Dear Scientists,
I have a problem with (probably) plugged column(s) after use of ammonium acetate buffer. Let me explain what I did so far.
On the beginning I worked on ACE Excel C18 column in reversed phase HPLC. I used gradient elution with 20 mM ammonium acetate buffer pH 6,9 (phase A) and ACN as phase B. Gradient started with 80/20 buffer/ACN and was going to final 10/90 buffer/ACN (and of course adequate time for equilibration), everything was done in 40 °C. After every sequence i was switching bottle with buffer to pure water and flush the system with high aqueous mobile phase (95/5 H2O/ACN) to remove the salts.
On some point I decided to rise pH a little bit to 7,5 (no sense, I know it now). Everything was fine till one morning when I saw that my buffer solution is cloudy. It happened during the night when analysis was running. I immediately switched bottle to pure water and flush all the system like I described before, with 95/5 H2O/ACN. I noticed really high pressure so I checked what was cousing it and it was deffinitely a column. So after I removed a buffer with water I flushed column with 10/90 H2O/ACN (didn't work), then with THF (didn't work as well) and then I tried 45/45/10 isopropanol/ACN/H2O and the pressure was still high. I reversed the column - nothing has changed. Then I checked how my column test mix chromatogram will look and it was terrible. So i tought that I will have to care about regenerate this column later, because I had deadline for my analysis, so I decided to get another column and stay with my buffer on pH around 7 and watch it all the time to prevent it get cloudy again.
I chose Kinetex C18 this time. Ran few sequences and pressure started to be really high again. No contaminations were spotted in the buffer this time. So i really have no idea what happened. I worked with stadnards of antipsychotic drugs, no biological samples was injected. I had no pre-column (but I want to start using it by now).
So any ideas how I can regenerate my columns? Or what caused this problem? Looking forward for your help because in other way I will probably end in psych. Thank you.
Becterial growth in your buffer solution (exclude the buffer salts precipitate), that why your buffer got cloudy and the column got block. 1. You need to replace your aqueous mobile phase with flesh buffer every day. 2. Before you use the buffer solution, you also need to filter the buffer solution with 0.22 um membranes. About the column regenetation, you are suggest to rinse your column with H2O/MeOH 50/50 (0.2-0.5 ml/min) over night in the opposide direction. I holp my suggestions will help you.
Becterial growth in your buffer solution (exclude the buffer salts precipitate), that why your buffer got cloudy and the column got block. 1. You need to replace your aqueous mobile phase with flesh buffer every day. 2. Before you use the buffer solution, you also need to filter the buffer solution with 0.22 um membranes. About the column regenetation, you are suggest to rinse your column with H2O/MeOH 50/50 (0.2-0.5 ml/min) over night in the opposide direction. I holp my suggestions will help you.
Reverse the column and flush 10 column volumes with 95/5 Water/Methanol and then gradient to 5/95 Water/Methanol. ACN is not very good a flushing out buffers.
If this doesn't work, you will have to take out your frits and clean them (sonicate 5 minutes in DI water). Always ultrafilter (0.22 um) your mobile phases and sample prior to injection!
Thank you very much for your suggestions. I forgot to mention that of course I filtered my buffer solution and prepared fresh buffer every day. That's why I'm so worried. I'll try your tips, thank you.
- Please get rid of the columns used so far. If they are contaminated with "cloudy" solution you may be wasting your time and money trying to clean them. Silica support absorb microbes really well! Do not use any fresh columns until you solve this problem. For now, with the column removed and a restriction capillary line placed in its position, pump pure HPLC grade water, then water/MeOH (50/50) and finally pure MeOH through your HPLC system for now to flush out any "critters". Leave it in MeOH until you solve the problem.
Freshly prepared buffer (every day) which is filtered should not get cloudy. That is the issue and clearly something is wrong. Do NOT run your HPLC with cloudy solution, ever. Question: Why is the solution cloudy? Bacterial growth is the most common reason for this. The bottles you are filtering into may be contaminated. What is the temperature near the instrument? Is it below 25C? It should be.
What if it is not due to bacterial contamination? Perhaps something is falling out of solution? Poor quality chemicals may be the cause. What grade ammonium acetate are you using? Is it fresh? What are you using to pH the final solution? Is it HPLC grade? How are you preparing the solution? Stir plate? How long do you stir before filtering? What kind of filter are you using? The wrong type of filter membrane or technique could result in failure of the filter. If you are pre-mixing the aqueous solution with the organic component, then that could rupture some types of filter membranes. - You need to run tests in buffer preparation to find out where the problem is.
How about using 0,1 mM NaOH with 20% EtOH? I read that sodium hydroxide is good sanitizing agent for removing any bacteria, yeast, fungi etc. It is most effective in minimum 0,5 M, but I can't use it in such high concentration because of pH (my columns have maximum pH of 10). Flushing with MeOH unfortunatelly didn't work.
Aleksandra: Columns are consumable items. Once you solve the problem, you really should start with a fresh column.
If you really want to spend time cleaning this column, then I can provide you with a more detailed procedure for RP columns which may help you. *The cleaning procedure is actually part of a scheduled October post on my HPLC Blog [ http://hplctips.blogspot.com/ ] which addresses this issue. If you send me your email address, then I will send you an early copy of the short article.
http://hplctips.blogspot.com
Bill: I don't want to spend time for cleaning, but I have to, because this ACE column was brand new and Kinetex was so far so good for my use that I feel sorry that they got destroyed (also I am a fresh PhD student, so you can imagine how my supervisor feel about getting rid of two of columns). I will send you my e-mail adress in PM and will be thankful if you will send me this procedure. Also thanks for link for your blog, looks really interesting.
IF it's salting out, you could try submerging the column in DI water and let it sit. Osmosis is a beautiful thing. I am tending to lean toward salting out rather than bacteria simply because ALL the steps you took to try to remedy the situation were high organic-low aqueous. It would have only made salting out worse. Your MP is more aqueous than organic, your flush with water after each run, but when your column plugged you went for a high organic approach... If your buffer has been stirring this whole time (which it should be), and if your column was never allowed to sit stagnant (zero flow, which I strongly discourage) for any extended period, then I don't see why you should be seeing bacterial growth. Even your column flush has organic in it, so that would help inhibit growth.
Another option is to set it up to run over the weekend on a slow flush, provided that it won't overpressure the system. I've never let a system set without any flow. Even if it's not going to be in use, I always keep it on a trickle flow of MeOH/H2O, even if I have to recirculate it.
Have you considered adding some ACN to your buffer solution to begin with, then adjust your gradient accordingly? I have run a method similar to this in the past where MP-A and MP-B were two different mixtures of buffer/ACN (80/20 and 70/30), and ran on a long, slow gradient. When prepping them, however, you have to keep stirring pretty vigorously and gentle heating (reservoir wrapped in a jacket) may also be required.
Be sure that the high pressure is duo to microbial contamination or salting out. before run the mobile phase in your HPLC system, mixed the same buffer solution and ACN in the same ratio as in the mobile phase: (80:20 and 10:90)
in some proportions the ingredients of buffer may precipitate. If you see precipitation, try adding small portion of methanol or other solvents to make a clear solution.
Sometimes the saturation of buffer is the reason. Check the concentration of the buffer, try a thinner buffer.
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