During an HPLC analysis of an analyte (reversed-phase) using a mixture of 1% trifluoroacetic acid/acetonitrile mixture, bubbles arises in the mobile phase and the baseline is not stable at all.
Any suggestions to overcome this problem.
Hi Fuad,
Firstly, I find the concentration of TFA in ACN quite high (we usually use 0.1% or 1 mL in 1 L). I am not sure if that is the problem though.
My questions would be:
- do you degas prior to putting your mobile phase onto the LC? If not, please do so.
- And what kind of temperature do you have in the lab?
I found that some mixes of mobile phase become "gassy" at high lab temperatures something I haven't observed at low temperatures.
Methanol is known to become "gassy" when mixed with water or aequeous solutions a phenomenon I haven't observed to the same extend with ACN.
In the event that you do degas your mobile phase and your lab is above 22C, I would try to lower the TFA concentration and see if the problem persists.
Good luck!
I agree with Sabine; it sounds like you are not degassing your mobile phase. I also agree with her comment about 1% TFA; it is very, very high.
Thanks Sabine and Mark a lot. Yes I think the problem is that TFA is high especially the temperature is 40 C (column temp).
so I will reduce TFA to 0.1%
thanks a lot.
Yes, 1% TFA is way too high a concentration. TFA is a very strong acid which can damage the steel and other materials used in the HPLC system. Please use no more than 0.1% (0.02 or 0.05, depending on desired pH or ION PAIRING need is usually fine).
Next, please review these basic articles to troubleshoot the stability problem and solve the issue.
- "Diagnosing & Troubleshooting HPLC Pressure Fluctuation Problems (Unstable Baseline)";
This second article may also help as when you have poor quality instrument conditions, you also tend to have poor quality separations.
- "HPLC Retention Time Drift, Change, Variability or Poor Reproducibility. Common Reasons for it. "
http://hplctips.blogspot.com/2014/01/diagnosing-troubleshooting-hplc.html
http://hplctips.blogspot.com/2015/11/hplc-retention-time-drift-change.html
Ivan wrote; "usually for HPLC used solvents bubbling of He or Ar through a solvent by use of sinter is the most common solution."
Please NEVER use argon gas to "degass" a mobile phase. It has the exact opposite result and adds gas to the solution (argon is soluble in the mobile phase).
If you wish to degass a mobile phase using a continuous source of pure, low pressure gas through a sintered disk or core, please only use helium. Helium is not soluble in the mobile phase (that is why we do use it alone).
Please refer to this linked article for more information and a real world example of how a lack of experience or proper training can result in long term chromatography problems.
"HPLC Solution Degassing, Sparging (Gas Choice Matters) "
http://hplctips.blogspot.com/2013/05/hplc-solution-degassing-sparging-gas.html
Aside degassing, and working under high temperatures in the lab, does it matter to degas 25% ACN prepared in 0.1% TFA?
It is good practice to continuously degas all mobile phases. Degassing results in better pump operation (no cavitation or loss of prime), accurate composition metering (no bubbles to cause check valves to get stuck), more stable baselines, less drifting, and better %RSD. Loss of prime and poor reproducibility are often the result of not degassing your mobile phase.
Actually, I got into the same trouble as yours. I found that when we use the mixture of solvent (water or organic solvents) and volatile additives (such as formic acid, acetic acid or TFA), the bubble phenomenon arises commonly even though I degas the mixture very carefully. In my experience, there are some caused as follows:
1. When we degas the mixture, volatile components in the mixture will evaporate but can not get out of the container. Under adequately high-temperature caused by a sonicator, these components still exist in gas state, but when the temperature suddenly decrease, the bubble will be observed when you run HPLC with this mixture > 1. After degassing, you should keep the container in the sonicator until the temperature is stable. 2. After mixing, you do not need to degas the mixture but you have to store the mixture at room temperature for one day. One day is enough to remove bubble inside the mixture and guarantee its quality.
2. Depending on the additive type you use and your purpose, I recommend you should use it at the as little concentration as possible, the higher the more trouble.
3. As to the column temperature, I have never found it affecting the bubble phenomenon. Could you give me some papers or detailed explanation to prove it, please?
Thank you so much for reading
We use 0.1% TFA in water/ACN mobile phases a lot with no such issues, but normally don't go so high in TFA as 1%. I am not sure if you are speaking of bubbles or foaming in the mobile phase bottles or in the lines or LC components. Others have mentioned degassing to help with baseline and avoiding bubbles coming out in LC components. I know we used to use HFBA a lot in mobile phase preps and would see some foam/bubbles form with it. I believe it was related to impurities in the HFBA, but don't recall for sure.
I would make sure mobile phase bottles never see soap or dishwasher, only water and solvents to rinse. Also, make sure all purities of additives and solvents/water are very good.
Good luck.
Randy
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