This document presents information on preparative high pressure liquid chromatography. It begins with an introduction to chromatography and classification of column chromatographic methods. It then discusses the differences between analytical and preparative HPLC and the objectives, instrumentation including pumps, injectors, columns, detectors, and fraction collectors of preparative HPLC. The document also covers topics such as method development and optimization, applications, hyphenation with other techniques, and commercially available instruments.
The document provides an overview of high performance liquid chromatography (HPLC). It begins with defining HPLC and explaining the basic principles of chromatography. It then describes the different types of HPLC based on the mode and principle of separation. The document also discusses HPLC instrumentation, including the solvent reservoir, pump, injector, column, and various detectors. It concludes by outlining some common applications of HPLC and discussing quantitative and qualitative analysis.
This document provides an overview of high performance liquid chromatography (HPLC). It discusses the principle, history, types, instrumentation, procedure, advantages, disadvantages and applications of HPLC. Key points include that HPLC uses high pressure to separate compounds faster and more efficiently than other chromatography techniques. Silica is commonly used as the stationary phase because it can withstand pressures over 300-400 atmospheres. The major components of HPLC instrumentation are the solvent reservoir, pump, injector, column, detector and data collector. HPLC has various applications in fields like analytical chemistry, pharmaceuticals, forensics and environmental analysis.
A brief review on development and validation of hplc method.adhirajain
the slides in the ppt gives a brief review on product development and its validation in HPLC method. Contents are with advantages, disadvantages, application , classification and methods for development.
Chromatography is a laboratory technique for the separation of a mixture. The mixture is dissolved in a fluid called the mobile phase, which carries it through a structure holding another material called the stationary phase.
High-performance liquid chromatography (HPLC) is an analytical chemistry technique used to separate, identify, and quantify components in mixtures. It works by forcing a pressurized liquid solvent through a column packed with adsorbent particles under high pressure. This allows for better separation than traditional column chromatography due to smaller particle sizes and detection methods. HPLC has applications in manufacturing, legal, research, and medical fields such as drug analysis, food testing, and pharmaceutical development.
The document provides guidance on developing an analytical method for determining assay and related substances in new drug formulations using HPLC-UV. It outlines the key steps, including selecting the detector and chromatographic conditions based on the drug's properties; forced degradation studies to identify degradation products; method validation including linearity, accuracy and comparison to pharmacopeial methods; and establishing system suitability criteria and finalizing the method. The goal is to develop a robust stability-indicating method for routine quality control testing according to ICH guidelines.
Briefly introduce what flash chromatography is and its purpose in scientific research.
Explain the key principles of flash chromatography, including the differences between normal phase and reverse phase chromatography.
Discuss the equipment required for flash chromatography and the different types of media that can be used.
Provide examples of when flash chromatography is commonly used in scientific research, such as in drug discovery or natural product isolation.
Discuss the advantages and limitations of flash chromatography compared to other chromatography techniques.
Highlight any novel or innovative applications of flash chromatography that you may have explored in your research.
Conclude with a summary of the key takeaways from your presentation, including any practical tips or advice for those looking to use flash chromatography in their own work.
1) HPLC provides improved performance over classical column chromatography due to smaller particle sizes (<5 microns), higher operating pressures (>4000 psi), and higher column efficiencies (>100,000 theoretical plates per meter).
2) There are two main modes of HPLC separation - normal phase which uses a polar stationary phase and non-polar mobile phase, and reverse phase which uses a non-polar stationary phase and polar mobile phase.
3) Key components of an HPLC system include pumps to deliver the mobile phase at high pressure, injectors to introduce samples, columns packed with stationary phase to perform the separation, and detectors such as UV/Vis to identify eluted components.
The document provides an overview of high performance liquid chromatography (HPLC). It begins with defining HPLC and explaining the basic principles of chromatography. It then describes the different types of HPLC based on the mode and principle of separation. The document also discusses HPLC instrumentation, including the solvent reservoir, pump, injector, column, and various detectors. It concludes by outlining some common applications of HPLC and discussing quantitative and qualitative analysis.
