Isosorbide TLC problem

[qfiviera]

Hey guys. I have a problem with Isosorbide mononitrate tablets related compounds test from USP 34 monography. It´s a TLC and quantitates Isosorbide. I can´t see the more diluted Isosorbide standard solution (0.0125 mg/mL of Isosorbide in absolut ethanol) when I developed it with permanganate solution. I don´t know what´s happening. Mobile phase is Ethanol:Toluene (8:2), but I remove it all from the plate. I´ve tried to develop it with fosfomolibdic acid, and potassium iodide-starch solution and I never see the spot. I passed from 20 uL to 40 uL and still couldn´t see it. Perhaps the path is too long, about 13.5 cm long and is too difuse to see it. Anyone has any experience? (sorry for my English, I speak Spanish)

[Blumenstock]

Dear Mr. Viera,

what about the diameter of the starting spot? In my eyes 40 µL is a rather huge volume on a thin layer plate! What about drying between the application of 40 µL?
Do you apply the volume manually or by an application device?

Sincerely yours

Tekamthi

[qfiviera]

I started with 20 uL as the monography says, and then 40 uL. And I dryied between each application so spots are not bigger than 0.5 cm. And I apply manually. Any other idea I will be grateful.

[tom jupille]

Are you using the correct type of plate? Activated or non-activated?

If this were my problem, I would begin at the beginning: spot the standard, let it dry, and then try to visualize it without developing. You should be able to see the spot at the origin. If not, the problem is either with your standard or your visualization reagent(s).

If you *can* see the spot at the origin, prepare and spot another plate and develop it for only a short time/distance (perhaps 1/4 of the usual), then dry and visualize. If you see response at the solvent front (or if the spot remains at the origin), then the problem is either with your solvent or your plate.

Prepare a fresh batch of solvent and repeat the previous step. If it looks the same, the problem is the plate (perhaps the silica used or the activation/wash used).

[qfiviera]

I developed it with permanganate solution

My mistake. That is the revealing solution (i don`t know if that is the correct expression in English, perhaps visualization solution or reagent). I sprayed with it and heat it for 5 minutes at 105 ºC.

And I have tryied to dry the plate before to activate the silica, or run it less distance (I thought it could be diffused for long paths) and another "revealing solution" like phosphomolibdic acid, or use one with fluorescence indicator and without it. Still the same problem. I see the concentrated spot but not the diluted one. And I`m worried because it is an USP technique.

I think Tom I`m having a concentration problem. And I`m preparing the permanganate at the moment. I can see my standard if it is concentrated (and it`s an USP one, recently bought). But the 0.0125 concentration is the problem.

I left that in pause at the moment and doing another things, but I still need to resolve it. I`m not having much choices left yet.

Thank you Tom and Blumenstock. Still needing ideas if anyone had one.
Happy new year.

[qfiviera]

Still having problems. Did anyone know about this USP monography? Did anyone did this test?

[rwjohnson]

Are you producing this product or is it a purchased raw material? If you are purchasing, then the supplier should be able to help with technical questions regarding the test. I used to work with USP products in a former job and often handled questions about our testing procedures.

Ron J

[qfiviera]

Raw material and finished product (tablets).

[tom jupille]

OK, back to the basics. Don't bother developing the plate (running the solvent) but focus on detecting the initial spot.

Make up a solution sufficiently concentrated that you can detect easily, and make serial dilutions. Now spot each of those next to one another on the same plate, dry, and visualize (spray with the permanganate solution and heat). That will let you estimate your detection limit. If it is far higher than it should be then as I indicated earlier, the problem is either in your standards or the visualization procedure.

Assuming you have double-checked your weighing procedure, experiment with the heating conditions (time/temperature) and with the permanganate concentration. The results may vary depending on things like the thickness of the bed or the activity of the silica.

Once you have the visualization working, you can proceed to tweaking the chromatography as necessary.

[qfiviera]

Thank you Tom, I`ll try that. But I think sometimes USP tests doesn`t work well. Does this happened to you? Not all tests, in general I don´t have problems, but with some I had, and I had to validate when this happen and I make changes in them.

[rwjohnson]

Yes, I have had some issues with compendial methods but they always worked out in the end. The labs that present the method each have a unique way of performing methods and this finds its way into the compendia. The revisions do get presented prior to becoming official, so everybody has a chance to challenge the change if it is only a proposed change and not an adopted method.

You can always provide improved procedures to USP. And as you eluded to, you can also implement "better" methods in your lab if they provide equivalent results.

Ron J