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Boardwalk :: Forum List Forum Category HPLC Troubleshooting
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 HPLC mobile phase life - 2005/11/28 19:08 Can anyone give me information or direct me to a reference on maximum LC mobile phase life? We typically use buffered mobile phases with about 25% organic modifier. We would like to reduce the daily mp prep time by making up larger quantities at a time for extended use.
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 HPLC mobile phase life - 2005/11/28 19:08 Take some advice from a lazy chemist who has learned the hard way. Make up small quantities frequently. You can make up large quantities, try to make them last as long as possible and get away with it for a period of time. Eventually it will come back to bite you in the butt! You will lose a days work, even lose a column or worse have someone challenge your results only to find the long lasting eluent introduced determinate error into your results. I started out with nothing but 4L bottles. I now never make anything up in greater than 2L quantities. If I wish to use the remaining half of yesterdy's eluent, I prepare a fresh liter, add yesterday's remnant, mix and vacuum filter, degas it into the 2L bottle. Think of it this way. If you do not take the time to do it right, you may have to take the time to do it twice.
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 HPLC mobile phase life - 2005/11/28 19:09 When using buffered mobile phases, please check pH of buffer before adding organic solvent. Some time ago I run into a customer "complaint". They reported some retention shift when using a new column for a validated method. Suddenly they found more peaks, but resolution was better. During my visit I asked them how they prepare the buffer, and I was told that they have a "mother" buffer solution and just take an aliquote out to dilute with water. The procedure how to prepare the "mother" buffer solution was on that bottle. I asked them to test pH. On the bottle lable was written, that buffer "is" pH6,0! When testing buffer we found pH5,1! And just when they prepared a new "mother" buffer solution, they purchased a new column. At the end it was embarassing for them, because it was clear that during method development they did not a propper job, and SOP was also done in the wrong way. And it was a big wellknown pharmaceutical company. I forgot to ask them what they will do with the "new" compounds they found with the wrong pH value of the buffer!!??!! Please follow Harvey's advise, and always check pH! It's just half an hour preparation, but can save you minimum one working day and avoids a lot of trouble! Good luck. Gerhard
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 HPLC mobile phase life - 2005/11/28 19:10 Gerhard brings up an interesting point relative to more than just HPLC mobile phase when he mentions pH. It is taken for granted by most people that the pH value from a meter is an absolute. Most forget that when they were introduced to pH it was by definition applicable to DILUTE, AQUEOUS solutions. Ph meters do not read pH. The read millvoltage and apply the 59mV to 60mV per pH unit, temperature dependant, relationships. When you do not have an aqueous solution, for example 40% methanol or 30% acetonitrile, that pH, millivolt relationship goes out the window. Proper mobile phase preparation requires adjusting pH before adding mobile phase. Most technicians do it after because mobile phase is described as the mixture adjusted to a pH. As a result some methods are now actually developed instructing technicians to adjust before use. If that is how a method was developed, good luck to anyone trying to use it that way. I have had to start from scratch, to re-invent the wheel, so that I can reproduce results, by adjusting the aqueous to a determined pH before adding the organic. If ever you are developing a method, dissolve the salt in water, adjust pH and then add organic. Do not mix old pH dependant eluent with fresh. Dispose of the old and replace it with the new.
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 HPLC mobile phase life - 2005/11/28 19:10 I think that greater than 20% organic of any common HPLC organic is surely biostatic and you could have extended expiration dates. I read someplace that 2% ACN, 10%MeOH, and 7%IPA were biostatic. We give all orgainic containing mobile phases a two week expiration and haven't had any problems. When doing standard and sample stability I also set aside some mobile phase and do one run with two week old mobile phase and make sure everything looks ok.

Amber bottles are useful for storing non-biostatic solutions like buffers without organic.
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Boardwalk :: Forum List Forum Category HPLC Troubleshooting

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