Separation of methoxypropylamine and morpholine...

[IC_Gal]

I'm trying find a method to separate methoxypropylamine and morpholine. I have tried cationic IC with a methane sulfonic acid mobile phase using the Dionex CS18 and had limited success. We cannot get them completly resolved. Does anyone have ideas regarding another column or mode of LC that we can use?

I do not have access to a GC at this point.

[Uwe Neue]

In would try a C18. I recommend Atlantis T3, since it is compatible with a completely aqueous mobile phase (which is what you will need to get retetnion) and still has a rather high hydrophobic retention. I expect morpholine to elute first.

[IC_Gal]

Thank you I will look into this....

I'm not as familiar with HPLC as I am with IC so I have a couple of questions. Could we use a UV-Vis detector for this?
If so what wavelength range would you recommend for detecting amines in a 100% aqueous mobile phase? Our sample matrix is water so this would work well for us I think.

[SIELC_Tech]

Here is method for morpholine and other simple amines. You need ELSD/CAD (TFA in mobile phase) or LC/MS (ammonium formate). You are not going to see too much in UV and any tiny impurity will throw you off track:
http://www.sielc.com/compound_070.html

Contact me if you have questions.

Vlad

P.S. here is relative comparison of mixed-mode approach (Obelisc R column) and reverse phase ( Atlantis T3, Zorbax AQ, Synergy) for retention of polar analytes:
http://www.sielc.com/pdf/SIELC_August_2008.pdf

[Uwe Neue]

You can work with a phosphate buffer and monitor the elution at low wavelength, 210 nm. This method is rather unproblematic. If you have other detection tools, tell us what you have and we can see if one or the other would be better than UV at 210.

[IC_Gal]

Unfortunately what I have available to me at this point is limited. The IC has a conductvity detector and I have an HPLC setup with a UV-Vis and that's it. If there is little response in the UV perhaps a chromophore can be added through derivatives? These samples may contain other amines (alkyl or alkanol type) and cations like NH4+, Na, K, Ca, Mg etc in them as they are industrial waters. IC would have been my first choice but none of the columns I've looked at has given a baseline separation of the two.

[Uwe Neue]

Derivatization is possible, but makes the two molecules to be separated even more similar. I bet that you will see the analytes well at 210 against a phosphate buffer.

[maria.rey]

The column manual for the Dionex CS18 column shows the conditions to obtain baseline separation between methoxypropylamine and morpholine. There are two potential reasons why these two peaks may not be completely resolved:
1. High concentration of morpholine vs low concentration of methoxypropylamine, which makes it not feasable to dilute the sample
2. Low peak efficiencies, potentially due to a column not currently meeting specifications or system problems which cause band dispersion of the sample.

If the reason is #1, the CS18 is a low capacity column, therefore not much could be done about this if the concentration ratios of the two analytes are very diverse. Optimizing the system so that it has the lowest possible noise will allow diluting the sample more, as the minimum detection limit will be lower and therefore dilution can be afforded. The sample injected should be as low in concentration as possible.

If the reason is #2, optimizing the system is a must. The column should be first run under "test chromatogram conditions" and the results should be similar to those in the data sheet shipped with the column.

[IC_Gal]

Hi Maria, thanks for your explanation(s)....

We do have high concentrations of one vs low of another so we cannot dilute the sample much further. I think this makes more sense as to why we are having trouble getting the baseline resolution of both with the CS18.

Do you know if one of the older column types eg CS12, CS14 etc will do this? We tried it on a CS10 but there is no resolution bewteen the two. I looked in some other column manuals but could not find an example that showed the separation of the two that we wanted.

[maria.rey]

Dear IC-Gal,

Before giving up the CS18, could you tell me what are the respective analytes' concentrations?

The noise level in the system could be optimized so that it is well below 2 nS. If you optimize the noise and maximize the peak efficiency, the minimum detection level of your analyte could be lowered, maximizing the dilution you can make. Resolution also improves as the peak efficiency gets better.

Another parameter you can try on the CS18 is temperature. Just remember to place the suppressor outside the elevated temperature if it is higher than 40C. The column can be used up to 50C.

If diluting further is not possible, then I would try a high capacity column, like the Dionex IonPac CS16. I am not sure though if the two peaks will be resolved on this column, but if you have it would be good to try with an MSA eluent gradient. Once again, temperature can be used as an additional parameter.

Good luck!
Maria.