This document provides an overview of high performance liquid chromatography (HPLC). It discusses the principle, history, types, instrumentation, procedure, advantages, disadvantages and applications of HPLC. Key points include that HPLC uses high pressure to separate compounds faster and more efficiently than other chromatography techniques. Silica is commonly used as the stationary phase because it can withstand pressures over 300-400 atmospheres. The major components of HPLC instrumentation are the solvent reservoir, pump, injector, column, detector and data collector. HPLC has various applications in fields like analytical chemistry, pharmaceuticals, forensics and environmental analysis.
A brief review on development and validation of hplc method.adhirajain
the slides in the ppt gives a brief review on product development and its validation in HPLC method. Contents are with advantages, disadvantages, application , classification and methods for development.
Chromatography is a laboratory technique for the separation of a mixture. The mixture is dissolved in a fluid called the mobile phase, which carries it through a structure holding another material called the stationary phase.
High-performance liquid chromatography (HPLC) is an analytical chemistry technique used to separate, identify, and quantify components in mixtures. It works by forcing a pressurized liquid solvent through a column packed with adsorbent particles under high pressure. This allows for better separation than traditional column chromatography due to smaller particle sizes and detection methods. HPLC has applications in manufacturing, legal, research, and medical fields such as drug analysis, food testing, and pharmaceutical development.
The document provides guidance on developing an analytical method for determining assay and related substances in new drug formulations using HPLC-UV. It outlines the key steps, including selecting the detector and chromatographic conditions based on the drug's properties; forced degradation studies to identify degradation products; method validation including linearity, accuracy and comparison to pharmacopeial methods; and establishing system suitability criteria and finalizing the method. The goal is to develop a robust stability-indicating method for routine quality control testing according to ICH guidelines.
Briefly introduce what flash chromatography is and its purpose in scientific research.
Explain the key principles of flash chromatography, including the differences between normal phase and reverse phase chromatography.
Discuss the equipment required for flash chromatography and the different types of media that can be used.
Provide examples of when flash chromatography is commonly used in scientific research, such as in drug discovery or natural product isolation.
Discuss the advantages and limitations of flash chromatography compared to other chromatography techniques.
Highlight any novel or innovative applications of flash chromatography that you may have explored in your research.
Conclude with a summary of the key takeaways from your presentation, including any practical tips or advice for those looking to use flash chromatography in their own work.
1) HPLC provides improved performance over classical column chromatography due to smaller particle sizes (<5 microns), higher operating pressures (>4000 psi), and higher column efficiencies (>100,000 theoretical plates per meter).
2) There are two main modes of HPLC separation - normal phase which uses a polar stationary phase and non-polar mobile phase, and reverse phase which uses a non-polar stationary phase and polar mobile phase.
3) Key components of an HPLC system include pumps to deliver the mobile phase at high pressure, injectors to introduce samples, columns packed with stationary phase to perform the separation, and detectors such as UV/Vis to identify eluted components.
CHROMATOGRAPHY and its types with procedure,diagrams,flow charts,advantages a...university
Chromatography is a technique used to separate mixtures by distributing components between a stationary and mobile phase. The document discusses various chromatography techniques including thin layer chromatography, paper chromatography, column chromatography, high performance liquid chromatography, and gas chromatography. It explains the basic principles, procedures, applications, advantages, and disadvantages of each technique. HPLC is described as a highly improved form of column chromatography that uses high pressure to force the mobile phase through the column, allowing for faster and more efficient separations.
Here are the basic steps to calculate the molarity of a standard solution:
1. Weigh out an accurately known amount of the solid reagent.
2. Dissolve the solid in a known volume of solvent. This gives us the total moles of solute and total volume of solution.
