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Speakers
Tony Taylor
Technical Director
Crawford Scientific
Moderator
Peter Houston
Editorial Director
LCGC Magazine
Charles White
UK Area Manager
YMC Europe GmbH
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Outline
Introduction
Uses of Preparative Chromatography
Column Considerations
Preparative HPLC Method Development
Method Development at Analytical Scale
Optimisation of the Throughput
Scaling Up
Productivity and Throughput
Product Recovery

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Introduction
Analytical and preparative scale HPLC separations have
many similarities but a number of differences.

The prime object of an analytical scale separation is to
produce a work of art in a chromatogram which has sharp,
well resolved, symmetrical peaks.

The aim of preparative scale HPLC is to produce a quantity
of pure compound as easily as possible in the most
economical way.
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Introduction
Animation 1.
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Uses of Preparative Chromatography
Scale Column
I.D. (mm)
Quantity of
Product
Typical
Column
Length (mm)
Purpose
Analytical 4.6 1 - 40 mg 250 Biological
materials for
activity testing
Semi-
Prep
10 30 100mg 3g 250 Reference
compounds
Prep 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
Table 1. Definition and scales of operation
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Uses of Preparative Chromatography
1000 mm x 400 mm i.d. columns and pre-
columns. Courtesy of YMC Co Ltd, Japan
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Uses of Preparative Chromatography
4000 mm x 1600 mm i.d. column.
(Courtesy of NovaSep, France)
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Column Initial Considerations
Size of the Column
Type of column
Do you have the required infrastructure (pumps,..) to
work with the selected column?
Before any preparative separation can be undertaken, the
choice of column must be made. This may sound obvious, but
you have to consider if you are to perform the separation many
times over a period of time, or are simply looking to do a one-
off separation.

Important considerations:
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Column Considerations
ID (mm) Column Lengths (mm) Guard Col
Length (mm)
10 50 100 150 250 10 or 20
20 50 100 150 250 10 or 20
25 100 150 250 10 or 20
30 150 250 20
50 250 300 500 1000 50 or 100
70 250 300 500 1000 100
100 250 300 500 1000 100
150 300 500 1000 200
200 500 1000 200
Table 2. Commercially available pre-packed column sizes
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Preparative Columns
Preparative HPLC columns consist of a tube (made of stainless
steel, glass or synthetic polymers) filled with micro-particulate
porous silica.

The performance of the separation is influenced by the packing
material:
Particle size and shape
Stationary phase

Silica
Organic polymers
Chiral stationary phases
Monolithic stationary phases



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Preparative HPLC Method Development
Strategy for preparative
HPLC method
development
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Preparative Separation Goals
With any preparative separation there is always balance
between the costs of the separation and subsequent
downstream processing, time to develop and perform
the separation and available systems to operate the
process
The aim of scaling up to preparative separations is to
provide the simplest, most cost-effective but most robust
method which can be performed without loss of any
product degradation within an acceptable timeframe.
Many preparative separations start life as an analytical
method, but thought should be given to the whole
process, especially if the method is to require larger
scales of operation than lab-based systems
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Analytical Separations Designed For Preparative
Applications
An analytical method which requires a complex gradient may
prove difficult to replicate on the large scale
A method which used large amounts of buffer salts and
modifiers may give problems at a larger scale
The use of complex eluents may have severe cost implications
Exotic phases may not be available for larger scale applications
Organic solvents may introduce problems of explosion-proofing
equipment
Removal of large volumes of water is more difficult than similar
volumes of cheap solvents
Elevated temperatures may prove costly to reproduce on a
large scale
Non-bonded silica is more economical than bonded phases
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Definition Separation Requirements
Of fundamental importance to preparative method development
is the identification of the problem and challenges associated.
You should bear in mind the following aspects:
Sample information
Analyte(s) of interest (type, number, concentration,
required level of purity, etc.)
Other separation strategies suitable for your sample
Detection method
Amount of material to be isolated
Required degree of accuracy, precision, etc
Method verification
Costs
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Practical Importance of Preparative HPLC
The following situations are practical examples where preparative
HPLC should be selected over other separation techniques:
Reversed phase HPLC is well suited for the separation of
peptides
The separation of oligonucleotides is dominated by a
combination of ion-exchange and reversed phase
techniques
The separation of racemic mixtures is, no doubt,
dominated by chiral HPLC
The separation of thermally labile compounds of
biological/biochemical origin (such as those commonly
found in toxicology and pharmaceutical analysis) is
efficiently achieved by HPLC means

