What is the best mobile phase for washing c18 column before turning off the bump (methanol, acetonitrile, ...etc )? and what is the suitable flow and washing period?. I use HPLC Agilent 1200
You can run the blank sample like methanol or acetonitrile, for two or three times with previously used method, then you run with the solvent given in the column brochure or 70:30 (water: acetonitrile) for 10 mins.with good flow rate.
Dear Mohamed,
C18 columns are best preserved in high organic solvents. After you run a gradient with no sample injection (cleaning gradient) which can be the same gradient as the one that is used for sample analysis), you can run the isochratic mode at 90% acetonitrile and turn off the HPLC so that your column is kept in high organic until next time it is used.
You can also keep everything flowing and wet by turning your flow rate to a minimum (not sure what the common flow rate used for Agilent 1200) and running the system isocratic at 90% organic for weekends and long periods where the system is not used to run samples. This is a much better practice if you can afford the cost of the organic solvent.
Best,
Hediye.
The best method is to use the correct solvent and duration that elutes any impurities from the column that may interfere with subsequent analyses. You will have to experiment with this depending on your sample matrices. Columns exposed to only organic-aqueous mixtures should be rinsed with pure aqueous phase (to remove salts) followed by the pure organic phase. Acetonitrile is a "stronger" solvent than methanol and I prefer to use it for dirty matrices like plasma or serum. Once you have the column equilibrated into a pure organic solvent like methanol or acetonitrile you can double the flow rate to decrease the rinsing time. I like to use at least 10 column volumes as the minimal amount of solvent for rinsing an HPLC column. If I see something is still eluting after 10 column volumes of rinse solvent, as is evident by a high detector signal, I will keep on rinsing them until the detector signal returns to baseline. I typically store columns long term in solvent mixtures containing about 20% organic phase without any modifiers (TFA, Formic Acid etc.). The column flow rate depends on the column dimensions and particle size and even the packing material, it is best to use the flow rate the column manufacturer recommends.
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.
It depends on your analyte. For small biomolecules like vitamin C or any water soluble compound, use pure millipore water,
For storing column use methnol
For removal of salt, if any buffers you use, purge the system before and after use with water,
If you see column block or increase pressure, use combination of water: ACN for 20 column wash until the base line is corrected
If you use peptide resolution using TFA, dont allow the system with TFA overtime this very low pH may spoil your column
The question is: Should washing the C18 only be preventively? Or have been strong impurities on the column?
Best regards
Burghard Cordes
Hi Sir
I''m glad to receave your request about washing HPLC column c18
Tthe best mobile phase for washing c18 column before turning off the bump is methanol, but I advice you to consult the manual
good luck
Dear Mohamed,
I agree with what Hediye and Bruce mentioned above. I am using an Agilent 1200 series HPLC. I typically would run 2 or 3 'no sample' injections with the gradient you have been using. This is followed by a high organic mixture (90%) for >10 column volumes (approx volume= Πr2h, where Π =3.142, r = radius, h=height or length of column). If you have time, again you could include 'no sample' injections and check these injections for any signal. If so, I would flush with more organic mixture until the signal drops and you get back to baseline. As the others have mentioned, it is good practice to leave your column relatively clean and in high organic solvent mixture (~90%) but not 100%. Have fun and goodluck
Hello,
Washing with 80:20 (water:ACN) for 3 hours at 1ml/min and 80:20 (ACN:water) works fine
Otherwise there are more precise procedure (siurce: chromacademy)
Reversed phase columns include C30, C18, C8, C4, C1, CN and phenyl stationary phases. To clean and regenerate a reversed phase column flush it with the following volumes of solvent.
20 column volumes of water/acetonitrile (95:5 v/v)
20 column volumes of acetonitrile
5 column volumes isopropanol
20 column volumes hexane
5 column volumes isopropanol
20 column volumes acetonitrile
20 column volumes of water/acetonitrile (95:5 v/v)
Re-equilibrate with the required mobile phase
Regards
The simplest method would be washing with high polar solvent like water first and then go for a slow gradient of organic solvent like ACN or Methanol until > 90 % concentration in low flow of 0.5 ml/min for approximately 3 hours before closing it. It removes maximum if not all of the impurities like salts, proteins and any organic compound residue if present in the column. All the best
I always used to finish with Methanol: 90% is good for overnight, but 100% Methanol if shutting-down and changing columns, since less dissolved air would be trapped. I used methanol in preference to Acetonitrile, as it is less dangerous to handle when you need to change the column or disconnect any other components in the solvent path (guard columns, filters, bubble traps, injectors, detectors or other peripherals. If storing the column, it is important to make sure the ends are firmly sealed, although if stored with methanol, any air is easy to displace after re-connecting.
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