3. Use the formula M = moles of solute / liters of solution to calculate the molarity.
For example, if we weighed 2.345 g of NaOH and dissolved it in 1 L of water, we would:
- Find the moles of NaOH using its molar mass (40.00 g/mol): moles = mass / molar mass =
HPLC is a type of liquid chromatography that is used to separate, identify, and quantify components in a mixture. It works by forcing a pressurized liquid mobile phase through a column packed with solid particles or porous material. Samples are injected into the column and the different components interact differently with the stationary phase, causing them to elute from the column at different rates and allowing separation. HPLC provides efficient, high resolution separations and is commonly used in fields like pharmaceutical analysis and quality control due to its ability to analyze complex mixtures.
The document provides an overview of high performance liquid chromatography (HPLC). It discusses the history and development of chromatography and HPLC. The basic components of an HPLC system are described, including the solvent reservoir, pump, injector, column, detector, and recorder/integrator. Various techniques are also summarized, such as reverse phase chromatography. The document outlines the principles of HPLC separation and provides examples of applications such as pharmaceutical analysis.
HPLC is a chromatographic technique used to separate components in a mixture. It works by pumping a pressurized mobile liquid phase through a column containing a stationary phase, which causes the components in a sample to separate as they are transported through the column at different rates. Key components of an HPLC system include solvent reservoirs, pumps to precisely deliver the mobile phase, an injector to introduce samples, columns for separation, detectors, and a data system to analyze results. HPLC offers advantages like high separation capacity, reproducibility, and ability to analyze a wide range of biological, medical, food, environmental, and industrial samples.
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.
This document discusses high performance liquid chromatography (HPLC). It begins by defining chromatography as a technique used to separate mixtures into their individual components using both a stationary and mobile phase. It then describes some key aspects of HPLC, including that it uses high pressure to force the mobile phase through a column with small particle sizes for better separation. The document outlines the basic components of an HPLC system, including the pump, injector, column, detectors, and computer. It also discusses some common terms and uses for HPLC, such as separating and analyzing compounds in research, quality control, and environmental monitoring.
Nilesh Dashrath Kamble presented a seminar on method development and validation in HPLC. The presentation discussed the steps involved in HPLC method development including column selection, mobile phase composition, pH range selection, and optimization of separation conditions. It also covered validation parameters such as accuracy, precision, specificity, limit of detection, and limit of quantification as per ICH guidelines. The presentation included an example method development for the simultaneous estimation of atorvastatin and telmisartan from a tablet formulation.
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.
Development and Validation of a RP-HPLC methodUshaKhanal3
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.
High Performance Liquid Chromatography (HPLC) is a separation technique that uses pumps to force a liquid mobile phase through a column packed with solid particles. Sample components interact differently with the stationary and mobile phases allowing separation. HPLC instruments consist of pumps, injectors, columns, detectors and computers. Samples are injected and the separated components are detected and data is analyzed to identify and quantify the components. HPLC is used in various fields to analyze complex mixtures like pharmaceuticals, chemicals, and biological samples.
This document discusses chromatography and PCR techniques. It provides details on:
- The principles and types of chromatography including TLC, HPLC, and their components and procedures. HPLC allows for quantitative analysis and is commonly used for pharmaceutical quality control.
- PCR amplification which uses DNA polymerase to exponentially replicate DNA sequences. It requires template DNA, primers, nucleotides, and DNA polymerase. Repeated heating and cooling cycles allow for target DNA replication.
- Applications of chromatography and PCR include pharmaceutical analysis, forensic analysis, detection of genetic disorders, microbial detection, and molecular biology research techniques. Both provide powerful tools for separation, detection, and analysis of biological molecules.
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.
The slides are informative of HIGH PERFORMANCE THIN LAYER CHROMATOGRAPHY & its thorough components further its advantages and applications. The comparison of HPLC and HPTLC is explained.
