Reproducibility problems on Hypercarb

[sahram]

Hello,

I am using Hypercarb-column on LC-MS(electrospray) with UV-detector, too. My method is gradient 0-25%ACN/25min, and my A-buffer is 10 mM NH3 plus 20 mM NH4HCO3. I have autosampler and column is thermostated (25 degrees celsius).

I cannot make any reproducible runs: the peaks come later and later after run after run, not much between two single runs, but the difference between the first and the sixth is too much. Also day to day difference is really big.

I also used isocratic method, but the difference was much bigger then.

Please, ask me for more info if required.

What I should try next to make my runs reproducible?

Thanks, Sahram

[Chris Pohl]

sahram,

There are a lot of different things that can cause problems of the sort you describe. What I would try first is a rewetting experiment: first rinse the column with 100% acetonitrile for an hour, then repeat your analytical test. Is the retention time higher than any previous measurement after this test? If so, the material was either contaminated and needed cleaning or it had become dewetted from use with a highly aqueous mobile phase. Porous graphite phases and porous polymeric phases are also subject to the same dewetting phenomena frequently mentioned in this forum in conjunction with use of C18 silica reversed phase columns under highly aqueous conditions. It's probably not a great idea to operate such a column with 100% aqueous eluents due to this effect.

[Kostas Petritis]

Sahram,

I would also go on and measure the pH of my mobile phase A and then measure the mobile phase of a freshly prepared mobile phase A. I do not know what the buffering capacity of your solution but maybe the pH is shifting a little bit due to dissolution of atmosheric CO2 in your mobile phase.

I would also try to let the system equilibrate for an hour and then run my gradients.

Finally, I do not think that it is a dewatting problem (but it is maybe contamination) as in my experience Hypercarb columns are quite resistant to dewetting, maybe for the same reason the C30 are resistant to dewetting...

[sahram]

Thank you very much for your answers.

Now I can give some more information.

The pH of buffer A (50mM Ammonia plus 20mM Ammoniumbicarbonate) is around 9 or so. I check the exact value today.

The reason for the components are: Ammonia gets better ionisation and Ammoniumbicarbonate gets the right molcular ion. Ammoniumbicarbonate alone splits my peaks in two and Ammonia alone make peaks broader. So this is really a compromise. I think it can be optimized to be better.

My buffer A is always freshlyl made (same day than run) and prefiltered. The system also has a degasser, so I shouldn't have any problems with it.

My sample is clean and solved in pure water. But this cleaness/dirtyness started to worry me, because in the future I should analyze by samples from dirty matrises. I hope to find good method to clean the samples.

Last night I rised the temperature from 25 to 50 degrees and reproducibility was better but the trend is still longer and longer retention after run after run. The intresting thing was that after ten samples I ran three blanco runs (no sample, only gradient) and after that the retention time came back (or actually, little less than before) and then started to rise again. This is really tireing.

I wonder if injection volume can be one factor to that how much is the shift between two single runs.

My flow is 0,5ml/min and injection volume is 1µl or 5µl. I would prefer 5µl but now 1µl has given better results.

[HW Mueller]

Your analytes are anions under the mentioned conditions and are mainly, or only retained by ion exchange? If so, your injection must be introducing some equilibrium shift in ions somewhere? Raising the ionic strength of your mobile phase might help, then. Actually, this might then be something for Chris as I don´t feel too sure with ion exchange. It is also too early in the morning, here, to think about whether dewetting would cause a retention time increase or decrease in this case.

[sahram]

I have now checked the pH of buffer A, and it was a bit suprise for me :pH is 8.82, I would have expected over 9. NH4HCO3 affects more than I tought.

I may increase ammonia concentration for future runs, because I really want to be sure my analytes are fully ionized. I think this pH affects more to ionization than separation itself.

Let's hope retention times become reproducible too.

I am also very interested in dewetting pheonomenon because I thougt graphitized carbon is resistant to it. Is dewetting same as 'hydrofobic collapse'?

