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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.

7. PRINCIPLE:
 Adsorption.
Similar to HPLC.
The only difference is sample goes from detector into fraction
collector.
PREPARATIVE HPLC:

9. OBJECTIVES OF PREPARATIVE HPLC
Purity
Yield
Throughput

10. 1. Solvent reservoir
2. Pump
3. Preparative injector
4. Preparative columns
5. Detectors
6. Programmer
7. Recycle valve
8. Fraction collector.
Figure 2 : Instrumentation Of Preparative HPLC.
INSTRUMENTATION:

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

18. Fig: Largest column in preparative HPLC with 4000 mm x 1600 mm i.d.

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

21. Techniques For
Preservation of
Column
Radial
compression
Packing
Technique.
Longitudinal
Compression
Packing
Technique.
For preservation of column bed, two techniques are used:

22. Figure 10: Radial Compression Packing
Technique.
Figure 11: Longitudinal Compression
Packing Technique.

23.  COMMERCIALLY AVAILABLE PREPARATIVE
COLUMNS:

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.