To clean your column, use a mixture of water and an organic solvent that matches the mobile phase containing your ion-pairing reagent. This helps remove any retained compounds and maintain column performance.
Here's a recommended cleaning procedure:
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Initial Wash: Start by flushing the column with a water/organic solvent mixture that reflects your mobile phase composition. For example, if your mobile phase is 10% acetonitrile in water (with an ion-pairing reagent), clean the column with 10% acetonitrile in water for at least 30 minutes. This will help dislodge any strongly retained compounds that are similar in polarity to your mobile phase.
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Intermediate Wash: Follow this with a mixture of water and acetonitrile in a 50/50 ratio for at least 30 minutes. This step broadens the range of compounds that can be removed.
Important Considerations:
- Flow Rate: Use a reduced flow rate during cleaning to avoid over-pressuring the column. Typically, half of the analytical flow rate is sufficient.
- Solvent Compatibility: Always check the solvent compatibility of your column's stationary phase. Consult the column manufacturer's instructions. Never use solvents that are known to damage or dissolve the stationary phase.
- Column History: Knowing the history of your column (e.g., the types of samples and mobile phases it has been exposed to) will help you choose the most appropriate cleaning solvents.
- Detection: Monitor the column effluent during the cleaning process. A UV detector can help you visualize when the column is clean (i.e., when the baseline returns to normal).
- Gradient Cleaning (if applicable): For columns used with gradient elution, consider running a gradient from low to high organic solvent concentration during the cleaning process. This can help elute a wider range of contaminants.
- Storage: After cleaning, flush the column with a storage solvent recommended by the manufacturer. This usually involves a high percentage of organic solvent (e.g., 80% acetonitrile in water).
Example Scenario:
Let's say you're using a C18 column with a mobile phase of 20% methanol in water and an ion-pairing reagent like trifluoroacetic acid (TFA). A suitable cleaning procedure would be:
- Flush with 20% methanol in water for 30 minutes.
- Flush with 50% methanol in water for 30 minutes.
- Flush with 80% methanol in water for storage.
Why this works:
Using a similar solvent composition to the mobile phase helps solubilize and elute compounds that may be retained due to similar hydrophobic interactions. The intermediate wash with higher organic solvent concentration helps remove compounds with increased hydrophobicity.
