Fundamentals and Techniques of Preparative HPLC Parto Zist Behboud www.partoz.com Tel: 42108
Today s Overview 1 Basic know-how for up-scaling 2 Practical examples of up-scaling 3 Recycle preparation 4 Column switching preparation
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
Sample loading amount Guide of maximum sample loading amount Column I.D. (mm) Assumptions Dissociation is suppressed Mobile phase has adequate solubility Separation from interference is accomplished Maximum amount (mg) Section area Flow rate (cm 2 ) (ml/min) 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
Sample loading amount and overload Sample loading amount- Mass of sample that can be loaded onto the column Features: Irrespective of loading amount, separation has to be maintained 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 5
Resolution Effect of particle size on resolution 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 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 Injection amount (g/cm 2 ) Column: Shim-pack PREP-ODS Mobile phase: water/methanol = 2/3 Linear velocity: 3.02cm/min Sample: Phenol, 2-Naphtol 10-6 10-5 10-4 10-3 10-2 10-1 Effect of particle size on resolution
Effect of sample solvent (1) Behavior of analyte and sample solvent in column Band broadening is caused by strong elution power of methanol Methanol solvent Water solvent analyte analyte Flow direction Flow direction Separation mode: Reversed phase Mobile phase : Mixture of water and methanol
TPN(10 3 ) Effect of sample solvent (2) Solvent effect Peak degradation caused by strong elution power of sample solvent Large injection volume requires weak sample solvent Sample solvent : water Sample solvent : water /methanol = 7/3 Column: Shim-pack CLC-ODS 6mmI.D. 150mmL. Mobile phase: water/methanol = 3/7 Flow rate: 1mL/min Sample: Caffeine 10mg Sample solvent: methanol Sample solvent : methanol 0.1 mg/ml 100 μl 0.2 mg/ml 50 μl Starting point of elution 1.0 mg/ml 10 μl Injection volume (μl) Effect of injection volume on TPN (under constant loading amount) Effect of injection volume on peak shape (under constant loading amount)
Effect of column temperature When column oven is employed at high flow rate Peak degradation may occur generally, separation is carried out at room temperature Column temp.:room temp.(26 ) Column temp.:40 Column temp.:50 Effect of column temperature on peak shape
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
Procedure of up-scaling Analytical scale Evaluate separation condition preparative scale Confirm separation on preparative column Increase loading amount and check peak degradation Yes Establish HPLC conditions No Fix fractionation parameters Check purity for obtained fraction Yes Automatic fractionation No
Analytical/Preparative compatible HPLC system Convenient system for up-scaling procedure Injector(Anal.) Pump Mixer(Anal.) Analytical column Data processor Column switching valve Detector Fraction collector Pump Mixer(Prep.) Preparative column Injector(Prep.)
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. Flow rate should be 20 times 芳香族化合物のスケールアップ例 column :Shim-pack PREP-ODS(H)kit A: 4.6 mm I.D. 250 mm L. 5μm B: 20 mm I.D. 250 mm L. 5μm Mobile phase :10 mm (sodiumu) phosphate ph=2.6) Flow rate : 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 /Methanol= 1/9 A: 0.8 ml/min B: 15 ml/min 9: Benzoic acid n-propyl 10: Naphthalene 11: Biphenyl 12: Benzoic acid pentyl 13: unknown 14: Phenanthlene 15: Anthracene 16:fluoranthene Increase in flow rate is determined by the ratio of column section areas
Essentials for upscaling (2) Employing same particle size for both conventional and preparative HPLC Increased loading amount corresponds to ratio of sectional areas and provides same resolution of peaks 4.6 mm I.D.column Injection 50 L (Benzene 25 mg) loading amount should be 20 times 20 mm I.D.column Injection 1mL (Benzene 500 mg) Comparison of chromatograms at maximum loading amount in respective column dimensions
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. mau 22 mm I.D.column Injection volume: 500 μl Flow rate: 10 ml/min uv 4.6 mm I.D.column Injection volume: 20 μl Flow rate: 1 ml/min 60 50 Curcumin 500000 450000 400000 Curcumin 40 350000 300000 30 250000 20 200000 150000 10 100000 0 50000 0 5.0 10.0 15.0 20.0 25.0 30.0 min -50000 2.5 5.0 7.5 10.0 12.5 15.0 min Preparative chromatogram Conventional chromatogram for purity test
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. Recycling Prep System The same separation efficiency, as if longer column is used, achieved by re-introducing the column eluate including target component band into the column. 18/23
Flow Diagram of Recycling Prep System 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 Solvent Delivery Unit Sample injector Column Detector Recycle valve Flow Diagram Flow out Fraction collector 19/23
n-, Iso-propylparaben Iso-propylparaben n-propylparaben Example of Separation Improved by Recycling The separation is improved by re-introducing non-separated peak band into the column repeatedly. 5.0 10.0 15.0 20.0 25.0 30.0 35.0 40.0 45.0 min 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 20/23
Advantages of Recycling Prep System 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. 21/23
Recycling System for Large Prep Scale 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 22/23
Software for Recycling Prep -Recycle-Assist-
Features of Recycle-Assist Easy to use recycling prep system 1. 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. 24/23
Simple Operation with GUI Easy monitor of system status Support total workflow Reduce risks of operation error Sample Injection Recycling Fractionation One screen displays from parameter setup and chromatogram. Automatic updating with system condition 25/23
Easy Setting of Recycling Prep Only one mouse click on chromatogram settable timing of recycling and fractionation by clicks Data 1 Click! Setting of Recycling Condition Click start point for recycling. Data 2 Click! Click stop point for recycling. Click! Data 2 Click fractionation timing. The method is created for automatic recycling preparative chromatography. 26/23
Transfer from Manual to Automatic Recycling Prep Chromatography 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. Click! Recycling starts. Manual recycling prep chromatography Click! Recycling is finished. Click! Fractionation Switches valve to [Recycle] Switches valve to [Drain] Automatic recycling prep chromatography Time event for manual recycling prep can be stored to use for automatic recycling prep. Fractionation starts by click (and stops). 27/23
Automatic Saving of Results Automatic saving of fraction information Chromatograms and fractionation conditions are automatically saved in the preparative data. Checking with chromatogram and text 28/23
Example of Recycle Separation
Recycle Separation of Surface-active Agent Recycle separation of TritonX-100 Column Mobile phase Flow rate Sample Injection volume : GPC-2025+2002+2001 (300 mm 20 mm I.D. each) : THF : 3 ml/min : TritonX-100 1% (w/v) : 1 ml 30/23
Recycle Separation of Protein 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 Injection volume : Egg Albumin 0.5% (w/v) Myoglobin 0.3% (w/v) : 12.5 ml Pump Injection 31/23
Comparison of General Prep System and Recycling Prep System 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 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. 32/23
Summary 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. 33/23
Thank you for your attention. 34/23