines the types of HPLC including partition, adsorption, ion exchange, size-exclusion, and affinity chromatography. It also describes the various components of an HPLC system including the solvent system, injection valve, column, and detector.
Chromatography is a laboratory technique used to separate components of a mixture through differential partitioning between a stationary and mobile phase. The key aspects are:
- Mixtures are separated based on how components partition between a mobile liquid or gas phase and a stationary solid or liquid phase.
- There are various types including adsorption chromatography which uses interactions between components and a solid stationary phase, partition chromatography which relies on differing solubilities in mobile and stationary liquid phases, and ion-exchange chromatography which separates based on charge.
- Factors like pH, salt concentration, temperature and column properties influence the separation in chromatography. It has many applications in analyzing compounds like drugs, proteins, sugars and more.
Advanced techniques and laborotory equipments for biologistsNawfal Aldujaily
Gas chromatography (GC) separates volatile compounds using an inert gas as the mobile phase. The sample is injected into a heated port to volatilize it. The gas mobile phase carries the volatilized sample through a heated column coated with a stationary phase that interacts with analytes. Components are separated based on differences in volatility and affinity for the stationary phase, then detected and recorded. GC is useful for separating volatile, non-polar compounds.
Chapter-6-ADSORPTION-PARTITION-CHROMATOGRAPHY.pdfNeelam Malik
This document provides an overview of adsorption partition chromatography. It defines chromatography as a technique for separating mixtures by passing them through a medium where components move at different rates. The document then discusses various types of chromatography based on the stationary and mobile phases used, including column chromatography, partition chromatography, and thin layer chromatography. It also provides details on the theories and processes of adsorption chromatography and partition chromatography.
This document provides an overview of high performance liquid chromatography (HPLC). It describes the basic components of an HPLC system including the solvent delivery system, injector, column, and detector. It explains how samples are separated based on the differences in how components partition between the mobile and stationary phases. Various types of chromatography are also summarized, including partition, adsorption, ion exchange, and size exclusion. Factors that can affect separations like column parameters, instrument parameters, and sample parameters are outlined.
This document discusses HPLC (High Performance Liquid Chromatography). It begins by providing an introduction to Talsaniya Roman, the author, and their presentation on HPLC. It then covers various topics related to HPLC in more detail over several pages, including the definition of chromatography, different types of HPLC classification like normal phase vs reversed phase, different stationary and mobile phases, detectors commonly used in HPLC like UV, refractive index, fluorescence, and conductivity detectors, and key components of an HPLC system like the pump, injector, and column. The document provides information on HPLC in a technical presentation format.
1. Quantitative analysis using chromatography relies on measuring either the height or area of analyte peaks. Peak area provides a better measure of concentration since it is unaffected by variations in column efficiency.
2. The concentration of an analyte is determined by comparing its peak area to that of a standard of known concentration. Internal standards are used to control for variable experimental conditions between runs.
3. Calibration curves relate the detector response of external standards to their known concentrations. As long as injection volumes are identical, they provide accurate and precise results. Internal standards use the ratio of analyte to internal standard peak areas to normalize for variable conditions.
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.
This document provides an overview of chromatographic techniques. It begins with definitions and a brief history, then covers principles, applications, classification, specific techniques (e.g. gas chromatography, liquid chromatography), terms, and properties of stationary phases. The document presents chromatographic methods and their use in separating mixtures like drugs, proteins, and other compounds. It concludes that supercritical fluid chromatography falls between HPLC and GC in performance for applications in pharmaceutical and bioanalytical analysis.
This document provides an overview of chromatographic techniques. It begins with definitions and a brief history, explaining that chromatography separates mixtures based on how components partition between a mobile and stationary phase. The document then covers key terms, classifications of chromatography by mechanism and phase, and specific techniques like gas-liquid chromatography, solid-liquid chromatography, and thin layer chromatography. It discusses important properties of liquid stationary phases and concludes that chromatography techniques like supercritical fluid chromatography fall between HPLC and GC in terms of separation capabilities.
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.
High Performance Liquid Chromatography (HPLC) drl.pptxprakhar rai pk
HPLC is used to separate, identify, and quantify components in mixtures. It works by distributing analytes between a flowing mobile phase and a stationary phase inside a column. Under high pressure, analytes are separated based on differences in how they interact with the phases. Key aspects of HPLC include the stationary and mobile phases used, fundamental concepts like retention time and selectivity, types of detectors, and steps to develop and optimize an analysis method for a target analyte or pharmaceutical compound.
This document provides an overview of high performance liquid chromatography (HPLC). It describes HPLC as a type of liquid chromatography that uses small particle sizes as the stationary phase to separate compounds dissolved in a solution. The key components of an HPLC system are a solvent delivery pump, injector, column, detectors, and data collection system. Different types of columns include normal phase, reverse phase, size exclusion, and ion exchange. Factors that affect HPLC separation include column length and temperature, flow rate, particle size, and mobile phase properties. HPLC is used for quantitative and qualitative analysis in various applications like analyzing drugs, pollutants, and food/drug products.
HPLC- high performance liquid chromatographyhirenthakkar4
HPLC- high performance liquid chromatography or high pressure liquid chromatography overall review
good animation & GIF for presentation
detectors in detail
basic instrumentation with detectors
Chromatography is an analytical technique used to separate mixtures by distributing components between two phases. It involves a stationary phase and mobile phase. High performance liquid chromatography (HPLC) is a type of chromatography that uses high pressure to pass a liquid mobile phase through a column packed with solid particles. HPLC can separate a wide range of compounds and is used for both qualitative and quantitative analysis in applications such as drug discovery, clinical analysis, and forensic chemistry.
