Determination of vitamin D and E

[Lorespain]

Hello! I'm having problems with these vitamins. I have done correctly the calibration curves for each one at 0,800 ml/min, 280nm and Hexane:ethylacetate (70:50) as mobil phase.
I've just started testing the vitamins in fish (method USP24) and vitamin D soesn't appear in the chromatogram and the retention times for tocoherol have changed.
My tutor ask me to try one extraction with a known amount of the vitamins (without fish) to see if we lose vitamins during the process. So I prepared a solution with 10mcg of Tocopherol and 0,2mcg of vitamin D in ethanol:water (50:50), I did the extraction diluting the vitaminis with hexane and after the evaporation under nitrogen I disolve them in 4 ml mobil phase. So the concentration must be now 10/4 mcg/ml vitamin E and 0,2/4 mcg/ml vitamin D.
The thing is that the peak areas obtained were higher than the areas corresponding to these concentrations of vitamin in the calibration curve and besides I can't determine which peak matchs with the differents forms of tocopherol since the retention times have changed comparing to these ones obtained when I did the calibration curves.
Can anyone help me?
Sorry for te long message

[tom jupille]

I can't help in detail, but I can point out that if your retention times have changed significantly, then your original calibration curves are no longer valid. You will have to re-run the calibrations.

[Lorespain]

Thank you tom jupille . Anyway I have another question. As I said before, after extraction and evaporation of hexane I disolve the residue in 4 ml mobil phase (50 hexane:50 ethylacetate) and I filter in a 0,45 mcm membrane filter. After filtration I could see that the solution was not homogeneous, like in two phases. Can be this posible?What is the explanation?

[tom jupille]

hexane and ethyl acetate are listed as "miscible", so I wouldn't expect two equal layers. If you vacuum filtered, is there a chance that evaporative cooling allowed some water vapor to condense? (wild speculation here!)

[Lorespain]

Hello again! The filtration isn't it vacuun.. And there are not two equal phases, it's like one phase but with a cm on the top of another one. I don't know if I am explaining well. Thank you for answer

[tom jupille]

Do you see the two layers with standard solutions, or only with actual sample extracts? Is there a chance that you are also extracting out lipids/waxes/?? which are more soluble in hexane than in your mobile phase?

[Lorespain]

I only see the two layers when I am working with the food sample or maybe i haven`t notice with standards..could be that using 50 hexane, 50 ethylacetate to disolve the residue, lipids don`t disolve properly because of polarity? It is posible that some water remains in the residue after evaporation? I am getting a bit crazy

[Lorespain]

I am going to try this changes to find out the mistake:
mobil phase Hexane 100% or hexane:isopropanol (95:5)
increased volume injection from 20µl to 40µl
increased wavelenght and change flow rate
use a higher volume of saponificated sample in extraction

Do you think this could be useful? Could you tell me another things to try to find out where´s the problem or optimize the process.

Thank you

[tom jupille]

.could be that using 50 hexane, 50 ethylacetate to disolve the residue, lipids don`t disolve properly because of polarity?

That would be my speculation. What happens if you do the initial extraction with the 50/50 EtOAc/hexane?

It is posible that some water remains in the residue after evaporation?

. That was my initial speculation, but seems relatively unlikely.

Do you think this could be useful? Could you tell me another things to try to find out where´s the problem or optimize the process.

First things first! If this were my problem, I would start by running my standards through the prep/cleanup process to establish recovery. Wavelength should be standardized for these compounds, so I wouldn't spend too much time in that direction. As far as flow rate, I like to run as fast as possible consistent with reasonable operating pressure (different people define "reasonable" differently).

Once I have established that I can get consistent results through the cleanup steps, I would repeat with spiked samples. Compare spiked and unspiked samples to make sure that I get consistent recovery. It's probably at this point that any problems with lipids, etc. will become evident, and may require modification of the sample prep -- at which point we start over!

[Lorespain]

Thank you for your advice I have been talking to my tutor and he ask me to run the LC with isopropanol 100% as mobil phase. I can't undertand because if the colum is Nucleodur NH2 high purity silica (polar) I would have to use non polar mobil phase, wouldn't I? And isopropanol is polar.
Can you explain to me this?

[tom jupille]

The polarity of the mobile phase must be adjusted to give reasonable retention of your analytes.

Check with your tutor; I suspect that what was meant was a hexane/isopropanol mixture (remember, you will need less isopropanol than ethyl acetate to give the same overall retention).

If the problem is residual water, isopropanol may be a better co-solvent than ethyl acetate in terms of maintaining a single phase.

[Lorespain]

Hi! My last day of work I was trying the extraction with Hexane:Isopropanol (95:5) and then changing the wavelenght in LC to 229nm because the spectrometry showed this was the best one for vitamins D and E. The thing is that the base line in the chromatogram wasn't straight, it was inclined and decreasing mV all the time. Do you have to stabilize the column or do anything when you change solvents or wavelenght?It's the first time I change them and I have no idea. I passed throught the colum the new eluyent during 30 minutes but the problem didn't solve.
Thank you for help me, I am a beginner in LC.

[tom jupille]

Did you change the extraction solvent or the mobile phase?

[Lorespain]

Yes, I tried the extraction with hexane 100% and then I disolved the residue with hexane:isopropanol (95:5). I used this mix as mobil phase. Now vitamin D appears at 229nm and at 280nm too. But I am having problems with tocopherol, peak of alpha-tocopherol is so small and vitamin D peak and delta-tocopherol overlap a little. Appart, when I run tocopherol alone baseline drifts, but not when I run it with vitamin D. Do you know why? What do you recomend me to improve the separation?