This document provides an overview of high performance thin layer chromatography (HPTLC). It discusses the principle, steps involved, applications, and references. HPTLC is an automated form of TLC that allows for the separation and analysis of multiple samples simultaneously. Key steps include selecting plates and mobile phases, applying samples, developing the plate, and detecting and visualizing results. HPTLC has various applications in pharmaceutical analysis, food testing, herbal product identification, and clinical and forensic analysis. It can be used to quantify compounds and perform multi-component analyses of drugs, food contaminants, and herbal constituents.
This document summarizes a student's research project analyzing the quantitative analysis of the drug lamotrigine using high performance liquid chromatography (HPLC). The student aims to partially validate an HPLC technique for quantifying lamotrigine. The document provides background on HPLC, including its history, theory of operation, instrumentation such as columns, detectors, and pumps. It also discusses validation components like accuracy, precision, linearity and limits of detection and quantification that the student will analyze to partially validate the HPLC method for quantifying lamotrigine.
High performance liquid chromatography (HPLC) involves injecting a liquid sample into a column packed with porous particles where the sample components are separated based on interactions with the packing particles. Components exit the column and are identified by a detector. Modern HPLC uses high pressure to achieve fast flow rates through columns packed with small, uniform particles for high separation efficiency. HPLC has various modes and is widely used in pharmaceutical, chemical, biochemical, and other industries for analysis, quantification, and purification.
The simplified electron and muon model, Oscillating Spacetime: The Foundation...RitikBhardwaj56
Discover the Simplified Electron and Muon Model: A New Wave-Based Approach to Understanding Particles delves into a groundbreaking theory that presents electrons and muons as rotating soliton waves within oscillating spacetime. Geared towards students, researchers, and science buffs, this book breaks down complex ideas into simple explanations. It covers topics such as electron waves, temporal dynamics, and the implications of this model on particle physics. With clear illustrations and easy-to-follow explanations, readers will gain a new outlook on the universe's fundamental nature.
CHROMATOGRAPHY and its types with procedure,diagrams,flow charts,advantages a...university
Chromatography is a technique used to separate mixtures by distributing components between a stationary and mobile phase. The document discusses various chromatography techniques including thin layer chromatography, paper chromatography, column chromatography, high performance liquid chromatography, and gas chromatography. It explains the basic principles, procedures, applications, advantages, and disadvantages of each technique. HPLC is described as a highly improved form of column chromatography that uses high pressure to force the mobile phase through the column, allowing for faster and more efficient separations.
Here are the basic steps to calculate the molarity of a standard solution:
1. Weigh out an accurately known amount of the solid reagent.
2. Dissolve the solid in a known volume of solvent. This gives us the total moles of solute and total volume of solution.
3. Use the formula M = moles of solute / liters of solution to calculate the molarity.
For example, if we weighed 2.345 g of NaOH and dissolved it in 1 L of water, we would:
- Find the moles of NaOH using its molar mass (40.00 g/mol): moles = mass / molar mass =
HPLC is a type of liquid chromatography that is used to separate, identify, and quantify components in a mixture. It works by forcing a pressurized liquid mobile phase through a column packed with solid particles or porous material. Samples are injected into the column and the different components interact differently with the stationary phase, causing them to elute from the column at different rates and allowing separation. HPLC provides efficient, high resolution separations and is commonly used in fields like pharmaceutical analysis and quality control due to its ability to analyze complex mixtures.
The document provides an overview of high performance liquid chromatography (HPLC). It discusses the history and development of chromatography and HPLC. The basic components of an HPLC system are described, including the solvent reservoir, pump, injector, column, detector, and recorder/integrator. Various techniques are also summarized, such as reverse phase chromatography. The document outlines the principles of HPLC separation and provides examples of applications such as pharmaceutical analysis.