This document provides an introduction to high performance liquid chromatography (HPLC). It begins with definitions of HPLC and basic chromatographic terms. It then discusses the instrumentation of HPLC including common components like the solvent delivery pump, injector, column, detector, and data system. Different types of chromatography are also outlined, including reverse phase, normal phase, ion exchange, and size exclusion. Key terms used in HPLC like retention time, peak width, and tailing factor are defined. The principles of HPLC separation and factors that influence separation are described.
The document discusses different types of chromatography. It begins with an introduction to chromatography, including its history and principles. It then describes various classifications of chromatography based on mechanism and phases. Specific techniques are defined, including adsorption chromatography, partition chromatography, gas-liquid chromatography, solid-liquid chromatography, and liquid-liquid chromatography. Key terms are explained. Applications and steps of chromatographic separation are outlined. Important properties of liquid stationary phases are also summarized.
Liquid chromatography uses two immiscible liquid phases to separate components of a mixture. The stationary phase is adsorbed to a support and the mobile phase flows through, carrying solutes between the phases. Separation occurs as solutes distribute differently between the phases based on their properties. Liquid-solid chromatography uses a solid stationary phase, like silica or alumina, which can selectively retain solutes based on interactions between functional groups. Quantitative analysis with chromatography requires optimizing the separation, identifying peaks, establishing a calibration curve to determine detection limits and linearity, and validating the method to ensure consistent, accurate results.
HPLC involves separating mixtures using differences in how components distribute between a mobile and stationary phase. It uses particles of small diameter as the stationary phase. Compounds are separated by injecting the sample mixture onto the column where the components pass through at different rates due to differences in how they partition between the mobile and stationary phases. HPLC can separate a wide variety of compounds and is used for quantitative and qualitative analysis of drugs, pollutants, and other analytes.
HPLC is a form of liquid chromatography that can separate compounds dissolved in solution. It works by injecting a sample into a column packed with tiny particles, then using a pump to force a liquid mobile phase through the column. This carries the sample components along the column at different speeds based on their interaction with the stationary phase, separating them. HPLC can separate a wide range of compounds and is used in pharmaceutical and chemical analysis applications.
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.
This document provides an overview of organic chemistry. It discusses how organic chemistry originated from distinguishing between compounds from living versus non-living sources. It then defines organic chemistry as the study of carbon compounds, as carbon can form diverse structures through its ability to form four strong covalent bonds. The document proceeds to discuss atomic structure, bonding theory including hybridization, and provides examples of sp3 and sp2 hybridization in molecules like ethane and ethene.
Chromatography is a technique used to separate chemical mixtures by exploiting differences in how components interact with stationary and mobile phases. It was first developed in 1900 to separate plant pigments. The key is separation of components to simplify analysis of unknown substances. Chromatography can be classified based on the physical means of contact between phases, the type of mobile and stationary phases used, and the type of interactions that occur. The efficiency of separation depends on column resolution, which is improved by increasing the difference in retention times and decreasing peak widths. Migration rates are determined by distribution constants between phases and affect retention times.
Advanced techniques and laborotory equipments for biologistsNawfal Aldujaily
Gas chromatography (GC) separates volatile compounds using an inert gas as the mobile phase. The sample is injected into a heated port to volatilize it. The gas mobile phase carries the volatilized sample through a heated column coated with a stationary phase that interacts with analytes. Components are separated based on differences in volatility and affinity for the stationary phase, then detected and recorded. GC is useful for separating volatile, non-polar compounds.
Chapter-6-ADSORPTION-PARTITION-CHROMATOGRAPHY.pdfNeelam Malik
This document provides an overview of adsorption partition chromatography. It defines chromatography as a technique for separating mixtures by passing them through a medium where components move at different rates. The document then discusses various types of chromatography based on the stationary and mobile phases used, including column chromatography, partition chromatography, and thin layer chromatography. It also provides details on the theories and processes of adsorption chromatography and partition chromatography.
This document provides an overview of high performance liquid chromatography (HPLC). It describes the basic components of an HPLC system including the solvent delivery system, injector, column, and detector. It explains how samples are separated based on the differences in how components partition between the mobile and stationary phases. Various types of chromatography are also summarized, including partition, adsorption, ion exchange, and size exclusion. Factors that can affect separations like column parameters, instrument parameters, and sample parameters are outlined.
This document discusses HPLC (High Performance Liquid Chromatography). It begins by providing an introduction to Talsaniya Roman, the author, and their presentation on HPLC. It then covers various topics related to HPLC in more detail over several pages, including the definition of chromatography, different types of HPLC classification like normal phase vs reversed phase, different stationary and mobile phases, detectors commonly used in HPLC like UV, refractive index, fluorescence, and conductivity detectors, and key components of an HPLC system like the pump, injector, and column. The document provides information on HPLC in a technical presentation format.
1. Quantitative analysis using chromatography relies on measuring either the height or area of analyte peaks. Peak area provides a better measure of concentration since it is unaffected by variations in column efficiency.
2. The concentration of an analyte is determined by comparing its peak area to that of a standard of known concentration. Internal standards are used to control for variable experimental conditions between runs.
3. Calibration curves relate the detector response of external standards to their known concentrations. As long as injection volumes are identical, they provide accurate and precise results. Internal standards use the ratio of analyte to internal standard peak areas to normalize for variable conditions.
