Chromatography Forum

We are working to develop UPLC methodology to speed up sugar and organic acid analysis for fermentation and molecular biology applications. Is anyone else out there attempting to use the BEH amide column? We are using a Waters application note as the base method: A) 80/20 ACN/H2O + 0.2% TEA and B)70/30 ACN/H2O + 0.2% TEA. The method is basically a linear gradient over 10 minutes to 60%/40% A/B, before re-equilibration back to 100%A. We love the selectivity and are able to resolve many of our really important peaks of interest in less than 20 minutes!! However, we are running into a problem. When we run the system just as described in application notes, we can only inject ~75-100 sugar standards onto the column before it overpressurizes. We haven't even been able to begin to work with real-world samples, yet. This has happened to two columns so far. We had the appropriate guard column inline on both columns. We have been unable to find references in the literature about pressure issues while attempting to validate the methodology. What is really strange about the situation is that we are unable to rescue the column in any way shape or form. We find it hard to believe that we are the only chemists running into this issue, given the excitement around potentially shortening BioRad 87H sugar type methodology from 30 + minutes down to less than 20. Any advice would be greatly appreciated. We are currently working with our vendor to resolve/troubleshoot the issue, and my colleague and I are both highly experienced chemists with several years of successful method development under our belts. This issue has us stumped.

FYI - We are using the properly filtered mobile phases and high quality eluent components. The system is new and we have been following the manufacturers recommendations for proper column use.

What are you column dimensions and flow rate? Just curious. Also, can you provide some more information on how the pressure increases? Specifically, I'm wondering whether it increases gradually over time (i.e. does pressure during the first couple minutes of the run continue to increase over time?) or does it remain normal for a while and then jump up after a set number of runs?

You can also try, as a test, reversing the column after, say, 50 injections and flush at a low flow rate (0.1 mL/min) in your strong phase for a while and turn it back around. If the pressure problems are still there, it will at least confirm it is the column, and not buildup of something elsewhere in the system.

Lastly, how are your samples being filtered?

Hello. Thanks for checking out my post and for replying. Much appreciated.

The column dimensions are 2.1 x 100mm. The flow rate we are using is 0.130 ml/min. Our base method is a Waters application note for analysis of Beer fermentation samples. The pressure starts out at about 800 psi, and then as the gradient proceeds to more water, linearly over 10 minutes, the pressure increases to about 1000 psi. As the column re-equilibrates back to the higher ACN mobile phase A, the pressure comes back down to the starting pressure, as expected. As more sugar standards, of appropriate concentration, in the appropriate solvent, are injected onto the column, the pressure of the higher water portion of the gradient is amplified with subsequent injections, until the system overpressurizes after ~100 injections or so. We haven't even injected real-world samples, yet. What is really strange, is that eventhough the column over pressurizes with any water containing solvent, "plugged" columns will still allow ACN to flow through without over pressurizing. The minute we attempt to equilibrate the system back to 100% A to run a sequence, the system overpressurizes. Good point about the column. The same thing has happened now on 2 columns. We thought that the first one was just a fluke. We are certain that it isn't a system problem, because when we put on a new guard column/column set, we are good to go for another 100 injections. I am afraid that this could be a method robustness issue. We are also worried about whether or not we have some sort of chemical reaction going on in the column between mobile phase components, sugars and maybe the column itself. We have been unable to "rescue" either column.

Hmm, that seems to be a really strange problem. I've used the BEH amide column for a couple of analyses (no sugar analysis, though), with all sorts of ACN/buffer mobile phases, including acidic buffers, alkaline buffers up to pH11, isocratic, gradient, even phosphate buffer with high % organic once. I've never experienced problems as you describe...
You're using a guard column on top of your analytical column, right? The backpressure of a "fouled" column does not decrease if you remove the guard column and use the analytical column alone?
I'd wonder if you'd still see the pressure increasing if you just run blank gradients without injecting anything. That way you could at least conclude if the mobile phase/system or your reference samples are the problem.
You're in contact with Waters? Did they tell you anything regarding this issue?