HPLC is a chromatographic technique used to separate components in a mixture. It works by pumping a pressurized mobile liquid phase through a column containing a stationary phase, which causes the components in a sample to separate as they are transported through the column at different rates. Key components of an HPLC system include solvent reservoirs, pumps to precisely deliver the mobile phase, an injector to introduce samples, columns for separation, detectors, and a data system to analyze results. HPLC offers advantages like high separation capacity, reproducibility, and ability to analyze a wide range of biological, medical, food, environmental, and industrial samples.
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.
This document discusses high performance liquid chromatography (HPLC). It begins by defining chromatography as a technique used to separate mixtures into their individual components using both a stationary and mobile phase. It then describes some key aspects of HPLC, including that it uses high pressure to force the mobile phase through a column with small particle sizes for better separation. The document outlines the basic components of an HPLC system, including the pump, injector, column, detectors, and computer. It also discusses some common terms and uses for HPLC, such as separating and analyzing compounds in research, quality control, and environmental monitoring.
Nilesh Dashrath Kamble presented a seminar on method development and validation in HPLC. The presentation discussed the steps involved in HPLC method development including column selection, mobile phase composition, pH range selection, and optimization of separation conditions. It also covered validation parameters such as accuracy, precision, specificity, limit of detection, and limit of quantification as per ICH guidelines. The presentation included an example method development for the simultaneous estimation of atorvastatin and telmisartan from a tablet formulation.
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.
Development and Validation of a RP-HPLC methodUshaKhanal3
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.
High Performance Liquid Chromatography (HPLC) is a separation technique that uses pumps to force a liquid mobile phase through a column packed with solid particles. Sample components interact differently with the stationary and mobile phases allowing separation. HPLC instruments consist of pumps, injectors, columns, detectors and computers. Samples are injected and the separated components are detected and data is analyzed to identify and quantify the components. HPLC is used in various fields to analyze complex mixtures like pharmaceuticals, chemicals, and biological samples.
This document discusses chromatography and PCR techniques. It provides details on:
- The principles and types of chromatography including TLC, HPLC, and their components and procedures. HPLC allows for quantitative analysis and is commonly used for pharmaceutical quality control.
- PCR amplification which uses DNA polymerase to exponentially replicate DNA sequences. It requires template DNA, primers, nucleotides, and DNA polymerase. Repeated heating and cooling cycles allow for target DNA replication.
- Applications of chromatography and PCR include pharmaceutical analysis, forensic analysis, detection of genetic disorders, microbial detection, and molecular biology research techniques. Both provide powerful tools for separation, detection, and analysis of biological molecules.
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.
The slides are informative of HIGH PERFORMANCE THIN LAYER CHROMATOGRAPHY & its thorough components further its advantages and applications. The comparison of HPLC and HPTLC is explained.
This document provides an overview of high performance thin layer chromatography (HPTLC). It discusses the principle, steps involved, applications, and references. HPTLC is an automated form of TLC that allows for the separation and analysis of multiple samples simultaneously. Key steps include selecting plates and mobile phases, applying samples, developing the plate, and detecting and visualizing results. HPTLC has various applications in pharmaceutical analysis, food testing, herbal product identification, and clinical and forensic analysis. It can be used to quantify compounds and perform multi-component analyses of drugs, food contaminants, and herbal constituents.
This document summarizes a student's research project analyzing the quantitative analysis of the drug lamotrigine using high performance liquid chromatography (HPLC). The student aims to partially validate an HPLC technique for quantifying lamotrigine. The document provides background on HPLC, including its history, theory of operation, instrumentation such as columns, detectors, and pumps. It also discusses validation components like accuracy, precision, linearity and limits of detection and quantification that the student will analyze to partially validate the HPLC method for quantifying lamotrigine.
High performance liquid chromatography (HPLC) involves injecting a liquid sample into a column packed with porous particles where the sample components are separated based on interactions with the packing particles. Components exit the column and are identified by a detector. Modern HPLC uses high pressure to achieve fast flow rates through columns packed with small, uniform particles for high separation efficiency. HPLC has various modes and is widely used in pharmaceutical, chemical, biochemical, and other industries for analysis, quantification, and purification.