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Select the Correct HPLC Mode
In principle the same separation mechanisms that are used
with analytical scale chromatography are also available with
preparative HPLC; however, the preparative HPLC universe is
dominated by reversed and normal phase applications.

The following factors should be considered when selecting the
appropriate HPLC mode for your separation:
Solubility
Molecular weight
Functional groups
Sample matrix
Detectability
Other separation alternatives
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HPLC
Mode
Typical Analytes Eluent System Temperature
Normal
Phase
Lipophylic analytes such as:
oils, fats, lipids, etc.
Organic solvents Usually performed at
elevated temperatures
Reversed
Phase



Aqueous
mixtures of
methanol,
acetonitrile and
additives.
Usually performed at
elevated temperatures
Ion
Exchange
Aqueous buffers,
ionic solutions
Usually performed at
elevated temperatures
Chiral Enantiomers Aqueous or
organic solvents
Usually performed at
lower temperatures than
other forms of prep HPLC
Size-
Exclusion
Polymers, proteins, nucleic
acids
Aqueous or
organic solvents
Usually performed at
elevated temperatures
Select the Correct HPLC Mode
Table 3. Preparative HPLC mode selection
Neutral compounds with
molecular weights < 2000;
weak acids and bases;
strong acids and bases (ion
pair); etc
Inorganic ions; acids and
bases
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Method Development at Analytical Scale
So, before selecting the analytical method of choice, you
need to consider if normal phase might be more appropriate
than reversed phase or if you need a high purity bonded
phase to avoid the use of buffer salts and modifiers, etc.

Important: you must consider the downstream requirements
as this can dictate the separation mechanisms which can be
used.
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Method Optimisation
Remember that the chromatographic column (the stationary
phase) used in scale up MUST be commercially available in
both analytical and preparative columns (or as a bulk
preparative material) and that they have true seamless
scalability as this will decrease the need for high pressure
equipment.

Important parameters to consider include:
Chromatographic resolution (R
s
)
Efficiency (N)
Retention factor ()
Temperature
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Temperature
Temperature plays an important role in HPLC; this is because
both the kinetics and thermodynamics of the chromatographic
process are temperature dependent.
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Particle Size
Column efficiency is dictated by the particle size of the packing
material; the smaller the particle, the more efficient the
separation, but at a much higher cost.
Particle Size (m) Column Efficiency (N)
Back Pressure
(psi) (bar)
5 20,000 3,300 228
7 14,300 1,680 116
10 10,000 830 57
15 6,700 370 26
20 4,500 250 17
40 2,300 60 4
50 2,200 35 2.4
Table 4. Influence of particle size on column efficiency and
back pressure
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Particle Size
The effect of particle size on the
elution profile of a C18 column
250 x 6.0mm i.d. eluted with
acetonitrile:water (50:50)
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Pore Size


The influence of molecular weight on pore size
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Gradient Operation
Controlling gradients in large scale separations is more difficult
than in the analytical lab. To a first approach, when developing
a generic preparative HPLC gradient process, the separation
conditions should be set as in the analytical separation.