HPLCaddict - it sure is a strange problem. Good to know that you have successfully used the column for analyses with various buffers. This is a positive. Yes. We are using the appropriate guard column in line with the analytical column. I have tried just putting either the analytical and the guard column inline by themselves and it seems to be that both suffer the same fate after an overpressurization event. I wish that I could figure out how to post a pic on this blog. The pressure trace is interesting. The overpressurization is amplified when the column is seeing higher water, then it goes back down once returned to the higher ACN mobile phase A. Once the column starts to overpressurize, it doesn't matter if the injections are blanks or sugar standards. The pressure continues to increase with each injection until failure, regardless. I am afraid that our issue is a problem with the analysis of sugars on the column, and/or partially caused by our mobile phase additive, TEA or maybe lack of equilibration time. Although, I have found literature showing analysis of similar compounds utilizing similar equilibration steps. Waters has been amazing. We are working very closely with them to identify the issue.

have you tried to flush the column with plain water (or 10%ACN) after a certain number of injections and then requilibrate it thoroughly?
I had the same idea as HPLCaddict, to test if the pressure rise of a new columns starts only from the eluent or if it is related to the injection count.

Thanks so much for the replies and suggestions, everyone. Much appreciated!!

We solved our issue, and have now injected more than 1400+ samples in the column!!

Onward with validation!!

nice to hear.

would you like to tell us how you solved your problem or what was the cause?

Dear JamieAlison,

I am having the same proble but on an HPLC column, could you tell me how did you solve the problem, please?

Thanks in advance,

Carol

About the increasing of Head pressure:
I'm working on wines, liquors, honey and soft drink sugars content.
Flow: 1 ml/min; eluent 80:20 ACN/ Water plus 0.2% Triethylamine. Samples at 25°C Column 35°C RI detector 35°C
I've destroyed two column because the increasing pressure.
Now, at the end of the day I clean up the column as follows:
First change to 80:20 ACN/water without Tryethylamine,
Then change to 50:50 60 min
then change to 50:50 ACN/Water plus 0.1% Trifluoroacetic acid and hold 60 min
New change to 50:50 ACN/Water 30 min abd finally store the column in 80:20 ACN/Water

I'm working like this three months and pressure increase has been no significant (Initially 1175psi, nowadays 1250 psi)
The column is 250 x 4.6 mm
Perhaps it can help
Guillermo

About the increasing of Head pressure:
I'm working on wines, liquors, honey and soft drink sugars content.
Flow: 1 ml/min; eluent 80:20 ACN/ Water plus 0.2% Triethylamine. Samples at 25°C Column 35°C RI detector 35°C
I've destroyed two column because the increasing pressure.
Now, at the end of the day I clean up the column as follows:
First change to 80:20 ACN/water without Tryethylamine,
Then change to 50:50 60 min
then change to 50:50 ACN/Water plus 0.1% Trifluoroacetic acid and hold 60 min
New change to 50:50 ACN/Water 30 min abd finally store the column in 80:20 ACN/Water

I'm working like this three months and pressure increase has been no significant (Initially 1175psi, nowadays 1250 psi)
The column is 250 x 4.6 mm
Perhaps it can help
Guillermo

Dear JamieAlison,

I am having the same proble but on an HPLC column, could you tell me how did you solve the problem, please?

Thanks in advance,

Carol

nice to hear.

would you like to tell us how you solved your problem or what was the cause?

Thanks so much for the guidance, Wiley One. Very helpful!!

Carol and Hollow,

We really are not 100% sure what the cause was. We think that it is temperature related, but cannot figure out if it is temp related, or temp and mobile phase component related. Nonetheless, for us, running at the lower temperature was the ticket. Although there are applications out there that describe using the column at 85C, this is clearly too hot and causes the column to have a shorter life.

We are going to work to determine the exact cause, but for now we have to run with what works and validate our method.

Best of luck!!

Jamie

Thanks for the replay, and let us know how it goes!