By Anonymous on Tuesday, January 27, 2004 - 11:25 am:
I am developing an assay for purity of Vitamin D3 - cholecalciferol. Currently I am using a 150mm x 4.6mm, 5um C18 column with Methanol/water as mobile phase. I've tried gradients and isocratic in ratios from 80:20 MeOH:water to 100% water. The problem is is that there appears to be some sort of impurity, a minor peak co-eluting with the cholecalciferol peak. I am unable to resolve the two, changing the mobile phase only seems to effect retention time.
I am using a UV PDA detector, the minor peak has a very similar UV spectrum to the main peak.
I am not familiar with this analyte, I've never done vitamin D before. Does anyone have any suggestions as to what this peak may be, how to resolve it etc? Bear in mind that I am more of a GCMS guy than HPLC, so please speak slowly and avoid big words.
Also, how toxic is this material, I was a little intimidated by the MSDS...
Thanks,
Mike
By Einar Pontén - SeQuant AB on Tuesday, January 27, 2004 - 04:08 pm:
Mike,
It's "easy". You only need to keep three things in mind. Efficiency, capacity, and selectivity
If you have an efficient system expressed as a large plate number then bandbroadening within and outside the column is okey as well as flow rate.
While changing the mobile phase composition stepwise or in a gradient run then you mainly control the capacity factor (retention).
Finally, the whole great world of chemistry can be utilised to influence the selectivity. I think you should focus on that part.
For instance, try to use a 5-20 mM buffer salt in the eluent at different pH values (2-7 on silica column), try acetonitrile as organic modifier and so on..
Good luck!
By Anonymous on Friday, January 30, 2004 - 06:05 am:
USE
75% ACETONIRILE AND 25% METHANOL ON C18 250 CM 5 um column you will get nice one at 17 minutes which is stability indicating or even you can use BP method for analysis
By Anonymous on Thursday, February 5, 2004 - 12:59 pm:
Is it a synthetic analog of D3 or a real D3?
Also keep in mind that D3 is not water soluble, so 100% water is not a suitable mobile phase for it or its related compounds.
By Anonymous on Wednesday, March 17, 2004 - 01:23 pm:
Mike,
The interfering peak is probably vitamin D2 (ergocalciferol) or pro-D3. Both of these will have spectra pretty much identical to vit D3. Most reverse-phase columns do not give very good resolution of D2 and D3. One column that does is the Vydac 210TP54 column. We use a 25 cm column with mobile phase of 90:5:5 Acetonitrile:Methanol:Water at 1.5 mL/min. This should give you good resolution of the peaks and allow you to quantitate with appropriate standards.
-Ken