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.
This document provides an overview of chromatographic techniques. It begins with definitions and a brief history, then covers principles, applications, classification, specific techniques (e.g. gas chromatography, liquid chromatography), terms, and properties of stationary phases. The document presents chromatographic methods and their use in separating mixtures like drugs, proteins, and other compounds. It concludes that supercritical fluid chromatography falls between HPLC and GC in performance for applications in pharmaceutical and bioanalytical analysis.
This document provides an overview of chromatographic techniques. It begins with definitions and a brief history, explaining that chromatography separates mixtures based on how components partition between a mobile and stationary phase. The document then covers key terms, classifications of chromatography by mechanism and phase, and specific techniques like gas-liquid chromatography, solid-liquid chromatography, and thin layer chromatography. It discusses important properties of liquid stationary phases and concludes that chromatography techniques like supercritical fluid chromatography fall between HPLC and GC in terms of separation capabilities.
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.
High Performance Liquid Chromatography (HPLC) drl.pptxprakhar rai pk
HPLC is used to separate, identify, and quantify components in mixtures. It works by distributing analytes between a flowing mobile phase and a stationary phase inside a column. Under high pressure, analytes are separated based on differences in how they interact with the phases. Key aspects of HPLC include the stationary and mobile phases used, fundamental concepts like retention time and selectivity, types of detectors, and steps to develop and optimize an analysis method for a target analyte or pharmaceutical compound.
This document provides an overview of high performance liquid chromatography (HPLC). It describes HPLC as a type of liquid chromatography that uses small particle sizes as the stationary phase to separate compounds dissolved in a solution. The key components of an HPLC system are a solvent delivery pump, injector, column, detectors, and data collection system. Different types of columns include normal phase, reverse phase, size exclusion, and ion exchange. Factors that affect HPLC separation include column length and temperature, flow rate, particle size, and mobile phase properties. HPLC is used for quantitative and qualitative analysis in various applications like analyzing drugs, pollutants, and food/drug products.
HPLC- high performance liquid chromatographyhirenthakkar4
HPLC- high performance liquid chromatography or high pressure liquid chromatography overall review
good animation & GIF for presentation
detectors in detail
basic instrumentation with detectors
Chromatography is an analytical technique used to separate mixtures by distributing components between two phases. It involves a stationary phase and mobile phase. High performance liquid chromatography (HPLC) is a type of chromatography that uses high pressure to pass a liquid mobile phase through a column packed with solid particles. HPLC can separate a wide range of compounds and is used for both qualitative and quantitative analysis in applications such as drug discovery, clinical analysis, and forensic chemistry.
This document provides an introduction to high performance liquid chromatography (HPLC). It begins with definitions of HPLC and basic chromatographic terms. It then discusses the instrumentation of HPLC including common components like the solvent delivery pump, injector, column, detector, and data system. Different types of chromatography are also outlined, including reverse phase, normal phase, ion exchange, and size exclusion. Key terms used in HPLC like retention time, peak width, and tailing factor are defined. The principles of HPLC separation and factors that influence separation are described.
The document discusses different types of chromatography. It begins with an introduction to chromatography, including its history and principles. It then describes various classifications of chromatography based on mechanism and phases. Specific techniques are defined, including adsorption chromatography, partition chromatography, gas-liquid chromatography, solid-liquid chromatography, and liquid-liquid chromatography. Key terms are explained. Applications and steps of chromatographic separation are outlined. Important properties of liquid stationary phases are also summarized.
Liquid chromatography uses two immiscible liquid phases to separate components of a mixture. The stationary phase is adsorbed to a support and the mobile phase flows through, carrying solutes between the phases. Separation occurs as solutes distribute differently between the phases based on their properties. Liquid-solid chromatography uses a solid stationary phase, like silica or alumina, which can selectively retain solutes based on interactions between functional groups. Quantitative analysis with chromatography requires optimizing the separation, identifying peaks, establishing a calibration curve to determine detection limits and linearity, and validating the method to ensure consistent, accurate results.
HPLC involves separating mixtures using differences in how components distribute between a mobile and stationary phase. It uses particles of small diameter as the stationary phase. Compounds are separated by injecting the sample mixture onto the column where the components pass through at different rates due to differences in how they partition between the mobile and stationary phases. HPLC can separate a wide variety of compounds and is used for quantitative and qualitative analysis of drugs, pollutants, and other analytes.
HPLC is a form of liquid chromatography that can separate compounds dissolved in solution. It works by injecting a sample into a column packed with tiny particles, then using a pump to force a liquid mobile phase through the column. This carries the sample components along the column at different speeds based on their interaction with the stationary phase, separating them. HPLC can separate a wide range of compounds and is used in pharmaceutical and chemical analysis applications.
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.
This document provides an overview of organic chemistry. It discusses how organic chemistry originated from distinguishing between compounds from living versus non-living sources. It then defines organic chemistry as the study of carbon compounds, as carbon can form diverse structures through its ability to form four strong covalent bonds. The document proceeds to discuss atomic structure, bonding theory including hybridization, and provides examples of sp3 and sp2 hybridization in molecules like ethane and ethene.
Chromatography is a technique used to separate chemical mixtures by exploiting differences in how components interact with stationary and mobile phases. It was first developed in 1900 to separate plant pigments. The key is separation of components to simplify analysis of unknown substances. Chromatography can be classified based on the physical means of contact between phases, the type of mobile and stationary phases used, and the type of interactions that occur. The efficiency of separation depends on column resolution, which is improved by increasing the difference in retention times and decreasing peak widths. Migration rates are determined by distribution constants between phases and affect retention times.
