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.
This document provides an overview of high performance liquid chromatography (HPLC). It describes the key components of an HPLC system, including the pump, injector, separation column, and detector. It explains how the different components of a mixture are separated through their interaction with the mobile and stationary phases in the column. The document also discusses various types of columns, detectors, and applications of HPLC in chemistry, biochemistry, and quality control.
DEVELOPMENT AND VALIDATION OF AN RP-HPLC METHOD FOR SIMULTANEOUS DETERMINATIO...rahul ampati
This document describes the development and validation of an RP-HPLC method for the simultaneous determination of ramipril and amlodipine in tablets. The method utilizes a gradient elution with a C18 column, mobile phase of acetonitrile and sodium perchlorate buffer, and UV detection. The method was validated per ICH guidelines and showed good linearity, accuracy, precision, specificity and robustness. Forced degradation studies demonstrated the method can separate ramipril, amlodipine and their degradation products. The method was successfully applied to determine the content of ramipril and amlodipine in three tablet batches.
This document provides an overview of high performance liquid chromatography (HPLC). It discusses the principle of HPLC, which separates mixtures by forcing a mobile liquid phase through a column containing a stationary phase. The document describes the main components of an HPLC instrument, including the mobile phase reservoirs, pump, sample injection system, column, detector, and recorder. It provides details on each component, such as the types of pumps, columns, detectors commonly used. The document concludes by listing references used to compile the information presented.
This document summarizes the uses of high-performance liquid chromatography (HPLC) in pharmaceutical sciences. HPLC is widely used in pharmaceutical industries for drug discovery, development, and manufacturing. It is used for separation, identification, and quantification of compounds. Some key applications of HPLC include drug purity testing, stability studies, impurity profiling, and analytical method development and validation. HPLC plays a vital role throughout the drug development process from early research to quality control of final drug products.
Chiral HPLC uses an asymmetric chromatographic system to separate enantiomers. There are three main methods: using a chiral mobile phase, chiral liquid stationary phase, or chiral solid stationary phase. The chiral species forms diastereomeric complexes with the enantiomers, allowing separation. Indirect separation is also possible by derivatizing the enantiomers to form diastereomers, which can be separated on a non-chiral system. Common stationary phases include proteins, Pirkle compounds, cellulose/amylose derivatives, macrocyclic glycopeptides, and cyclodextrins. Applications include separating drug enantiomers and fullerenes.
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.
This document provides an overview of high performance liquid chromatography (HPLC). It describes the key components of an HPLC system, including the pump, injector, separation column, and detector. It explains how the different components of a mixture are separated through their interaction with the mobile and stationary phases in the column. The document also discusses various types of columns, detectors, and applications of HPLC in chemistry, biochemistry, and quality control.
DEVELOPMENT AND VALIDATION OF AN RP-HPLC METHOD FOR SIMULTANEOUS DETERMINATIO...rahul ampati
This document describes the development and validation of an RP-HPLC method for the simultaneous determination of ramipril and amlodipine in tablets. The method utilizes a gradient elution with a C18 column, mobile phase of acetonitrile and sodium perchlorate buffer, and UV detection. The method was validated per ICH guidelines and showed good linearity, accuracy, precision, specificity and robustness. Forced degradation studies demonstrated the method can separate ramipril, amlodipine and their degradation products. The method was successfully applied to determine the content of ramipril and amlodipine in three tablet batches.
This document provides an overview of high performance liquid chromatography (HPLC). It discusses the principle of HPLC, which separates mixtures by forcing a mobile liquid phase through a column containing a stationary phase. The document describes the main components of an HPLC instrument, including the mobile phase reservoirs, pump, sample injection system, column, detector, and recorder. It provides details on each component, such as the types of pumps, columns, detectors commonly used. The document concludes by listing references used to compile the information presented.
This document summarizes the uses of high-performance liquid chromatography (HPLC) in pharmaceutical sciences. HPLC is widely used in pharmaceutical industries for drug discovery, development, and manufacturing. It is used for separation, identification, and quantification of compounds. Some key applications of HPLC include drug purity testing, stability studies, impurity profiling, and analytical method development and validation. HPLC plays a vital role throughout the drug development process from early research to quality control of final drug products.
Chiral HPLC uses an asymmetric chromatographic system to separate enantiomers. There are three main methods: using a chiral mobile phase, chiral liquid stationary phase, or chiral solid stationary phase. The chiral species forms diastereomeric complexes with the enantiomers, allowing separation. Indirect separation is also possible by derivatizing the enantiomers to form diastereomers, which can be separated on a non-chiral system. Common stationary phases include proteins, Pirkle compounds, cellulose/amylose derivatives, macrocyclic glycopeptides, and cyclodextrins. Applications include separating drug enantiomers and fullerenes.
This document describes the development and validation of an RP-HPLC method for the simultaneous estimation of anti-tuberculosis drugs isoniazid, rifampicin, pyrazinamide, and ethambutol in human plasma. It provides background on tuberculosis and the common drugs used to treat it. The document reviews several literature methods for analyzing these drugs and discusses the drug profiles. It states that the objective is to develop a sensitive analytical method to quantitatively determine the drugs and metabolites in biological fluids to evaluate pharmacokinetics and pharmacodynamics.
This document discusses high performance thin layer chromatography (HPTLC), which is an automated form of thin layer chromatography that allows for efficient separation of samples in a short time. HPTLC has several advantages over other chromatographic methods, including its ability to simultaneously process multiple samples, lower analysis times and costs, simple sample preparation, and use of a scanner for detection and documentation of results. The document outlines the basic principles and steps of HPTLC, including sample preparation, selecting the chromatographic layer, pre-washing and conditioning plates, applying samples, mobile phase selection and development, and detection of spots using scanning.
HPLC is a type of liquid chromatography that uses high pressure to force a sample through a column packed with porous particles. This allows for faster separations compared to traditional chromatography. Key parameters in HPLC include retention time, which measures how long components spend in the column; capacity factor k', which is a ratio of time spent in the stationary vs mobile phase; selectivity factor α, which is the ratio of k' values and describes separation of adjacent peaks; and theoretical plates N, which estimates column efficiency based on peak widths and retention times. Optimizing these parameters can improve resolution of components in the mixture.
UPLC refers to ultra performance liquid chromatography. It enhances speed, resolution, and sensitivity compared to HPLC by using particles less than 2μm in diameter. UPLC operates at very high pressures and provides better separation and faster analysis. It has applications in determining pesticides, analyzing pharmaceutical impurities, and more. UPLC offers advantages like reduced run time and solvent usage but also has disadvantages like higher back pressures reducing column life.
General considerations and method development in ce,ChowdaryPavani
This document provides an overview of capillary electrophoresis (CE). It defines CE, describes its principle and instrumentation. CE involves separating components of a sample based on their differential rate of migration in an applied electric field. Key points covered include electrophoretic mobility, electroosmotic flow, sample introduction techniques, and common applications such as protein, DNA and pharmaceutical analysis. CE provides high resolution separations due to its small capillary diameter and long separation length.
This document summarizes a presentation on chiral separations by HPLC. It discusses chirality and why it is important for drugs. It describes different types of chiral stationary phases used for chiral chromatography, including polysaccharide and immobilized polysaccharide phases. The document provides examples of method development on these phases under normal phase, polar, and reversed phase conditions. It discusses factors that influence separations like solvents, additives, and injection solvents. The document demonstrates chiral separations of several drug compounds under various chromatographic conditions.
