Transcript Preparative Chromatography
Flash Chromatography
150 & 300 ml/min Flash Pumps New 200 ml/min Binary Flash Pump
Flash 300
SPECIFICATIONS
• • • • • Flow Rate . . . . . .. . 0.1 – 300.0 ml/min Max. Pressure . . . . 200 p.s.i.
Flow Accuracy . . . + 3% (10 – 250 ml/min) Dimensions . . . . . . 7.0" W x 12.0" D x 7.5" H Weight . . . . . . . . . . 20 lbs
Flash 300 – Performance Data
• • • • • • • • • • • • •
Flash 300 Features
Dual-Head Design for High Flow & Low Pulsation Ceramic Pistons and High Performance UHMW, HPLC-grade Seals Ruby Ball / Sapphire Seat Check Valves Universal Voltage Input: 90 – 260 VAC / 50 – 60 Hz Constant Flow Performance with Varying Back Pressure Accurate Flow Rate and No Loss of Prime with Hexane, Methylene Chloride and other Light Solvents Combination Prime-Purge Valve / ”T” / Single-Port Outlet (1/8”) Additional Outlet Check Valve to Prevent Backflow during Priming All-Stainless Steel Fluid Path (unless otherwise indicated – pistons, check valves & seals) Fluid Path Isolated from Electrical Components Interactive Digital Keypad, RS-232 PC Control & Monitoring, Remote Run/Stop Fine-Tune Flow Calibration Set-Up Function: 2% increments to
± 10% at 300 ml/min EZChrom
Driver available
Compatible with Gradient Flash
• • • • Use the SCU 470 for stand alone gradient 600ml/min isocratic 300ml/min gradients VUV 14 or Model 500 detector optional
Simplicity and Function
Calibration Button Power Switch Run / Stop Button Pump Inlet / Outlet Check Valve Holders One Capsule per Holder Pump Inlet / Outlet Check Valve Holders One Capsule per Holder Inlet (flexible tubing provided) Prime-Purge Valve Check Valve to Prevent Back Flow During Priming
Calibrate for Any Solvent
Flash Pump FlowRate vs Comp Setting
15.000
10.000
5.000
0.000
-5.000
-10.000
1 2 3 4
Flowrate (ml/min)
5 6 7 8 9 10
Flash 150 Binary Pump
• • • • • • 2 pumps in one cabinet 150 ml/min for gradient 300 ml/min isocratic 150 p.s.i. max Low cost for the flow range Allows use of larger columns than other Binary pumping system
NEW
Binary Flash 200
SPECIFICATIONS
• • • • # Pumps . . . . . .. .. 2 Flow Rate . . . . . .. . 0.1 – 200.0 ml/min each pump Max. Pressure . . . . 200 p.s.i. Flow Accuracy . . . + 3% (10 – 200 ml/min) Dimensions . . . . . . 7.0" W x 12.0" D x 7.5" H • Weight . . . . . . . . . . 20 lbs
• • • • • • • • • • • • •
Binary Flash 150 & 200 Features
Two Single-Head Pumps with Electronic Fast-Refill for Low Pulsation in a Compact Package Ceramic Pistons and High Performance UHMW, HPLC-grade Seals Ruby Ball / Sapphire Seat Check Valves Universal Voltage Input: 90 – 260 VAC / 50 – 60 Hz Constant Flow Performance with Varying Back Pressure Accurate Flow Rate and No Loss of Prime with Hexane, Methylene Chloride and other Light Solvents Combination Prime-Purge Valve / ”T” / Single-Port Outlet (1/8”) Additional Outlet Check Valve to Prevent Backflow during Priming All-Stainless Steel Fluid Path (unless otherwise indicated – pistons, check valves & seals) Fluid Path Isolated from Electrical Components Interactive Digital Keypad, RS-232 PC Control & Monitoring, Remote Run/Stop Fine-Tune Flow Calibration Set-Up Function: 2% increments to
± 10% at max flow EZChrom
Driver available
Flash Chromatography Today Demands
• Higher throughput • • Higher purity requirements More Compounds purified in less time • A wider variety of compound types to separate
Organic Chemists Face the Following Challenges
• • • • • • •
Little time to develop chromatography Minimal interaction with instrumentation Low solubility of compounds in water Aqueous solvents incompatible with the next reaction Difficult to remove water and alcohol Very familiar with TLC no time to optimize separation conditions
Organic Chemists Require
• • • • • • •
High quantities of material High loads Systems for samples soluble in NP solvents Must deal with low solubility and high volumes Ever increasing purity requirements Disposable columns Simple to use hardware
Today's Synthetic Reality
• • • • • Modern synthesis is a multi step process Purification between steps increases purity and yield Except in biological synthesis samples are not RP compatible HPLC is too expensive and sophisticated for synthesis purification Old style flash is not fast enough nor giving high enough purity
Unique Problems with Flash
• • •
Sample Load Sample Solubility Incompatibility of sample solvent with purification method Let the Flash 150, 200 & 300 solve these problems
Volume Load Effects
•
Compared to analytical chemistry
– Chemists require high sample loading (10mg-100’s gram) •
End goal may only be 10 mg but may require 50-100 grams of starting material
– Sample solubility problems require the use of solvents that
are strong (cause elution of product)
1.80
Loading Volume and Band-Broadening
1.00
0 1.80
1.00
0 1.80
A B Desired product 150 mg in 1 mL
1.0
2.0
3.0
4.0
5.0
6.0
7.0
8.0
9.0
10
150 mg in 8 mL
1.0
2.0
3.0
4.0
5.0
6.0
7.0
8.0
9.0
10 • • • •
Same sample loaded with constant mass:
– 150 mg load – Three different cartridges
