HPLC method development and data analysis
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this presentation informs you the criteria about to uses of solvent and column for separation of the compounds and also useful for HPLC data analysis.
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HPLC method development by DrA.S Charan
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HPLC method development
This document outlines the five main steps for developing an analytical HPLC method: 1) selecting the initial HPLC method and conditions, 2) selecting the initial chromatographic conditions, 3) optimizing selectivity, 4) optimizing system parameters, and 5) validating the method. Key aspects of each step are discussed, including selecting the type of chromatography, column, detector, and mobile phase based on the analytes. The goal is to develop a validated method that provides adequate resolution and selectivity within a desired analysis time.
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a brief explanation of chiral stationary phases and mobile phases and designing the setup for better resolution
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Sample preparation is an essential part of HPLC analysis to provide a reproducible and homogenous solution suitable for injection onto the column. The goal of sample preparation is to remove interferences and ensure the sample is compatible with the HPLC method without damaging the column. Sample matrices can be organic or inorganic solids, semisolids, liquids or gases, with liquids being easiest to prepare. Solid and semisolid samples require reducing particle size through processes like blending or grinding. Filtration is also important to remove particles that could damage the column. Common pretreatment methods for liquid samples include liquid-liquid extraction and solid phase extraction, while newer techniques are used for solid samples like supercritical fluid extraction. Derivatization can improve
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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.
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The presentation focuses on how to choose the appropriate mode of separation, the correct column and highlights the importance of the correct mobile phase. This approach will be applied to a wide selection of compound types ranging from proteins, peptides, glycans to small pharmaceutical molecules and their metabolites. It will also look at specific application areas for monoclonal antibody analysis, namely: titer, aggregation, charge and oxidation variant. Platform methods for biologics characterization are also discussed.
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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.
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HPLC method development by DrA.S Charan
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Mobile phase selection
Column selection
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This document outlines the five main steps for developing an analytical HPLC method: 1) selecting the initial HPLC method and conditions, 2) selecting the initial chromatographic conditions, 3) optimizing selectivity, 4) optimizing system parameters, and 5) validating the method. Key aspects of each step are discussed, including selecting the type of chromatography, column, detector, and mobile phase based on the analytes. The goal is to develop a validated method that provides adequate resolution and selectivity within a desired analysis time.
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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.
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Sample preparation is an essential part of HPLC analysis to provide a reproducible and homogenous solution suitable for injection onto the column. The goal of sample preparation is to remove interferences and ensure the sample is compatible with the HPLC method without damaging the column. Sample matrices can be organic or inorganic solids, semisolids, liquids or gases, with liquids being easiest to prepare. Solid and semisolid samples require reducing particle size through processes like blending or grinding. Filtration is also important to remove particles that could damage the column. Common pretreatment methods for liquid samples include liquid-liquid extraction and solid phase extraction, while newer techniques are used for solid samples like supercritical fluid extraction. Derivatization can improve
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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.
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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.
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2. Column particle sizes have decreased over time from 100 μm to below 2 μm, increasing theoretical plate counts. Column dimensions and particle sizes are selected based on the application.
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3. Optimizing these parameters through adjusting mobile phase or column properties can enhance separation and analysis of chromatographic runs.
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This document discusses factors affecting the stability of drugs and drug metabolites in biological matrices. It outlines various external factors like light, temperature, pH, oxidation, and enzymes that can impact stability. It also discusses structural factors such as ester pro-drugs and chiral interconversion. Specific examples are provided to illustrate how these different factors can influence stability and the importance of controlling for these factors during sample collection, storage, and processing. Approaches to improve stability are also described.
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HPLC is a form of liquid chromatography used to separate compounds dissolved in solution based on how they partition between a stationary and mobile phase. The key components of an HPLC system are a pump, injector, column, and detector. Method development involves selecting parameters like the mobile phase, column type, detection method, and chromatography conditions to optimize separation of the sample components. HPLC offers advantages like high sensitivity, rapid analysis times, and use for both analytical qualitative and quantitative analysis.
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3. Common problems discussed include issues with the mobile phase, pump, injector, detector, and peaks/baseline, along with potential causes and solutions for each.
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2. Column particle sizes have decreased over time from 100 μm to below 2 μm, increasing theoretical plate counts. Column dimensions and particle sizes are selected based on the application.
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Ion exchange chromatography is a technique that separates ions and polar molecules based on their charge. It works by using an ion exchange resin with charged functional groups that interact with and retain analyte ions of the opposite charge from a mobile phase. Common stationary phases use functional groups like sulfonate, carboxylate or quaternary amine. The document discusses the history of ion exchange chromatography and provides examples of its applications including protein purification, water analysis, separation of amino acids, vitamins and drugs. Factors that affect separations like pH, ionic strength, temperature and mobile phase modifiers are also summarized.
