Chromatography Forum

Hallo, I am trying to purify a transmembrane protein ( pI around 9, MW 34kDa) which is member of a class of similar proteins (with similar pI and MW). The idea would be to separate fractions coming out the HPLC and check the presence of the protein with Western Blotting.
I found on a paper this method applied to a similar protein:

the protein was obtained from mitochondria extracted with chloroform:
methanol:water (75:23:2 by vol) containing 20 mM ammonium
formate, pH 3.7. These protein were dissolved in formic
acid (60%) and purified by reverse-phase HPLC on a PRP-3
column at 40°C (Hamilton, Reno, NV) in 0.1% trifluoroacetic
acid with an acetonitrile gradient.

I have a UPLC Acquity system and I am wondering (since I have never worked with intact proteins) if the whole protein could in some way obstruct the system.
Does anyone has experience with that column? Is there anything more suitable to my HPLC system? Thanks in advance!

The PRP-3 column is made of polymeric material (PSDVB) rather than silica-based (such as a C18). Recovery of hydrophobic, basic proteins is typically better with this type of support due to lack of silanols. The PRP column may also be operated at higher temperatures than a silica-based material, and elevated temperatures sometimes improve recovery.

Another option is HILIC.

Yes to HILIC. For references on HILIC of similar proteins using a decreasing organic solvent gradient containing a number of volatile solubilizing agents, see:
1) J. Carroll, I.M. Fearnley and J.E. Walker, PNAS 103 (2006) 16170;
2) J. Carroll, M.C. Altman, I.M. Fearnley and J.E. Walker, PNAS 104 (2007) 14330

A J Alpert? My book purports you to be the very person that coined the term HILIC.

Ron J

Guilty as charged. What book is that?

It is "HPLC Columns: Theory, Technology and Practice" by Ewe Neue.

That is pretty cool to have credit for naming a technique. I will have to go back and read the published literature that was referenced.

Ron J

I had to give it a new name. At the time that I was developing HILIC, people were separating sugars using amino-silica columns under HILIC conditions and speculating that the mechanism involved transient formation of Schiff bases. This begged the question of why it worked just as well with sugars with no reducing ends. A term with the word "hydrophilic" drove home a point about the properties that really were involved. I also wanted to make the point that you could perform normal-phase type separations with a lot of water present; otherwise, I would have just called it normal phase chromatography. You might be amused by a brief reminiscence about the genesis of the concept: A.J. Alpert, J. Chromatogr. A 1218 (2011) 5879.

Uwe Neue was a frequent contributor to this Forum until his untimely death last year. At the time he wrote that book that you refer to, he was engaged in a study with David McCalley that experimentally verified the speculation about the partitioning mechanism of HILIC. He knew the subject he was writing about. Good book you've got there.

I had to give it a new name. At the time that I was developing HILIC, people were separating sugars using amino-silica columns under HILIC conditions and speculating that the mechanism involved transient formation of Schiff bases. This begged the question of why it worked just as well with sugars with no reducing ends. A term with the word "hydrophilic" drove home a point about the properties that really were involved. I also wanted to make the point that you could perform normal-phase type separations with a lot of water present; otherwise, I would have just called it normal phase chromatography. You might be amused by a brief reminiscence about the genesis of the concept: A.J. Alpert, J. Chromatogr. A 1218 (2011) 5879.

Uwe Neue was a frequent contributor to this Forum until his untimely death last year. At the time he wrote that book that you refer to, he was engaged in a study with David McCalley that experimentally verified the speculation about the partitioning mechanism of HILIC. He knew the subject he was writing about. Good book you've got there.

That's an interesting nugget of HILIC history, Andy. I wonder, under non-aqueous conditions, why a Schiff's base would -not- form between primary amine stationary phase and aldehyde of open-form carbohydrate?

Water in the mobile phase makes separation of biomolecules possible in this mode. Were you the first to report this, Andy?

Per your second question: Hardly. There were 70 references cited in my 1990 paper that introduced HILIC, a few of which dealt with the the role of the aqueous part of HILIC mobile phases. The paper discusses them in detail. Now, I believe that I was the first to demonstrate that this method would work for polar biomolecules in general.

Regarding the formation of Schiff bases between reducing sugars and an amino-silica column: The first papers to examine this issue, in the early and mid 1980's, did show that recovery of reducing sugars decreased the longer they spent on an amino-silica column. It's plausible that Schiff base formation was responsible for this. Unfortunately, the authors of those papers concluded that the same reaction explained the retention and separation of sugars. The two phenomena are surely independent. People who were interested in how chromatography columns worked generally were not able to get positions in academic departments, leaving it to staff scientists in the column manufacturing companies to explain how their columns worked. I regret to say that in the resulting papers, the ratio (handwaving speculation)/(systematic experimentation) was dismayingly high.