Conductometry measures the conductivity or resistance of a solution between two electrodes. Only ionizable molecules conduct electricity, and the magnitude of conductivity depends on the amount of ions present. Conductivity is affected by electrolyte type, concentration, and temperature. Conductometry is used to determine ion concentrations through titrations, where changes in conductivity indicate the equivalence point. It can be applied to dilute, colored, or turbid solutions where other methods cannot be used. Typical titration curves show changes in conductivity as strong/weak acids and bases are neutralized.
This document provides an introduction to analytical separation techniques and chromatography. It discusses classical and instrumental methods of analysis, with instrumental methods using physical properties and efficient separation techniques. Chromatography is introduced as a physical method that separates analytes distributed between two phases, one stationary and one mobile. Key terms like mobile phase, stationary phase, and supporting medium are defined. Different types of chromatography are classified based on the physical means of separation, type of mobile/stationary phases, and type of interaction between analyte and stationary phase. Important chromatography concepts like elution, resolution, migration rates, distribution constants, and theoretical plates are also introduced.
Amines are organic compounds containing a nitrogen atom with a lone pair of electrons. They can be classified as primary, secondary, or tertiary depending on the number of organic substituents attached to the nitrogen. Amines are named systematically and have basic properties due to the lone pair on the nitrogen. Common reactions of amines include alkylation, acylation, diazotization, and reduction of diazonium salts.
Classical analytical methods involve separating sample components through precipitation, extraction, or distillation and then qualitatively analyzing the separated components using color, odor, solubility, or other physical properties. Quantitative analysis in classical methods involves gravimetric or volumetric techniques. Instrumental methods exploit other phenomena like conductivity, light absorption, and mass spectrometry for separation and quantification and have largely replaced classical techniques due to greater efficiency and precision. Instrumental methods are based on physical and chemical properties like conductivity, light absorption, and mass that have been used analytically for over a century.
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.
How to Add Chatter in the odoo 17 ERP ModuleCeline George
In Odoo, the chatter is like a chat tool that helps you work together on records. You can leave notes and track things, making it easier to talk with your team and partners. Inside chatter, all communication history, activity, and changes will be displayed.
How to Fix the Import Error in the Odoo 17Celine George
An import error occurs when a program fails to import a module or library, disrupting its execution. In languages like Python, this issue arises when the specified module cannot be found or accessed, hindering the program's functionality. Resolving import errors is crucial for maintaining smooth software operation and uninterrupted development processes.
Walmart Business+ and Spark Good for Nonprofits.pdfTechSoup
"Learn about all the ways Walmart supports nonprofit organizations.
You will hear from Liz Willett, the Head of Nonprofits, and hear about what Walmart is doing to help nonprofits, including Walmart Business and Spark Good. Walmart Business+ is a new offer for nonprofits that offers discounts and also streamlines nonprofits order and expense tracking, saving time and money.
The webinar may also give some examples on how nonprofits can best leverage Walmart Business+.
The event will cover the following::
Walmart Business + (https://business.walmart.com/plus) is a new shopping experience for nonprofits, schools, and local business customers that connects an exclusive online shopping experience to stores. Benefits include free delivery and shipping, a 'Spend Analytics” feature, special discounts, deals and tax-exempt shopping.
Special TechSoup offer for a free 180 days membership, and up to $150 in discounts on eligible orders.
Spark Good (walmart.com/sparkgood) is a charitable platform that enables nonprofits to receive donations directly from customers and associates.
Answers about how you can do more with Walmart!"
This presentation includes basic of PCOS their pathology and treatment and also Ayurveda correlation of PCOS and Ayurvedic line of treatment mentioned in classics.
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.
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.
Exploiting Artificial Intelligence for Empowering Researchers and Faculty, In...Dr. Vinod Kumar Kanvaria
Exploiting Artificial Intelligence for Empowering Researchers and Faculty,
International FDP on Fundamentals of Research in Social Sciences
at Integral University, Lucknow, 06.06.2024
By Dr. Vinod Kumar Kanvaria
বাংলাদেশের অর্থনৈতিক সমীক্ষা ২০২৪ [Bangladesh Economic Review 2024 Bangla.pdf] কম্পিউটার , ট্যাব ও স্মার্ট ফোন ভার্সন সহ সম্পূর্ণ বাংলা ই-বুক বা pdf বই " সুচিপত্র ...বুকমার্ক মেনু 🔖 ও হাইপার লিংক মেনু 📝👆 যুক্ত ..
আমাদের সবার জন্য খুব খুব গুরুত্বপূর্ণ একটি বই ..বিসিএস, ব্যাংক, ইউনিভার্সিটি ভর্তি ও যে কোন প্রতিযোগিতা মূলক পরীক্ষার জন্য এর খুব ইম্পরট্যান্ট একটি বিষয় ...তাছাড়া বাংলাদেশের সাম্প্রতিক যে কোন ডাটা বা তথ্য এই বইতে পাবেন ...
তাই একজন নাগরিক হিসাবে এই তথ্য গুলো আপনার জানা প্রয়োজন ...।
বিসিএস ও ব্যাংক এর লিখিত পরীক্ষা ...+এছাড়া মাধ্যমিক ও উচ্চমাধ্যমিকের স্টুডেন্টদের জন্য অনেক কাজে আসবে ...
