Fundamentals and Techniques of

Preparative HPLC

Parto Zist Behboud

www.partoz.com

Tel: 42108

Today’s Overview

• Basic know-how for up-scaling1

• Practical examples of up-scaling2

• Recycle preparation3

• Column switching preparation4

Basic know-how for up-scaling

• Sample loading amount

• Sample loading amount and overload

• Effect of particle size on resolution

• Effect of sample solvent (1)

• Effect of sample solvent (2)

• Effect of column temperature

◆ Guide of maximum sample loading amount

Dissociation is suppressed

Mobile phase has adequate solubility

Separation from interference is

accomplished

Column I.D.

(mm)Section area

(cm2)Flow rate(mL/min)

Maximum amount

(mg)

2.0 0.03 0.1 3

4.6 0.17 0.8 17

20 3.1 15 300

50 20 90 2000

*GPC column requires lower flow rate than indicated above

Assumptions

Sample loading amount

5

◆ Sample loading amount- Mass of sample that can be loaded onto

the column

Irrespective of loading amount, separation has to be maintainedFeatures:

1) Maximum sample loading amount is in proportion to column volume.

2) Generally, in Prep LC sample is overloaded onto column to increase fraction yield.

Column: Shim-pack PREP-ODS(L)

50mmI.D.×250mmL.

Mobile phase: Water/Methanol = 2/3

Flow rate: 100mL/min

Sample: Phenol, 2-Naphtol

The relationship between peak formation and sample loading amount

Sample loading amount and overload

Column: Shim-pack PREP-ODS

Mobile phase: water/Methanol = 2/3

Linear velocity: 3.02cm/min

Sample: Phenol, 2-Naphtol

Smaller particle provides higher resolution hence higher cost

Larger loading amount provides lower performance.

Minimal separation can be accepted under overloading condition

Preparative HPLC employs large particle size → lower cost

Injection amount (g/cm2)

Res

olu

tio

n

● 20 mm I.D.×250 mm L. 5μm○ 20 mm I.D.×250 mm L. 15μm△ 30 mm I.D.×250 mm L. 15μm× 50 mm I.D.×250 mm L. 15μm

Effect of particle size on resolution

10-110-210-310-410-510-6

Effect of particle size on resolution

Separation mode: Reversed phase

Mobile phase : Mixture of water and methanol

Flow direction

Methanol solvent

analyte

Flow direction

Water solvent

analyte

Band broadening is caused by strong elution power of methanol

◆ Behavior of analyte and sample solvent in column

Effect of sample solvent (1)

◆ Solvent effect → Peak degradation caused by strong elution power of sample solvent

Column: Shim-pack CLC-ODS

6mmI.D.×150mmL.

Mobile phase: water/Methanol = 3/7

Flow rate: 1mL/min

Sample: Caffeine 10mg

0.1 mg/mL×100 μL

0.2 mg/mL×50 μL

1.0 mg/mL×10 μL

Sample solvent : methanol

Injection volume (μL)

TP

N(1

03 )

Sample solvent : water

Sample solvent :water /methanol = 7/3

Sample solvent:

methanol

Effect of injection volume on TPN

(under constant loading amount)

Effect of injection volume on peak shape

(under constant loading amount)

Large injection volume requires weak sample solvent

Starting point of elution

Effect of sample solvent (2)

Column temp.：room temp.（26 ℃）

Column temp.：40 ℃

Column temp.：50 ℃

Effect of column temperature on peak shape

◆ When column oven is employed at high flow rate

Peak degradation may occur

･･･generally, separation is carried out at room temperature

Effect of column temperature

Practical Examples of Up-Scaling

• Procedure of up-scaling

• Analytical/Preparative compatible

HPLC system

• Same particle size for both conventional

and preparative HPLC

• Confirming purity of collected fraction

Evaluate

separation condition

Increase loading

amount and check

peak degradation

Establish HPLC conditions

Confirm separation on

preparative column

Fix fractionation

parameters

Check purity for

obtained fraction

Automatic fractionation

Analytical scale preparative scale

Yes

Yes No

No

Procedure of up-scaling

Analytical/Preparative compatible

HPLC system

◆ Convenient system for up-scaling procedure

Mixer(Anal.)

Detector

Injector(Anal.)

Pump

Fraction collector

Preparative columnPumpMixer(Prep.)

Injector(Prep.)

