Translator for HPLC HINTS and TIPS for Chromatographers

Friday, August 24, 2012


Many vendors offer an HPLC Pump "Seal Wash" option. If you often operate your instrument with high concentrations of aqueous salt buffers (e.g. Protein, Peptide Separations), then an optional seal wash system might be something you want on your HPLC system. When combined with daily flushing of the HPLC system to remove buffers, it can extend the life of and reduce the maintenance needed on your HPLC system. 

NOTE: If your HPLC system has a piston seal wash feature installed, then failure to utilize it (leaving it "dry"), may result in decreased lifetime of the wash seals and piston(s) due to the added friction they exert. If you have a seal wash system, but do not need it (i.e. running only NP solvents), then replace it with a non-seal wash system or utilize the seal wash feature to prevent damage.

To prevent the build up of salt crystals inside of the narrow bore tubing, pump and other HPLC components we strongly recommend that you wash the system down each day, after use. We routinely see HPLC systems with white fluffy crystals built up around the pump heads, pistons and various fittings from lack of maintenance on a daily basis. High concentrations of mobile phase buffer in your system (e.g. 0.1 M is considered 'high', but all buffers should be flushed out) can damage the pump pistons, pump seals, injector parts and are corrosive to the stainless steel used. The resulting damage can lead to expensive repairs.

  • Two types of flushing techniques can be employed to reduce the damage caused by these salt buffers and extend the life of the system. Flushing the entire HPLC flow path with a solution which does not contain any buffers (to rinse it) and optionally, flushing the back of the pump pistons using a "seal wash" system.

(1) Flushing the HPLC Flow Path: Potential damage from salts can be avoided if you remember to always flush down the entire flow path of your HPLC each day (and anytime it may sit unused) with a proper mixture of HPLC grade water and some organic (to prevent the growth of bacteria and/or mold). Flush the column down first with an appropriate solution and then remove it from the flow path. Next flush the entire HPLC system down to rinse it of any remaining deposits (sometimes the column can be left in-line and flushed with the system. Consult your column manufacturer for advice). The exact mixture to use will depend on the exact type of mobile phase you are using. You want to select something which will dissolve the buffer used in your mobile phase into the solution plus incorporate some organic solvent component to reduce the surface tension and also deter the growth of bacteria over time. For example: A common Reverse Phase (RP) wash solution of 80% HPLC Grade water and 20% Methanol can be used in many applications. If you have an automated HPLC system, then this entire process can be stored as a "Flush" method and programmed to run at the end of each day's sequence or series of runs so you do not have to remember to do it manually.

(2) Seal Wash System Use: When run with buffers, the HPLC pump's pistons are coated with buffer solution. Over time, the liquid evaporates and a film of buffer salts is deposit on the pistons. These salts accumulate and can scratch the piston surface allowing air to enter the system and/or leaks to occur (drips from behind the piston seal). Premature replacement of the pump head seals and pistons often results from this damage. Washing the internal flow path of the HPLC system (as described in section #1 above) does not wash away these salt deposits. A "seal wash" system can be employed to assist to deal with the problem. The seal wash pump's inlet line can be placed in a bottle with fresh wash solution and through either an automatic timer feature set in the pump's software or through the operator manually turning the wash pump on and off, it can wash the back of the piston area to rinse these deposits away. The rinse solution used to wash the pistons will again depend on the type of mobile phase you are using (just like the HPLC flushing solvent). For most RP applications, I recommend a mixture of HPLC Grade water and Methanol (50/50 to 80/20). Other common seal wash solutions are: 90% HPLC Grade water and 10% IPA or 80% HPLC Grade water and 20% ACN. For most applications, I prefer using Methanol over IPA because it is much better at dissolving many of the buffers used. A third option would be to use a wash solvent which is the same as your mobile phase, but without any buffers added (try to include at least 20% organic content). Again, you must review your own method to determine which wash solution is best as their is no such thing as a 'universal' wash solution that can be used with all methods.

If you are running Normal Phase (NP) applications, then the seal wash can also be employed to keep the pistons 'wet' during operation and avoid excessive high pitched piston squeal noise, which is common when running dry solvents (e.g. Hexane). Manufacturers often provide special piston seals designed for use with normal phase solvents, but sometime the incorporation of the mobile phase as a seal wash solvent can lubricate the pistons well. IPA can often be employed as a NP seal wash solvent choice too. In any case, always make sure that the tubing used in your seal wash pumps is fully compatible with the wash solution you choose.

  • Seal Wash SEALS: One final reminder about HPLC system which use a"Seal Wash". Some designs (not all) incorporate a separate piston seal, behind the main pump head seal, to seal the rinse solution inside the wash area. Just like the piston seals at the front of the pump head, these wash seals require regular replacement. If your HPLC system uses a wash seal, be sure and have some extras on hand so they can be replaced when you service the pump head. Failure to replace these worn seals usually results in liquid leaking out the back of the pump head. This may be mistaken for a seal failure at the front of the pump head, so you need to be aware of their use to diagnose and repair any leaks correctly.