High performance liquid chromatography stationary phases can separate polar and nonpolar compounds using reversed-phase or normal phase columns. Aqueous normal phase chromatography uses silicon-hydride stationary phases to retain both polar and nonpolar analytes in a single isocratic run through dual retention mechanisms. This distinguishes it from other chromatographic methods that can only separate compounds of specific polarities.
HPLC
Chromatography
Mobile Phase & Stationary Phase
CLASSIFICATION OF CHROMATOGRAPHY
Characteristics of HPLC
Purpose
Superiority of HPLC
TYPES OF HPLC TECHNIQYES
Principle
PHASING SYSTEM & (normal vs reversed phase)
INSTRUMENTATION
Flow diagram of HPLC instrument
Advantages of HPLC
high performance liquid chromatoghraphy (HPLC)ummiabah
This document provides an overview of high performance liquid chromatography (HPLC). It discusses what HPLC is, types of separations, columns and stationary phases, mobile phases and their role, injection, and detection methods. It also covers variations like ion chromatography and size exclusion chromatography. The key aspects of HPLC covered are selecting the appropriate type of separation, column, mobile phase, and detector for the analytes of interest and optimizing the separation.
This document provides an overview of high performance liquid chromatography (HPLC). It discusses various modes of separation in HPLC including normal phase, reversed phase, ion exchange, and size exclusion chromatography. The document also describes HPLC instrumentation components such as solvent delivery systems, pumps, sample injection systems, chromatographic columns, and detectors. It provides details on the development, optimization, and validation of HPLC methods.
The document provides an overview of high performance liquid chromatography (HPLC), including its components, applications, types, and instrumentation. HPLC is an analytical technique used to separate components of a mixture using various chemical interactions between the analyte and chromatography column. It provides enhanced separations in a short time and is commonly used for pharmaceutical, environmental, clinical, and food analyses. The key components of an HPLC system include the solvent system, pumping system, sample injector, column, and detectors.
UPLC provides faster, more sensitive chromatographic separations compared to HPLC. It works by using smaller particle sizes (<2.5um) in the column packing which allows for higher pressure and flow rates based on the van Deemter equation. This provides benefits like reduced run times, decreased sample volume needs, and improved resolution. However, it also requires more robust instrumentation to handle the increased pressures and columns have reduced lifespan. UPLC has applications in fields like pharmaceutical analysis, metabolomics, and impurity profiling due to its enhanced resolution and sensitivity capabilities.
This document provides an overview of high performance liquid chromatography (HPLC). It discusses the basic principles of chromatographic separation and defines key terms like retention time and resolution. It also describes different HPLC techniques including normal phase, reversed phase, ion exchange, size exclusion, and ion-pair chromatography. The document outlines the typical instrumentation used in HPLC including the pump, injector, columns, detectors, and data collection systems. It provides details on how each component works and its role in the chromatography process.
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.
HPLC is a form of column chromatography that separates compounds based on their polarity and interaction with the stationary phase. It utilizes a pump to push the mobile phase and analytes through a column under high pressure. Various detectors can then provide the retention time of analytes as they exit the column. Key aspects of HPLC include the types of columns, mobile phases, and detectors used, which are selected based on the compounds being analyzed. HPLC is commonly used to analyze biological, pharmaceutical, environmental, and forensic samples.
This document provides an overview of high performance liquid chromatography (HPLC). It begins by defining HPLC and explaining that it uses high pressure to pump the mobile phase, yielding faster separation than traditional column chromatography. The document then discusses the basic principles of chromatography and liquid chromatography. It provides details on the types of HPLC based on mode of separation, principle of separation, elution technique, scale of operation, and type of analysis. The key components of an HPLC instrument are described including the solvent reservoir, pump, injector, column, detectors, and data recording system. Various columns, stationary phases, and pumps used in HPLC are also outlined.
UPLC is an improved version of HPLC that provides higher resolution, speed, and sensitivity. It uses smaller particle sizes of 1.7μm in its columns compared to 4μm in HPLC columns. This allows for faster separations using shorter columns or higher flow rates. UPLC also uses less solvent and reduces analysis times. It has various applications like analysis of natural products, metabolites, bioanalysis, ADME screening, dissolution testing, method development and validation, forced degradation studies, impurity profiling, and analysis in manufacturing and quality control.
Hplc instrumentation in detail (Practical) Hplc pump inj_columnPratikShinde120
This document discusses HPLC instrumentation and techniques. It describes the key components of an HPLC system including the solvent delivery system, pumps, sample introduction methods, and detectors. For solvent delivery, it explains the mobile phase reservoirs, degassing, and tubing used. It discusses different types of pumps like reciprocating, syringe, and dual piston pumps. For sample introduction, it covers manual injection methods like septum and valve, as well as automated injection. It also provides details on various detectors like UV-Vis, fluorescence, refractive index, and conductivity.
In this slide contains types of HPLC Columns, Plate theory and Van Deemter Equation.
Presented by : Malarvannan.M (Department of pharmaceutical analysis).
RIPER,anantpur.
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.
High Performance Liquid Chromatography..Somnath Patil
1) High Performance Liquid Chromatography (HPLC) is a technique used to separate components of a mixture. It uses differences in how fast components move through a column containing a stationary phase under the influence of a liquid mobile phase.
2) There are different types of liquid chromatography including normal phase, reverse phase, ion-exchange, size-exclusion, and adsorption chromatography which separate components based on properties like polarity, ionic charge, and molecular size.
3) HPLC instruments consist of pumps to push the mobile phase through the column at high pressure, injectors, columns, detectors, and data systems. Reversed phase is most commonly used for pharmaceutical analysis using C18 columns.
This document describes the development and validation of an RP-HPLC method for the simultaneous estimation of anti-tuberculosis drugs isoniazid, rifampicin, pyrazinamide, and ethambutol in human plasma. It provides background on tuberculosis and the common drugs used to treat it. The document reviews several literature methods for analyzing these drugs and discusses the drug profiles. It states that the objective is to develop a sensitive analytical method to quantitatively determine the drugs and metabolites in biological fluids to evaluate pharmacokinetics and pharmacodynamics.
This document discusses high performance thin layer chromatography (HPTLC), which is an automated form of thin layer chromatography that allows for efficient separation of samples in a short time. HPTLC has several advantages over other chromatographic methods, including its ability to simultaneously process multiple samples, lower analysis times and costs, simple sample preparation, and use of a scanner for detection and documentation of results. The document outlines the basic principles and steps of HPTLC, including sample preparation, selecting the chromatographic layer, pre-washing and conditioning plates, applying samples, mobile phase selection and development, and detection of spots using scanning.
HPLC is a type of liquid chromatography that uses high pressure to force a sample through a column packed with porous particles. This allows for faster separations compared to traditional chromatography. Key parameters in HPLC include retention time, which measures how long components spend in the column; capacity factor k', which is a ratio of time spent in the stationary vs mobile phase; selectivity factor α, which is the ratio of k' values and describes separation of adjacent peaks; and theoretical plates N, which estimates column efficiency based on peak widths and retention times. Optimizing these parameters can improve resolution of components in the mixture.