Analytical column

Data processor

Column switching valve

Essentials for up-scaling (1)

◆ Same particle size for both conventional and preparative HPLC

Increased flow rate to give same linear velocity thereby results in similar peak resolution.

A: 4.6 mm I.D.×250 mm L. B: 20 mm I.D.×250 mm L.

芳香族化合物のスケールアップ例

column ：Shim-pack PREP-ODS(H)kitA: 4.6 mm I.D.×250 mm L. 5μmB: 20 mm I.D.×250 mm L. 5μm

Mobile phase ：10 mM (sodiumu) phosphate pH=2.6）/Methanol= 1/9

Flow rate ： A: 0.8 mL/minB: 15 mL/min

samples ：1: Phthalic acid

2: Caffeine acid

3: Sarytilic acid

4: Benzoic acid

5: 2-Naphtol

6: Benzene

7:unknown

8: Benzoic acid iso-propyl

9: Benzoic acid n-propyl

10: Naphthalene

11: Biphenyl

12: Benzoic acid pentyl

13: unknown

14: Phenanthlene

15: Anthracene

16:fluoranthene

Flow rate should be 20 times ･･

Increase in flow rate is determined by the ratio of column section areas

4.6 mm I.D.column

Injection 50 L

(Benzene 25 mg）

20 mm I.D.column

Injection 1mL

(Benzene 500 mg）

Comparison of chromatograms at maximum loading amount in respective column dimensions

loading amount should be 20 times･･･

Increased loading amount corresponds to ratio of sectional areas and provides same resolution of peaks

◆ Employing same particle size for both conventional and

preparative HPLC

Essentials for up-

scaling (2)

Essentials for up-scaling (3)

◆ Confirming purity of collected fraction

Fraction reanalysis- e.g. re-inject Curcumin fraction in Crucuma aromatica Salisbn onto analytical column to confirm that impurity has been effectively removed.

4.6 mm I.D.columnInjection volume： 20 μLFlow rate： 1 mL/min

22 mm I.D.columnInjection volume： 500 μLFlow rate： 10 mL/min

Curcumin

5.0 10.0 15.0 20.0 25.0 30.0 min

0

10

20

30

40

50

60

mAU

2.5 5.0 7.5 10.0 12.5 15.0 min

-50000

0

50000

100000

150000

200000

250000

300000

350000

400000

450000

500000

uV

Preparative chromatogram Conventional chromatogram for purity test

Curcumin

Shimadzu Preparative HPLC

Introduction of Shimadzu Preparative

HPLC

What is “Recycling Prep System” ?

When separation is insufficient,

Target components cannot be fractionated with high purity.

Use of longer column or multiple columns in series,

Separation is improved, but it will be too expensive.

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The same separation efficiency, as if longer column is used,

achieved by re-introducing the column eluate including target

component band into the column.

Recycling Prep System

Flow Diagram of Recycling Prep System

19/23Flow Diagram

ColumnSample injector

Detector

Recycle valve

Fraction collector

Solvent Delivery Unit

Flow out

What is necessary for Recycling Prep?

Selectable line between recycle line and flow-out line by recycle valve

Low-volume recycling flow line to improve separation efficiency by

minimized diffusion

Solvent delivery unit can re-introduce eluent from column

Example of Separation Improved by Recycling

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5.0 10.0 15.0 20.0 25.0 30.0 35.0 40.0 45.0 min

Iso

-pro

pylp

ara

ben

n-p

rop

ylp

ara

ben

n-,

Iso

-pro

pylp

ara

ben

Column : Shim-pack Prep-ODS(H) (250 mm × 20 mm I.D.)

Mobile phase : 0.1% Acetic acid/Acetonitrile=35/65

Flow rate : 13 mL/min

Detection : 254 nm

Column temp. : Room temperature

Sample : n-propylparaben, iso-propylparaben (each 0.1%)

Injection volume : 200 μL

The separation is improved by re-introducing non-separated peak band

into the column repeatedly.

Advantages of Recycling Prep System

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Improve separation by re-introducing insufficiently separated peak

into column.

Not necessary to use longer column or multiple columns in series

to improve separation. Preparative column is expensive. >> Reduce column cost.

Save solvent consumption during recycling separation

Not necessary to develop “perfect method “ for separation The recycling separation is especially effective in GPC, which does not

allow separation conditions to change easily.

* Gradient analysis is not applicable.

But,

Isocratic is the most effective method to improve peak resolution in certain elution area.

