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Wash the column with the current mobile phase without buffer, salt or acid for 10-15 min. If the ratio of organic solvent and water is changed, salt may precipitate. Wash impurities in a column to achieve a stable baseline: Use a solvent with strong elation properties in which the sample dissolves well.
In general, you clean a column by flushing with a solvent that is stronger than the mobile phase. In this case, that would be water (or a water/methanol mix). The catch is that if your separation depends on having an additive adsorbed to the silica surface, you could be looking at long reequilibration times.
Wash with 4 column volumes of up to 70% ethanol or 30% isopropanol (see Table 21 for recommended flow). Rinse with at least 2 column volumes of distilled water (see Table 21 for recommended flow) until the UV-baseline and fluent pH are stable.
In your case wash the column with 70% water. 15% methanol. 15% acetonitrile. Divert the column fluent to waste not to contaminate your detector(s). Wash the column slowly over to 100% methanol and wash for at least 15 minutes. Wash the column over to 100% acetonitrile and wash for at least 15 minutes.
After the column has been flushed, it is usually best to store the column in the flushing solvent (acetonitrile or methanol) until it is used again. To re-equilibrate to normal operating conditions, equilibrating with 1020 column volumes of mobile phase usually is sufficient.
In your case wash the column with 70% water. 15% methanol. 15% acetonitrile. Divert the column fluent to waste not to contaminate your detector(s). Wash the column slowly over to 100% methanol and wash for at least 15 minutes. Wash the column over to 100% acetonitrile and wash for at least 15 minutes.
Remove buffer with HPLC grade water. Remove contaminating substances with a strong solvent, for example pure acetonitrile. Place the wash solvent reservoir for needle wash (optional: needle seat flush) into the solvent cabinet. Use an appropriate solvent based on the sample and mobile phase properties.
Reverse the direction. Attach the column fitting that is normally at the outlet to the tubing that is normally connected to the inlet. Select the appropriate wash solvent. Wash out contaminants. Reconnect the column in the normal direction.
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