UPLC refers to ultra performance liquid chromatography. It enhances speed, resolution, and sensitivity compared to HPLC by using particles less than 2μm in diameter. UPLC operates at very high pressures and provides better separation and faster analysis. It has applications in determining pesticides, analyzing pharmaceutical impurities, and more. UPLC offers advantages like reduced run time and solvent usage but also has disadvantages like higher back pressures reducing column life.
General considerations and method development in ce,ChowdaryPavani
This document provides an overview of capillary electrophoresis (CE). It defines CE, describes its principle and instrumentation. CE involves separating components of a sample based on their differential rate of migration in an applied electric field. Key points covered include electrophoretic mobility, electroosmotic flow, sample introduction techniques, and common applications such as protein, DNA and pharmaceutical analysis. CE provides high resolution separations due to its small capillary diameter and long separation length.
This document summarizes a presentation on chiral separations by HPLC. It discusses chirality and why it is important for drugs. It describes different types of chiral stationary phases used for chiral chromatography, including polysaccharide and immobilized polysaccharide phases. The document provides examples of method development on these phases under normal phase, polar, and reversed phase conditions. It discusses factors that influence separations like solvents, additives, and injection solvents. The document demonstrates chiral separations of several drug compounds under various chromatographic conditions.
High performance liquid chromatography stationary phases can separate polar and nonpolar compounds using reversed-phase or normal phase columns. Aqueous normal phase chromatography uses silicon-hydride stationary phases to retain both polar and nonpolar analytes in a single isocratic run through dual retention mechanisms. This distinguishes it from other chromatographic methods that can only separate compounds of specific polarities.
HPLC
Chromatography
Mobile Phase & Stationary Phase
CLASSIFICATION OF CHROMATOGRAPHY
Characteristics of HPLC
Purpose
Superiority of HPLC
TYPES OF HPLC TECHNIQYES
Principle
PHASING SYSTEM & (normal vs reversed phase)
INSTRUMENTATION
Flow diagram of HPLC instrument
Advantages of HPLC
high performance liquid chromatoghraphy (HPLC)ummiabah
This document provides an overview of high performance liquid chromatography (HPLC). It discusses what HPLC is, types of separations, columns and stationary phases, mobile phases and their role, injection, and detection methods. It also covers variations like ion chromatography and size exclusion chromatography. The key aspects of HPLC covered are selecting the appropriate type of separation, column, mobile phase, and detector for the analytes of interest and optimizing the separation.
This document provides an overview of high performance liquid chromatography (HPLC). It discusses various modes of separation in HPLC including normal phase, reversed phase, ion exchange, and size exclusion chromatography. The document also describes HPLC instrumentation components such as solvent delivery systems, pumps, sample injection systems, chromatographic columns, and detectors. It provides details on the development, optimization, and validation of HPLC methods.
The document provides an overview of high performance liquid chromatography (HPLC), including its components, applications, types, and instrumentation. HPLC is an analytical technique used to separate components of a mixture using various chemical interactions between the analyte and chromatography column. It provides enhanced separations in a short time and is commonly used for pharmaceutical, environmental, clinical, and food analyses. The key components of an HPLC system include the solvent system, pumping system, sample injector, column, and detectors.
UPLC provides faster, more sensitive chromatographic separations compared to HPLC. It works by using smaller particle sizes (<2.5um) in the column packing which allows for higher pressure and flow rates based on the van Deemter equation. This provides benefits like reduced run times, decreased sample volume needs, and improved resolution. However, it also requires more robust instrumentation to handle the increased pressures and columns have reduced lifespan. UPLC has applications in fields like pharmaceutical analysis, metabolomics, and impurity profiling due to its enhanced resolution and sensitivity capabilities.
This document provides an overview of high performance liquid chromatography (HPLC). It discusses the basic principles of chromatographic separation and defines key terms like retention time and resolution. It also describes different HPLC techniques including normal phase, reversed phase, ion exchange, size exclusion, and ion-pair chromatography. The document outlines the typical instrumentation used in HPLC including the pump, injector, columns, detectors, and data collection systems. It provides details on how each component works and its role in the chromatography process.
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.
HPLC is a form of column chromatography that separates compounds based on their polarity and interaction with the stationary phase. It utilizes a pump to push the mobile phase and analytes through a column under high pressure. Various detectors can then provide the retention time of analytes as they exit the column. Key aspects of HPLC include the types of columns, mobile phases, and detectors used, which are selected based on the compounds being analyzed. HPLC is commonly used to analyze biological, pharmaceutical, environmental, and forensic samples.
This document provides an overview of high performance liquid chromatography (HPLC). It begins by defining HPLC and explaining that it uses high pressure to pump the mobile phase, yielding faster separation than traditional column chromatography. The document then discusses the basic principles of chromatography and liquid chromatography. It provides details on the types of HPLC based on mode of separation, principle of separation, elution technique, scale of operation, and type of analysis. The key components of an HPLC instrument are described including the solvent reservoir, pump, injector, column, detectors, and data recording system. Various columns, stationary phases, and pumps used in HPLC are also outlined.
UPLC is an improved version of HPLC that provides higher resolution, speed, and sensitivity. It uses smaller particle sizes of 1.7μm in its columns compared to 4μm in HPLC columns. This allows for faster separations using shorter columns or higher flow rates. UPLC also uses less solvent and reduces analysis times. It has various applications like analysis of natural products, metabolites, bioanalysis, ADME screening, dissolution testing, method development and validation, forced degradation studies, impurity profiling, and analysis in manufacturing and quality control.
Hplc instrumentation in detail (Practical) Hplc pump inj_columnPratikShinde120
This document discusses HPLC instrumentation and techniques. It describes the key components of an HPLC system including the solvent delivery system, pumps, sample introduction methods, and detectors. For solvent delivery, it explains the mobile phase reservoirs, degassing, and tubing used. It discusses different types of pumps like reciprocating, syringe, and dual piston pumps. For sample introduction, it covers manual injection methods like septum and valve, as well as automated injection. It also provides details on various detectors like UV-Vis, fluorescence, refractive index, and conductivity.
In this slide contains types of HPLC Columns, Plate theory and Van Deemter Equation.
Presented by : Malarvannan.M (Department of pharmaceutical analysis).
RIPER,anantpur.
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.
High Performance Liquid Chromatography..Somnath Patil
1) High Performance Liquid Chromatography (HPLC) is a technique used to separate components of a mixture. It uses differences in how fast components move through a column containing a stationary phase under the influence of a liquid mobile phase.
2) There are different types of liquid chromatography including normal phase, reverse phase, ion-exchange, size-exclusion, and adsorption chromatography which separate components based on properties like polarity, ionic charge, and molecular size.
3) HPLC instruments consist of pumps to push the mobile phase through the column at high pressure, injectors, columns, detectors, and data systems. Reversed phase is most commonly used for pharmaceutical analysis using C18 columns.
New microsoft office power point presentationHARSHITHA REDDY
This document provides an overview of chromatography and high performance liquid chromatography (HPLC). It defines chromatography as a method to separate mixtures into individual components based on differences in how they move through a stationary and mobile phase. The document describes the basic principles, instrumentation, and applications of HPLC. Key components of HPLC systems discussed include pumps, injectors, columns, detectors, and various chromatography techniques like adsorption, partition, and ion exchange chromatography.
1. The document discusses high performance liquid chromatography (HPLC), including its principles, types, instrumentation, and applications. HPLC is a technique used to separate compounds in a mixture using high pressure to force the mixture through a column packed with a stationary phase.
