Problem with base peak seperation

Discussions about HPLC, CE, TLC, SFC, and other "liquid phase" separation techniques.

23 posts Page 1 of 2
Hi,

I am working on my LC-MS method development for a mixture of standards and I want to separate them in a reverse phase chromatography.

The main challenge for me is that I could not use acidic modifiers since one of my analytes that is indole-3-carbinol (I3C) will be converted to another compound inside the column under the acidic conditions. In addition, I am restricted to column temperature of 20 centigrade since even in the absence of acidic modifier at higher temperature than 20, my analyte (I3C) will be transformed into another compound.

Therefore, basically for getting a good peak resolution among 4 different reference standards, I am limited to the solvent strength and gradient factors. My main question is what strategy do you recommend I take? At the moment, I know methanol is working better for my method than ACN. I tried many different gradient program but the peaks can not be separated well. I could see only the changes in the retention time but not the separation. May I ask what do you recommend? Should I mainly work on my gradient or there are other parameters I can manipulate to get a good separation?

P.s: Most of my analytes are eluting out from the column when the gradient goes from 60 % MeOH to 100 % MeOH without any modifier.

Thank you in advance
Hi,kouroshh1,

Seems that you're doing what you can to improve the selectivity, use MeOH instead of ACN, remove modifier and lower temperature.

Next would be to use a shallower gradient, say starting at 40% MeOH. At what time/% MeOH does the last peak elute?

[You Can try adding some water to the neat MeOH supply to help with this].

Next...a different stationary phase.

Let's see how others weigh in.
MattM
you could also look at phenolic phase columns, or F5 columns. They should retain things like indole carbinol, but with different selectivity to C18. If you're really lucky, one of the column suppliers will have some information in their database or an application note.
Thank you mattmullaney and Imh for your recommendation

I tried with 40 % MeOH but I didn't get a good separation. Then I started from 50 % MeOH. Please find my chromotogram and the information about the gradient here:

http://tinypic.com/r/6ydx8h/9

May I ask what do you think about my chromotogram?

The resolution of the 1 and 2 standard is not that good and I am trying to find a right gradient to be able to seperate them from each other but I was not successful so far. Do you recommend to add water to the methanol eluent to reduce its strength and then use it as an organic eluent (for instance 90 % methanol as solvent B)? Do you have any recommendation to be able to separate the first 2 peaks from each other with good selectivity?

I also tried F5 column from phenomenex but the selectivity was very poor and the peaks were coeluting.

Thank you in advance
Hi, kouroshh1,

Yes, if you add water to the MeOH, you'll be able to run a shallower gradient. I'm having trouble with TinyPic here, will try again later.

Imh's idea is apt, I agree...will look into method development with that phase.

EDIT: Try MeOH first with waterr when using the F5 phase, the selectivity should be greater.
MattM
Hello again,

Please let me know if you still have a problem to open the file in TinyPic then I will upload again.

Sure, I will try F5 phase again

Thank you for your time and help
Hi Again, K,

Got TinyPic to work here at last. What are the column dimensions and the flow rate? (Unless you're doing better with the F5 phase)?

It's not too bad with the fusion as it is...starting at 40:60 A/B where B is, say, 80:20 MeOH/water should help.

I'll wait for your response in case I'm missing something here.
MattM
Hello again Mattmullaney,

The column dimension is as follow:

Particle Size (µm): 4
Pore Size (Å): 80
Length (mm):250
Internal Diameter (mm): 2

The flow rate:0.3 ml/min

Sure, I will also try very soon with starting at 40:60 A/B where B is, say, 80:20 MeOH/water should help.

Thank you for your reply.
:P Hoping for Best on this end! My thanks for the parameters. Will take another look at your chromatograms after spending some time with the family here.
MattM
Thank you :) I hope you would enjoy your time besides your family. Actually today I have tried the column with F5 phase with the same gradient I showed in a previous picture on Got TinyPic and it worked pretty well.

My only problem at the moment is that I have a big column conversion of indole 3 carbinol even though I have tried both ACN and MeOH in an absence of modifier and in addition to that the column temperature was set to 20 centigrade. I do not know what can I really do to stop this column conversion condensation reaction.

I tried with basic eluent and it was very helpful. However, the intensity was very low and without a proper intensity I can not do a proper quantification.

May I ask if I take the area peak of the left peak in a chromotogram in Got TinyPic from indole 3 carbinol, would it be acceptable even though there are 2 peaks belongs to indole 3 carbinol present in a chromotogram?

Enjoy your weekend
Kourosh
Hi, kouroshh1,

My luck with tinypic has disappeared again, this is most frustrating. Seems to me to be the pop-up ads.

This indole-3-carbinol is worse than "my" old acrolein-DNPH troubles...as I recall from Tom Waeghe's (sp?) reference, there isn't a whole heck of a lot that may be done with these condensation reactions, unfortunately.

I'd say, though, if it is possible to determine an estimate of the response factor of that condensation product (don't have access to Waeghe's reference from this PC, my laptop is charging at the moment), then yes, the second peak may be factored into the quantification. Wouldn't be the first time for something like this to happen (peak groups assigned to a single source).