Sahram

[Chris Pohl]

sahram,

Kostas has already indicated that dewetting can't be the source of the problem. I was presuming that this phenomenon would occur based on experience with reversed phase media based on silica and polymers and knowing that a graphite base material should exhibit a similarly high contact angle necessary to produce the dewetting phenomenon (phase collapse is the old, incorrect name for the phenomenon). However, Hypercarb is far from a pure hydrophobic material, exhibiting both anion exchange properties and oxidation/reduction properties (I guess this makes it a mixed mode material but I can't say whether or not it's the greatest advance in the last 25 years). Apparently, contamination of the surface with polar substituents responsible for the anion exchange and oxidation/reduction properties also significantly lowers the contact angle compared to what would be expected for a graphite surface.

Since we can eliminate dewetting as a possible cause of your problem, I suggest examining other possible causes. One possible cause of problems is the chemistry of ammonium bicarbonate. Bicarbonate solutions can be relatively unstable, especially ammonium bicarbonate. Are you sparging your buffer solution during use? If so, drifting buffer composition might be the cause of the problem. Does the buffer solution have any overpressure or are you operating without any pressurization of your buffer solution?

The other possibility is connected to the oxidation/reduction properties of this material. The oxidized form of Hypercarb exhibits more retention by anion exchange than the reduced form so perhaps you're drifting retention is due to in column oxidation. Is it possible that your eluent is contaminated with oxygen?

[Kostas Petritis]

Sahram and Chris,

My observations about the absence of dewetting when using Hypercarb are purely emperical and might not be the case of what other people are experiencing. I have used though the material with 100% water and different additives (and pH) including ion-pairing reagents or low ionic force buffers or high ionic force buffers and never observed any dewetting.

(Chris, I guess that Hypercarb is not the greatest advance in the last 25 years, although I could make it sound like one with arguments I used in one of my previous posts (see below)).


http://www.sepsci.com/chromforum/viewtopic.php?t=807

[James Hillis]

Dear all, just to agree with what has been said before. I have had lots of problems with ammonium bicarbonate mobile phases. pH adjusted ammonium acetate tends to offer a more stable alternative.

[Supercritical]

I think Chris has something when he mentions surface contamination. I would be thinking about this. Understand about the use of your buffers (peak shapes), but does the reproducibilty get better just using ammonia?

Also agree with James on the bicarb.

BTW Having used Hypercarb (& similar) and Primesep columns, I can assure you that they are NOT similar. Not even close. No comparison. I'm amused that discussion has spilled over onto this topic.

[ConstantinS]

Dear Chris,

possibly, you remember our discussions on "ion exclusion" topic. I want to know is it possible to contact with you by e-mail?
My address is chromplus*yandex.ru

Best regards,
Constantine S. Sychov

[Chris Pohl]

supercritical,

I was a joke (although I guess not a very good one). My point was only that there are lots of mixed mode columns (such columns have been comercially available for more than 15 years).

ConstantinS

You are welcome to email me directly: chris.pohl@dionex.com

[Supercritical]

Chris, no problem!

I agree, mixed mode columns have been around for years. Some intentionally made, others (most) not. Most of them are difficult to control.

However, I have never found a mixed mode column that can do what the primesep line does. Nothing even close. Enlighten me if you know of another column technology that does.

[Chris Pohl]

supercritical,

You might be interested in the new mixed mode column Dionex is introducing a at Pittcon (Paper 980-9), more about it in your thread "Biggest Breakthrough in HPLC in 25 years!"

[HW Mueller]

One can do stuff on the Hypercarb that you can´t do on other columns (oxalic.....), I have done crazy stuff on Pinkerton (leukotrienes) that are not even nearly repeatable on other columns. They are absolutely and precisely controllable in this. Still, partially because of this, most the time these are a pain in the neck.....
Chris´s assessment in the other chain is very much agreeable.