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1. Presented By : Miss. Bharti G. Jadhav.
(First Year M. Pharm.)
(Quality Assurance.)
Under Guidance of : Mr. Abhay R. Shirode.
(Assistant Professor.)
Bharati Vidyapeeth’s College of Pharmacy,
C.B.D., Belapur, Navi Mumbai- 400614.
PREPARATIVE HIGH PRESSURE LIQUID
CHROMATOGRAPHY.
2. 1. Introduction to chromatography.
2. Classification of column chromatographic methods.
3. Preparative chromatography.
4. Preparative HPLC.
5. Objectives.
6. Instrumentation.
7. Method development and optimisation.
8. Applications.
9. Hyphenation with other analytical chromatographic
techniques.(Review of reported studies)
10. Commercially available instruments for preparative high
pressure liquid chromatography.
HIGHLIGHTS:
3. Chromatography is a powerful separation method. The term
chromatography( Greek word Chroma= “Color” and Graphein=
“To write”) meaning color writing.
INTRODUCTION TO CHROMATOGRAPHY:
What is mean By
Chromatography
4. General Classification Specific
Method
Stationary Phase Type Of Equilibrium
Liquid chromatography
( Mobile Phase: Liquid)
Liquid-Liquid Liquid adsorbs on solid Partition between immiscible
liquids
Liquid bonded
phase
Organic species bonded to a
solid surface
Partition between liquid and
bonded surface
Solid-Liquid Solid Adsorption
Ion exchange Ion exchange resin Ion exchange
Size exclusion Liquid in interstices of a
polymeric solid
Partition
Gas chromatography
(Mobile Phase: Gas)
Gas-Liquid Liquid adsorbs on solid Partition between gas and
liquid
Gas bonded
phase
Organic species bonded to
solid surface
Partition between liquid and
bonded surface
Gas-Solid Solid Adsorption
Supercritical Fluid
chromatography
(Mobile Phase:
Supercritical Fluid)
Organic species bonded to
solid surface
Partition between
supercritical fluid and solid
surface.
Table 1: Types of Column Chromatographic Methods.
5. • Powerful technique for the isolation and purification of variety
of chemicals, pharmaceutical compounds, natural products and
biological molecules.
• To increase throughput and separation power, the first
preparative HPLC system was developed in the 1970’s.
• Types of HPLC: Based on the scale of operation.
PREPARATIVE CHROMATOGRAPHY:
6. Analytical HPLC. Preparative HPLC.
1. Sample goes from detector into waste. 1. Sample goes from detector into
fraction collector.
2. Use quantification and/or identification
of compounds.
2. Use for isolation and/purification of
compounds.
3. Column has internal diameter-1-5mm 3. Column has internal diameter-1-10cm
4. Column particles are 5um or smaller. 4. Column particles are 7um or larger.
5. HPLC pump provide up to 10mL/min. 5. HPLC pump provide >>10mL/min.
6. Solubility of sample in mobile phase
usually not important.
6. Solubility of sample usually very
important.
7. Mobile phase is not recover. 7. Mobile phase recovery is possible.
Table 2: Difference Between Analytical And Preparative HPLC.
13. 1. SOLVENT RESERVOIR:
Material of construction: Glass or stainless steel
For biologically sensitive, or labile substances: Coating of
biocompatible material.
14. 2. PREPARATIVE PUMP:
Figure 3: Dual Preparative Pump. Figure 4: Industrial Preparative System.
• Requires high eluent flow rate 10 and 100 ml/min and
large internal diameter of columns.
• A larger piston head is required to work at flows of 10-
100ml/min.
15. • Should inject sample within
the range of 0.1 to 100 ml.
• Rheodyne injector is used.