2. The key components of an HPLC system are the solvent reservoirs, pump, injector, column, and detector. HPLC can be used for both analytical and preparative purposes to separate, purify, identify, and quantify compounds.
3. Common applications of HPLC include separation of volatile and non-volatile compounds, qualitative and quantitative analysis, and determination of retention times. Reversed phase HPLC using C18 columns is frequently utilized.
This document provides an introduction and overview of high performance liquid chromatography (HPLC). It defines HPLC as a technique used to separate mixtures of compounds through a column. The principal of HPLC is based on adsorption or partition chromatography depending on the stationary phase. The document outlines the key components of an HPLC system including the solvent reservoir, pump, injector, column, detector and recorder. It also discusses stationary and mobile phases, sample injection methods, and types of pumps used in HPLC.
The document discusses chromatography and high performance liquid chromatography (HPLC). It defines chromatography as a method used to separate components in a complex mixture using two phases, a stationary phase and a mobile phase. It then discusses various types of chromatography, including normal phase and reversed phase chromatography, based on different factors like separation principle, elution technique, scale of operation, and type of analysis. The document also discusses key components of HPLC like pumps, injectors, columns, detectors and provides details on their functioning. It highlights advantages of HPLC like high resolution, sensitivity, reproducibility and its importance in qualitative and quantitative analysis.
This document discusses high performance liquid chromatography (HPLC). It begins by defining chromatography and describing the basic principles of HPLC. It then discusses the types of HPLC separations based on modes, principles, elution techniques, scale of operation, and type of analysis. The document also covers the principles, types, and advantages of liquid chromatography. It provides details on the instrumentation of HPLC including solvent reservoirs, degassing, pumps, injectors, columns, detectors, and data handling. In summary:
HPLC is a type of column chromatography used to separate mixtures by distributing components between a stationary and mobile phase. It can be used for qualitative and quantitative analysis. The document outlines the various components of an HPLC system
a brief introduction to countercurrent chromatography with its principle. working and modes of operation along with little bit of history, the types of CCC and its applications
This document discusses a GPAT online class on pharmaceutical analysis part 4 presented by Dr. P Ramalingam. The class covers chromatography types and principles as well as HPLC instrumentation. Chromatography is introduced as a method to separate mixtures based on distributing components between a stationary and mobile phase. Key terms like chromatogram, eluent, eluate and stationary/mobile phases are defined. Common chromatography techniques like liquid chromatography, gas chromatography, paper chromatography, and thin-layer chromatography are described based on their stationary and mobile phases. The rest of the document focuses on HPLC, including reverse phase chromatography, HPLC components like pumps, columns, and detectors, and separation mechanisms.
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_A practical guide for the beginner users.pdfSherif Taha
This lecture presents an introduction to the beginner user on the usage of high-performance liquid chromatography. The main topics are; selecting a buffer solution, and the stationary & mobile phases.
High performance liquid chromatography (HPLC) is described. HPLC is a chromatographic technique used to separate mixtures of compounds. It involves pumping a pressurized mobile phase through a column containing a stationary phase. Compounds in a sample injected into the mobile phase are separated as they are carried through the column at different rates depending on their interactions with the stationary phase. HPLC is useful for both qualitative and quantitative analysis and has advantages such as high resolution, sensitivity, accuracy, and reproducibility. Common applications and components of an HPLC system are also outlined.
High performance liquid chromatography (HPLC) is described, including the basic principles and components of HPLC systems. HPLC uses high pressure to pass a liquid mobile phase through a column packed with solid adsorbent particles or porous beads. This allows for separation of mixtures based on differences in how components partition between the stationary and mobile phases. Key components reviewed are the solvent reservoirs, pump, injector, column, and detectors. Common applications of HPLC mentioned are qualitative and quantitative analysis of both volatile and non-volatile compounds.
High performance liquid chromatography (HPLC) is summarized as follows:
HPLC is a technique used to separate mixtures by distributing the components between a stationary and mobile phase. It can be used for both qualitative and quantitative analysis. HPLC utilizes high pressure pumps to pass a mobile phase through a column packed with adsorbent particles, allowing separation of components based on differences in their partitioning behavior between the mobile and stationary phases. Common detectors used in HPLC include UV/Vis, refractive index, fluorescence, and mass spectrometry.
High performance liquid chromatography (HPLC) is described, including the basic principles and components of HPLC systems. HPLC uses high pressure to pass a liquid mobile phase through a column packed with solid adsorbent particles or porous beads. This allows for separation of mixtures based on differences in how components partition between the stationary and mobile phases. Key components reviewed are the solvent reservoirs, pump, injector, column, and detectors. Common applications of HPLC mentioned are qualitative and quantitative analysis of both volatile and non-volatile compounds.
The document discusses High Performance Liquid Chromatography (HPLC). It provides background on the origins and development of HPLC from the early 1900s. By the 1980s, HPLC was commonly used for separation, identification, purification and quantification of chemical compounds. The document then discusses the basic principles, components, and instrumentation of HPLC systems. It explains how HPLC uses high pressure to force a mobile phase through a column containing a stationary phase to separate compounds based on their interactions with the phases. Common applications of HPLC across various fields like pharmaceuticals, environmental analysis, forensics and food are also summarized.
HPLC-High Performance Chromatography with principle,types and intrumentation.ToobaDedmari
The document summarizes key aspects of high performance liquid chromatography (HPLC). It describes the principle of HPLC, which separates compounds using columns and solvents at high pressure. It outlines the main components of an HPLC system including the solvent reservoir and degassing system to remove gases from solvents, high precision pumps to move solvents, and sample injection systems. It provides details on the types of pumps (syringe, reciprocating, constant pressure) and chromatography modes (reverse phase, normal phase, isocratic and gradient elution).
High performance liquid chromatography (hplc) by Muhammad ShakaibMuhammad Shakaib
High performance liquid chromatography (HPLC) is a technique used to separate components in a mixture. It works by differential migration of solutes through a column containing a stationary phase as a pressurized mobile phase flows through. Solutes are detected as they elute from the column. Key aspects of HPLC include the use of a high pressure pump to deliver the mobile phase, columns packed with microparticulate stationary phases, and detectors to identify eluted components. HPLC is useful for separating compounds that are not volatile enough for gas chromatography.
Similar to HPLC-MS Use-Care and Maintenance, Troubleshooting (20)
Literature Review Basics and Understanding Reference Management.pptxDr Ramhari Poudyal
Three-day training on academic research focuses on analytical tools at United Technical College, supported by the University Grant Commission, Nepal. 24-26 May 2024
Redefining brain tumor segmentation: a cutting-edge convolutional neural netw...IJECEIAES
Medical image analysis has witnessed significant advancements with deep learning techniques. In the domain of brain tumor segmentation, the ability to
precisely delineate tumor boundaries from magnetic resonance imaging (MRI)
scans holds profound implications for diagnosis. This study presents an ensemble convolutional neural network (CNN) with transfer learning, integrating
the state-of-the-art Deeplabv3+ architecture with the ResNet18 backbone. The
model is rigorously trained and evaluated, exhibiting remarkable performance
metrics, including an impressive global accuracy of 99.286%, a high-class accuracy of 82.191%, a mean intersection over union (IoU) of 79.900%, a weighted
IoU of 98.620%, and a Boundary F1 (BF) score of 83.303%. Notably, a detailed comparative analysis with existing methods showcases the superiority of
our proposed model. These findings underscore the model’s competence in precise brain tumor localization, underscoring its potential to revolutionize medical
image analysis and enhance healthcare outcomes. This research paves the way
for future exploration and optimization of advanced CNN models in medical
imaging, emphasizing addressing false positives and resource efficiency.