বাংলাদেশ অর্থনৈতিক সমীক্ষা (Economic Review) ২০২৪ UJS App.pdf
instrumental cha 4
1. CHAPTER FOUR
4. HIGH-PERFORMANCE LIQUID
CHROMATOGRAPHY (HPLC)
Although gas chromatography is widely used, it is limited
to samples that are thermally stable and not easily
volatilized.
Nonvolatile samples, such as peptides and carbohydrates,
can be analyzed by GC, but only after they have been
made more volatile by a suitable chemical derivatization.
For this reason, the various techniques included within
the general scope of liquid chromatography are among the
most commonly used separation techniques.
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2. Liquid chromatography is a separation technique which uses liquid as
mobile phase and that involves the placement (injection) of a small
volume of liquid sample into a tube packed with porous particles
(stationary phase) where individual components of the sample are
transported along the packed tube (column) by a liquid moved by
gravity.
It includes all planar chromatographic techniques.
The components of the sample are separated from one another by
the column packing that involves various chemical and/or physical
interactions between their molecules and the packing particles.
The separated components are collected at the exit of this column
and identified by an external measurement technique, such as a
spectrophotometer that measures the intensity of the color, or by
another device that can measure their amount.
HPLC is among the most popular liquid chromatographic techniques.
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3. High-performance liquid chromatography, HPLC, is a type
of chromatography that employs a liquid mobile phase and
a very finely divided stationary phase.
To obtain satisfactory flow rates, the liquid must be
pressurized to several hundred or more pounds per square
inch.
Based on its usage of high pressure for pushing the mobile
phase as well as sample components it is sometimes
called as high pressure liquid chromatography.
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4. 4.1. Principles of HPLC
In high-performance liquid chromatography (HPLC), a liquid sample,
or a solid sample is dissolved in a suitable solvent and carried through
a chromatographic column by a liquid mobile phase.
It is a technique for the separation of components of mixtures by
differential migration through a column containing a microparticulate
solid or liquid, which is coated on the wall of column, stationary
phase.
Solutes are transported through the column by a pressurized flow of
liquid mobile phase, and are detected as they are eluted.
Separation is determined by solute/stationary-phase interactions,
including liquid–solid adsorption, liquid–liquid partitioning, ion
exchange and size exclusion, and by solute/mobile-phase
interactions. In each case, however, the basic instrumentation is
essentially the same except nature of packing of column.
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5. 4.2. Instruments for HPLC
A common high-performance liquid chromatography
consists of the following five major components:
Solvent delivery system
Sample injection valve
Column
Detection and recording system
Microcomputer with control and data-processing software
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6. Figure 4.1. Schematic diagram of a high-performance liquid chromatograph (HPLC)
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7. A. Mobile Phases
The mobile phase or eluent, is either a single solvent or a blend of two or
more having the appropriate eluting power and resolution for the sample
components.
These are determined by its overall polarity, the polarity of the stationary
phase and the nature of the sample components
Unlike a GC carrier gas, which plays no part in chromatographic retention
and selectivity, the composition of an HPLC mobile phase is crucial in both
respects.
For normal-phase separations (stationary phase more polar than mobile
phase), eluting power increases with increasing solvent polarity, whilst for
reversed-phase separations (stationary phase less polar than mobile
phase), eluting power decreases with increasing solvent polarity.
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8. Choosing Mobile phase:
It ranges from a nonpolar liquid to aqueous buffers mixed
with an organic solvent.
The mobile phases are aqueous solutions containing
methanol, water-miscible organic solvents and ionic
species, in the form of a buffer.
Solvent strength and selectivity are determined by kind
and concentration of added ingredients and ions in this
phase which compete with analyte ions for the active site
in the packing.
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9. Properties of the mobile phase must be
Dissolve the sample.
Have a strong solvent strength leads to reasonable retention times.
Interact with solutes in such a way that leads to selectivity.
Beyond these criteria selection of mobile phase depends on the
polarity index, which is a quantitative measure of solvents polarity.
Table 4.1. provides the polarity index, P’, of several commonly used
mobile phases, in which large values of P’ correspond to more polar
solvents. Mobile phase of intermediate polarity can be fashioned by
mixing two or more solvents in table 4.1.
For example, a binary mobile phase made by combining solvents A
and B has the polarity index P’AB, of
P’AB = P’AῳA+ P’B ῳB
Where P’A and P’B are polarity indices of solvent A and B, and ῳA and
ῳB are the volume fractions of the two solvents, respectively.
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10. Isocratic Versus Gradient elution
Isocratic elution
mobile phase polarity stays constant throughout
elution process.
use of a constant mobile phase composition to elute
solutes.
Gradient Elutions
mobile phase composition varies throughout elution
time.
mobile phase polarity varies throughout elution process
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11. The elution order of solutes in HPLC is governed by
polarity.
In a normal-phase separation the least polar solute
spends proportionally less time in the polar stationary
phase and is the first solute to elute from the column.
Retention times are controlled by selecting the mobile
phase, with a less polar mobile phase leading to longer
retention times
If, for example, a separation is poor because the solutes
are eluting too quickly, switching to a less polar mobile
phase leads to longer retention times and more
opportunity for an acceptable separation
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12. When two solutes are adequately resolved, switching to a
more polar mobile phase may provide an acceptable
separation with a shorter analysis time.
In a reverse-phase separation the order of elution is
reversed, with the most polar solute being the first to
elute.
Increasing the polarity of the mobile phase leads to
longer retention times, whereas shorter retention times
require a mobile phase of lower polarity.