For a generic gradient the following rules should be followed:
First rule of thumb: under column overload conditions the
main component usually elutes at approximately two-thirds
of the concentration of polar modifier observed in the
analytical separation
Second rule of thumb: elution should be started at a
concentration that corresponds to the predicted
concentration separation minus 10%. Then apply a change
in concentration corresponding to 5% over 15 minutes

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Generic gradient method development for preparative HPLC.
The results of a gradient HPLC separation (gradient conditions)
show that a valuable analyte elutes at a concentration of
approximately 30% of the polar modifier. Design an equivalent
large scale gradient separation under column overload conditions.
Gradient Operation -Example
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Solvent Selection
The key issues in selecting solvents are:
Selectivity
Viscosity and backpressure
Cost efficiency
Impurities
Recycling
Toxicity & flammability
Costs of waste disposal
Costs of storage

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Solvent Selection
High viscosity Comments Consider for
Water Need high purity to avoid rogue
peaks
Acetonitrile Low viscosity, decreased UV
adsorption, expensive, toxic
Difficult
separations
Methanol High viscosity, low price, good
solubility for salts
Non-critical
separations
2-Propanol Less toxic than methanol, higher
viscosity
THF High viscosity, good miscibility,
high solubility for salts
Special
selectivity
Hexane Flammable, low cost, low viscosity Normal phase
Ethyl acetate Flammable, low viscosity Normal phase
Table 5. Common solvents for preparative chromatography
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Optimisation of the Throughput
Preparative HPLC provides two ways of achieving purification
of large sample amounts: scale-up of the analytical system
and column overloading.
Scale-Up of the Analytical System




Column Overloading
Larger column diameters
Higher eluent flow rates
Larger sample volumes (constant concentration)


Concentration Overloading
Volume Overloading
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Concentration Overloading
When the sample of interest has good solubility in the mobile
phase, then concentration overload is the technique of choice.
Here, sample concentration is increased while the injected
sample volume remains constant.
Analytical HPLC run versus preparative HPLC concentration
overloading
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Concentration Overloading
The effect of
increasing sample
concentration on a
50 x 4.6mm id
column packed
with C18 material
and eluted at
1mL/min with
acetonitrile:water
(60:40)
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Volume Overloading
When the sample of interest has poor solubility in the mobile
phase, then volume overload is the technique of choice. Here,
sample concentration is kept constant while the injected
sample volume is increased.
Analytical HPLC run versus preparative HPLC volume overloading
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Volume Overloading
The effect of
increasing injection
volume whilst
maintaining sample
concentration on a
50 x 4.6mm id
column,
packed with C18
material and eluted
at 1mL/min with
acetonitrile:water
(60:40)
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Which Overload Method?
Volume Overloading Concentration Overloading
Determined by injection
volume
Determined by solubility of
compound in mobile phase
Appropriate when sample
has poor solubility
Appropriate when sample has
good solubility
Throughput determined by
column diameter
Throughput determined by
selectivity
Linear (analytical) area of
adsorption isotherm
Non-linear (preparative) area of
adsorption isotherm
Small particle sizes
improve loadability
Particle size has very little
influence on concentration which
can be loaded.
Table 6. Summary of column overloading parameters
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Sample Solvent Considerations
To achieve maximum loading, the solvent used to dissolve the
sample is a major driving force but in practice a combination of
overload methods is used.
Optimisation of the analytical method.
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Detector Overloading
UV detector
overload in
preparative HPLC
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Scaling Up
Preparative HPLC leads to separations with reduced
chromatographic resolution. There are, however, resolution
requirements that must be met for the separation to be practical.

When performing method development and optimisation for
preparative HPLC, the following steps should be followed:
Selection of the appropriate mode of chromatography
(normal phase, reversed phase, etc.)
Optimisation of the separation (stationary phase, mobile
phase, temperature, etc.)
Optimisation of the throughput (sample amount, column
overloading)
Scaling up

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Methods of Scaling Up
Column scale up

Analytical
Prep
2
Analytical
Prep
Analytical
Prep
Length
Length
Diameter
Diameter
Volume
Volume
Factor Up Scale
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Linear relationship
between column
length and
loadability
Methods of Scaling Up
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Effect of column diameter on loadability
Methods of Scaling Up
Colum
n Size
(mm)
Load
(mg)
Flow
(mL/
min)
20X 250 x
20
200 9.5
5X 250 x
10
50 2.4
1X 250 x
4.6
10 0.5
a 250 x
4.6
1 0.5
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Linear Scale Up
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Simple Calculations Example 1
It was found that for a C
18
25cm3.0mm5m the optimum
eluent flow rate was 0.6 mL/min and the amount of sample loaded
was 15L. Calculate the equivalent flow rate in a
C
18
25cm20.0mm5m column
Flow
PC
Flow
AC