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This document discusses various concepts related to high performance liquid chromatography (HPLC) peak analysis including:
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3. Optimizing these parameters through adjusting mobile phase or column properties can enhance separation and analysis of chromatographic runs.
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This document discusses factors affecting the stability of drugs and drug metabolites in biological matrices. It outlines various external factors like light, temperature, pH, oxidation, and enzymes that can impact stability. It also discusses structural factors such as ester pro-drugs and chiral interconversion. Specific examples are provided to illustrate how these different factors can influence stability and the importance of controlling for these factors during sample collection, storage, and processing. Approaches to improve stability are also described.
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HPLC is a form of liquid chromatography used to separate compounds dissolved in solution based on how they partition between a stationary and mobile phase. The key components of an HPLC system are a pump, injector, column, and detector. Method development involves selecting parameters like the mobile phase, column type, detection method, and chromatography conditions to optimize separation of the sample components. HPLC offers advantages like high sensitivity, rapid analysis times, and use for both analytical qualitative and quantitative analysis.
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This document discusses high performance liquid chromatography (HPLC). It begins by defining HPLC and explaining that it uses high pressure to forcibly pump the mobile phase, allowing for high performance and speed separations of mixtures into individual components. The document then discusses the basic components of an HPLC system including the solvent reservoir, pump, injector, column, detectors, and recorder. It explains the functions of these components and provides examples. It also discusses different types of HPLC including normal phase, reverse phase, size exclusion, and ion exchange. The document provides several examples and applications of HPLC.
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The document describes the process of developing and validating a reverse phase high performance liquid chromatography (RP-HPLC) method. It involves determining method goals and analysis requirements based on the sample properties, conducting research on existing methods, selecting an analysis technique, optimizing the separation conditions through a systematic approach, and validating the method. Key steps include choosing the detector and mobile phase, optimizing variables like column type, temperature, flow rate and solvent composition to improve resolution and separation time, and testing the method's accuracy, precision, specificity and robustness.
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1. The document discusses troubleshooting strategies and common problems in HPLC.
2. It outlines a 5-step troubleshooting strategy of identifying the problem, determining the cause, isolating the exact cause, rectifying the problem if possible, and returning the system to use.
3. Common problems discussed include issues with the mobile phase, pump, injector, detector, and peaks/baseline, along with potential causes and solutions for each.
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Columns in HPLC
This document provides information about different types of columns used in high performance liquid chromatography (HPLC). It discusses normal phase and reverse phase chromatography columns. It describes various column packing materials, particle sizes, dimensions, costs and specifications. It provides details on columns from several major manufacturers like Waters, Phenomenex, Agilent, GE Healthcare and others. Preparative chromatography is also briefly mentioned. Resources for further information are listed at the end.
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The document discusses analytical method development for HPLC. It notes that method development requires selecting requirements, instrumentation type, and why. Existing methods may be unreliable, expensive, or time-consuming, necessitating new method development. Key steps in development include defining goals, establishing sample preparation, selecting detector and mode of separation, performing preliminary separations, optimizing conditions, and validating the method. Method development is informed by factors like number of analytes, sample matrix, and analyte properties.
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What is % purity?
Different methods to determine % Purity.
Determination of % purity of Organic Compound By Using DSC:
•1) Polycyclic aromatic Hydrocarbons e. g Benzo[c]phenanthrene, BcPh, C18H12
•2) Ibuprofen medicine
•3) Phenacetin samples at different purity ratios
•4) Methamphetamine Paracetamol and Mixture of both.
•5) AZT (C10H13N5O4; 3´-azido-2,3´-dideoxythymidine)
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LC-MS combines liquid chromatography with mass spectrometry. It involves removing the detector from the LC column and interfacing the column directly with the mass spectrometer. The two key components are the ion source, which generates ions, and the mass analyzer, which sorts the ions. Common ion sources used include electrospray ionization, atmospheric pressure chemical ionization, and atmospheric pressure photoionization. Popular mass analyzers are quadrupole, time-of-flight, ion trap, and Fourier transform ion cyclotron resonance. LC-MS has applications in fields like molecular weight determination, structural determination, pharmaceutical analysis, food safety testing, and environmental analysis.
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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.
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This document provides an overview of high performance liquid chromatography (HPLC). It discusses how HPLC uses various chemical interactions between substances and a chromatographic column to separate components of a mixture. The key components of an HPLC system are described, including the solvent reservoir, pump, injectors, column, and detectors. Different modes of chromatographic separation are explained depending on the nature of the stationary phase. Applications of HPLC include analysis, separation, and purification of proteins, peptides, drugs and other compounds.
