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, method development, applications, and commercially available instruments for preparative HPLC. The instrumentation section describes the major components of a preparative HPLC system including the solvent reservoir, pump, injector, columns, detectors, fraction collector and more. Method development and optimization factors like mobile phase selection, temperature, retention, selectivity, and column overloading techniques are also covered.
HPTLC- Principle, Instrumentation and Software (Abhishek Gupta)Abhishek Gupta
HPTLC is the improved method of TLC which utilizes the conventional technique of TLC in more optimized way
It is also known as planar chromatography or Flat-bed chromatography.
As we all know chromatographic fingerprinting of botanicals is a quite recent concept. This presentation will help to the beginners to understand basic thories and fundamantals of thin layer chomatography. The presentation will also provide basic experiemental understanding to perfrom HPTLC fingerprinting of samples/extracts/formulations.
HPTLC- Principle, Instrumentation and Software (Abhishek Gupta)Abhishek Gupta
HPTLC is the improved method of TLC which utilizes the conventional technique of TLC in more optimized way
It is also known as planar chromatography or Flat-bed chromatography.
As we all know chromatographic fingerprinting of botanicals is a quite recent concept. This presentation will help to the beginners to understand basic thories and fundamantals of thin layer chomatography. The presentation will also provide basic experiemental understanding to perfrom HPTLC fingerprinting of samples/extracts/formulations.
Ion pair chromatography for pharmacy studentsabhishek rai
Ion-PairChromatography
A GENERALISED OVERVIEW
Chromatography
HPLC
Reverse Phase Chromatography
Ion Pair Chromatography
Ion Pair Reagent
Mechanism of Ion Pair Chromatography
Ion Pair Wash Procedure
Detectors are the brain of any chromatograhic system. It help us to record the chromatogram based on certain characteristics of the analyte and help us in identifying that compound both qualitatively and quantitatively.
The drug or drug combination may not be official in any pharmacopoeias.
A proper analytical procedure for the drug may not be available in the literature due to patent regulations.
Analytical methods may not be available for the drug in the form of a formulation due to the interference caused by the formulation excipients.
Analytical methods for the quantitation of the drug in biological fluids may not be available.
Analytical methods for a drug in combination with other drugs may not be available.
The existing analytical procedures may require expensive reagents and solvents. It may also involve cumbersome extraction and separation procedures and these may not be reliable.
fluid chromatography (SFC) can be used on an analytical
scale.
It is a combination of High performance liquid chromatography (HPLC)
and Gas chromatography (GC).
It can be used with non-volatile and thermally labile analytes.
It can be used with the universal flame ionization detector.
It is important to producing narrower peaks due to rapid diffusion.
It is important for the chiral separations and analysis of high-molecularweight
hydrocarbons.
Supercritical fluids are suitable as a substitute for organic solvents in a
range of industrial and laboratory processes.
HPLC[ HIGH PERPROMANCE LIQUID CHROMATOGRAPHY OR HIGH PRESSURE LIQUID CHROMAT...Dr. Ravi Sankar
GASSCHROMATOGRAPHY[GC], ADVANCED STUDY OF THE FOLLOWING AND THEIR APPLICATIONS, INTRODUCTION, THEORY, COLUMN OPERATION,INSTRUMENTATION AND DETECTION,APPLICATIONS AND ADVANTAGES OF GC,PRINCIPLE OF SEPARATION IN GC, HOW GC MECHINE WORKS? COLUMN, DETECTORS.
BY P.RAVISANKAR, VIGNAN PHARMACY COLLEGE, VADLAMUDI, GUNTUR, A.P, INDIA.
The Preparative and Process Chromatography | Find out - Market Size | Market ...Akash Jaiswal
The Preparative and Process Chromatography market size, in terms of value, is expected to reach $8,950 million by 2019 from $6,351.5 million in 2014, at a CAGR of 7.1%. The Process Chromatography market is segmented on the basis of products and services, end user, and geography.
Ion pair chromatography for pharmacy studentsabhishek rai
Ion-PairChromatography
A GENERALISED OVERVIEW
Chromatography
HPLC
Reverse Phase Chromatography
Ion Pair Chromatography
Ion Pair Reagent
Mechanism of Ion Pair Chromatography
Ion Pair Wash Procedure
Detectors are the brain of any chromatograhic system. It help us to record the chromatogram based on certain characteristics of the analyte and help us in identifying that compound both qualitatively and quantitatively.
