By [68-23-5] on Tuesday, February 4, 2003 - 05:10 am:
Dear Friends,
1 - I am having degradation of my major peak (Norethynodrel) with Acetonitrile (HPLC grade).
2 - Other Acetonitrile (different mark) do not causes it.
3 - Is this problem common with (for) you?
Best Regards,
By Anonymous on Friday, February 7, 2003 - 05:46 am:
Hi 68-23-5
Part answer yes and part answer no because any solvent do not degrade your main peak but sometimes it happen that due to contaminated solvent or due to expose to air. Solvent give problem in baseline stability or extra peak or hump or shoulder peak to peak of intrest. I had problem with solvent like Glacial acetic acid and chloroform.
By dinakar on Wednesday, May 26, 2004 - 01:31 am:
Dear friends,
I am a first time user of HPLC.I intend to fractionate my protein on a c-18 column.In this regard I have a couple of questions:
1.I originally intended to use a agarose based column but was told that silicon based columns r much better?
2.Is it necessary that I pass my proteins through say DEAE-Sepharose or some other column prior to HPLC.
3.The technician at our place seems to be apprehensive of using my buffer system for equilibration and elution(TGEN).Says he doesn't know how it might react with the coulumn.Is he justified?
4.will the use of acetonitrile-milliq water for elution have any effect on the biological activity of the protein in the fraction?
early response solicited
By Anonymous on Sunday, May 30, 2004 - 05:27 pm:
I will be a little bit harsh here and assume that you are also a first-time worker with proteins, otherwise you would know that the use of acetonitrile-water is an excellent way to denature proteins.
So, to deal with your questions in reverse order:
4. Yes, ACN/water will almost surely denature your protein.
3. If your buffer was originally designed for an ion-exchange or size-exclusion separation, then it will almost surely be useless on a silica-based reversed-phase column. An analogy would be the use of diesel fuel in a gasoline engine.
2. Whether other separation techniques are required prior/subsequent to HPLC depends on your protein and what you are trying to separate is from.
1. Silica (not "silicon", by the way) based C18 columns are almost always used with mobile phases composed of acetonitrile or methanol mixed with water. This makes them "better" at separating denatured proteins, but almost entirely unsuitable for working with proteins in their native conformation.