Translator for HPLC HINTS and TIPS for Chromatographers

Saturday, October 29, 2016

Notes on Cleaning bound Protein from RP HPLC columns:

First, a few comments:

  • ·         Before proceeding with any column regeneration or cleaning procedures, always refer to the specific advice provided by the column manufacturer. Approved maintenance and cleaning instructions can often be found in the product guide which comes with the new column. Their guidelines supersede these!
  • ·         Columns are consumable items. After a suitable amount of use, the time and materials required to regenerate them may cost more than the purchase of a replacement column. Always have a new, spare column on hand.
  • ·         Protect your detector. Before washing or cleaning the column, disconnect the column outlet line and direct the column to waste only.
  •      Column Storage solutions are not the same as column wash solutions. Never store a column in buffer or ion pairing containing solutions.

For RP supports, if buffers have been used, always start by washing the column down with ultra-high purity water and some organic solvent (e.g. Water/MeOH, 95%/5%) to remove all salts. Use about 10 column volumes to flush these off.

Many polymeric resins (e.g. PS-DVB) can be effectively cleaned using 0.1 M Sodium Hydroxide solution or a mobile phase solution containing equal parts of isopropanol (IPA) and 3 M Guanidine hydrochloride at ~ 50 °C. Optionally, some success has been reported using other solutions such as: 5M Urea (pH 7) buffer solution; 1 M NaCl (pH 7) and even mixtures containing some methylene chloride solvent. Check with the manufacturer!

For RP silica based supports, we often use a series of wash solutions. In most cases, pure water or pure organic solvents such as MeOH or ACN will not remove bound protein. An acid, base or ion pairing reagent is often needed. One of the first general wash solutions to start with is a 1% Acetic acid solution in Methanol (50/50). If desired a stronger acid such as Trifluoroacetic acid (TFA) can be swapped for the acetic acid (where possible, start with a weaker acid). This can be followed with a later solution where IPA replaces the MeOH (50/50). In both cases, adjust the pH to ~ 2.5. 5 M Urea solution has also proven effective in removing bound protein from silica supports too. Another salt solution that has shown some promise is 1 M sodium phosphate solution, pH 7.0. Run the salt solutions for about one hour at a moderate flow rate. Follow up with rinses of water and MeOH (80/20), then 90% MeOH/Water.


  1. If after trying these wash solutions and the column is still plugged or gives us poor results, should we dispose of the column or continue washing?

    1. Columns are consumable items. At some point you must decide if the time and materials used (Some solvents are very expensive) to try and clean them are worth the effort. It may not make financial sense to spend days and lots of money trying to clean a column. If the column is marginal at best, that will directly impact your analytical results. Do you want poor quality results which can not be relied upon because you elected to use a contaminated column?

      Developing high quality methods which fully dissolve all of the samples (at all times), retain, then elute them off the column prevents column fouling. Follow up each analysis method with a separate column wash and re-equilibration method. This should incorporate a stronger solution than your mobile phase (to remove any late eluters). Use freshly prepared, filtered mobile phase solutions. Filter samples. Don't overload the column (do a loading study) with sample. Check the column with a separate std on a regular basis to monitor its efficiency. These types of steps will reduce the need to spend time washing a fouled or contaminated HPLC column.