An eluotropic series of solvents, which lists them in order
of increasing polarity, is a useful guide to solvent selection
for HPLC separations.
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13. Table 4.1. An eluotropic series of solvents for HPLC
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14. B. HPLC Columns
HPLC typically includes two columns which are an
analytical column responsible for the separation and a
guard column.
The guard column is placed before the analytical column,
protecting it from contamination.
Guard Columns: Two problems tend to shorten the
lifetime of an analytical column.
First, solutes binding irreversibly to the stationary phase
degrade the column’s performance by decreasing the
available stationary phase.
Second, particulate material injected with the sample
may clog the analytical column.
To minimize these problems, a guard column is placed
before the analytical column.
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15. It is also used to remove particulate matter and
contamination, protect analytical column and control the
temperature to be: < 150 °C.
Guard columns usually contain the same particulate
packing material and stationary phase as the analytical
column, but are significantly shorter and less expensive;
a length of 7.5 mm and a cost one-tenth of that for the
corresponding analytical column.
Because they are intended to be sacrificial, guard
columns are replaced regularly.
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16. Analytical Columns: This is a column that true separation
is takes place.
The most commonly used columns for HPLC are
constructed from stainless steel with internal diameters
between 2.1 mm and 4.6 mm, and lengths ranging from
approximately 30 mm to 300 mm.
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17. Stationary Phases
In liquid–liquid chromatography the stationary phase is a
liquid film coated on a packing material consisting of 3–10
µm porous silica particles.
HPLC stationary phases are predominantly chemically-
modified silicas, unmodified silica or cross-linked co-
polymers of styrene and divinyl benzene.
The surface of silica is polar and slightly acidic due to the
presence of silanol (Si-OH) groups.
It can be chemically modified with reagents, such as
chlorosilanes, which react with the silanol groups
replacing them with a range of other functionalities.
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18. The resulting bonded phases, which are hydrolytically
stable through the formation of siloxane (Si-O-Si-R)
bonds, have different chromatographic characteristics
and selectivities to unmodified silica.
The properties of a stationary phase are determined by the nature of
the organosilane’s alkyl group.
If R is a polar functional group, then the stationary phase will be
polar. Examples of polar stationary phases include those for which R
contains a cyano (–C2H4CN), diol (–C3H6OCH2CHOHCH2OH), or amino (–
C3H6NH2) functional group.
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19. The combination of a polar stationary phase and a
nonpolar mobile phase is called normal-phase
chromatography
In reverse-phase chromatography, which is the more
commonly encountered form of HPLC, the stationary
phase is nonpolar and the mobile phase is polar.
The most common nonpolar stationary phases use an
organochlorosilane for which the R group is an n-octyl (C8)
or n-octyldecyl (C18) hydrocarbon chain.
Most reverse phase separations are carried out using a
buffered aqueous solution as a polar mobile phase.
Liquid chromatography using a nonpolar stationary phase
and a polar mobile phase is called reverse-phase
chromatography.
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20. Table 4.2. Stationary phases for HPLC
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21. C. HPLC Detectors
Detection systems are used to detect solutes.
Unlike gas chromatography, there is no universal
detector, a detector which responds to most types of
solutes.
Many types of detectors have been investigated, and the
most widely used detectors for HPLC are:
Absorbance (UV with Filters, UV with Monochromators and
Photo-Diode Array), IR Absorbance, Fluorescence,
Refractive-Index, Evaporative Light Scattering Detector
(ELSD), Electrochemical, Mass-Spectrometric detector.
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22. Ideally, detectors should have the following characteristics:
a rapid and reproducible response to solutes that is
independent on flow rate;
high sensitivity, i.e. able to detect very low levels of
solutes;
Good stability in operation;
a signal directly proportional to solute concentration o
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23. insensitivity to changes in temperature and flow rate;
high reliability and ease of use and
non-destructive for the sample.
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24. Table 4.3. Common Liquid Chromatographic Detectors
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25. 4.3. Modes of HPLC
Modes of HPLC are defined by the nature of the stationary
phase, the mechanism of interaction with solutes, and the
relative polarities of the stationary and mobile phases.
Almost any type of solute mixture can be separated by
HPLC because of the wide range of stationary phases
available, and the additional selectivity provided by
varying the mobile phase composition.
Both normal-and reversed phase separations are possible,
depending on the relative polarities of the two phases.
Although these are sometimes referred to as modes of
HPLC, the nature of the stationary phase and/or the
solute sorption mechanism provide a more specific means
of classification, and modes based on these and the types
of solutes to which they are best suited are summarized
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26. 4.3.1. Adsorption HPLC
In adsorption chromatography, the stationary phase is the
surface of a finely divided polar solid.
With such a packing, the analyte competes with the
mobile phase for sites on the surface of the packing, and
retention is the result of adsorption forces.
Separations are usually normal-phase with a silica gel
stationary phase and a mobile phase of a nonpolar solvent
blended with additions of a more polar solvent to adjust
the overall polarity or eluting power, e.g. n-hexane +
dichloromethane or di-ethyl ether.
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27. Solutes are retained by surface adsorption; they compete with
solvent molecules for active silanol sites (Si-OH), and are eluted in
order of increasing polarity.
In general, the polarities of common organic functional groups in
increasing order are:
Aliphatic hydrocarbons < olefins < aromatic hydrocarbons < halides <
sulfides < ethers < nitro compounds < esters = aldehydes = ketones <
alcohols = amines < sulfones < sulfoxides < amides < carboxylic acids <
water.