Gradient is not used in the GPC.

Isocratic elution is usually used, in separation which needs recycling.

Recycling System for Large Prep Scale

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Recycling preparative system for large-volume preparative column (Max. 50

mm I.D.) (Flow rate: Max. 150 mL/min)

LC-20AP pump covers wide range flow rate from analytical flow range to

large-scale prep range

Many fractionation support functions of FRC-10A

Easy operation by dedicated Software Recycle-Assist (described later)

Save space by small system deign

Upgradable to Gradient preparative/Recycling preparative switching system

LC-20AP Manual Recycling

Preparative System

LC-20AP Automatic Recycling

Preparative System

Software for Recycling Prep

-Recycle-Assist-

Features of Recycle-Assist

24/23

Easy to use recycling prep system1. Simple operation environment by GUI

Graphical user interface (GUI) supports the entire workflow, with showing

flow line status

This easy operation concept reduces risks of operation error, not wasting

valuable samples

2. Just one mouse click → Go Recycle Prep

Settable recycling parameters with simple mouse click on chromatogram

Easily Settable timing of recycling and fractionation

3. Seamless transfer from manual to automatic recycling

preparative operation

Recycling and fractionation in real time while checking the separation

“Record” function enable to store manual prep event for automatic

preparative operation.

Simple Operation with GUI

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Sample Injection

Recycling

Fractionation

One screen displays from

parameter setup and chromatogram.Automatic updating with system

condition

Easy monitor of system

status Support total workflow

Reduce risks of operation error

Easy Setting of Recycling Prep

26/23

Click start point for recycling.

Click stop point for recycling.

Click fractionation timing.

The method is created for automatic recycling

preparative chromatography.

Click!

Click!

Click!

Setting of Recycling Condition

Data 1

Data 2

Data 2

Only one mouse click on chromatogramsettable timing of recycling and fractionation by clicks

Transfer from Manual to Automatic

Recycling Prep Chromatography

27/23

Automatic recycling

prep chromatography

Manual recycling prep

chromatography

Switches valve to [Recycle]

Click! Recycling starts.

Switches valve to [Drain]

Click! Recycling is finished.

Click!

Fractionation

Seamless transfer of manual recycling prep condition to automatic

recycling prep condition Manual recycling prep by click of icons while monitoring the chromatogram.

“Record” function allows to store time event that was setup for manual recycling. The stored event can be used for

automatic recycling prep condition.

Time event for manual recycling prep can be

stored to use for automatic recycling prep.

Fractionation starts by click (and

stops).

Automatic Saving of Results

28/23

Automatic saving of fraction information Chromatograms and fractionation conditions are automatically saved in the preparative

data.

Checking with

chromatogram and text

Example of Recycle Separation

Recycle Separation of Surface-active

Agent

30/23

Recycle separation of TritonX-100

Column : GPC-2025＋2002＋2001 (300 mm × 20 mm I.D. each)

Mobile phase : THF

Flow rate : 3 mL/min

Sample : TritonX-100 1% (w/v)

Injection volume : 1 mL

Recycle Separation of Protein

31/23

Recycle Separation of Albumin (Egg) and

Myoglobin

Albumin

Myoglobin

Column : GS-510P (500 mm × 21.5 mm I.D.)

Mobile phase : 100 mmol/L phosphate (sodium) buffer (pH=6.9)，

100 mmol/L sodium sulfate

Flow rate : 5 mL/min

Sample : Egg Albumin 0.5% (w/v)Myoglobin 0.3% (w/v)

Injection volume : 12.5 mL Pump Injection

Comparison of General Prep System and

Recycling Prep System

32/23

General prep Recycling prep

Column 4 1

Flow rate 10 mL/min＊ 13 mL/min

Repetition - 5 times

Analysis time 36 min. 41 min.

Solvent consumption 351 mL 104 mL

* Flow rate has limitation for column pressure limits.

To get the same peak resolution performance

To get 70,000 theoretical plate number…….

Column: ODS column (250 mm × 20 mm I.D.)

Column pressure: less than 20MPa

Summary

33/23

Recycling prep system is applicable to a wide range of

applications from semi-prep to large-volume prep

Low volume recycling line by small system design

Easy operation by Recycle-Assist

Easy transfer from manual to automatic recycling by Recycle-

Assist software.

High resolution is efficiently achieved by recycling preparative

system.

34/23

Thank you for your attention.