D
PC
D
AC

2
0.6mL/ min
20mm
3.0mm

2
26.67mL/ min
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Simple Calculations Example 2
It was found that for a C
18
25cm3.0mm5m the optimum
eluent flow rate was 0.6 mL/min and the amount of sample loaded
was 15L. Calculate the equivalent flow rate and sample load in
a
C
18
25cm20.0mm10m column
Flow
PC
Flow
AC

D
PC
D
AC

dp
AC
dp
PC

0.6mL/ min
20mm
3.0mm

5.0 m
10 m

13.3mL/ min
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Simple Calculations Example 3
It was found that for a C18 25cm3.0mm5m the optimum
eluent flow rate was 0.6 mL/min and the amount of sample loaded
was 15L. Calculate the sample load in a
C
18
25cm20.0mm10m column
Load
PC
Load
AC

D
PC
D
AC

L
PC
L
AC

15 L
20mm
3.0mm

25cm
25cm

666.7 L
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Productivity Considerations
Defined as the total amount of output (service, product, profit,
etc.) per unit input (labour, equipment and capital), productivity
is a measure of the effectiveness of productive effort.

The lack of productivity will make destroy any project however
good. The following pointers should be considered when
performing preparative HPLC method development:
There are situations where HPLC cannot be easily replaced
by any other single separation technique
Column load is a decisive productivity factor
Sometimes it may be better to purify a valuable product in
smaller portions rather than compromising separation targets
The initial investment
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Column ID (mm)
4.6 20 50 150
Scale Up Factor 1 18.9 118 1060
Sample purity (%) 50 50 50 50
Product yield (%) 98 98 98 98
Solvent consumption/run (mL) 20 378 2360 21200
Sample consumption/run (g) 3 56.7 354 3180
Product yield (g) 0.147 2.78 17.35 155.8
Injections per 50 g 340 18 3 1
Time consumption (min) 6800 360 60 20
Solvent consumption (litre) 6.8 6.8 7.1 21.1
Table 9. Effect of column size on production costs.
Productivity Considerations Production Costs
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Productivity Considerations Economic Factors
Column
Dimension
Flow Rate
(mL/min)
Volume /
50g
Costs Yield / Run
4.6 mm 1 6.8 L 400.00 1.47 g
50 mm 118 7.1 L 420.00 174 g
150 mm 1060 21.1 L 1,250.00 1560 g
4.6 mm i.d. 50 mm i.d. 150 mm i.d.
Solvent 400.00 420.00 1,250.00
Waste disposal 40.00 40.00 120.00
Personnel (200
/h) 22,700.00 1,200.00 70.00
Materials 800.00 2,500.00 15,000.00
Total 23,940.00 4,160.00 16,440.00
50g Product
sales (1000 /g) 50,000.00 50,000.00 50,000.00
Table 10. Economic factors affecting preparative separations.
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Robustness and Method Verification
Hopefully your work at the analytical scale proved that you had
a robust method, but it is always essential to verify this at the
final preparative scale because occasionally minor issues arise
due to:






Once the method has been verified as producing the required
product in the expected yield and purity and in a reproducible
manner, you have reached your goal of a preparative
separation.
Changes in the grade of the solvent
User-packed columns as not as efficient (or reproducible)
as you hoped
You have changed a non-scalable factor

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Product Recovery
Preparative HPLC fraction collection principle
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Sample Collection
The layout of a splitter to allow
destructive detectors to be used
for preparative separations
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Types of Collector
Manual Fraction collector Valve-based collection
Various types of fraction collection systems
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Summary
Preparative HPLC:
- Analyte separation and purification
Column Considerations
- Size and type of column
Preparative Method Development
- Clearly define your goals
- Consider analytical development first
- Scale Up of the Analytical System
- Optimisation of the Throughput (column overloading)
Product Recovery
- Sample collection
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