Problem with Indole-3-carbinol chromotography

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I purchased the Indole-3-carbinol reference standard and made a stock solution of 10 ppm in absolute methanol. Then from that I made a working standard solution of 100 ppb or ng/ml in pure water but still some trace of methanol present.

Subsequently, I applied the 100 ppb Indole-3-carbinol into LCMS where the MRM scan mode was used. After doing LCMS using 100 % H2O as a aquas solvent and 100 % methanol as an organic solvent, I noticed that I could not see 1 single peak. It was 2 peaks with 2 minutes retention time difference belong to the same analyte Indole-3-carbinol. I did MS and MS/MS analysis and I realized both peaks are indeed belong to Indole-3-carbinol.

I checked at the structure of Indole-3-carbinol and I couldn't find a chiral center so no diastereomer could cause the appearance of 2 peaks.

I checked the Pka value of Indole-3-carbinol and it is around 15. Unfortunately, I couldnt make high pH mobile phase since our column is not resistance to the high pH. I know when we have a basic analyte we should go 2 units above the pH.

Apart from Pka, my question is that do you have any solution for my problem? Why I see 2 peaks instead of one peak because I only applied Indole-3-carbinol into the system and nothing more.


Thank you
Kourosh
Hi kouroshh1,

One suggestion for the splitting of the Indole-3-carbinol peak(s) is that the HPLC column's inlet frit could be partially blocked. Or the HPLC column may have a void (missing stationary phase).
MattM
Hi Mattmullaney,

I only have problem with this standard and for other standards i could see only one single peak. The retrntion time for indole 3-Carbinol, 2 peaks is 12 and 14 minuets respectively and the chromotogram is smearing from 12 to 14 minuets. That is the reason initially I thought there is a problem with a pH of mobile phase. The first peak intensity is around 6000 cos and the second peak 20000 cps.
Hello,

Can you post the following please:

- Column
- Injection volume
- Gradient
- Flow

Did you rule out carry-over? Wash solvent?
Hi kouroshh1,

The information Rndirk asks after will be helpful as the retention factor(s) may be determined for the two peaks. "Smearing" suggests some sort of equilibrium problem, though this is likely not due to the analyte (see below).

Have you tried flow injection analysis without a column in place to check the health of the injector?

I confirmed the pKa of your analyte...I'll not that if you used something like ammonium formate or formic acid to your eluent, you're working at a pH well away from the pKa of Indole-3-carbinol, so you ought to have one species present...provided that the concentration of the additive is high enough.

Knowing the type/mfg. of the column also will be helpful-also the identity of the additive to the eluent.
MattM
Dear All,

Thank you for all your comments and help. Below please find the information you have asked me:

Column: Synergi Fusion reverse phase from Phenomenex
- Injection volume: 10 microliter
-solvents: A: H2O 100 % in 20 mM Acetic acid
B: Methanol 100 % in 20 mM Acetic acid
- Gradient:

time (min)/ A%/ B%
0/ 95/ 5
3/ 95/ 5
5/ 55/ 45
20/ 0/ 100
23/ 0/ 100
23.5/ 95/ 5
35/ 95/ 5

- Flow: 0.3 ml/min

Indole 3- carbinol peaks comes at retention time of 13 and 15 as 2 separate peaks which is smeared with fronting.

Actually I have fronting of the peaks not tailing. Today I tried 1 microlter of injection volume of 200 ppb of my standard instead of 10 microliter but unfortunately I could still see 2 peaks smearing with fronting peak.

In addition, i diluted my 200 ppb standard to 20 ppb with pure Milli-Q water and ran it on LCMS but again I saw 2 peaks with fronting peaks.

I am really puzzled that why I see the peaks like that. I have a mixture of different analytes and only for this specific compound I could see 2 peaks instead of 1.

I havent tried the flow injection yet. I can give it a try. I optimized my standards using infusion and inject it directly into MS to optimize the parameters such as entrance potential and collision energy.

I would really appreciate it if you can give me some advice on what should i do?
Hi kouroshh1,

So both peaks are fronting (it is not tailing of the first peak blending into fronting of the second peak).

My advice is, please do the flow injection--this will help us decide if the trouble is related to the column. If you have a second Fusion column swapping it in may also be helpful in identifying the column as the source of the problem. Lastly, have you tried a diluent injection just to see if the trouble isn't due to autosampler carryover--and what is your rinse solution?