This mode is not used extensively, but is suitable for mixtures of
structural isomers and solutes with differing functional groups.
Members of a homologous series cannot be separated by adsorption
chromatography because the nonpolar parts of a solute do not
interact with the polar adsorbent surface.
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28. 4.3.2. Partition HPLC (Partition chromatography)
Partition chromatography is the most widely used type of HPLC.
In partition chromatography, liquids are used as the stationary phase
which is immiscible with the liquid mobile phase.
The stationary phase may be adsorbed on the surface of a silica or
alumina support materials or chemically bonded with silica.
Partition chromatography can be classified based on two criteria:
A. the means by which the stationary phase is attached with support
materials
B. the polarities of the mobile and stationary phases
A. Based on the first criteria partition chromatography can be divided
into two:
1. Liquid-liquid partition chromatography
2. Modified or bonded phase partition chromatography (liquid-
bonded phase chromatography
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29. In Liquid-liquid partition chromatography, the liquid
stationary phase is adsorbed on the surface of the support
materials, silica or alumina.
In Modified partition or bonded-phase chromatography
(BPC) that includes, most HPLC stationary phases that are
chemically-modified silicas, or bonded phases, by far the
most widely used being those modified with nonpolar
hydrocarbons.
The liquid stationary phase is chemically bonded with
silanol group.
The solute sorption mechanism is described as modified
partition, because, although the bonded hydrocarbons are
not true liquids, organic solvent molecules from the
mobile phase form a liquid layer on the surface
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30. The preparation of bonded phase stationary phase
involves the following step:
Si-OH is the silanol group i.e. support material.
The R group will determine the polarity of the stationary
phase.
The most popular phase is octadecyl (C18 or ODS), and
most separations are reversed-phase, the mobile phase
being a blend of methanol or acetonitrile with water or
an aqueous buffer.
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31. B. Based on the polarities of the mobile and stationary phase
Partition chromatography can be divided into two based
on the polarities of the mobile and stationary phase.
Normal phase partition chromatography
Reversed phase partition chromatography
In normal phase partition chromatography, the mobile
phase is nonpolar and the stationary phase is polar.
Nonpolar solutes have weak interaction and polar solutes
have strong interaction with stationary phase.
The elution order is that nonpolar solutes elute first and
polar solutes elute late.
Gradient elution can be carried out by increasing the
polarity of the mobile phase.
.
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32. The elution order is that nonpolar solutes elute first and
polar solutes elute late.
Gradient elution can be carried out by increasing the
polarity of the mobile phase.
In reversed phase partition chromatography, the mobile
phase is polar and stationary phase is nonpolar.
Polar solutes have weak interaction with the stationary
phase while nonpolar solutes have strong interaction with
stationary phase.
The elution order is that polar solutes elute first and
nonpolar solutes elute late.
Gradient elution can be carried out by decreasing the
polarity of the mobile phase.
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33. 4.3.3 Ion-exchange chromatography
Ion-exchange resins are used as stationary phases for
liquid chromatography (ion-exchange chromatography) to
separate charged species.
Ion-exchange chromatography (IEC) stationary phases
for the separation of mixtures of ionic solutes, such as
inorganic cations and anions, amino acids and proteins,
are based on either microparticulate ion-exchange resins,
which are cross-linked co-polymers of styrene and divinyl
benzene, or on bonded phase silicas.
Both types have either sulfonic acid cation-exchange sites
(-SO3
-H+) or quaternary ammonium anion-exchange sites (-
N+R3OH-) incorporated into their structures.
In most cases, conductivity measurements are used to
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34. In this column or chromatography type the sample
components are separated based upon attractive ionic
forces between molecules carrying charged groups of
opposite charge to those charges on the stationary phase.
Separations are made between a polar mobile liquid,
usually water containing salts or small amounts of
alcohols, and a stationary phase containing either
acidic or basic fixed sites.
Ion exchange chromatography involves the separation
of ionizable molecules based on their total charge
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35. This technique enables the separation of similar types of
molecules that would be difficult to separate by other
techniques because the charge carried by the molecule of
interest can be readily manipulated by changing buffer pH.
Strong ion exchangers are often preferred resins for many
applications because their performance is unaffected by
pH.
However, weak ion exchangers can be powerful separation
tools in cases where strong ion exchangers fail because
the selectivities of weak and strong ion exchangers often
differ .
Depending on the pH of their environment, proteins may
carry a net positive charge, a net negative charge, or no
charge.
The pH at which a molecule has no net charge is called its
isoelectric point, or pI.
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36. Figure 4.2. Charges in exchanger resign and solutes of interest
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37. The counter ions to these fixed charges are mobile and
can be displaced by ions that compete more favorably for
the exchange sites.
Ion-exchange resigns are divided into four categories:
strong acid cation exchangers, weak acid cation
exchangers, strong base anion exchangers and weak base
anion exchangers.
The ion-exchange reaction of a monovalent cation, M+, at
a strong acid exchanger site is:
-SO3
-H+
(s) + M+
(aq) -SO3
- M+
(s) + H+
(s)
Al3+ > Ba2+ > Pb2+ > Ca2+ > Ni2+ > Cd2+ > Cu2+ > Co2+ > Zn2+ > Mg2+ > Ag+ > K+ > NH4
+ > Na+ > H+ > Li+
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38. Note that highly charged ions bind more strongly than ions
of lower charge.
Within a group of ions of similar charge, those ions with
small hydrated radius or those that are more polarizable
bind more strongly.