The drug or drug combination may not be official in any pharmacopoeias.
A proper analytical procedure for the drug may not be available in the literature due to patent regulations.
Analytical methods may not be available for the drug in the form of a formulation due to the interference caused by the formulation excipients.
Analytical methods for the quantitation of the drug in biological fluids may not be available.
Analytical methods for a drug in combination with other drugs may not be available.
The existing analytical procedures may require expensive reagents and solvents. It may also involve cumbersome extraction and separation procedures and these may not be reliable.
fluid chromatography (SFC) can be used on an analytical
scale.
It is a combination of High performance liquid chromatography (HPLC)
and Gas chromatography (GC).
It can be used with non-volatile and thermally labile analytes.
It can be used with the universal flame ionization detector.
It is important to producing narrower peaks due to rapid diffusion.
It is important for the chiral separations and analysis of high-molecularweight
hydrocarbons.
Supercritical fluids are suitable as a substitute for organic solvents in a
range of industrial and laboratory processes.
HPLC[ HIGH PERPROMANCE LIQUID CHROMATOGRAPHY OR HIGH PRESSURE LIQUID CHROMAT...Dr. Ravi Sankar
GASSCHROMATOGRAPHY[GC], ADVANCED STUDY OF THE FOLLOWING AND THEIR APPLICATIONS, INTRODUCTION, THEORY, COLUMN OPERATION,INSTRUMENTATION AND DETECTION,APPLICATIONS AND ADVANTAGES OF GC,PRINCIPLE OF SEPARATION IN GC, HOW GC MECHINE WORKS? COLUMN, DETECTORS.
BY P.RAVISANKAR, VIGNAN PHARMACY COLLEGE, VADLAMUDI, GUNTUR, A.P, INDIA.
The Preparative and Process Chromatography | Find out - Market Size | Market ...Akash Jaiswal
The Preparative and Process Chromatography market size, in terms of value, is expected to reach $8,950 million by 2019 from $6,351.5 million in 2014, at a CAGR of 7.1%. The Process Chromatography market is segmented on the basis of products and services, end user, and geography.
HPLC Principle,Instrumentation and ApplicationAlakesh Pradhan
HPLC Chromatography and its principle
Liquid chromatography
High Performance Liquid Chromatography ( HPLC )
The components of the high performance liquid chromatograph (HPLC).
The separation process.
The chromatogram
Veeprho Pharmaceuticals s.r.o is a supplier of Atorvastatin Impurities like Trans Atorvastatin Impurity, Atorvastatin Lactone Impurity and Atorvastatin Related compound A, B,C, D, E etc. to pharmaceuticals industry.
A separation technique in which the mobile phase is a gas. Gas chromatography is always carried out in a column.
Separating mixtures of gases or volatile materials based primarily on their physical properties.
Silica Gel | Aluminium Oxide Column chroamtographySORBEAD INDIA
Buy Silica Gel Powder for Silica Gel, Aluminium Oxide, Paper and Flash Column Chromatography us in Pharmaceutical Industries – Bulk Drugs & API, Nutraceuticals, Herbal Extracts products manufacturers, Research Laboratories, Laboratories Chemical Repackaging, Contract Research Laboratories. Column Chromatography is one of the most useful methods for purification & separation (Isolation) of individual desire compound from mixture of unwanted compounds.
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.
Following are the list of attributes to be followed;
1.0 Objective:
The purpose of the study is to develop analytical method for determination of Assay & Related substances in new formulation product by HPLC using UV-Visible detector.
2.0 Introduction:
This document will help analyst to develop robust Analytical method for routine testing.
3.0 Scope:
This guideline will provide information about analytical method development to be carried out as per ICH Guidelines.
4.0 Physico-Chemical details of API:
Following items to be checked viz;
IUPAC name, Molecular formula, Chemical structure, Molecular weight, Solubility, Appearance, Melting Point, pH solubility, pKa, LogP, BCS Classification, Polymorphism, Isomerism, Density, Hygroscopicity, Impurities listed as per process either in DMF or official monograph
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. 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.