Many instrument vendors offer an HPLC Pump with "Piston 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 to include on your HPLC system. When combined with daily flushing of the HPLC system to remove buffers, a piston seal wash system can extend the life of and/or 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 on a regular basis (leaving it "dry"), may result in decreased lifetime of the pistons, wash and piston seals, plus leaks due to the added friction. If you have a piston seal wash system, but do not need it (i.e. running only NP solvents), then replace it with a non-seal wash system (manufacturers offer kits) or begin utilizing the piston seal wash feature to prevent damage.
To prevent the build up of buffer salt crystals inside of the narrow bore tubing, LC pump and other HPLC components we strongly recommend that you wash the system down each day, after use. We routinely see HPLC systems with large amounts of white fluffy crystals built up around the pump heads, pistons and various fittings from lack of daily maintenance. High concentrations of mobile phase containing salt buffers in your system (e.g. 0.1 M is considered 'high', but all buffers should be flushed out) can damage the pump's pistons, pump seals, injector parts and are corrosive to the stainless steel used. The resulting damage can lead to expensive repairs and lost time.
(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 with a proper mixture of HPLC grade WATER plus some organic solvent (to prevent the growth of bacteria and/or mold). Flush the column down first with an appropriate solution to remove any buffers 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 with the column manufacturer for advice). The exact flushing mixture to use will depend on the exact type of mobile phase you are using, but pure water is often a good initial choice. You want to select a solution which will dissolve ALL of the buffer used in your mobile phase back 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 "RP System 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) Piston "Seal Wash": When running with buffers, the HPLC pump's pistons are coated with buffer solution. Over time, the liquid evaporates leaving a film of buffer salts deposited on the pistons. These salts accumulate and can scratch the piston surface or get stuck in the piston seals allowing air to enter the piston chamber and/or leaks to occur (drips from behind the piston seal). Early replacement of the pump head's piston 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 all of these salt deposits. A piston"seal wash" system can be used to help wash the back-side of the piston washing away remaining deposits stuck to the piston. The piston seal wash pump's inlet line can be placed in a bottle containing 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 (some systems just use gravity), it can wash the back of the piston area to rinse these deposits away. The rinse solution used to wash the pistons will 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 might include: 80% HPLC Grade water and 20% 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). Before starting, you must review your own methods to determine which general system wash and piston wash solution(s) are best as their is no such thing as a 'universal' wash solution that can be used with all methods.
If you are using Normal Phase (NP) applications, then the piston seal wash system can also be employed to keep the pistons 'wet' during operation and avoid excessive wear and noise (and that high pitched piston squeal noise), which are 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 help lubricate the pistons too. IPA can often be employed as a NP piston seal wash solvent as it is one of the best solutions to use in maintaining the seal over time (IPA is an excellent seal wash solvent for many NP applications). In any case, always make sure that the tubing used in your seal wash pumps is chemically compatible with the wash solution you choose.
- Two types of flushing techniques can be employed to reduce the damage caused by these salt buffers and extend the life of the HPLC system. Flushing the entire HPLC flow path with a solution which does not contain any buffers ('water' to rinse it) and optionally, flushing the back side of the pump pistons using a piston "seal wash" system. Let us consider these two systems.
(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 with a proper mixture of HPLC grade WATER plus some organic solvent (to prevent the growth of bacteria and/or mold). Flush the column down first with an appropriate solution to remove any buffers 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 with the column manufacturer for advice). The exact flushing mixture to use will depend on the exact type of mobile phase you are using, but pure water is often a good initial choice. You want to select a solution which will dissolve ALL of the buffer used in your mobile phase back 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 "RP System 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) Piston "Seal Wash": When running with buffers, the HPLC pump's pistons are coated with buffer solution. Over time, the liquid evaporates leaving a film of buffer salts deposited on the pistons. These salts accumulate and can scratch the piston surface or get stuck in the piston seals allowing air to enter the piston chamber and/or leaks to occur (drips from behind the piston seal). Early replacement of the pump head's piston 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 all of these salt deposits. A piston"seal wash" system can be used to help wash the back-side of the piston washing away remaining deposits stuck to the piston. The piston seal wash pump's inlet line can be placed in a bottle containing 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 (some systems just use gravity), it can wash the back of the piston area to rinse these deposits away. The rinse solution used to wash the pistons will 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 might include: 80% HPLC Grade water and 20% 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). Before starting, you must review your own methods to determine which general system wash and piston wash solution(s) are best as their is no such thing as a 'universal' wash solution that can be used with all methods.
If you are using Normal Phase (NP) applications, then the piston seal wash system can also be employed to keep the pistons 'wet' during operation and avoid excessive wear and noise (and that high pitched piston squeal noise), which are 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 help lubricate the pistons too. IPA can often be employed as a NP piston seal wash solvent as it is one of the best solutions to use in maintaining the seal over time (IPA is an excellent seal wash solvent for many NP applications). In any case, always make sure that the tubing used in your seal wash pumps is chemically compatible with the wash solution you choose.
- Piston Seal Wash SEALS: One final note about HPLC systems which use a "Seal Wash" system. 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.
Very useful for analysis
ReplyDeleteIsnt 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?
ReplyDeleteThe 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).
DeleteSince 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.
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!
ReplyDelete"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.
DeleteNeedle 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").
This was a very helpful post. Thank you! I always thought twice about canceling the Seal Wash Kit line item from my orders, but now I know for sure that I don't need it for my applications!
ReplyDeleteThank you for the post! I started to use HPLC so too many questions come up.
ReplyDeleteSo thankful to find your blog!
Thank you for this helpful information!
ReplyDelete