Probably the best way to do this is to obtain a proper standard material of the condensation product, though you can make estimates another way, maybe, with free-flow injection (no column in place) of the indole-3-carbinol.

EDIT: Just as a refresher, here is the bulk of Dr. Waeghe's information recopied here:

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

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

And I think you already know from the nice table (can't figure out how to attach that here), that I3C suffers far less from interconversion to DIM at pH values > 6.5 or so.

EDIT2: I forgot, basic conditions seem to harm the quantitative output, poor ionization. This I3C is truly a pain in the behind. Still should be no problem once a response factor is obtained for DIM. Not sure if this may be purchased, but it would be best to do so, if possible.
MattM
kouroshh1 wrote:
Thank you :)
May I ask if I take the area peak of the left peak in a chromotogram in Got TinyPic from indole 3 carbinol, would it be acceptable even though there are 2 peaks belongs to indole 3 carbinol present in a chromotogram?

Enjoy your weekend
Kourosh


As long as the ratio of those 2 peaks remains constant over the course of your sequence, your standards are matrix matched and you see the same phenomenon in them, your calibration curve and QC's are acceptable, yes.

Maybe not for a characterization type study but if all you want to do is quantitate that analyte your results should be accurate disregarding one of the peaks. I would watch for a split curve though because if it is a transformation and it is ongoing, they won't.
kouroshh1 wrote:
Hi,

I am working on my LC-MS method development for a mixture of standards and I want to separate them in a reverse phase chromatography.

The main challenge for me is that I could not use acidic modifiers since one of my analytes that is indole-3-carbinol (I3C) will be converted to another compound inside the column under the acidic conditions. In addition, I am restricted to column temperature of 20 centigrade since even in the absence of acidic modifier at higher temperature than 20, my analyte (I3C) will be transformed into another compound.

Therefore, basically for getting a good peak resolution among 4 different reference standards, I am limited to the solvent strength and gradient factors. My main question is what strategy do you recommend I take? At the moment, I know methanol is working better for my method than ACN. I tried many different gradient program but the peaks can not be separated well. I could see only the changes in the retention time but not the separation. May I ask what do you recommend? Should I mainly work on my gradient or there are other parameters I can manipulate to get a good separation?

P.s: Most of my analytes are eluting out from the column when the gradient goes from 60 % MeOH to 100 % MeOH without any modifier.

Thank you in advance


I saw that c-gram once and it looked fine, now I can't, but why is there such a concern over separation? You are going to extract those mass transitions out for quantitation individually and if you don't have separation it won't make a difference. Sure a co-eluting peak may affect the final area cts but if it's happening with all samples who cares? If your cal curve is acceptable and your QC's are all 100% is there any question what their concentration is? Once again I come from the world of strictly quantitation and not physical chemistry or characterization, some of those studies may have different guidelines.
Hi Mattmullaney and Kenn,

Actually, I tried infusing Indole 3 carbinol (I3C) into the MS directly by using syringe and I did not have a problem. The intensity was high and no condensation product could have been seen.

The problem is when the I3C reference standard is running through the LC the condensation will occur. Unfortunately, when the condensation product from I3C is reaching into the ion source it will be fragmented into I3C mass transitions before reaching into the first quadruple. So, in my multiple reaction monitoring (MRM) method as a precursor ion (Q1) I have 130 m/z and as a product ion Q3 I have 103.1 m/z in a positive polarity. Due to the in-source fragmentation, I only get the transition for I3C that is 130 m/z----> 103 m/z. So it is very hard to identify the mass of the second peak because it has a same mass transition as I3C unless as you said we purchase the standard which have not done it yet and compare it against the second peak.


Unfortunately, the ratio of those 2 peaks does not remains constant over the course of your sequence.

Regarding the second part, in my MRM method if I have several compounds and some of them are co-eluting together, it is acceptable to count the area peak of each analyte and do the quantification. Am I understood correctly? I can select each peak based on its MRM transition. So, would it be acceptable to calculate the area peak of each peak independently because it has its own MRM transition?

Thank you for your time and help
kouroshh1 wrote:
Hi Mattmullaney and Kenn,

Actually, I tried infusing Indole 3 carbinol (I3C) into the MS directly by using syringe and I did not have a problem. The intensity was high and no condensation product could have been seen.

The problem is when the I3C reference standard is running through the LC the condensation will occur. Unfortunately, when the condensation product from I3C is reaching into the ion source it will be fragmented into I3C mass transitions before reaching into the first quadruple. So, in my multiple reaction monitoring (MRM) method as a precursor ion (Q1) I have 130 m/z and as a product ion Q3 I have 103.1 m/z in a positive polarity. Due to the in-source fragmentation, I only get the transition for I3C that is 130 m/z----> 103 m/z. So it is very hard to identify the mass of the second peak because it has a same mass transition as I3C unless as you said we purchase the standard which have not done it yet and compare it against the second peak.


Unfortunately, the ratio of those 2 peaks does not remains constant over the course of your sequence.

Regarding the second part, in my MRM method if I have several compounds and some of them are co-eluting together, it is acceptable to count the area peak of each analyte and do the quantification. Am I understood correctly? I can select each peak based on its MRM transition. So, would it be acceptable to calculate the area peak of each peak independently because it has its own MRM transition?

Thank you for your time and help
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