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.

To prevent the build up of salt crystals inside of the narrow bore tubing, pump and other HPLC components I strongly recommend that you wash the system down each day, after use. I 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 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 'critters'). 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) 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: A second level of flushing buffers from the system involves the use of "seal wash" pump. These pumps are often small peristaltic pumps with silicone tubing connected to them (Some are simply gravity fed systems where the wash solvent bottle must be kept elevated and waste tubing kept low to function). The tubing is connected to a metal ring which surrounds the back of the main pump's piston in such a way that it can wash liquid over it and remove these deposits. When run with buffers, the main HPLC pump pistons receive a thin coating of the solution each time they complete a full stroke. Over time, the liquid evaporate and a film of buffer salts deposit on the sapphire pistons. These salts accumulate and can scratch the pistol surface allowing air to enter the system or leaks to results. 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 which occur outside on the piston surface. 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 to remove these deposits. The 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 80% HPLC Grade water and 20% Methanol. 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. 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 piston squeak 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 silicone tubing used in your seal wash pumps is fully compatible with the wash solution you choose.


  1. Very useful for analysis

  2. Isnt the use of acetonitrile not the best idea for a seal wash solution since it can degrade the seals over time, making them brittle and more likely to fail? That's why I always use water and IPA. Thoughts?

    1. The best seal wash rinsing solution would depend on the mobile phase composition used. The purpose of the seal wash solution is to remove buffer crystals so a pure organic solution of acetonitrile (ACN) should not be used alone. To dissolve the buffers, a suitable aqueous phase solution should be used (e.g. mixture with some organic such as 20% MeOH or ACN. *IPA has poorer solubility so is not a top choice for RP methods).

      Since the RP piston seals are designed for use with 100% organics such as ACN, no accelerated wear would be expected if you used pure ACN as the seal wash solution. However, you would not be using the seal wash system for what it was designed for, to dissolve and rinse buffer salts away from the seals.

  3. Which seal wash solvent should I use if I am running normal phase aplication. My mobile phase is 3:900:100 = Water:heptane:IPA. And which solvent should i use for needle wash? Thank you for your help!

    1. "The purpose of the Seal Wash solution is to remove buffer crystals". Your NP solvents do not contain buffers so no seal wash system is needed (a pump w/o the seal wash option would be used). Many manufacturers sell special NP only piston seals for use with their pumps. However, if your pump has a seal wash system installed, then you need to use it. For your specific example we would choose a Seal Wash solution which would keep the seals "wet" (to seal) and not dry out (which may result in leaks and/or produce a very annoying high pitched sound!). You mobile phase is essentially Heptane/IPA. IPA can mix with both water and Heptane so would be the best choice. If used, run it using a very slow flow rate.

      Needle wash solution is chosen based on sample solubility. Both your mobile phase and Needle wash solution are usually the same liquid because you want to rinse off any residue. If you have a very sticky sample OR if you have not selected a very good mobile phase which FULLY dissolves your samples (go back and develop a better method), then the needle wash solution should incorporate stronger solutions which DO dissolve ALL of the sample material off (Note, these stronger solution rinses can often be followed with mobile phase rinses to reduce contamination when you put the needle and loop back in the flow path). A well developed HPLC method where the sample dissolves into the mobile phase and the instrument is well maintained would rarely need a "needle wash" step(s) as part of the method. BTW: When used with proper HPLC methods, some brands and models of HPLC autoinjectors do not need needle washes at all because their design provides for complete and continuous flushing of the injectors flow path during the method. This means the needle and loop are always full of fresh, clean mobile phase and the only kind of needle washing that might be needed would be a needle "dip" to rinse off the outside surface of the needle (or use one of the latest "needle washing stations").