Hypercarb variable retention times

[trammells]

I have been using hypercarb columns for approximately 7 months now without much problem. However, I have allowed a column of dimensions (1mm inner diameter X 100 mm length) to sit after washing with high acetonitrile. It sat for approximately three months while I used a larger hypercarb column.

Recently, I have attempted to use the smaller column for the same assay as previous using the following solvents: A 50 mM ammonium acetate + .1% formic acid B .1% formic acid in acetonitrile. The retention times are now extremely variable between runs and appear and the order of elution shifts. I know that these columns require longer re-equlibriating between runs than other columns but the amount of time taken between runs is equivalent when accounting for column volumes to that I have used with the larger column.

Has anyone had a similar problem to this and how have they fixed it?

[Alp]

trammells

It would be interesting to know what you are injecting and how it was prepared. Is it an extract from blood, urine, food, soil...etc?

First thing I learned is it takes a few forevers to condition this column. Several times more than I would for a C19 for instance.
I am also seeing wild shifts in retention. I have been using a hypercarb 50x2.1, 3um for 2 days. I thought I had nailed chromatography for an analyte yesterday. I was using analyte dissolved in ACN. Mobilephase was 95/5 A/B.
A=ACN/IPA 2:1
B= 0.1% NH4OH
RT seemed fairly stable (within 5 to 10 seconds) and I planned to finish up today.
I capped the column without rinsing so I would not have to repeat the conditioning again.

Today I let the mobilephase run on the column 20 minutes before starting (0.2 mL/min).
My first injection gave very nice looking peaks at RT of 3.4 and 4.4 minutes. Yesterday they were at 2.3 and 2.6 minutes.
I continued to inject and the RT's got lower and lower and are now hovering around 2.9 and 3.5 minutes.
I cannot explain it. I could try all the mobilephase in one bottle (as I am isocratic at this point) to see if it is a mixing issue, but I think it is not.
Maybe 5% water content is bordeline or I can play with the % NH4OH.

I hope you have luck with your method. You might want to check up on the section on cleaning your column. I found a PDF online that mentions technique for cleaning the column.

good luck

Alp

[idlewildq]

used hypercarb before. gave nightmarish rt, as it keep getting less and less overtime, especially if used with esi. saw a paper or two discussing about the oxidation of graphitized carbon, thereby changes the retention properties.

[trammells]

Hello,

Thank you for the support. I have also read that ESI can reduce the column. I tried regenerating with .05% hydrogen peroxide. It may have worked for two of the standards I am using. The problem seems really complex.
The last samples injected were plasma samples resuspended in water after drying them down in methanol with nitrogen flow. Of course, I separated the particulate matter from the aqueous before injection onto the column.
The strange fact about all of this is that I have another larger column with a 2.1 mm ID and 100 mm length that is still running fine with a large variety of samples injected (plasma, cell culture, tissue). It's the same method for both.
I have basically restored and mostly maintained retention for two analytes, niacin and niacinamide. However, any standard with a ribose group or phosphorylated member is only retained for approximately 1 minute on the column with 10% B (A: 50 mM Ammonium Acetate + .1% formic acid, B: .1% formic acid in ACN).
I have read that pH can be of extreme importance in maintaining the column. So previously I used a very harsh wash with highly concentrated TFA followed by 100 mM HCl, followed by water and then ACN. I have read that TFA can conjugate or in some way alter remaining silica in the column. So I have tried washing with THF. I have yet to test the column after that wash. I will keep you posted on what happens with that. Thank you for responding to my post and any input would be greatly appreciated.

[Alp]

I would like to know more about how ESI can promote oxidation of the carbon in a hypercarb. What is the paper? Or is it a website?

Is some voltage/current making its way from the nebulizer tip to the column and promoting oxidation? What if the line is grounded at the glass capillary-peek "line in" junction?

I ran straight aqueous with 0.05% NH4OH the other day to see if it would help by conditioning the column in aq base because I had so little in the mobile phase.
After running this for 3 hours at 0.05 mL/min I capped the column until the next day when I ran my mobile phase 95/5 A/B. A= 2:1 ACN/IPA. B= 0.1% NH4OH aq.
retention time originally was about 2.3 and 2.6 minutes a day or two before.
After the basic-aq wash and a 30 minute conditioning with mobile phase I saw NO peaks. It all got retained. This is strange.
I tried various modifications with lower organic, adding methanol, nothing eluted.
I then ran an acidic buffer for a short time, then the original mobile phase again. The peaks returned at about the same intensity they did in the past but with slightly different retention.
As the column conditioned further, retention became closer to what I had seen originally (2.3 and 2.7 minutes). I repeated again today and the retention is the same.
Interesting that by rinsing with basic aq I can cause the column to retain a compound under conditions that the day or two before it was chromatographing in under 3 minutes.

