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General Elution Protocol
Normal Phase Chromatography with Sep-Pak Cartridges
To perform normal phase chromatography with Sep-Pak cartridges, use a gradient of nonpolar solvents with polar Silica, Florisil, NH2, Diol, CN, Alumina A, B, or N as a packing material*.1 You may condition the cartridge with six to ten hold-up volumes of nonpolar solvent, usually the sample solvent.
2 Load the sample into the cartridge.
3 Elute unwanted components with a nonpolar solvent.
4 Elute the first component of interest with a polar solvent.
5 Elute remaining components of interest with progressively more polar solvents.
6 When you recover all of your components, discard the used
cartridge
in an appropriate manner.
*Depending upon your chromatographic conditions, you may also use CN
as a packing material for normal phase chromatography.
Reversed-Phased Chromatography with Sep-Pak Cartridges
To perform reversed-phased chromatography with Sep-Pak cartridges, use a gradient of strongly to weakly polar solvents with nonpolar C18, tC18, C8, tC2, Diol, NH2 or CN as a packing material.1 Solvate the bonded phase with six to ten cartridge hold-up volumes of methanol or acetonitrile. Flush the cartridge with six to ten hold-up volumes of water or buffer. Do not allow the cartridge to dry out.
2 Load the sample dissolved in strongly polar solvent.
3 Elute unwanted components with a strongly polar solvent.
4 Elute weakly held components of interest with a less polar solvent.
5 Elute more tightly bound components with progressively more nonpolar solvents.
6 When you recover all of your components, discard the used
cartridge
in an appropriate manner.
Ion Exchange Chromatography with Sep-Pak Cartridges
To perform ion exchange chromatography with Sep-Pak cartridges, use a gradient of pH or ionic strength with Accell Plus CM, Accell Plus QMA or NH2 as a packing material.1 Condition the cartridge with six to ten hold-up volumes of deionized water or weak buffer.
2 Load the sample dissolved in a solution of deionized water or buffer.
3 Elute unwanted weakly bound components with a weak buffer.
4 Elute the first component of interest with a stronger buffer (change the pH or ionic strength).
5 Elute other components of interest with progressively stronger buffers.
6 When you recover all of your components, discard the used
cartridge
in an appropriate manner.
Important Considerations in Protocol Design
Flow Rate
Appropriate flow rates must be used with Sep-Pak cartridges so as to permit proper bed conditioning, sample loading, and elution, without the problem of incomplete equilibrium or sample breakthrough. In general, cartridges may be conditioned at flow rates up to 25 mL/min. Sample loading and elution is best done at flow rates below 10 mL/min. Recovery may still be adequate at flow rates up to 20 mL/min (testing should be done to verify this), but higher flow rates may not give acceptable results. For ion exchange applications, slower flow rates (1-2 mL/min) are recommended. For Sep-Pak Light cartridges, which have smaller internal diameters, all the above flow rates should be reduced by a factor of 3x.Sample Capacity
Sample sizes must be scaled to suit the size of the cartridge bed and the separation mode and strategy to be employed. A typical normal or reversed-phase cartridge may have capacity for up to 100 mg of very strongly retained substances. Note that this quantity includes every substance that may be strongly retained in any given sample, not just the component of interest! Less strongly held compounds may begin to elute before large volume samples have completely passed into the bed. Such breakthrough and capacity issues need to be understood as you develop your method. An understanding of k' (retention) for the components of your sample matrix will guide you in determining optimal loading conditions.
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