Problem with Indole-3-carbinol chromotography

[kouroshh1]

Dear All,

I read all your feedback very carefully and based on that I have tried different experiments.

First, I would like to share with you the picture of my indole 3 carbinol chromatography where I have seen 2 peaks instead of 1. Please open the link below to look at my peaks.

http://tinypic.com/r/t5srgm/9

In addition, today I had done some experiments based on your feedback and fortunately finally, I was able to observe one single peak. I used the same method such as the same gradient and modifier which I explained previously. However, the things I changed was my column temperature from 30 to 40 centigrade but still I could see 2 peaks. However, the interesting thing was that when I changed the temperature from 40 to 50 centigrade then I could see one single peak. Unfortunately since i left the office I couldnt make a copy of the file but I can upload the picture later here.

I am still confused why increasing of the temperature helped to solve the problem. Is it related to viscosity of the solvent? So if the solvent become less viscose then the solvent has more strength to elute the compound? Can someone please help me to find a reasonable explanation?

[mattmullaney]

Hi kouroshh1,

Strange to me, your hypothesis may be correct with regard to the merging of the two peaks. Likely the retention time also moved downward --the indole-3-carbinol peak eluted in less time than 13 minutes?

More applied heat to column = less viscous the eluent is, yes. Temperature does not affect the elution strength of the mobile phase, it speeds up the kinetics of the chromatographic process...the binding to and letting go of the analyte molecules to the stationary phase.

Very glad to hear that the autosampler was not the trouble...and that Rndirk's idea worked out well for you!

Happy holidays!

[Tom Waeghe]

The chromatogram looks like a classic on-column degradation situation or possible a peak splitting due to polar analyte with too strong a sample solvent.

See https://mcardle.oncology.wisc.edu/bradf ... s/9543.pdf

[kouroshh1]

Thank you mattmullaney and Tom for your comments. I couldn't remember what was the retention time but i can let you know about it next Tuesday together with the picture of the peak.

I checked the log p of indole 3 carbinol which is Log p=1.3. So, it seems it is quite non polar than being polar. In addition, besides running Indole 3 carbinol I was also testing 4 other standards and the peak shape of other analytes were fine. So, the column to me it seems it is working pretty well because I didnt have a problem with other standards peak shape. What do you think? Am I thinking correctly?

Please below find the chromatogram where 5 different standards including indole 3 carbinol were seperated. The temperature of the column here is 30 centigrade.

http://tinypic.com/r/2vnev4p/9

[Tom Waeghe]

http://lpi.oregonstate.edu/mic/dietary- ... ailability

If all the other peaks are normally shaped (no pun intended) and the indole-3 carbinol shows the peaks that it shows and the ratio of which changes with temperature, it suggest that you may be seeing on column conversion.

You could try separation at 20° if your instrument will allow it or carry out replicate injections at 30° C, 35° C, 40° C etc. and see if the ratio of the leading peak to the "fronting" peak changes such that the 2nd peak becomes larger.

[kouroshh1]

Hi Thomas,

Sure next week i will do an experiment as you suggested. The interesting thing was that indole 3 carbinol nominal mass is roghly 147 and in positive mode it should become (M+H)+ =148.

However, when i wanted to optimize the MS parametes for this analyte i realized 148 is not exist (or if it is present, it is in extremely low abundance). The precursor ion which our detector could haved identfied was (M+H-H2O)+=130 with very high abundance. Therefore, in my MRM method i used 130 m/z as a Q1.

So, regarding your theory i.e column conversation i should think what kind of equilbration is happening. I purchased the standard (97% pure) from sigma. There is no chiral center so no diasteromer can be formed. I am really puzzled.

[mattmullaney]

Hi kouroshh1,

I'm not enough of an MS guy, would not the acid-catalyzed compounds shown in Figure 2 of the link Tom W. (from Oregon State) sent be possible species that could form on-column under acidic conditions?

[kouroshh1]

Hi Mattmullaney,

Actually with our LCMS system i performed MS and MSMS analysis and both peaks has MS1 of 130 m/z and MSMS of 103 and 77 m/z in positive mode. So both peaks at retention time of 13 and 15 has exactly the same mass which is the nominal mass belong to indole 3 carbinol. So, that is my confusion. I hope I could have explained clearlyabout what i think.

[mattmullaney]

Hi kouroshh1,

Please note, what I do not know about MS would fill a library, unfortunately.

I do understand the confusion at this level; as both chromatographic peaks have the same MS (and MS/MS) peaks, everything points to both peaks being indole-3-carbinol.

Is it possible that those acid-catalyzed indole-3-carbinol compounds could afford similar MS spectra to indole-3-carbinol to begin with?

It reminds me of the GC-MS of polydimethylsiloxanes...after the polymer chain grows to a certain length, one cannot determine the difference between them using MS alone.

[I do understand there's a difference also between EI and the ionizaation techniques you use in LC-MS and Lc-MS/MS.]

In any case, Have Happy Holidays!! And I do hope the solutions being sought come about.

[kouroshh1]

Hello again,

Thank you for your very clear explanation. I will spend more time next werk and will investigate the possibility of formation of those acid-catalyzed compounds from indole-3-carbinol.

I will get back to you with more precise answer next werk. I think Tom suggestion regarding testing my standard at different temperatures with some replicates would be very helpful.

Happy holidays too
Kourosh

[HPLCaddict]

Even at the risk of disgracing myself with this ridiculously easy explanation: Your standard is 97% pure - is it possible that the 3% missing represent an isomer of indole-3-carbinol?

[kouroshh1]

But there is no chiral center in the molecule so what kind of isomer could be formed?

[mattmullaney]

Hi again K.,

Sorry, I'm not a fast typist...the 3% could also be one of those condensation products noted in that Oregon reference of Tom W.'s. Reminds me of DNPH-derivatized acrolein--if the standard solution is not carefully made, they can transform into a polymer in situ...caused me a bit of trouble a few years ago.

[kouroshh1]

Hello again

Yes, you are right. It is possible. I am planning to run the full scan mass spectrometry next week then i will look if any of those condensation products are present (being formed) based on the mass. However, my mobile phase contains 20 mM acetic acid and these kind of reaction may have been occurred while my standard was mixed with the mobile phase in the LC system.

In addition, I dissolved my standard in absolute methanol then i dilued it with milli q water 100% without any additive. So, these kind of transformation is likely that occurred while my analyte was standing in the vial in the LC system tray.

[mattmullaney]

Hi again, kouroshh1!

Agreed on all counts...I hope that the next experiments with temp and ionization (full scan) variation ferret this out.

LC stationary phase can be a good catalyst/helper for chemical processes to occur--much surface area...