DEEP LEARNING FOR SMART GRID INTRUSION DETECTION: A HYBRID CNN-LSTM-BASED MODELgerogepatton
As digital technology becomes more deeply embedded in power systems, protecting the communication
networks of Smart Grids (SG) has emerged as a critical concern. Distributed Network Protocol 3 (DNP3)
represents a multi-tiered application layer protocol extensively utilized in Supervisory Control and Data
Acquisition (SCADA)-based smart grids to facilitate real-time data gathering and control functionalities.
Robust Intrusion Detection Systems (IDS) are necessary for early threat detection and mitigation because
of the interconnection of these networks, which makes them vulnerable to a variety of cyberattacks. To
solve this issue, this paper develops a hybrid Deep Learning (DL) model specifically designed for intrusion
detection in smart grids. The proposed approach is a combination of the Convolutional Neural Network
(CNN) and the Long-Short-Term Memory algorithms (LSTM). We employed a recent intrusion detection
dataset (DNP3), which focuses on unauthorized commands and Denial of Service (DoS) cyberattacks, to
train and test our model. The results of our experiments show that our CNN-LSTM method is much better
at finding smart grid intrusions than other deep learning algorithms used for classification. In addition,
our proposed approach improves accuracy, precision, recall, and F1 score, achieving a high detection
accuracy rate of 99.50%.
International Conference on NLP, Artificial Intelligence, Machine Learning an...gerogepatton
International Conference on NLP, Artificial Intelligence, Machine Learning and Applications (NLAIM 2024) offers a premier global platform for exchanging insights and findings in the theory, methodology, and applications of NLP, Artificial Intelligence, Machine Learning, and their applications. The conference seeks substantial contributions across all key domains of NLP, Artificial Intelligence, Machine Learning, and their practical applications, aiming to foster both theoretical advancements and real-world implementations. With a focus on facilitating collaboration between researchers and practitioners from academia and industry, the conference serves as a nexus for sharing the latest developments in the field.
TIME DIVISION MULTIPLEXING TECHNIQUE FOR COMMUNICATION SYSTEMHODECEDSIET
Time Division Multiplexing (TDM) is a method of transmitting multiple signals over a single communication channel by dividing the signal into many segments, each having a very short duration of time. These time slots are then allocated to different data streams, allowing multiple signals to share the same transmission medium efficiently. TDM is widely used in telecommunications and data communication systems.
### How TDM Works
1. **Time Slots Allocation**: The core principle of TDM is to assign distinct time slots to each signal. During each time slot, the respective signal is transmitted, and then the process repeats cyclically. For example, if there are four signals to be transmitted, the TDM cycle will divide time into four slots, each assigned to one signal.
2. **Synchronization**: Synchronization is crucial in TDM systems to ensure that the signals are correctly aligned with their respective time slots. Both the transmitter and receiver must be synchronized to avoid any overlap or loss of data. This synchronization is typically maintained by a clock signal that ensures time slots are accurately aligned.
3. **Frame Structure**: TDM data is organized into frames, where each frame consists of a set of time slots. Each frame is repeated at regular intervals, ensuring continuous transmission of data streams. The frame structure helps in managing the data streams and maintaining the synchronization between the transmitter and receiver.
4. **Multiplexer and Demultiplexer**: At the transmitting end, a multiplexer combines multiple input signals into a single composite signal by assigning each signal to a specific time slot. At the receiving end, a demultiplexer separates the composite signal back into individual signals based on their respective time slots.
### Types of TDM
1. **Synchronous TDM**: In synchronous TDM, time slots are pre-assigned to each signal, regardless of whether the signal has data to transmit or not. This can lead to inefficiencies if some time slots remain empty due to the absence of data.
2. **Asynchronous TDM (or Statistical TDM)**: Asynchronous TDM addresses the inefficiencies of synchronous TDM by allocating time slots dynamically based on the presence of data. Time slots are assigned only when there is data to transmit, which optimizes the use of the communication channel.
### Applications of TDM
- **Telecommunications**: TDM is extensively used in telecommunication systems, such as in T1 and E1 lines, where multiple telephone calls are transmitted over a single line by assigning each call to a specific time slot.
- **Digital Audio and Video Broadcasting**: TDM is used in broadcasting systems to transmit multiple audio or video streams over a single channel, ensuring efficient use of bandwidth.
- **Computer Networks**: TDM is used in network protocols and systems to manage the transmission of data from multiple sources over a single network medium.
### Advantages of TDM
- **Efficient Use of Bandwidth**: TDM all
Comparative analysis between traditional aquaponics and reconstructed aquapon...bijceesjournal
The aquaponic system of planting is a method that does not require soil usage. It is a method that only needs water, fish, lava rocks (a substitute for soil), and plants. Aquaponic systems are sustainable and environmentally friendly. Its use not only helps to plant in small spaces but also helps reduce artificial chemical use and minimizes excess water use, as aquaponics consumes 90% less water than soil-based gardening. The study applied a descriptive and experimental design to assess and compare conventional and reconstructed aquaponic methods for reproducing tomatoes. The researchers created an observation checklist to determine the significant factors of the study. The study aims to determine the significant difference between traditional aquaponics and reconstructed aquaponics systems propagating tomatoes in terms of height, weight, girth, and number of fruits. The reconstructed aquaponics system’s higher growth yield results in a much more nourished crop than the traditional aquaponics system. It is superior in its number of fruits, height, weight, and girth measurement. Moreover, the reconstructed aquaponics system is proven to eliminate all the hindrances present in the traditional aquaponics system, which are overcrowding of fish, algae growth, pest problems, contaminated water, and dead fish.
Introduction- e - waste – definition - sources of e-waste– hazardous substances in e-waste - effects of e-waste on environment and human health- need for e-waste management– e-waste handling rules - waste minimization techniques for managing e-waste – recycling of e-waste - disposal treatment methods of e- waste – mechanism of extraction of precious metal from leaching solution-global Scenario of E-waste – E-waste in India- case studies.
A review on techniques and modelling methodologies used for checking electrom...nooriasukmaningtyas
The proper function of the integrated circuit (IC) in an inhibiting electromagnetic environment has always been a serious concern throughout the decades of revolution in the world of electronics, from disjunct devices to today’s integrated circuit technology, where billions of transistors are combined on a single chip. The automotive industry and smart vehicles in particular, are confronting design issues such as being prone to electromagnetic interference (EMI). Electronic control devices calculate incorrect outputs because of EMI and sensors give misleading values which can prove fatal in case of automotives. In this paper, the authors have non exhaustively tried to review research work concerned with the investigation of EMI in ICs and prediction of this EMI using various modelling methodologies and measurement setups.
Using recycled concrete aggregates (RCA) for pavements is crucial to achieving sustainability. Implementing RCA for new pavement can minimize carbon footprint, conserve natural resources, reduce harmful emissions, and lower life cycle costs. Compared to natural aggregate (NA), RCA pavement has fewer comprehensive studies and sustainability assessments.