Alp

[trammells]

Hello,

Here is the paper that referred to oxidation/reduction qualities and what they did to restore the redox state.

Retention studies of 2-2-difluorodeoxycytidine and 2-2-difluorodeoxyuridine
nucleosides and nucleotides on porous graphitic carbon: Development of a
liquid chromatography–tandem mass spectrometry method

doi:10.1016/j.chroma.2009.02.002

[Alp]

Thanks. I googled it. Interesting stuff. Of course, running a gradient with from 0 to 25mM bicarbonate WOULD require excessive conditioning. I imagine once bicarb got into the column, it stayed there even when the mobile phase went back to 0% bicarb. I wonder if they got bubbles (CO2) when they added the 10% formic acid after each gradient.

I came across something myself. Google "oxidation of graphitic surfaces" which kind of makes me wonder if I want to put peroxide on my hypercarb. I only browsed through, it certainly requires a good read.

Alp

[trammells]

Update:

I have still failed to restore retention order on the column. I have tried the recommended washes from Thermo Fisher (both acid/base wash and organic wash). I also decided to ground my column in case there is some effect from using ESI. Still no restoration. I am at wits end. If anyone has a fantastic suggestion, please feel free to share.

[Alp]

You have not said if the chromatography is isocratic or if you are running a gradient?

If you are isocratic, try to mix the mobilephase in one bottle and run it an the A line for a number of injections or on the B line. See if the RT remains stable. You probably would be happy if the RT settled down to something consistent at this point in time, even if it does not match what you were getting previously.
I tried this myself and got very different chromatography from what I was seeing when I used isocratic A:95% ACN B: 5% AmAc pH 10 (not sure on the pH).
This leads me to suspect I may require some pump maintenance.

There are probably several tests you can do to check if your pump is delivering the flow and percentage it is supposed to be.

Please, keep us informed of your results.

Alp

[trammells]

I am using a gradient. I have found a way to stabilize the retention time without increasing re-equilibiration time which was undoubtedly the problem with variable retention. Basically, between runs, a high amount of formic acid is injected onto the column in 10% B and run for 7 minutes before beginning the next run.

The real concern that I have is the retention order loss. It looks like the negative analytes I am interested in are eluting far earlier than they should be. I have tried the column on a completely different mass spectrometer to control for solvent issues before. I had very similar results as far as retention order breakdown of negatively charged analytes. I am now in the process of speaking with the analytic chemists in the UK that Fish Scientific employs. I will keep you informed if we are able to resolve this bizarre problem.

[trammells]

Essentially the response I have received from Fisher Scientific is that some unknown event changed the surface of the column, destroying it's properties. Basically, they are suggesting that I buy a new column.

[Alp]

I took the time to look at some of the early posts in this thread.

"The last samples injected were plasma samples resuspended in water after drying them down in methanol with nitrogen flow. Of course, I separated the particulate matter from the aqueous before injection onto the column.
The strange fact about all of this is that I have another larger column with a 2.1 mm ID and 100 mm length that is still running fine with a large variety of samples injected (plasma, cell culture, tissue). It's the same method for both."

The Hypercarb column may have a retention mechanism that is easily disrupted by some of the molecules and peptides, lipids that are found in plasma, blood, cell extracts. I would not be surprised if some of the components injected may be irreversibly retained.

Is it possible you are injecting the wrong type of stuff onto the column? Samples derived from biological matrices can be very "dirty" even after clean up. Dilute and shoot (no clean up) are the dirtiest, probably followed by protein precipitation. I have even seen some dirty Liquid/Liquid extracted plasma samples (Hexane/Etac or maybe cyclohexane/chloroform type).

I wish you luck. The column certainly has some challenge in it's usage.

Alp

[Andy Alpert]

HyperCarb acts like an anion-exchange material in regard to how it interacts with some acidic compounds. These can't be eluted with the usual acidic media with ~ 60-70% ACN that works with a C-18 column. Instead, try 200 mM pyrrolidine in 70% ACN.

[trammells]

Hello to everyone who answered. Thank you very much for your input. However, I believe that some unknown event must have changed the surface of the column making it unusable. I believe the input will be helpful with our other column as well as the PGC HPLC-Chip we are in the process of purchasing. If the methods suggested here are tried later, I will definitely post updates. Thank you again for your time.