Columns are shipped dry, but those with hydrophobic character need to be solvated in order to interact efficiently and reproducibly with aqueous matrices. Sample capacity is severely reduced on a dry column.

At low vacuum ( -3 in. Hg) add 1.5 ml of methanol or acetonitrile per 100 mg of sorbent to the sample preparation column. Release the vacuum or begin flushing immediately upon completion. The more air which passes through the column before sample loading, the less solvated the sorbent will be.

Apply deionized or distilled water or remove excess solvent which will interfere with hydrophobic binding. Use 1 ml H2O per 100 mg sorbent. Momentary high vacuum (5 to 8 in. Hg) may be necessary to restart flow. At - 2.5 in. Hg the column will resist air displacement (vacuum may be left on without drying the sorbent). If the sorbent is accidently dried, resolvate and reflush.

When using ion exchange columns, apply 1ml of buffer to the column after flushing to ensure that the sorbent pH is optimal for the sorbent analyte interaction desired. Where ion exchange interactions are involved, follow guidelines concerning pKa, pH and ionic binding. Use the same vacuum guidelines as described for flushing.


Sample Preparation and Application

Retention mechanisms may be hydrophobic, polar, or ionic. Add internal standard to the sample if quantitation is desired. Optimize sample application by removing particulates if necessary (centrifugation or filtering) and/or diluting viscous matrices with water or buffer to ensure proper pH for desired interactions. The analyte and sorbent should be uncharged for optimum hydrophobic retention. On ion exchange sorbents, analytes must be oppositely charged to the sorbent [anions (-) on anion exchange sorbents (+); cations (+) on cation exchange sorbents (-)]. During sample application, the analyte binds by displacing a counterion on the sorbent.

Applyu sample at a rate of 1ml/min. Again, a momentary increase in vacuum may be needed to initiate sample flow.

Washing the Sorbent and Eluting

Ideal washing removes as many interferences as possible while retaining the analyte(s). Ideal elution recovers 100% of the analyte while leaving behind interferences. Make certain your column is dry when changing between aqueous solutions and organic solvents.

Hydrophobic and Polar Analytes

The best approach towards using these types of sorbents is to search for a solvent mixture which will wash the most interferences from the sorbent wihtout loss of analyte. Note that wash pH may greatly affect cleanup and/or recovery. Keep analyte and sorbent pKa in mind if applicable. Elute with the strongest organic solvent, or by raising the percentage of organic, possibly in combination with pH change to disrupt binding.

Ion Exchange

Ionic bonds are strong enough to allow the analyte to remain bound while interferences are washed away with high percentages (up to 100%) of polar or nonpolar organic solvents. The pH will also affect sample cleanup. Remember to remain 2 pH units from the relevant pKa of your analyte and sorbent, both of which need to remain charged for ionic retention. Elute with aqueous buffers containing a stronger counterion than your analyte (classic ion exchange) or by changing pH to disrupt the ionic attraction. Make sure the elution solvent has enough organic character to voercome any adsorption due to packing material.

Colpolymeric Exchange

For ionically bound analytes, use washes of high organic strength to remove interferences retained by hydrophobic (solvent strength dependent) interactions. If your analyte is also capable of hydrophobic binding, remove polar interferences ionically similar to your analyte by using aqueous or weak aqueous/organic washes while disrupting ionic (pH and ionic strength dependent) binding. Elute by simultaneously disrupting ionic and hydrophobic interactions.