I loaded a polar fraction on a glass column packed with C18 and washed it isocratically with 10% methanol for 6 days. The color of this fraction remained on top of the column, then I washed it with 50% methanol-water and 100% methanol, but the color did not come down. What should I do to clean the column?
Please look at the following below links which may help you in your analysis:
http://phx.phenomenex.com/lib/gu54810610.pdf
https://mz-at.de/fileadmin/user_upload/shimadzu_hplc-columns.pdf
http://www.allpump.co.kr/alltech/c6001spe.pdf
https://www.chromacademy.com/channels/hplc/principles/hplc-column-cleaning-and-regeneration/
Thanks
It depends on what you have in your column and for what it has been used before. As a general procedure, you may follow this.
Step 1: Flush the column with 70% water; 15% methanol; 15% acetonitrile for 15-20 minutes. Usually this should matches with your MP.
Step 2: Flush at a lower rate with 100% methanol for 15 minutes
Step 3: Flush at a lower rate with 100% hexane for 15 minutes
Then you may store the column for future use.
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