HPLC Mystery

[tashian]

Hi,

We have some mystery with a compound that we are trying to determine, and since I could not come up with any ideas, I though I might ask the experts here:

We are working with a compound called S-methyl methionine, or SMM. We use a standard compound (SMM purchased at Sigma), using an amino acid separation method, which is also used by our collaborators.

The thing is that our collaborators have it eluted at 4.5 minutes, and we have it at 7.38. Additionally, their peak is higher for the same concentration. We are using the exact same chromatographic method, same column and conditions.

When both of us run an amino acid standard, we receive the same profile, with roughly the same retention times for all amino acids (+- one minute), but for SMM we see this huge difference in the retention time.

I have been dealing with this for a while now, but couldn not come up with a reasonable explanation for this.

I would be very happy to have your ideas of it... Many thanks!

[mattmullaney]

Hi Tashian,

Good to see you're still out there. The first thing to consider is, are the two HPLCs, yours and your collaborators, the same HPLC system? If even this is so, there still could be plumbing differences that may explain a portion of such difference in retention times. If there is no issue with the flow rate(s) for the two systems, there may be different mixing (in particular if the method is a gradient elution)...along this line, more method details may be helpful to add for us to help troubleshoot.

The "regular" amino acid method, is it a gradient elution or isocratic?

Are the columns roughly the same "age" in terms of usage?

Please see what you think and thank you.

[tashian]

Hi MattM,

Thanks for you reply!

The amino acid method is indeed a gradient, using something generally similar to this- see figure 4 pp. 216:

https://books.google.co.il/books?id=hxG ... ak&f=false

I will bring the specific details for our column and conditions, but basically it's a gradient of B: 0-3 min 2.5%, 3-13 2.5-45%, and then wasing with 100% B and regeneration; we're using a novapak column.

Our column is indeed newer, and we are not using the same instrument- nevertheless in this case my question is then how come the retention time difference for all other amino acids is way smaller then for this specific compound?

Thanks again

[mattmullaney]

Hi Again,

My first thought is WOW, are you using the 3.9 x 300 mm size NovaPak column? If so, are you and your collaborator using the same elution program--including for regeneration of the eluent composition after the top of the gradient?

The SMM is eluting for you at 7.4 min and for them at 4.5 min, under what amounts to isocratic conditions. If your collaborator's LC system contains a higher percentage of methanol/acetonitrile from a previous run than yours, this explains both the differerence in elution time(s) as well as their SMM peaks' height being higher than yours.

[mritunjai078]

Hi,
You have not explained the composition of mobile phase.
Some compound retention or very much dependent on pH of mobile phase. Is pH of mobile phase is same at both place.

Regards

[tashian]

Hi,

So as for regeneration time- I will double check, it might be somewhat different. But in such case- wouldn't the different eluent composition (percentage of methanol/acetonitril) effect the whole chromatogram, and impair also the separation of amino acids? we are getting a good separation and a generally similar profile- except for that SMM issue. If the concentrations are different between our runs and theirs- wouldn't it have more consequences? could it be that only one compound is effected?

As for pH- this is indeed important in this case, and we did adjust it the same way they do.

[mattmullaney]

Hi Tashian,

You are correct, but it is also true that the retention times of early-eluting peaks (like SMM) will be affected far more than the later eluting ones, those won't change nearly as much in retention time.

If you're using a 3.9 x 300 mm column, one column-volume is roughly 2.4 mL...in terms of time and supposing a flow rate of 1 mL/min this means that 2.4 min is a fair estimate of t0. In the case of your collaborator's lab, they aren't getting very much retention of SMM at all...the k'* value is 0.9, and you are observing k'* of 2 for SMM.

See what you can find...but something is clearly different between your two labs separation conditions. See if you can overlay the amino acid separations both labs do...and see if there is an overall difference in the retention times for all of the analytes...but again, the early-eluting ones should be eluting earlier than yours if something as I suggest is the culprit.

Best Wishes!