I would like to know what is the best storage condition of a C18 column for a mobile phase of acetonitrile as organic solvent. Is there any other recommended ratio? What's the best storage condition for long term storage and short tern storage(a day or two)?
The best way, to storage a C18 column for short (weekend) or long period, is to wash the column with a mobile phase water and acetonitrile(H2O/ACN HPLC grade) (70:30V/V) for 20 minutes (to remove any salts) and then flush water and acetonitrile(H2O/ACN HPLC grade) (30:70V/V) for 20 minutes (to store the stationary phase in the best conditions). after doing this the column can be capped and stored for the desired time.
I recommend to wash the column with ACN or MeOH properly. After washing remove your column from HPLc properly and fit its end ferrules. Now you can keep in your cupboard.
gudluck
As a general rule, avoid storing the column with water or salts, this can cause fungus or precipitate. Room temperature and dry environment are preferred always.
As mentioned above, wash out any modifiers (like trifluoroacetic acid, formic acid) and buffers and salts. Use an aqueous system to wash salts so they don't precipitate. Wash the column with organic solvent, and then put the end caps on the column.
You want to wash out the modifiers so they don't hydrolyze the C18 from the silica. Wash the salts out so they don't have an opportunity to precipitate. The final wash is with the organic solvent (acetonitrile or methanol are fine) so most of the retained material elutes off. Storing the column in organic solvent prevents phase collapse, where the C18 associates with itself rather than the mobile phase and your compounds (usually happens >90% water).
If I leave the column overnight, I like to wash off the modifiers- I found in my experience that the columns last longer. I would just leave the columns in the mobile phase if I'll be running it again within a work day.
If you use only ACN as your system you should just let pass about 30 volumes of ACN, and then disassemble the column, put the caps and store it. But it is normally writen in the instruction manual for the column.
Always store reverse phase columns wet and well capped in water and acetonitrile (10:90).
Usually RP-C18 columns are packed with 70% Methanol or Acetonitrile Which is mention in the leaflet available in the column box otherwise you can get these information on the manufacturer web site
Your instruction sheet that comes with the column (C18) usually suggest methanol/water in a 50-50 mix.
After completion of ACN mobile phase., it is better to wash with methanol+water mobile phase or methanol alone for 30 min to 1 hr., remove from LC., screw end cap on both the sides and store in appropriate column box you received from the manufacturer.,
If you are using buffer (like phosphate buffer) and acetonitrile, it may precipitate and may decrease the efficiency of column. So, it is recommended to wash the column with distil water (HPLC grade) for short time to wash the buffer (may be 15-20 mins) and then by 50% methanol for 30-45 mins and finally by 100% methanol for 30 mins. It can be stored for short to long term. If you are doing experiment on next day, no need to wash, just you can keep in mobile phase.
The best way, to storage a C18 column for short (weekend) or long period, is to wash the column with a mobile phase water and acetonitrile(H2O/ACN HPLC grade) (70:30V/V) for 20 minutes (to remove any salts) and then flush water and acetonitrile(H2O/ACN HPLC grade) (30:70V/V) for 20 minutes (to store the stationary phase in the best conditions). after doing this the column can be capped and stored for the desired time.
Use always Aqueous and org solvent mix ratio, for example after completion of analysis wash the column with water & Methanol in 9:1 ratio for a specified time (about 50-60 minutes) then again wash the column with Methanol:water in 9:1 ratio. It provides a good equilibrium condition for your column, store the column always in higher amounts of solvent with little aqueous condition instead of 100% solvent. This will prolongs your column life also. All the best
The manufacturer should have recommendations. Phenomenex, for example, recommends 65% acetonitrile, 35% water.
After washing the colum with isocratic H20/ACN (70/30) to wash salts you should wash with isocratic H20/ACN (30/70) so that your column is kept in high organic until next time it is used. Always avoid storing the column in high aqueous phase (> 90% water) in order to prevent the phase collapse.
The best way to store a C18 reverse phase column is to wash it with 50% Methanol (Methanol:Nanopure water) then store it in the same mix. When the column is shipped to you brand new, it comes in a Methanol: water mix (50-50).
To ensure long term performance, I would set a gradient to cool the column down to RT at the end of the sample set, while washing it with an isocratic H20/ACN (80/20) to wash salts for 10 CV. Then you wash isocratic H2O/ACN(10/90) for 5 CV, then down to isocratic H20/ACN (20/80) for another 5 to 10 CV. This will ensure that you removed salts, removed non-polar grease, and returned back to a well conditioned initial condition for C18 separation. Yes, never store a column in 100% water(collapses), or 100% organic solvent (swells).
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