Method Development

[Candy]

Hi Everyone

I am looking for a method for analyisis of a drug by the name Rapamycin.
This is the best I've got so far please assist:
Mobile Phase A: Water
Mobile Phase B: Methanol
Column temperature: 50 degrees
Flow rate 0.400ml/min
Injection:10ul
Column:Ascentis Express Column-C18 10cm X 4.6mm, 2.7 um

This gives me a front tailing and low resolution.
How do I improve this? I have tried changing the mobile phase to Acetonitrile and still didnt work.

[danko]

Candy,
Are you really running 4 mL/min on that column? If yes, correct it to ca. 1 mL/min.
What is Rapamycin dissolved in? (very important in the context of your observations)

Best Regard

[Alex Buske]

Its containing nitrogen, so a buffer will help. Fronting sound like column overloading. On the other hand there is plenty of literature on this: http://scholar.google.de/scholar?q=Siro ... i=scholart

Alex

[Candy]

At danko I will try that just now, It is dissolved in MeCN 0.5% of acetic acid.
Thanks alex will check the site, i tried buffer too it didnt seem to make much difference.

[danko]

Yes, try the flow rate thing first (1 mL/min) and if it helps, try to increase it until you observe problems. I would expect flow rate between 1 and 2 mL/min to be optimal.
Regarding the solvent, I believe the organic part is too big. Try 80% (or less) methanol instead.

Best Regards

[Candy]

Hi Everyone

Nothing seem to help the fronting, I have tried the buffer, changed the flow rate 0.2-1.5ml/min, Changed mobile Phase from 80%MeOH to 60%.
I even joined another column of the same(Column-C18 10cm X 4.6mm, 2.7 um) but makes no difference.

Anyone know how I can attach a file so you cn be able to see my results

I am realy stuck and need help urgently

[Mattias]

Did you see a big shift in retention time, going from 80 to 60% MeOH in the mobile phase? That should make a huge difference in retention time!

Where does the peak elute? If it is very early, it could just as well be the acetic acid in your sample (instead of Rapamycin).

If it is Rapamycin, I think you need to decrease the concentration of your sample or inject even lower volume to get a good peak shape.

[Candy]

Yes there was a huge shift from 23-3min, I have changed injection volume up to 3 microlitres, there was no change.
Are you suggesting that I use pure water instead?

I am currently injecting 200ul/ml, do you perhaps think I shoul reduce the concentration of Rapamycin?

[Mattias]

There should be no problem to inject 10 µl of 0.2 mg/ml solution of that large column. I don't think the MeOH in the sample is the problem either, the injection volume is too low. It could anyway be worth a shot to use more water in the sample.

Someone suggested a symmetry problem due to the nitrogen in the molecule, but that should give tailing - not fronting.

I am out of ideas I am afraid.

[shaun78]

Anything locking you into using MeOH as the organic?

At 60 or 80% organic, ACN will be a stronger solvent than MeOH. Might give you a tighter sample band.

Also, a 10 uL injection on a column with a 2.7 um particle is rather large. When I scaled a method down from a 4.6 x 150, 5 um column and a 10uL injection to an Acentis column similar to the one you are using, the injection volume also had to drop to about 2-3 uL. Though I do see that you changed the injection volume to 3 uL with no change ...

If this is a scale down method from a "normal" HPLC column, see equation 2 here: http://www.restek.com/aoi_pharm_A020.asp

Lastly, peak fronting can also be caused if the injection solvent is much stronger than the mobile phase. Maybe change the sample solvent to 80/20 ACN/Water instead of the 100% ACN you have it in now.

[gcguy]

A quick search found this

http://www.zirchrom.com/pdf/277.pdf

[Mattias]

Shaun78> Scaling is not applicable for change in particle size, only column diameter. Here we have a standard 4.6 mm column which should be able to handle 10 µl.

I think the key to the peak shape issue is found in the reference from gcguy. It states that the temperature needs to be very high for a good peak shape (75°C in the reference). Must be some conformation issue that occurs at lower temperatures?

[HW Mueller]

The structure of Rapamycin given in Wikipedia doesn´t show a basic nitrogen, but maybe an acidic OH??
If this thing is negatively charged in the mobile phase there could also be some ion exclusion going on, causing fronting, but I think the explanation of sample solvent mobile phase mismatch mentioned above is the most likely cause.

[Mattias]

If understood it correctly, Candy decreased the injection volume to 3 µl without any improvement of peak shape. If you could do betting in chromatography, I would place my money on the column temperature!

[HW Mueller]

An injection of 3µL is no guarantee that there is no mismatch effect, nor, what I forgot above, that it neccessarily prevents overloading the column.