Embedded machine learning-based road conditions and driving behavior monitoringIJECEIAES
Car accident rates have increased in recent years, resulting in losses in human lives, properties, and other financial costs. An embedded machine learning-based system is developed to address this critical issue. The system can monitor road conditions, detect driving patterns, and identify aggressive driving behaviors. The system is based on neural networks trained on a comprehensive dataset of driving events, driving styles, and road conditions. The system effectively detects potential risks and helps mitigate the frequency and impact of accidents. The primary goal is to ensure the safety of drivers and vehicles. Collecting data involved gathering information on three key road events: normal street and normal drive, speed bumps, circular yellow speed bumps, and three aggressive driving actions: sudden start, sudden stop, and sudden entry. The gathered data is processed and analyzed using a machine learning system designed for limited power and memory devices. The developed system resulted in 91.9% accuracy, 93.6% precision, and 92% recall. The achieved inference time on an Arduino Nano 33 BLE Sense with a 32-bit CPU running at 64 MHz is 34 ms and requires 2.6 kB peak RAM and 139.9 kB program flash memory, making it suitable for resource-constrained embedded systems.
3. Chromatography
Chromatography is a method by which a
sample mixture of solids, liquids or gases is
resolved into its components which are
detected on the basis of their physical
properties. There are various types of
chromatographic techniques e.g.
Gas chromatography, Liquid chromatography
(LC) etc.
Faisal Ghazanfar, PCSIR 3
6. Applications of HPLC
Find out up to ppm (part per millions) levels compounds in samples such
as caffeine, aspirin, steroid etc
Quantitative and qualitative analysis
Faisal Ghazanfar, PCSIR 6
8. Liquid chromatography (LC)
Any method of chromatography in which the
mobile phase is a Liquid. The liquid used as
the mobile phase is called the “eluent”.
The stationary phase is usually a solid or a
liquid.
In general, it is possible to analyze any
substance that can be stably dissolved in the
mobile phase
Categorized by nature of the stationary
phase and separation process such as Normal
separation, Reverse separation.
Faisal Ghazanfar, PCSIR 8
9. Faisal Ghazanfar, PCSIR 9
Block Diagram for HPLC
(Isocratic system )
9
Pump
Sample injection unit
(injector)
Column
Column oven
(thermostatic
column chamber)
Detector
Eluent
(mobile phase)
Drain
Data processor
Degasser
Stationary Phase
10. Animation of HPLC
Simple separation
Isocratic system of HPLC
Chromatogram
Single pump
Faisal Ghazanfar, PCSIR 10
11. Major parts of HPLC
Eluent (Mobile phase)
Solvent Delivery Pump
Sample Injection Unit
Column
Detectors (optical & mass)
Auto sampler (New addition)
MS (New detector)
Data processor (computer)
Faisal Ghazanfar, PCSIR 11
12. Block Diagram for HPLC (Gradient system)
Faisal Ghazanfar, PCSIR 12
Pump
Degassing
Assy.
Solvent
Proportioning
Assy.
(Gradient)
Pressure
Sensor
Pump
Pulse
Damper
Vacuum
Pump
Computer
Data
Station
&
Printer
Detector
Assembly
Column
A B c D
Pre-
Filter
(Mobile phase)
Knobs
Waste
Drain
Valve
waste
waste
Column Oven
Injection
Head
(Sample in)
OR
Sample in
from
Autosampler
Loop
14. Faisal Ghazanfar, PCSIR 14
Comparing Chromatography to the Flow of
a River...
14
Base
Water flow
Light leaf
Heavy stone
www.mtsu32.mtsu.edu
15. Mobile phase
The liquid used as the mobile phase is
called the “eluent”.
May be a mixture of A,B and A,B,C,D
Sample flow through mobile phase.
The ratios are form gradient that useful
and beneficial in detection of compound.
Isocratic system (constant eluent composition)
Gradient system (variable eluent composition)
It form base line.
Mixing mainly depend on mixer/pump.
Faisal Ghazanfar, PCSIR 15
16. Separations in HPLC
Normal Phase:
Polar stationary phase and a non-polar, non-aqueous mobile
phase, and works effectively for separating analytes readily soluble
in non-polar solvents. If polarity of mobile phase increases,
adsorption (stickness on surface) increases and cause increase in
retention time, so it is not in common using as compare to reverse
phase. (NP-HPLC)
Reversed Phase:
A non-polar stationary phase and an aqueous, moderately
polar mobile phase. (RP-HPLC or RPC). No adsorption with polar
solvents because stationary phase is non-polar.
Advantage:
it allows us to use relatively cheap and non-hazardous solvents like
methanol, ethanol, acetonitrile and even water. Not suitable for Strong
Acid and Strong Bases.
Faisal Ghazanfar, PCSIR 16
www.en.wikipedia.org
19. Isocratic system of LC
Faisal Ghazanfar, PCSIR 19
• The Eluent (Mobile phase)
composition is constant in LC
– problems:
– Low analysis time (quick)
– Poor separation
20. Gradient system of LC
Gradient system
Varying eluent composition e.g.
gradient starting at 10% methanol and
ending at 90% methanol after 20
minutes
Faisal Ghazanfar, PCSIR 20
28. Pump piston replacement
animations
Replacement of
Seal of head
Check Valve at inlet and outlet with seal
Piston replacement of high Pressure pump
Change of spring
Change of piston
How open pump and close
Faisal Ghazanfar, PCSIR 28
29. Vacuum Degasser operation
The Vacuum Degasser consists of a vacuum
chamber, degassing tube, variable speed
vacuum pump, microprocessor controller,
sensor, and check valves. The mobile phase
flows into a degassing tube, which is inside a
vacuum chamber. Decreased pressure in the
chamber causes the outward movement of gas
dissolved in the mobile phase across the tube
wall, in accordance to Henry’s Law (cold drink)
thus degassing the mobile phase
Faisal Ghazanfar, PCSIR 29
33. Pulse damper maintain
constant flow
Pumps have intervals in their pumping
cycle when flow and pressure
momentarily decrease. This "off time" is
the interval when the piston has finished
its solvent delivery stroke and is starting
to refill. To maintain constant flow, such
pumps require a pulse compensator or
damper that stores energy during the
pumps' delivery stroke and returns an
appropriate amount of work to the fluid
during the pump off time. This help to
reduce base line noise.
Faisal Ghazanfar, PCSIR 33
35. Faisal Ghazanfar, PCSIR 35
Pump Care
• Flush with water after running a buffer,
• Replace seals in a timely manner.
• Maintain check valves.
• Do not allow solids (ppt.) in the mobile
phase.
37. Rotary Sample Loop Injector
Injector needles are
used ranging from 10
µL to 500 µL to inject
a sample onto the
sample loop
Upon a 60° rotation
the pump introduces
the sample onto the
column in a reverse
direction that it was
loaded.
Faisal Ghazanfar, PCSIR 37
Reference 2
39. Routine Care of Injectors
Never use a pointed or
bevel tip needle.
Rinse after the use of
buffer solutions.
Avoid abrasive particles
by filtering samples
before injection.
Use burr-free tubing to
avoid metals shavings
from getting into the
injector.
