separate two amide

[ym3142]

does anybody here know how to separate the following two amides: CH3CH2CH(NH2)CONH2; and ClCH2CH2CH2CONHCH(CH2CH3)CONH2. To me they are much structural different but so far I have been obtain LC behave exactly the same for them: 1) void time with LUna C18 with MP 95% 20 mM pH 2.7 KH2Po4 buffer and 5% MeOH; void time with CN column and MP 100% 20 mM pH 2.7 KH2Po4 buffer; 2) both gave the same 3 , 6, 10 min under whatever conditions I was using e.g. organic percentage and flow with 20 mM KH2PO4, 10 mM Octanesulfonic acid ph 2.7; 3) exactly the same retention under Gemini column under different percentage of orgnic MeOH and 1%Triethylamine aqueous buffer pH ~12.

I am almost assuming I got the same chemical by mistake. Any ideas?

[HW Mueller]

Did you always inject both compounds simultaneously? If you did it may be that one of them was at tm (to) under every condition. (Column in your 2)?)

[ym3142]

HW, thanks for asking. I did both : injected each separately and the mixture.

[Uwe Neue]

How do you know that you see the analytes, and not system peaks?

In the first case, the peaks were in the void volume. No information.

In the second case, with the ion-pair reagent, you saw the same retained peak independent of the analyte. Maybe you detected the ion-pair reagent rather than the analytes. One of your compounds would ion-pair, the other one should not, and you should see a different behaviour with small differences in the conditions.

The peak that you see at pH 12 could be a system peak, either from the column or from the triethylamine.

You may need to think about some specific detection tools for your analytes.

[HW Mueller]

The results were the same with single and simultaneous injection? If yes, you are seeing either only one compound or none as Uwe suggested, or you are only injecting one compound as you suspected. I understood your 3) as meaning that you thought that both compounds are under the same peak under all conditions (I assumed that you know that it is not normally possible to get invarient retention when changing modifyers). If, instead, you meant the rt is invariant than you got your answer from Uwe.

[ym3142]

it seems to be easy for me to post all my results(retention):
3) gemini c18, 250x4.6, A: buffer aqueous TEA(1%) B: MeOH; 223 nm
90% B, I(for impurity I), 3.787, II(for impurity II), 3.795, I+II(the mixture) 3.784;
80% B, I 4.356, II, 4.365, I+II, 4.363, ~0.197 AU
60% B,I 6.967, II, 6.967, I+II 6.791, ~0.14 AU
50% B, I 8.646, II 8.622, I+II 8.642, ~ 0.07 AU

2) luna c18, 150x4.6, A: buffer 20mMKH2PO4, 10mM Octanesulfonic acid, pH 2.7; B: MeOH, 201 nm
20% B, I, 10.643, II 10574, ~0.04 AU
when the above Buffer pH 5.7
20% B, I, 10.198, II, 10.212 ~0.04AU
when the above pH2.7 Buffer dilute to 20% concentration,
20% B, Flow 1.5, I 7.831, II 7.795; I+II 7.737, ~0.04AU
25% B, Flow 1.5, I+II 6.528 single ~0.06AU
30% B, Flow 1.5, I+II 4.366 single ~0.07AU
30% B, Flow 1.2, I+II 5.454 single ~0.06AU
30% B, Flow 1.0, I+II 6.605 single ~0.06AU

basically, I agree the two chemicals should not be so similar in such many conditions. Something wrong should be with my samples.

[Uwe Neue]

I stick with my previously voiced opinion. You are looking at ghost peaks, and not your compounds. Don't forget that at the low wavelength that you are looking at, you will see anything... Do you have access to MS? You could collect the peaks and do MS.

[ym3142]

My questions is 1) so is it common for a ghost peak to move around under different conditions?
2) is it common for a ghost peak present itself as the only major peak for instance of 20 min run?
3) is it common for a ghost peak to be as strong as 70 to 200mAU though at 200nm?
4) if I test under the same conditions by injecting the diluent, the ghost peak will be present with similar retention and intensity, right? For both ion pairing and basic condition. ( I do not have easy access to MS). I will try.
I did test on injecting water(was the diluent) with Luna c18 150x4.6, 1ml/min, Buffer(20mM KH2P4) : MeOH=98:2 to give no peak at all though my impuirty reference sample gave peak at 3.24 min with 0.12 AU at 201nm.
thanks again,

[Kostas Petritis]

In reply to your questions:

1) Yes that is very common...
2) The number of system peaks depend on the complexity and nature of your mobile phase. You can have only one, or more that are co-eluted or more that are not co-eluted...
3) Yes it is. System peaks is a result of equilibrium distrurbance. The higher the disturbance the more intense the system peak. It also depends on the mode of detection, UV at 200 nm can create huge system peaks...
4)From what I said before, your forth assumption is partially wrong. The intensity will be different (lower). And you might or not see the system peak if ever the perturbation occured by the diluent is not significant... The retention time of the system peak though will be the same...

[ym3142]

HI, Kostas,
Thank you for your reply. Just to clarify: under one specific condition my sample solution gave a ghost at, say, retention time 10 min. So this ghost was caused by the diluent (perturbation) instead of the sample. So if I inject the diluent itself which was used to dissolve the sample, so similar peak with similar retention time and intensity should be present in the C-gram obtained under exact the same LC condition, is this right?
I did not see a peak in the diluent injection in the retention time of the peak of the sample injection , meaning the peak from the sample was really for the sample chemical, right?

[HW Mueller]

Not necessarily, it is also very likely that by dissolving the analyte in water you inadvertendly introduced something else, maybe shaking the solution saturated it with air, so that you are seeing air?

At least you can run a spectrum of the peak and compare it with that of analyte (If you don´t know what the spec is supposed to look like you may inject analyte without a column......careful, though, you need to have a highly pure sample. This will also tell you how big the peak (area!) should be for a given injection if the flow rates with and without the column are the same or corrected.)

You didn´t tell us what you ment, under your 3), regarding the invarient rt. This could negate some of the suggestions.

[ym3142]

Repeated experiment under ion-pairing reagent gave I at 7.4 min and II at 5.7 min, which made sense.
Sorry for wasting your time if you feel so. I learnt a lot in the process and wish you too. Thanks for all participation and have nice Labor Day.

[Kostas Petritis]

Excel,

I see that I was not clear enough. System peaks from the diluent and analyte peak can have different retention times. I suggest you that you read one of my past papers (K. Petritis et al. J. Sep. Sci. 2001, 24, 397-405) where I speak a lot about system peaks and I reference some forgotten papers addressing the system peak problem (Crommen and Guiochon have published a lot on this)...

Figure 3 in that paper replies your question...

[HW Mueller]

I am utterly confused by amides, I or II, impurities I and...... But: If that means that your "amides" were at 7.4 and 5.7 then you got your answer from Uwe. Sorry to be repetitive.

[ym3142]

sorry 1 for confusing, here amides=impurities.

Sorry 2 to everyone, when I said

Repeated experiment under ion-pairing reagent gave I at 7.4 min and II at 5.7 min, which made sense.

I meant the problem was solved. Earlier post I said the two amides gave the same retention, which did not make sense, which was caused by the sample preparation error. I was injecting the same sample though labeled differently. The repeatedly prepared samples did give different Rt and reinjection of old samples still gave the same Rt, which was the same as one of the Rt of the new samples.

Please let me know if you got more questions and I also need to read into Kostas' paper about the ghost, I did not get yet.
Have nice day, everyone.