Peakshape deformation with same method and condition

[ArneyHey]

Hello all, i recently started to work on an HPLC and now stucked at a problem i cant solve on my own. I use a Luna Omega polar C18 column (3µm, 4.6mm ID, 150mm L) with a guard column. I try to detect my substrate (futalosine) which is quite hydrophobic and therefore dissolve in DMSO. I dissolve my substrate prior measurement into 5% ACN and have a DMSO concentration below 5%. For my method i use a flow of 1ml/min 0-40% B (A= H2O, B=ACN) with an injection volume of 10 µl. My Kprime is at around 5 and in general my substrate gets retained long enough and i archieved a really good peak for a day. But the next day i measure the same sample again and since then even with freshly prepared sample my elution peak is really disorted. I tried varying the injection volume without any succes and sometimes i noticed that my sample flow through the dead volume. Does anyone had similiar problems or could provide me with some suggestions?

[Multidimensional]

Sounds like you are fouling your HPLC column. Try and filter each sample first, then using a gradient wash method after each analysis to remove contaminants. If your sample really does occasionally elute at T0, then this too implies you have fouled the column OR are using a method that has not been developed properly. Get some outside help from an exp chromatographer.

[Multidimensional]

Two other possible things. (1) Maybe too much DMSO is being injected (that will definitely screw up the peak shape). Make sure you remove DMSO from the injection solution. It can show up as 'column fouling' too. (2) You may also have a mobile phase composition issue where the solvent(s) you think you are pumping, in fact are not going through the column. This could be due to intermittent pump cavitation, a sticking check valve, a sticking or leaking gradient valve etc. Poor quality degassing can also result in similar problems too.
These are very basic things to check so try and bring in someone with industrial experience in HPLC (not someone out of college with little to no industrial experience ) to go over your method and instrument to find the cause(s). They should be able to do so very quickly.

[ArneyHey]

Thank you for the answer, i ran the method with another substrate as a test and this one however elutes with a nice peak shape at the same time as expected. I strongly assume that the DMSO which is used to dissolve my other substrate is interfering during the elution. Does anyone has experience with DMSO during HPLC?
Best regards

[Consumer Products Guy]

injection volume of 10 µl.

I tried varying the injection volume without any success

When you varied the injection volume, did you try smaller volume, like 5µl or 3µl ?

[ArneyHey]

Yes but the peak shape staid the same. Right now i try to prepare different samples with different DMSO concentrations (lowest at 0.05% so far) to see if DMSO is the disturbing factor.

[lmh]

how long are you re-equilibrating the column before the next injection?

[Multidimensional]

Unless the RP mobile phase consists of DMSO, please do NOT inject samples with DMSO into the system (basic fundamantal of LC). You are fouling the column when you do so. *DMSO is used in a lot of peptide, bio applications and it is very important that users understand they need to redissolve the sample in the mobile phase BEFORE injecting onto the column.

• Inject samples in THE MOBILE PHASE

• Wash the column with solution that is stronger than the mobile phase, but also dissolves ALL material 100% after each analysis

[Multidimensional]

You do not have an injection volume problem, you have a training problem.
Learn and follow the basic fundamentals of liquid chromatography and you will move forward with training.

[TylerSmith123]

Hi Arney,

I'm no expert at your analyte, but after looking it up, what pH do you keep your mobile-phase at? If your analyte is running right through and eluting following your dead time then you're seeing no retention. If your analyte is charged during injection, it will subsequently go straight through your column and exclusively interact with the polar water molecules. It looks like your analyte has a few functional groups. I'd aim to make a mobile phase with a buffer that keeps your analyte uncharged as it moves through your analysis. Try adding some 0.1% acid to both of your phases (or a small test-bottle for a run) and see how it goes. Peak shape will also be affected if your analyte is swapping between ionized and de-ionized. Otherwise, the other tips are great, I'd simply recommend dissolving your samples in as-close to your mobile-phase concentration as possible, if not then just use your organic solvent if you're having trouble with your aqueous solubility.

Good luck!

[ArneyHey]

Thank you all for the reply. Yes unfortunately my substrate is diluted in DMSO so i always dilute it prior injection with 5%ACN (i start with 100% H2O -> 40% ACN in my method). I reequilibrate my Column with 5CV H2o before another run. I ordered another batch of substrate and will see if i can dissolve it without using DMSO. I used only H2O and ACN so far but will switch to a potassium phosphate buffer pH 7.2 for a Test (Mobile phase 1).

[ArneyHey]

a little update: my run with the newly prepare potassium phosphate at pH 7.2 finally resulted into a nice peak. I guess my substrate was very unstable in Water as a mobile phase

[TylerSmith123]

That's excellent news and a simple solution. Don't be afraid to come back to ask a question if you have any other issues with your analyses.