Interesting retention time shift

Discussions about HPLC, CE, TLC, SFC, and other "liquid phase" separation techniques.

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I have attached a chromatogram of the same standard injection from last week, and from this week.

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These are, as I mentioned, the exact same standard solution, utilizing the exact same mobile phase and column. The parameters are as follows:

40/40/20 - Acetonitrile/Methanol/2% Ammonium Acetate pH 5.3 buffer
1.0mL/min
30ºC column temperature
Column: EC 4/3 Nucleoshell Bluebird RP18 2.7um column (50x4.6mm)

I use a guard column that I replaced, changed nothing but pressure (decreased system pressure by about 10bar).

I have double checked the pH of the mobile phase...as this molecule is very sensitive to pH, and it came out to be 5.27, which is acceptable.

I used to run this sample on a longer 150x4.6mm column and the retention times I'm seeing now are more similar to what I would have seen running the longer column. I have been running this column with amazing success for the past 4 - 5 months with the reduced retention times...and all of a sudden...retention time shift. It is also consistent. Multiple injections of not only this standard, but other standard concentrations and sample preparations are giving now consistent longer retentions.

I am not convinced there's an issue with the standard solution. Furthermore, there is a set of isomers that is nearly identical to these...they elute slightly later. That standard solution is also showing a required 25min run time to elute both isomers when it should only be 6 minutes with this column.

I'm not seeing any pressure increase or pressure fluctuations between injections, no leaks, no air. I am at a loss as to what could be causing this change...literally nothing has changed between last week and this week in regards to running these samples yet I'm seeing significantly longer retention. The only thing I can think of is something happened that significantly altered the column chemistry significantly...but I've used many columns for many injections and never seen such a sudden change of retention time. My experience has always been a gradual shift if the column starts to get used more and more frequently.

Any ideas as to where I can start looking would be helpful.
Well I'm running a different analysis and noticed that the Acetonitrile has two layers in it, I didn't check it earlier....I think one of the interns didn't add the right solvent into the mobile phase bottle. I didn't notice this earlier and this may be the cause. I'll report back Monday with the result but I think that might have been it.
Hello. Check for the pressure: it should be higher with methanol.
Best regards,
Dmitriy A. Perlow
Could be a drift of the pH value over a few days?
Hi Zoraku,

Reading your original post, you have a ternary mixture that comprises your isocratic eluent, "40/40/20 - Acetonitrile/Methanol/2% Ammonium Acetate pH 5.3 buffer".

Is this eluent prepared on the LC by mixing these three solvents? And you checked the pH (observed 5.27 vs. 5.3)--was this only for the aqueous portion of the eluent (if you are mixing three solvents)?

The ACN has formed a two-phase system in its bottle--that is scary to me on a couple of levels.
MattM
If the eluent is indeed prepared by mixing in a single bottle, you're asking for problems in my opinion. You're mixing a high concentration of salt in only 20% water, which could lead to phase separation or precipitation..
Hi all!

I appreciate everyone's comments!

The mobile phase is a 3 part mobile phase mixed by the system, not together in one bottle. It turned out that there was a very visible phase separation of solvents in my Acetonitrile mobile phase. Whatever was poured into the mobile phase was NOT acetonitrile and was more dense.

Whatever this solvent was, sat at the bottom of the bottle...where the stone is at, so it was not actually running 40/40/20 of Acetonitrile/Methanol/Acetate buffer but 40% of some unknown organic solvent that was more dense than acetonitrile.

I have a feeling the root cause is the intern in my lab grabbed the wrong solvent and added it into the acetonitrile without realizing what he was doing and didn't notice it....it's hard to find good help these days :)

Anyway, I ended up just dumping the bottle out and putting a fresh bottle of acetonitrile on the system, flushed it, and everything is working perfectly as expected!
Glad your problem is solved.

My guess for your unknown "organic solvent" denser than acetonitrile, is water. It forms 2 phases with acetonitrile given there's enough salt, and your components had more retention.
Rndirk wrote:
Glad your problem is solved.

My guess for your unknown "organic solvent" denser than acetonitrile, is water. It forms 2 phases with acetonitrile given there's enough salt, and your components had more retention.



Yes, I actually analyzed it on the GC/MS and I found water and acetic acid both present. We use acetic acid to adjust the pH of the acetate buffer. My thinking is that the intern had accidentally combined some of the buffer into the acetonitrile bottle opposed to the bottle that is supposed to be for the buffer.
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