My thanks for the gradient program and flow rate-the final details that would be helpful are the column dimensions so that the apparent retention factors can be calculated.

Cutting the volume and concentration of the standard did not eliminate the fronting behavior-You're working around pH 5 or so, I still think this is not a problem here. You're injecting the standard dissolved in mostly water, that also should not be a problem.
MattM
Hi Mattmullaney,

Thank you for your time and help. Actually, it is tailing of the first peak blending into fronting of the second peak. I couldn't explain it correctly last time and you described it very well.

Unfortunately I dont know how can i attach the picture of my chromatogram otherwise it could have been helpful.

You are welcome. I am happy that my information helped.

I measured the pH of my aquas solvent with 20 mM Acetic acid today and it was 3.

The column dimension is as follow:

Particle Size (µm): 4
Pore Size (Å): 80
Length (mm): 100
Internal Diameter (mm): 2

My rinse solution is 100 % ACN. Regarding diluent injection, I have tried ACN (10 µl) but no carry over could be seen.

Sure I will do a flow injection but if you have thought about something else please let me know.
Hello Again kouroshh1,

Okay, so the behavior of the two peaks is tailing of the first peak blending into fronting of the second peak. This could be caused by a chemistry problem unrelated to the column. Best way to ferret this out is swap in a new column, if you have one.

Oh, mixing MeOH in with water will affect the pH of the eluent by ca. 0.2-0.3 pH unit, so the eluent pH is ca. 3.2-3.3 in effect. Good to know.

Column ID is 2 mm, by 100 mm, so the column void volume is ca. 0.214 mL, divided by 0.3 mL/min affords t0 = 0.713 min. At 13 and 15 minutes, the two Indole-3-carbinol peaks are being well-retained...maybe even too long, but that is not so important Unless, Perhaps, something happens to the Indole-3-carbinol if it resides on the column too long. This may not be so likely, but it is a possibility to note in addition to the column stationary phase/frit being a possible cause of the trouble. Another possibility, is the temperature in the lab very different from that the separation is being carried out at?
MattM
Hello again Mattmullaney,

Actually besides using synergi fusion RP column, I have also tried using synergi polar RP column from Phenomenex (which has a polar endcapped, ether-linked phenyl phase) and it was the same story as you described very well i.e tailing of the first peak blending into fronting of the second peak. So it was no difference between the 2 different columns.

The temperature of the column was 30 centigrade and it was not varied. Do you recommend me to increase the temperature of the column?

Do you think since i have a basic compound such as indole 3 carbinol the pH of the mobile phase shouldn't be 2 unit above the Pka of my analyte?
Hi again,

Indole-3-carbinol is a pretty weak base, I think--it exists in one form from pH 1 - 12, so I don't think you're observing chemical equilibrium effects from a conversion between acid and base forms. That kind of thing won't happen
as you're at a pH well away from the pKa--the pH does not have to be more alkaline than it is. Anyhow, how would you achieve pH 17 in water? [Not possible.]

The temperature in the lab is probably not a problem, not too much of a mismatch most likely.

Hmmm, two different stationary phases, yet the same trouble with two partially-merged peaks? This observation makes me wonder about the health of the autosampler. Flow injection and an examination of the autosampler rinse solvent seem to be in order.
MattM
Hi Again,

You are right we can not achieve pH= 15 :)

I was trying to rationalize the thing it might have happened but I couldn’t succeed to bring a reasonable reason.

I will take your advice and will check the health of the autosampler to make sure there is no carry over effect.

Thank you again for all your helps.

Kourosh
Like MattM, I don't think there is any issue with pH.

It would help to add a chromatogram to visualize the problem, see this link explaining how.

I would try a couple of things:

- Replace the wash solvent (100% ACN) with 50/50 water/methanol.
- Start the gradient at a higher % organic.
- Inject the standard without a pre-column (if any)
- Increase the column temperature to 40°C

Make sure the column is well-conditioned.
Hi kouroshh1,

Understood, and hang in there, I think the problem is close to being resolved.

Hi Rndirk,

My thanks for your apt comments, I wasn't as certain about raising the separation temperature, it is a good suggestion I think after some more thinking on it. My thought is that the trouble is most likely the autosampler...and we'll see!
MattM
Hi kouroshh1,

Found this at LC-GC, it may help (?):

http://www.chromatographyonline.com/pea ... 0?pageID=1
MattM
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