fundamentals and techniques of preparative...
TRANSCRIPT
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.
18/23
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
20/23
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
22/23
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
25/23
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.