Faisal Ghazanfar, PCSIR 39
45. Column Heaters
Faisal Ghazanfar, PCSIR 45
•Some separations of complex mixtures must be temperature
optimized to achieve separations of overlapping peaks.
•Increased column temperature will also shorten retention times
for a given column dimension.
•Eliminates retention time variation due to room temperature
fluctuations.
46. How life of column will be
longer
Filter your sample and mobile phase. Make sure the pH
of the mobile phase is within the working range of the
column.
Flush the column with methanol or acetonitrile if it is
not going to be used for sometime.
Faisal Ghazanfar, PCSIR 46
47. Faisal Ghazanfar, PCSIR 47
Removing Buffers from a Reverse
Phase Column
• DO NOT FLUSH WITH 100% WATER AS YOUR FIRST
STEP -
• Substitute water for the buffer but leave the
remaining proportions the same. Run through about
5 column volumes.
• Wash through 10 column volumes of a strong organic
solvent, example - Methanol.
• If you plan to store the column, read the directions
in manual.
• If the phase collapses, a 50-50 water, organic
solvent wash for 30 minutes can restore it.
51. Nuts and ferrule
Check the condition of the nut and ferrule
After repeated use, nuts (and especially
ferrules) will gradually become deformed
to the point of being incapable of
creating the seal they were designed to
make. Always keep an extra supply of all
the nuts and ferrules you are using so that
you can replace them quickly and avoid
unnecessary down time.
Faisal Ghazanfar, PCSIR 51
52. Fittings of pipes of HPLC
SS fittings
Polymer based fittings
Column
Ferrule connections with column
Incompatible fittings may cause leakage
Faisal Ghazanfar, PCSIR 52
55. Faisal Ghazanfar, PCSIR 55
Differential Refractive Index Detector
(Deflection-Type)
55
Light
Sample cell
Reference cell
Light-receiving
unit
56. Faisal Ghazanfar, PCSIR 56
Optical System of Differential
Refractive Index Detector (Deflection-
Type)
56
W lamp
Slit
Sample cell
Reference cell
Photodiode
The slit image moves if the
refractive index inside the
flow cell changes.
60. MS detector
Ionization for GC/MS
Electron Ionization/Impact (EI)
Chemical Ionization (CI)
Ionization for LC/MS
Electro-spray (ESI)
Atmospheric Pressure Chemical Ionization (APCI)
All are fitted in a stack (ion source housing)
MS
Quadruple
Dynode (Detection of ions)
Faisal Ghazanfar, PCSIR 60
70. Faisal Ghazanfar, PCSIR 70
Sheath(nitrogen)
nebulized/spray
Auxilaryoutside ESI to
guide spray gas
Sweep gas prevent in
stack to enter other gas
71. Ethene spectrum
Faisal Ghazanfar, PCSIR
(nis
tdemo) E
thene
10 12 14 16 18 20 22 24 26 28 30 32 34 36 38 40
0
50
100
14 24
25
26
27
28
29
H
H
H
H
Name: Ethene
Formula: C2
H
4
MW: 28
28 999 | 27 545 | 26 501 | 25 73 | 29 22 |
largest peaks:
73. Troubleshooting HPLC
Preliminary checks
How removes Leaks
Retention Time
Base line
Pressure
Peaks
Faisal Ghazanfar, PCSIR 73
74. No peaks or very small peaks
Possible cause Solution
Detector off Check detector
Broken connections to
recorder/computer
Check connections
No sample/
Wrong sample
Check sample. Be sure
it is not deteriorated.
Check for bubbles in the
vials
Wrong settings on
recorder or detector or
software
Check attenuation.
Check gain
Faisal Ghazanfar, PCSIR 74
75. No Flow
Possible cause Solution
Pump off Start Pump
Flow interrupted
Check reservoirs. Check position of
the inlet tubing. Check loop for
obstruction or air. Check degassing
of mobile phase. Check
compatibility of the mobile phase
components.
Leak
Check fittings. Check pump for leaks
and precipitates. Check pump seals.
Air trapped in the system
Disconnect column and prime pump.
Flush system with 100% methanol
or iso-propanol.
Faisal Ghazanfar, PCSIR 75
76. Column and fitting leaks
Problem Possible cause Solution
Column end leaks
Loose fitting
Tighten or replace fitting
Cut tubing and replace
ferrule; disassemble
fitting, rinse and
reassemble.
Leak at detector Detector-seal failure
Replace detector seal or
gaskets.
Leak at injection
valve
Worn or scratched
valve rotor
Replace valve rotor
Leak at pump Pump seal failure
Replace pump seal; check
piston for scratches and, if
necessary, replace
Faisal Ghazanfar, PCSIR 76
77. Check Retention time
(sample time to travel from column)
Possible cause Solution
Contamination buildup
Flush column occasionally with strong
solvent such as methanol
Equilibration time insufficient for
gradient run or changes in isocratic
mobile phase
Pass at least 10 column volumes through the
column for gradient regeneration or after
solvent changes
First few injections - active sites
Condition column by injecting concentrated
sample
Inconsistent on-line mobile-phase
mixing
Ensure gradient system is delivering a
constant composition; compare with
manually prepared mobile phase; partially
premix mobile phase (1ml check by
software)
Selective evaporation of mobile-phase
component
Cover solvent reservoirs; prepare fresh
mobile phase
Varying column temperature
Thermostat or insulate column; ensure
laboratory temperature is constant.
Faisal Ghazanfar, PCSIR 77
78. Checking retention time….
Decrease Retention
Times
Column overloaded with
sample
Decrease sample amount or use
larger-diameter column.
Increasing flow rate Check and reset pump flow rate.
Varying column temperature
Thermostat or insulate column;
ensure laboratory temperature is
constant
Increasing Retention
Times
Decreasing flow rate
Check and reset pump flow rate;
check for pump cavitations;
check for leaking pump seals
and other leaks in system
Changing mobile-phase
composition
Cover solvent reservoirs; ensure
that gradient system is
delivering correct composition.
Faisal Ghazanfar, PCSIR 78
79. Base line
Problem Possible cause Solution
Noise
Air bubbles in mobile phase Degas or use back pressure restrictor on detector
Positive-negative - difference in
refractive index of injection
solvent and mobile phase
Normal with many samples; use mobile phase as
sample solvent
Drifting
baseline
Negative direction (gradient
elution) - absorbance of mobile-
phase A
Use non-UV absorbing mobile phase solvents; use
HPLC grade mobile phase solvents; add UV
absorbing compound to mobile phase B.
Positive direction (gradient
elution) - absorbance of mobile
phase B
Use higher UV absorbance detector wavelength; use
non-UV absorbing mobile phase solvents; use HPLC
grade mobile phase solvents; add UV absorbing
compound to modile phase A.
Positive direction - contamination
buildup and elution
Flush column with strong solvent; clean up sample;
use HPLC grade solvents
Wavy or undulating - temperature
changes in room
Monitor and control changes in room temperature;
insulate column or use column oven; cover refractive
index detector and keep it out of air currents.
Faisal Ghazanfar, PCSIR 79
80. Base line….
Baseline noise
Continuous - detector lamp problem
or dirty cell
Replace UV lamp( each should last 2000 h; clean and
flush flow cell.
Gradient or isocratic proportioning -
lack of solvent mixing
Use proper mixing device; check proportioning
precision by spiking one solvent with UV absorbing
compound and monitor UV absorbance detector output.