3. PREPARATIVE INJECTOR:
Figure 6: Positions Of Rheodyne Injector.
Figure 5: Rheodyne Injector.
16. Column is the heart
of the liquid chromatography.
Sample distribution plate is
used to distribute the sample
across the column.
It consists of a disc
with series of radial slots.
4. PREPARATIVE COLUMNS:
Figure 7: A typical Preparative Column
17. Scale
Column
I.D. (mm)
Quality of
Product
Typical Column
Length (mm)
Purpose
Analytical 4.6 1 - 40mg 250
Biological materials for
activity testing
Semi-Prep 10 - 30 100mg - 3g 250 Reference compounds
Preparative 50 - 70 5 - 10g 250 - 1000
Intermediates for lab
synthesis
Pilot 100 - 300 20g - 5kg 300 - 1000
Pharmaceutical
development
Process >300 kg - tons 500 - 1000 Large scale production
19. It depends on the particle size, scale of the separation and on
the nature of the material to be separated.
There are two type of Column Packing’s.
Particle size more than 20mm- Dry Packing.
Particle size less than 20mm- Slurry Packing.
Packing of Preparative Columns:
Packing of
preparative
columns
20. Once column diameter approaches 5 cm, additional difficulties
arises.
Formation of channels/bridges in the column bed.
Chances of friction between particles and wall of the column.
Figure 9: Difference between Analytical and Preparative Column.
Difficulties Aries During Packing of Analytical Column
24. In preparative HPLC eluent should be
diluted with more mobile phase and
then passed through the detector.
Detectors are same as that of HPLC.
5. PREPARATIVE DETECTOR:
25. In preparative HPLC sample goes from detector to fraction
collector.
The fraction collector diverts the flow either to waste or, to a
fraction container via the fraction collection needle which
can achieve by using diverter valve.
6. FRACTION COLLECTOR:
Figure 14: Fraction Collector System.
26. The preparative scale fraction collector is designed for flow rates up
to 100 mL/min.
Figure 15: Eluent Flow Rate.
Designing of Fraction Collector:
27. • Based on a signal plot.
• Highest flexibility.
Manual
fraction collection
• Based upon detector response.
Peak-based
fraction collection
• Compound with the desired
mass is selectively collected
Mass-based
fraction collection
Time-based
fraction collection
Fraction Collection Methods:
• Based on time of interval
28. Scale Up
Optimization of Throughput
( Column Overloading)
Optimization of Separation
(Mobile Phase, Stationary Phase, Temperature, Retention, Selectivity.)
Selection of Appropriate Mode of Separation
Definition of Separation Problem
METHOD DEVLOPMENT AND OPTIMIZATION OF
PREPARATIVE HIGH PRESSURE LIQUID
CHROMATOGRAPHIC METHOD:
29. The first step in preparative method development is to identify the
problem and challenges associated.
Sample information.
Analyte(s) of interest. (type, number, concentration, required
level of purity)
Other separation strategies suitable for your sample.
Detection.
Amount of material to be isolated.
Required degree of accuracy, precision etc.
Method verification.
DEFINITION OF SEPATION PROBLEM:
30. The following factors should be considered when selecting
the appropriate HPLC mode for your separation.
Solubility.
Molecular weight.
Sample matrix.
Detectability.
Other separation alternatives.
SELECTION OF APPROPRIATE MODE OF
SEPARATION:
31. 1. Mobile Phase:
Viscosity of mobile phase.
sample solubility in mobile phase.
pH
volatility of solvents/buffers.
solvent cost.
2. Stationary Phase:
The chemistry of the stationary phase controls,
Selectivity.
Production rate.
3. Temperature:
Increase in temperature,
Improves resolution and solubility.
Decreases the viscosity of the mobile phase.
Increase in production rate.
Optimisation of Separation:
32. 4.Retention:
• Minimum retention factor(k) necessary for isolating the product
and providing the desired purity, cycle time is decreased and the
production rate increased.