For a strong base anion exchanger the general order is:
SO4
2- > I- > HSO4
- > NO3
- > Br- > NO2
- > Cl- > HCO3
- > CH3COO-
> OH- > F-
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39. 4,3.4.Molecular (Size) Exclusion Chromatography
Size- exclusion, or gel, chromatography is the newest of
the liquid chromatographic procedures.
This is suitable for mixtures of solutes with relative
molecular masses (RMM) in the range 102–108 Da.
Stationary phases are either microparticulate cross-linked
co-polymers of styrene and divinyl benzene with a narrow
distribution of pore sizes, or controlled-porosity silica
gels, usually end-capped with a short alkyl chain reagent
to prevent adsorptive interactions with solutes.
Exclusion is not a true sorption mechanism because
solutes do not interact with the stationary phase
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40. They can be divided into three groups:
Those larger than the largest pores are excluded
completely, and are eluted in the same volume as the
interstitial space in the column, Vo.
Those smaller than the smallest pores, can diffuse
throughout the entire network and are eluted in a total
volume, Vtot.
Those of an intermediate size separate according to the
extent to which they diffuse through the network of
pores, of volume Vp and are eluted in volumes between Vo
and Vtot.
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41. In size- exclusion chromatography, fractionation is based on size of
the molecules.
There are two commonly known packings for size- exclusion
chromatography:
Gel filtration is a type of size- exclusion chromatography in which
the packing is hydrophilic and is used to separate polar species.
Gel permeation is a type of size- exclusion chromatography in which
the packing is hydrophobic. It is used to separate nonpolar species.
In size exclusion the HPLC column is consisted of substances which
have controlled pore sizes and is able to be filtered in an ordinarily
phase according to its molecular size.
Small molecules penetrate into the pores within the packing while
larger molecules only partially penetrate the pores (Figure 4.3).
The large molecules elute before the smaller molecules.
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42. Size exclusion chromatography (SEC), also called gel
filtration chromatography, separates molecules based on
their sizes.
SEC resins are gels that contain beads with a known pore
size.
When dissolved molecules of various sizes flow into
the column, smaller dissolved molecules flow more
slowly through the column because they penetrate
deep into the pores, whereas large dissolved molecules
flow quickly through the column because they do not
enter the pores.
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43. Figure 4.3. Working principle of molecular (size) exclusion chromatography
SEC is of particular value in characterizing polymer mixtures and in separating biological
macromolecules such as peptides and proteins. It is also used for preliminary separations prior
to further analysis by other more efficient modes of HPLC.
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44. To summarize, the chromatographer will choose the best
combination of a mobile phase and particle stationary
phase with appropriately opposite polarities.
Then, as the sample analytes move through the column,
the rule like attracts like will determine which analytes slow
down and which proceed at a faster speed.
After selection of an appropriate mode, column and detector
for the solutes to be separated, the composition of the mobile
phase must be optimized to achieve the required separation.
A trial and error approach or a computer aided investigation
can be adopted.
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45. Summary of major components of HPLC and their function
1. Pump: The role of the pump is to force a liquid (called the mobile phase)
through the liquid chromatograph at a specific flow rate, expressed in milliliters
per min (mL/min).
Normal flow rates in HPLC are in the 1- to 2-mL/min range.
Typical pumps can reach pressures in the range of 6000-9000 psi (400- to 600-
bar).
During the chromatographic experiment, a pump can deliver a constant
mobile phase composition (isocratic) or an increasing mobile phase
composition (gradient).
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46. 2. Injector: The injector serves to introduce the liquid sample into the flow
stream of the mobile phase.
Typical sample volumes are 5- to 20-microliters (µL).
The injector must also be able to withstand the high pressures of the liquid
system.
An autosampler is the automatic version for when the user has many samples
to analyze or when manual injection is not practical
3. Column: Considered the “heart of the chromatograph” the column’s
stationary phase separates the sample components of interest using various
physical and chemical parameters.
The small particles inside the column are what cause the high backpressure at
normal flow rates.
The pump must push hard to move the mobile phase through the column and
this resistance causes a high pressure within the chromatograph.
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47. 4. Detector: The detector can see (detect) the individual
molecules that come out (elute) from the column.
A detector serves to measure the amount of those
molecules so that the chemist can quantitatively analyze
the sample components.
The detector provides an output to a recorder or
computer that results in the liquid chromatogram (i.e.,
the graph of the detector response).
5. Computer: Frequently called the data system, the
computer not only controls all the modules of the HPLC
instrument but it takes the signal from the detector and
uses it to determine the time of elution (retention time)
of the sample components (qualitative analysis) and the
amount of sample (quantitative analysis).
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48. Application of HPLC
High performance liquid chromatography is now one of
the most powerful tools in analytical chemistry.
It has the ability to separate, identify, and quantitate the
compounds that are present in any sample that can be
dissolved in a liquid.
Today, compounds in trace concentrations as low as parts
per trillion (ppt) may easily be identified.
Fields in which HPLC is used
HPLC can be, and has been, applied to just about any
sample, such as pharmaceuticals, food, cosmetics,
environmental matrices, forensic samples, and industrial
chemicals.
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49. Biogenic substances like; Sugars, lipids, nucleic acids,
amino acids, proteins, peptides, steroids, amines, etc.
Medical products for drugs, antibiotics, etc.
Food products like; vitamins, food additives, sugars,
organic acids, amino acids, etc.
Environmental samples; for analysis of inorganic ions,
hazardous organic substances, etc.
Organic industrial products like; synthetic polymers,
additives, surfactants, etc.
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