Question about graphitized carbon column

[squall]

Hi Everyone,

I met a strange problem recently when I was using graphitized carbon column for separation. Since I am still in the method test step, so I just used peptide mixtures for LC-MS analysis. The mobile phase I used are A: 0.1% FA in water (pH=4), B: 0.1% FA in ACN. I also tried the 40mM ammoniu formate (pH=4) in buffer A, but no big difference observed. The gradient I tried were 3%B-80%B or 3%B-100%B in 10, 20, 30min. At the beginning, I tried some standard peptide mixtures (angiotensin, bradykini, etc), but under all above conditions, I just never saw the signals! Even I put the concentration to 10pmol, 100pmol injection, I still didn't see evident signals. With the same settings(mobile phase, gradient), all peptides have good signal in C18 column. Then I switched to BSA digest, it seems only 3 peptides appeared, all the other 10 abudant ones I found in C18 also not detected.

I used direct injection to mass spec, so no trap column used, according to my results, the only explaination I can have is most of the peptides are either still on the column even I use 100%ACN for 20min washing or those peptides were diluted by the carbon column? I am really confused.

I am really new to use graphitized carbon column, can any experienced users give me some suggestions on my strange observations? Is this normal or not for peptide case? Although I know the graphitized carbon is used for hydrophilic compounds, such as glycans. But I have limitted glycan materials, so I am still using peptides for testing. Do I make anything wrong?


Thanks

[HW Mueller]

Graphitized carbon can have positive ion sites, especially under acidic conditions, which could have interfered. Also, 100% ACN can precipitate proteins/peptides.

[Andy Alpert]

Following up on HW Mueller's posting:
Instead of formic acid, try using either ammonium hydroxide or pyrrolidine in Mobile Phase B. This is necessary for eluting phosphorylated peptides from HyperCarb, for example. We use 200 mM in 60% ACN, although you may need appreciably less than that for your particular set of peptides.

[squall]

Thanks a lot, Mueller and Andy! Besides 100% ACN, I also used 80% ACN and other gradients, if my peptides were precipated, that should also happen to C18 column, right? But I found all peptides in C18.

If I use ammoniu hydroxide or other things, is that still compatible for my direct on-line LCMS?

The thing confused me is there are some application notes from Thermo hypercarbon, which just use regular mobile phase and gradient, it seems not much trick there, but they just have nice results. That's the most confusing part for me

[HW Mueller]

It is possible that the precipitable peptides were long since gone by the time you reached high ACN concentration with the C-18 column, but not with the hypercarb. Just a possibility.

[adam]

Yeah, and wouldn't peptides stick to hypercarb very strongly.

What is the benefit of using a graphite column with peptides? Is this commonly done?

[Andy Alpert]

Graphite retains some peptides that regular C-18 material does not. This is especially true of polar peptides such as glycopeptides and phosphopeptides. In general, graphite acts like an anion-exchange material to retain those while C-18 does not. If a peptide has more than one phosphate or sialic acid residue, though, then you need a high pH to assure their elution in addition to a high level of ACN.

[idlewildq]

personnal experience with PGC: it can be used in both aqueous normal phase and reverse phase, which means at certain amount of ACN it's behaving like reverse phase but after a certain threshold it start to behave like normal phase (compound dependent).

also when PGC attached to ESI, after some time, may notice drop in retention time. read about it somewhere that it is caused by oxidation of graphite by electrons (not fully grounded) ESI. i do have such occurence in the lab.