Gradient or isocratic proportioning -
malfunctioning proportioning
valvesl
Clean or replace proportioning precision valves;
partially remix solvents.
Occasional sharp spikes - external
electrical interference
Use voltage stabilizer for LC system; use independent
electrical circuit.
Periodic - pump pulses
Service or replace pulse damper; purge air from pump;
clean or replace check valves.
Random - contamination buildup
Flush column with strong solvent; clean up sample; use
HPLC grade solvent
Spikes - bubble in detector
Degas mobile phase; use back pressure restrictor at
detector outlet.
Spikes - column temperature higher
than boiling point of solvent
Use lower column temperature.
Faisal Ghazanfar, PCSIR 80
81. Pressure
Problem Possible cause Solution
Decreasing Pressure
Insufficient flow
from pump
Loosen cap on mobile
phase reservoir
Leak in hydraulic
lines from pump to
column
Tighten or replace fittings;
tighten rotor in injection
valve
Leaking pump
check valve or seals
Replace or clean check
valves; replace pump
seals.
Pump cavitations
Degas solvent; check for
obstruction in line from
solvent reservoir to pump;
replace inlet-line frit
Faisal Ghazanfar, PCSIR 81
82. Pressure…..
Fluctuating
pressure
Bubble in pump Degas solvent;
Leaking pump check
valve or seals
Replace or clean check valves; replace
pump seals
High Back
Pressure
Column blocked with
irreversibly absorbed
sample
reverse-flush column with strong solvent
to dissolve blockage
Column particle size too
small (for example 3
micrometers)
Use larger particle size (for example 5
micrometer)
Mobile phase viscosity
too high
Use lower viscosity solvents or higher
temperature
Plugged frit in in-line
filter or guard column
Replace frit or guard column
Plugged (block)inlet frit Replace end fitting or frit assembly
Faisal Ghazanfar, PCSIR 82
83. Parts of column
Entrance
Guard column
Frits
Outer jacket (AL, SS)
Main column
Exit
Faisal Ghazanfar, PCSIR 83
84. Ghost peaks
Possible cause Solution
Contamination
Flush column to remove
contamination; use HPLC-grade solvent
Elution of analytes retained
from previous injection
Flush column with strong solvent at end of
run; end gradient at higher solvent
concentration
Reversed-phase
chromatography –
contaminated water
Check suitability of water by running different
amounts through column and measure peak
height of interferences as function of
enrichment time; clean water by running it
through old reversed-phase column; use
HPLC-grade water.
Faisal Ghazanfar, PCSIR 84
85. Peaks…..
Problem Possible cause Solution
Negative
peaks
Refractive index detection –
refractive index of solute less
than that of mobile phase
Reverse polarity to make peak positive
UV-absorbance detection –
absorbance of solute less than
that of mobile phase
Use mobile phase with lower UV absorbance; if
recycling solvent, stop recycling when recycled
solvent affects detection
Peak
Doubling
Blocked Frit
Replace or clean frit; install 0.5-um porosity in-
line filter between pump and injector to
eliminate mobile-phase contaminants or
between injector and column to eliminate
sample contaminants
Coelution of interfering
compound from previous
injection
Flush column with strong solvent at end of ran;
end gradient at higher solvent concentration
Column overloaded
Use higher-capacity stationary phase; increase
column diameter; decrease sample amount
Faisal Ghazanfar, PCSIR 85
86. Peak doubling
Faisal Ghazanfar, PCSIR 86
To resolved doubling problem
of peak:
A new batch of mobile phase
was made and new auto
sampler wash solvent was used
with no improvement. The
entire system was flushed
thoroughly with acetonitrile in
an effort to clean the column
and wash the pump, auto
sampler and detector; this did
not appear to help……………firit?
Reverse column run?
90. Peaks…..
Problem Possible cause Solution
Peak Doubling
Column void or channeling
Replace column, or, if
possible, open top end fitting
and clean and fill void with
glass beads or same column
packing; repack column
Injection solvent too strong
Use weaker injection solvent
or stronger mobile phase
Sample volume too large
Use injection volume equal to
one-sixth of column volume
when sample prepared in
mobile phase for injection
Un-swept injector flow path Replace injector rotor
Peak Fronting
Channeling in column Replace or repack column
Column overloaded
Use higher-capacity stationary
phase; increase column
diameter; decrease sample
amount
Faisal Ghazanfar, PCSIR 90
91. Peak tailing
Faisal Ghazanfar, PCSIR 91
Shopping in a corridor example.
Column normally filled with silica.
Cause due to secondary interaction of analytes
(silanol interaction) with some thing to the
stationary phase
92. Peaks…..
Problem Possible cause Solution
Tailing Peaks
Basic solutes - silanol interactions
use a stronger mobile phase; use base-
deactivated silica-based reversed-phase
column;
Beginning of peak doubling is also a tailing
peaks
See peak doubling
Chelating solutes - trace metals in base silica
Use high purity silica-based column with low
trace-metal content;
Silica-based column - degradation at high
temperature
Reduce temperature to less than 50 C
Spikes
Bubbles in mobile phase
Degas mobile phase;
ensure that all fittings are tight
Column stored without caps
Store column tightly capped; flush reversed-
phase columns with degassed methanol
Faisal Ghazanfar, PCSIR 92
93. Problem Possible cause Solution
Tailing Peaks
Silica-based column –
degradation at high
temperature
Reduce temperature to less than 50 C
Silica-based column – silanol
interactions
Decrease mobile-phase pH to suppress
silanol ionization; increase buffer
concentration; derivatize solute to
change polar interactions
Void formation at head of
column
Replace column, or, if possible, open
top end fitting and clean and fill in void
with glass beads or same column
packing; rotate injection valve quickly;
use injection valve with pressure
bypass; avoid pressure shock
Faisal Ghazanfar, PCSIR 93
Peaks…..
94. Symptoms of Dirty columns
HIGH BACK PRESSURE. (15-20% to do reverse flush)
CHANGING RETENTION TIMES.
BROAD PEAKS AND TAILING.
LOSS OF COLUMN RESOLUTION
Solution:
Regeneration of column /flushing of column
(purging column with pure acetonitrile)
(5% methanol + 95% water, 01hr)
Faisal Ghazanfar, PCSIR 94
95. Column fritt
Faisal Ghazanfar, PCSIR 95
Filter like material, ring
shape of mcro-m
(size<0.5um), placed on
both sides of column.
System back pressure
increases and, in some
instances, peaks become
distorted or split.
the sample to be
distributed evenly across
the top of the column
96. Column frit
When plugging of the inlet frit occurs there are two
ways to restore column performance. The easiest and
fastest way is to back flush particulates off the inlet
frit. This may not always work, but since it is so easy to
do, it is worth a try.
Faisal Ghazanfar, PCSIR 96
97. How do back flushing of inlet
frit
1. Connect the column to the chromatograph so
that the now is in the reverse direction. Do not
connect the column to the detector because
dislodged particulates from the inlet frit may
flow into the detector flow cell.
2. Back-flush the column with approximately 10
column volumes (V-pi*r^2*h) of mobile
phase.(~1ml/min upto 25ml)
3. Connect the column to the chromatograph so
that the flow is in the proper direction.
4. Check to see if chromatographic performance is
acceptable.
5. If problem still their, replace frit or guard
column
Faisal Ghazanfar, PCSIR 97