• Concentration of the product in collected fractions decreases
when retention increases, Column efficiency increases, but the
cycle time and solvent consumption are increased as well.
• k= 1.2-2.0 for isocratic separation, k = 3-4 for gradient
separations.
5. Selectivity:
• Increasing the selectivity value up to 2 or 3 significantly
improves the throughput of separation.
• Selectivity can be optimised by changing the solvent
composition as well as pH and nature of buffer added to the
mobile phase.
33. Two ways of performing column overloading:
1. volume overloading. 2. concentration overloading.
Table 4: Difference between Volume and Column Overloading.
OPTIMIZATION OF THE THROUGHPUT:
Volume Overloading. Concentration Overloading.
1. Determined by injection volume. 1. Determined by solubility of the
compound in mobile phase.
2. Appropriate when sample has poor
solubility.
2. Appropriate when sample has good
solubility
3. Throughput determined by column
diameter.
3. Throughput determined by selectivity.
4. Analytical area of adsorption
isotherm.
4. Preparative area of adsorption
isotherm.
5. Small particle size improves 5. Particle size has very little influence
34. 1. Purification in medicinal or high-throughput chemistry.
2. Purification in natural product chemistry.
3. Purification of by-products for impurity analysis.
4. Recovery collection.
5. Automated fraction re-analysis.
APPLICATIONS OF PREPARATIVE HPLC:
35. 1. Preparative High-Performance Liquid
Chromatography–Mass Spectrometry for the High-
Throughput Purification of Combinatorial Libraries.
• Marcus Bauser* Medicinal Chemistry VII, Business
Group Pharma, BAYER AG, 42096 Wuppertal,
Germany
Preparative HPLC when coupled/hyphenated with
other analytical techniques:
(review of reported studies)
36. 2. Excellent combination of counter-current chromatography and
Preparative high-performance liquid chromatography to separate
galactolipids from pumpkin.
• A. Berthod, G.G. Leitao, I.A. Sutherland and W.D. Conway
3. Preparative high pressure liquid chromatography-flash
chromatography.(Puriflash)
37. Commercially available instruments for
preparative high pressure liquid
chromatography:
1. Japan analytical Industry:
Features:
Reduces expenses of purchasing
columns.
Achieves the same separation as
if using a longer column.
38. 2. Waters:
Features:
Flexible solvent delivery options
allowing binary or quaternary
based pumps providing low-
pressure multi-solvent blending or
high pressure gradient mixing of
flow rates up to 150 ml/min.
Easy to use.
highly sensitive UV/Visible or
Photodiode Array detectors are
use.
39. CONCLUSION:
Preparative chromatography is powerful technique
for the isolation and purification of variety of
chemicals, including pharmaceutical compounds,
natural products and biological molecules.
If we optimise all the parameter like column
loadability, selectivity, flow rate, particle size, we
can scale up the technique from analytical to
preparative scale.
40. REFERENCES:
1. Taylor T., White C.A. ,The CHROMacademy Essential
Guide - Basics of Preparative HPLC;105-115.
2. Kazakevich Y., LoBruto R., John Wiley & Sons. “HPLC for
Pharmaceutical Scientists” ISBN-13:2007; 937 – 980.
3. Truei Y., Tingyue Gu, Tsai G., Large-Scale Gradient Elution
Chromatography Advances in Biochemical
Engineering/Biotechnology.1992,( 47);1-44.
4. Breslav M., Leshchinskaya V., Preparative High performance
Liquid Chromatography Optimisation;1903-0909.
5. Ganetsos G., Barker P.E.,Preparative and Production scale
Chromatography, Chromatographic Science,1993(61);786.
6. Berthod A., Leitao G.G., Sutherland I.A., Excellent
combination of counter-current chromatography and
preparative high-performance liquid chromatography to
separate galactolipids from pumpkin,8 May 2009, 19 (1216);
4176–418.