Chromatogram A:
– Loading volume equivalent to 3% of
column void volume (36 mL) Chromatogram B:
– Loading volume equivalent to 22%
column void volume Chromatogram C:
– Loading volume equivalent to 50%
column void volume High volume Loads destroy the separation C 150 mg in 18 mL
1.00
0 1.0
2.0
3.0
4.0
5.0
Minutes
6.0
7.0
8.0
9.0
10
Large Volume Loads Reduce Resolution A B
p
C
100 90 80 70 60 50 40 30 20 10 0 0 5 10 15 20 25 30 35
Load Vol./Void Vol. (% )
40 45 50 55 • • •
Illustrates the effect of sample volume on separation of component B from A Sample load volume, as a percentage of void volume (Load Vol./Void Vol.), was plotted vs. resolution Resolution degrades when sample load increase to 20% of the column void volume
Keep load at <20% of column volume
Flash 150, 200 & 300 Solution to Loading Problems
• Allows bigger columns so sample can be less than 20% of column void
120 100 80 60 40 20 0
High Mass Loading Destroys Separation Mass loaded (mg)
5.5
6.0
• 1600 1400 1200 1000 800 700 600 400 300 200 100 50 6.5
7.0
•
Sample volume was kept constant
– 2 mL in 80/20
acetonitrile-water Effect of sample load increase shown:
– As mass increases, peak
fronts shift
– Peak tails overlap as
mass increases Load determines Column Size required
1.0
1.5
2.0
2.5
3.0
Desired component
3.5
4.0
Minutes 4.5
5.0
Impact of Sample Mass on Separation
6.5
6.0
5.5
5.0
4.5
4.0
3.5
3.0
2.5
2.0
0.0
0.5
1.0
1.5
2.0
2.5
Sample Mass/Packing (%)
3.0
3.5
Retention at Peak Max Retention at Peak Tail Asymmetry (10%) •
Ratio between loading mass and amount of packing material, Sample Mass/Packing (%), is shown vs. retention behavior:
•
When loading mass increases over 1.5% of the packing material, peak asymmetry increase significantly
• •
Retention shifts to front as loading mass increases As loading mass increases, peak tails overlap (without shifting)
Solvent and Co-Elution Reduces Loading
40% 30%
120 mg (A+B) in 0.8 mL DCM On 12+M Co-elution A B
20% 10% Area A3 (%) Area B3 (%) [A3+B3] (%) 0% 0 5 10 20 25 30 15
Fraction number
• • • • •
A detailed fraction analysis for an affected purification Total sample load 120 mg for components A and B Sample dissolved in 0.8 mL dichloromethane 95:5 Hexane-Ethyl Acetate as eluting solvent Fractions are collected: 1.3 mL/fraction Fraction purity was analyzed using HPLC and plotted vs. fraction number
Flash 150, 200 & 300 Advantage
• Allows column size to be matched to load with out sacrificing time due to low flows
Organic Synthesis Trends
•
Organic chemists’ face rapidly changing, conflicting needs
– More Synthesis – Greater output – Higher synthesis
purity
– Greater synthesis
yields Higher product purity More products/day Synthesis catch-22 Increased product yields
•
A synthesis catch-22 dilemma
Flash 150, 200 & 300 Changes the Game
• • Bridges the flash to prep HPLC gap Allows a new approach to flash based on: – Higher pressures – Gradient – More efficiency
Effect of Particle Size on Efficiency
• 13-20 micron particles give optimal prep efficiency
Effect of Efficiency on Resolution
• • Must square the number of theoretical plates to double resolution Loading reduces high plate column efficiency faster than lower plate columns
Capacity K’
• • • • Measured in column volume Think in terms of column volume’s of retention Solvent strength effects k’ Gradient changes k’ with time from infinity to less than 1
Factors Affecting Capacity
• • • • • • Surface Area Porosity Particle density Size Active coating coverage Solvent strength
Effect of Particle Size on Pressure
• • • Doubling particle size reduces pressure by a factor of 4 at the same linear velocity Flash 200 & 300 allow 16 20 micron particles to be used =Higher efficiency in the same time with more surface area Particle size 5 10 20 40 80 Pressure 2000 500 125 31 8 Pressure Vs Particle Size 2500 2000 1500 1000 500 0 0 Pressure 20 40 60
Particle size
80 100
Flash 150, 200 & 300 Advantage
• • • The 200psi pressure limit allows smaller particles and higher resolution Capacity can be dramatically Improved Beat the CATCH 22 by using the Flash 200 & 300 benefits
Gradient Effects
• • • Can further dramatically increase through put by allowing full use of the column for purification.
Can actually shorten separation time.
Can concentrate samples
Flash 300 & 200 & 150 Advantage
• • • Can use SCU470 for stand alone gradient control High flow in isocratic mode = 600ml/min for 2 Flash 300 and 400ml/min for Flash 200 Gradient is now reasonable for Flash Separations
SSI Offers
• • • • Single channel systems Increase capability at moderate costs A series of pumps for flash chromatography System capability
Flash 150, 200 & 300 Summary
• • • • • • Allows bigger columns so sample can be less than 20% of column void Allows column sized to be matched to load with out sacrificing time due to low flows The 200psi pressure limit allows smaller particles and higher resolution Allows gradients Easy calibration for a wide variety of solvents Simplicity of design for reliable performance