Lab Tech Guy on HPLC wash solvents

2. ©2009 Mourne Training Services Dear Lab Tech Guy, I am working in QC, doing mainly HPLC analysis. I am often confused by the composition of seal wash and needle wash. Do you have any advice on what is the right composition of both that increase efficiency of HPLC? Yours, Virender (Bangalore, India) ?

3. ©2009 Mourne Training Services Dear Virender, To answer your question I will briefly describe the purpose of both seal wash and needle wash. The most suitable composition for each follows logically from their purpose. Seal wash is used to rinse the backs of the piston seals in the pump of a HPLC system. The piston is the part of the pump which moves back and forward in the pump head drawing in and pushing out the mobile phase solvent with each movement. Because it is a moving part, a seal around this piston is required to prevent the liquid leaking out the back of the pump. Piston Seal

4. ©2009 Mourne Training Services Over a period of time small amounts of mobile phase solvents seep through the seal to the back of the pump head. If these solvents contain buffers then the salts may precipitate out forming deposits which can shorten the life of the seal. Seal wash is used when the mobile phase contains buffers. The wash flushes the back of the piston seals removing any deposits and maximising the lifetime of the seal. Seal Wash? It follows that the composition of the seal wash should be aqueous to dissolve buffers. A small amount of organic solvent is added to prevent bacteria growth and also to reduce the surface tension of the water (this helps the wash solvent cling to surfaces). Typical seal wash composition is 90% water and 10% organic solvent. The organic solvent may be methanol, acetonitrile or isopropyl alcohol (IPA).

5. The needle in the HPLC system is used to introduce the sample into the mobile phase so that it can be separated on the HPLC column. The needle wash is used to clean the needle after an injection. The design of the needle in the injector system varies for different manufacturers. In some designs the inside of the needle is part of the flow path of the mobile phase and thus it is flushed continuously by the mobile phase between injections. In this case it is the outside of the needle which is cleaned by the needle wash. In other designs the needle is separate from the mobile phase flow path and thus the needle wash is used to clean the inside and outside of the needle. In all cases the composition of the needle wash needs to be matched to the sample since this is what you want to clean off the needle. Therefore the selection of a suitable needle wash is part of the development of a particular HPLC method. Typically a composition of needle wash which matches the proportions of aqueous and organic solvents in the mobile phase will be appropriate (assuming you are using reversed phase HPLC). ©2009 Mourne Training Services

6. If the needle is not cleaned correctly then you may experience carryover between injections where sample from the previous injection which has not been cleaned from the needle will interfere with the next injection. Needle wash procedures will also help to prolong the life of the seals in the injection system. Appropriate selection of seal wash and needle wash solvents will help you in the quest for trouble free operation of your HPLC system and ensure that your analysis is not a washout. Yours chromatographically, ©2009 Mourne Training Services You’ve run the analysis, now eat the snack (?)