I´ve been using an Agilent Zorbax CDB C18 15 cm Column for the last months to determine and quantify sesamol(a sesame lignan) disolved in water, employing methanol-water 70:30 as mobile phase. It has always worked fine, well defined and resolved peaks even 2 weeks ago when I used it. Now, I've tried to use it since yesterday but the peaks doubled, were wider, not resolved. Tried cleaning it with acetonitrile 100%, increasing water concentration up to 100 % for the whole night at counterflow and 35 °C and then keeping methanol:water at the usual ratio for another hour, but the peaks still appear wider and sometimes as double peaks. Does anyone have experienced similar behavior and knows a good cleaning protocol that could help me with is? Thank you!
Hi Manuel.
You need use solvent with less polarity to clean up HPLC column. You could decrasing mobile phase polarity for remove apolar compounds wich still in column. You could use water/methanol/acetonitrile/isopropanol, you could make this with 30 ml for each solvent with flow 0.2 ml/min, return for water in reverse order of solvent beginning with isopropanol and finishing with water. I have used in some cases THF, but this solvent could atack Peek tubes. In attachment has a guide wrote by Agilent wich describe clean up procedure for reverse phase HPLC column.
I hope this help you.
I´ve tried with similar polarity solvents, but still hasn´t work. I would like to try with more non-polar solvents, maybe chloroform o DMSO. In your experience, would that be convenient? Thanks!
Hello Manuel, Peak splitting rarely has to do anything with a dirty column as a whole. Peak doubling is a classic sign of a dirty column frit due to particulate matter or some insoluble impurity. It can also happen that column bed has collapsed. Did you use guard columns by chance? If yes, time to change it. I am not sure, how old is your column, but every column has a life time. Beds eventually settle with time. As a last rescue effort, you can reverse the flow direction (i.e. the inlet becomes the outlet and do not attach any tubing). Let the mobile phase flow at 2 mL/min. Retest and check if peak splitting is still present or not. You cannot use any arbitrary solvents, chloroform might eat away pump seals etc.
Hi Manuel.
Farooq is correct, chloroform is very agressive for HPLC sistem and column, it is possible lost column efficience and not regenerate chromatoghraphic capacity. It is not convencional procedure, but could work, clean up your column in reverse flow (with same solvents I mentioned above). If you have some experience and feeling secure for this you could open your column carefuly remove frit and clean up in sonic bath with dilute nitric acid (maybe 10% for 30 minutes), but this is last way for save your column.
Good luck. I hope this regenerate your column.
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