ION-EXCLUSION CHROMATOGRAPHY
This document discusses ion exclusion chromatography, which uses an ion exchange stationary phase to separate ionic and nonionic substances. Ionic substances pass quickly through the column while nonionic substances are retained longer. Separation depends on whether substances are ionized and repelled by the resin or able to enter the resin network if nonpolar or partially ionized. Detection methods include conductivity detectors and UV-visible or fluorescence detectors. Applications include separation of carboxylic acids, inorganic anions, amino acids, and determination of water in organic solvents.
HPLC
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
Hplc
The document discusses various aspects of high performance liquid chromatography (HPLC), including the types of separations, columns and stationary phases, mobile phases, injection, and detection methods. It provides details on normal phase, reversed phase, adsorption, and size exclusion chromatography. The roles of the stationary phase, mobile phase, column, and detector are explained. Common detection techniques like UV-VIS, diode array detection, refractive index, and evaporative light scattering are outlined.
Tieng viet hplc hl09
This document discusses high performance liquid chromatography (HPLC). HPLC uses a liquid mobile phase to separate components in a liquid or dissolved sample. The mobile phase passes through a column containing a stationary phase, which interacts with sample components to cause separation. Common stationary phases include bonded silica for reverse phase chromatography and ion exchange resins. Key aspects of HPLC covered include the instrument, mobile and stationary phases, sample introduction, detectors, and quantitative applications.
Ion pair chromatography final
1) Ion pair chromatography is a type of column chromatography that uses ion pairing agents to neutralize charged analytes and allow their separation on a reversed-phase column.
2) By adding counter ions with the opposite charge to the mobile phase, ion pairs form between the counter ions and analytes, neutralizing their charge and increasing their hydrophobicity.
3) The use of ion-pairing reagents as mobile phase additives allows the separation of ionic and highly polar substances that cannot otherwise be separated by reversed-phase chromatography.
Gaas' hplc
This document provides information on chromatography techniques. It discusses the basic components and processes involved in chromatography. The key techniques described are column chromatography, planar chromatography, gas chromatography, liquid chromatography, and affinity chromatography. It also summarizes different modes of chromatography such as normal phase chromatography, reverse phase chromatography, ion exchange chromatography, size exclusion chromatography, and affinity chromatography.
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HPLC method development and data analysis
- 2. Molecular characteristics of the sample. Selection of Stationary phase & Mobile Phase Troubleshooting & Optimization Data Analysis: Qualitative & Quantitative. Steps for HPLC method development
- 3. What are the molecular characteristics of the analyte or sample? CHASM 1. Charge Positive/negative 2. Hydrophobicity 3. Affinity “lock and key” sites 4. Solubility & stability pH, ionic strength, organic solvents 5. Molecular weight
- 4. Bulk (SiO2)x Bulk (SiO2)xSurface Surface Selection of Stationary Phase Normal Phase Reverse Phase SurfaceBulk (SiO2)x Ion Exchange
- 5. Normal Phase Reverse Phase Ion Exchange Stationary Phase Polar Non polar Silica having functional group Mobile Phase Non polar (hexane,heptan) & slightly polar (isopropanol, ethylacetate) Polar (water, methanol, acetonitrile, tetrahydrofuran) Depending on analyte, may salt gradient or pH gradient Sample Movement Non polar fastest Polar fastest More cation/anion Separation based on Different polarities (functionality) Different hydrocarbon content Ion exchange. Selection of Mobile Phase
- 7. Solvent used in Reverse Phase HPLC
- 8. Retention strength in Reverse Phase HPLC
- 9. Reverse Phase Normal Phase 1. Neutral & Nonpolar. compound (MW<2000 Da). 2. Homo-logs & Benzologs. 3. Weak Acid & Base. 4. Protein & Peptide. 1. Too hydrophilic/ hydrophobic. 2. Not soluble in water. 3. Isomers . Choice of columns
- 10. Data Analysis Qualitative Quantitative By Retention time By Spectral profile By Peak Area measurement
- 11. Use of reference standards for peak identification
- 12. Use of spectral detectors
- 13. Area Estimation
- 14. Amount of analyte Peak Area Response Factor Amount (Std.) Response Factor Peak Area (Std.) Calculation Std. Amount = 2,000 µg/ml Std. Peak Area = 100,000 Analyte Peak Area = 57,000 Amount (analyte) = ? Response Factor = 100,000/2,000 = 50 Amount (analyte) = 57,000/50 = 1,140 µg/ml
- 15. Troubleshooting Unusual Peak Shapes: 1. Broad Peak 2. Ghost Peak 3. Negative Peak 4. Peak Doubling 5. Peak Fronting & tailing Fluctuating Retention time:
- 16. Broad Peak
- 17. Ghost Peak
- 18. Negative peak
- 19. Peak doubling
- 20. Peak fronting & tailing