Chromatography Forum

Hi everyone.

I recently purchased a capillary monolithic column (PS-DVB) from LC packings. The problem I got is that the protein (cytochrome C ) or peptide (angiotensin II) can not be eluted out from the column when I run linear gradient (30min 5%~90%B, A:95% water, 5% ACN and 0.05%TFA; B:10%water, 90%ACN and 0.05%TFA). But when I tested the column in isocratic mode, the protein and peptide can be eluted out when I use 50% B. Before I run linear gradient mode. I use 95% A to equilibrium the column over night. I repeat this experiment for several times and get the same result. Is there anyone who can tell me what is the problem in my experiment? Thanks a lot.

What happens if you run an isocratic experiment at 60% ACN, or 70%, 80%, and so on? Could it be that these compounds have a "U-shaped" retention curve on this column?

Or, could it be that your gradient pump is malfunctioning?

Your problem sounds strange...

May I ask what are the retention times of the protein and peptide in isocratic mode? Are they retained or eluted in the void volume? What do you use for their detection and if UV at what wavelength?

I think you are using a too shallow gradient and hence can not see the peak from the protein when it elutes, try a 5 minute gradient.

Sure, I also tried isocratic mode in 60%, 70%, 80%. It works fine. I can see the sample I injected in approximately the same retention time. That indicates the protein or peptide will not be retained in such cases. In the isocratic mode, the retetion time is 2.00 min which is correspondent to the dead volume in the system.

I am using the mass spectrometer as my detector. The strange thing is that when I ran HPLC in gradient mode, I got M.W.of the exactly same sample other than that I got in isocratic mode. The electrospray generates multi-charged ions. So I am talking about the deconvoluted spectrum. In isocratic mode, I got single peak which is 12358 Da, I gradient mode, I got three series peaks, from 12012Da to 12479Da. It looks like the protein bind with something in gradient mode. The peaks I got in gradient mode acturally come out when I used 90% B to rince the column. It did not come out in normal time.

Moreover, the back pressure changed from time to time. With the same buffer (5% B), the backpressure is 2000psi some time and it's 2800psi in some other time. I don't know why.

I do test 10 minutes gradient. It's not helpful.

Perhaps your pump's gradient delay volume is not being accounted for, and the run is being ended before the entire gradient has reached the column?

Is the column kept at 60 C; and did you check the flow coming out of the column (should be 2.5 ul/min for a 200 um ID monolith)? Using your solvent system I would recommend to take the gradient up to 50 %B instead of 90%B; cytochrome C does not have much retention on this monolithic column.
Is it possible to run the same experiment once with a packed capillary wide pore reversed phase column to rule out any possible problems with the gradient formation?

Thanks for you reply. I really appreciate.

I put column in a water bath which is held in 60 degree. And I do see the backpressure pressure become lower. So I think my column should be heated properly. I do test the flow rate from ESI tip. It's approximately 2.5ul/min. I am sorry I did not measure it precisely. The reason why I set gradient to 90%B is that I did not got protein coming out when the gradient goes to 50% B But in isocratic mode, the protein came out. Acturally, even I set the gradient going to 90% B the protein (Cytochrome C) still did not come out before the gradient finished. It came out after the gradient finish and at that time I use the 90% B to rinse the column. Moreover, the MW I measured was change. It is higher than the MW I got in isocratic mode.

Thanks again.

zf12345

The precipitation of proteins on a stationary phase is a delicate matter.

Why use a monolithic (hydrophobic PS-DVB) stationary phase. Isn't that to make everything more complicated?

have you just checked your pumps to ensure they work well and correctly?

Our pump should be OK. Since my lab mate got good result by using the same pump but with another reversed phase column.

Hi Einar Pontén, Thanks for your reply. Protein percipitation probably a problem. Can you recommend me some Hydrophilic interaction column which can provide comparable resolution as Monolithic hydrophobic column can provide. Thanks again.

Assuming your HPLC is working properly at the flow rates used, there should be no problem running smaller intact proteins like cytochrome c on this monolithic column.

I suggest to contact your local LC Packings/Dionex representation in order to get the column replaced.

Dear zf12345
I am sorry I am not aware of any hydrophilic monolithic capillary column. Isco introduced monoliths but there are not marketing around from them. Does LC Packings have another alternative?

Maybe you should accept the offer to get a new column. It might just be a problem with the one to got.