Help - unseparable compounds

[Fee]

With acetic acid added to my mobile phase I had round spots but the problem stayed the same : my product were in all spot, no matter the Rf!

Thank you for your tips, I will try the basic mobile phase, and possibly the ion-reagent pair.

Another detail : I am now almost sure that the one of those molecule has a dilauryl 3,3'-thiodipropionate moity. Acid hydrolysis of the extract gave me this compound, which is an antioxydant used in food and cosmetic industry... Since it is quite weird to find that kind of compound, I can now confirm that this is not dirt because I have another extract (that I analyzed some months ago) that contains a least one of the two compound I am looking for.

Thank you again for your help.

Fee

[yangz00g]

I woud assume one compound quickly degrades or metabolizes to the other in the media. It's no very common to see such a interesting problem from pure analytical point of view.

[ym3142]

yangz00g 's suggestion can explain both A and B fraction became a mixture of compound a and compound b after collecting and being tested though original separation gave pure peak 1 and pure peak 2, considering it is quite possible that isomer D and isomer L can equilibrate in the solution with a specific percentage.( Each peak corresponds to a compound. ) But this won't explain how

(about 10 fractions equal to 10 different peaks).

all contain the same two Fee claimed compounds.
Fee, did you ever describe how do you collect, treat and test the fractions?
And what make you believe there were two molecules?
If GC/MS has always been your final determining method for the claiming of two molecules, I believe you GC/MS was contaminated. Or your Rotovapor, or anything you were commonly using from your collecting to your GC/MS testing.
Did you ever get some other results(different from this) in your GC/MS test after you treated the sample exact the same proceure and instruments as in this project since you had this problem?

good luck!

[HW Mueller]

This has all the earmarks of cross-contamination, as someone mentioned. "Dirt" which is analyte elsewhere is the ugliest dirt. Your stuff is carried into everything: All HPLC fractions, all TLC fractions.......
Tony, I see many substances that streak badly in TLC, though giving normal HPLC. As much as I like TLC, it seems that the TLC´s stationary phases are not up to par with those of HPLC, also there are binders, high ratio of support to stat. phase....

[Peter Apps]

Hello Fee

Something is not clear so far - you say that you detect both compounds in nearly every fraction but you do not say whether their concentration is the same in every fraction.

If their concentration stays the same from fraction to fraction the peaks that you see by GC-MS cannot be the compounds that you are seeing as HPLC peaks. Even with huge amounts of post-detector band spreading the concentration would be highest in the fractions that correspond to the HPLC peaks (allowing for time lags from detector to collector etc).

What do you see if you run the fractions back through the HPLC ?, do you then detect the two compounds in every fraction ?

650 is a very high molecular weight for a GC separation - these are compounds with more than 40 carbon atoms. I would be surprised to find a natural molecule that big that was thermally stable enough to analyse by GC. What kind of MS are you using - the common benchtop quads do not even go up to that MW.

Regards Peter

[Fee]

Ym3142: I don't think that both compound are isomers because both have a different molecular weight according with GC-MS analysis (one is 646 and the other is 662). I suspect thoses masses to be the molecular weight because in every MS I have a strong peak corresponding to (M+ - CH3).

How I collected the fraction : For silica gel chromatography, I used a gradient from 100% hexane to 100% EtOAc. Compound 662m/z appeared at when gradient was 90:10 Hexane:EtOAc (fraction no 2). When gradient changed to 80:20 Hexane:EtOAc, compound 646 appeared (fraction no 4) (compound 662 was stil there).
Then, I continued the gradient until 50:50 hexane:EOAc, the proportion of
compound 662 compared with compound 646 decreased a little bit until fraction no 16 when proportion of compound 662 increased again and got higher than proportion of compound 646. While gradient was changing to 20:80 Hexane:EtOAc, compound 662 increased and compound 646 decreased. When gradient reached 20:80 hexane:EtOAc, the opposite situation followed : from fraction 23 to fraction 26 the proportion of compound 646 increased while compound 662 decreased. But when mobile phase reached 10:90 Hexane:EtOAc, proportion of compound 662 was higher than proportion of compound 646 (fraction no 27). And when mobile phase was 100% EtOAc, none of both compounds were present in the subsequent fractions (I continued the gradient from 100% EtOAc to 100% MeOH).
For HPLC analysis, I collected 13 fractions (from a phenyl-hexyl semi-prep column, using water and acetonitrile as mobil phase, UV detection at 273 nm), I extracted HPLC fraction with CHCl3, dried the organic phase on anhydrous sodium sulfate, concentrated under N2 and injected in the GC-MS.

About the repetability of my analysis : Yesterday I took my old lab book and I read everything I did since the beginning of these experiments. I found this : 1) At least compound 662 was present in other plant extract. 2) I seems that acid hydrolisis gives the dilauryl 3,3'-thiodipropionate. 3) When electronic ionisation of GC-MS (a quadrupole that can analyse masses up to 800m/z) is set at 20eV instead of typically used 70eV, mass spectrum of compound 662 contains all the characteristic ions peaks of dilauryl 3,3'-thiodipropionate, along with others ions that might be relative to compound 662 (or to other moities of compound 662?).

RMN analysis of a misture of both compounds gives a dilauryl 3,3'-thiopropionate moity with an very substituted aromatic moity but no structure could be proposed.

I have also injected on another GC, using a FID detector and I got the same results as GC-MS analysis.

Peter: I don't understand this :

the peaks that you see by GC-MS cannot be the compounds that you are seeing as HPLC peaks.

Otherwise, you mean that I should reinject the fractions purified from HPLC again in HPLC and see what happen ?

Thank you everyone for your comments. This is really usefull to me.

Fee

[Bruce Hamilton]

Well, my suggestion would be to work from the back to the front, as your varability of composition for the 100 nC6 to 100 EtOAc points to abnormal behaviour. The fact that other peaks do separate suggests either abnormal behaviour or contamination. If abnormal behaviour, a different solvent system could be tried.

Forget the CHCl3 extraction of Phenylhexane prep fractions, analyse them directly on the GC-FID and HPLC to confirm the elution profile is still as noted.

Analyse the two fractions with greatest differences between the peaks ( when each is the larger peak ) by both GC and HPLC to confirm your peak identification is consistent for both techniques.

Confirm that your solvents are mixed/mixing as you believe. The easiest way to check is to do a simple density ( weight of xx ml ) of the fractions, as the amount of dissolved material will not greatly impact on the density. Density should confirm the composition, noting that hydrocarbons do result in a slight increase ( around 2% ) in volume, so just look for the increasing density trend.

I assume that the method has worked well previously, but I'd not normally start with 100% n-Hexane, I'd condition and start with some ethyl acetate ( 5% - 10% ).

Do a blank run ( don't load any sample ), but follow your process exactly. If you don't want to do that, consider putting warm 1:1 nC6:EtOAc through the column before and after a run - just to ensure the column is clean. Remember to re-equilbrate with initial solvent.

Load much smaller and more dilute sample onto the column ( ensuring your loading solvent isn't too polar or different - compared to the initial elution solvent ), and/or warm the column to ensure solubility is maintained and complete.

You could look at the effect of residual water, as these dichloromethane-soluble compounds might have a functionility that is affected by the presence of small amounts of water.

My reasoning is that if some other compounds have separated, the chromatography is working, so there is another source or property that is affecting the elution of these two. Given the variable results, post-run contamination also hasn't been excluded. Try to simplify your processing.

Hope this is helpful, and please keep having fun.

Bruce hamilton