to separate dexamethasone and Betamethasone

[ym3142]

I saw Dr. Pyrex reported his success with hypercarb column to separate the dexamethasone and betamethasone though he did not provide the detail.
I was able to separate with Rs 1.5 using Luna C18 3u ACN.
anyone else here made some new progresses on this subject?

And also my question is to separate things having so many carbon with only one spacially different methyl group, what is the possible approaches ?

Dr. Pyrex(hope spell correctly), could you please let me know how well you could separate these?

[dr_Pyrex]

I am really impressed reading your success in separation of dex- and betamethasone. What are retention times of substances with your HPLC conditions? Is it gradient or isocratic elution?
In method development I've started with Luna C18 (3um and 5um) columns. I used ACN (or MeOH) and 0,05M ammonium acetate gradient. I had poor separation of dex- and betamethasone (RT about 10 min).
Then I found posters and publications when HyperCarb columns were used.
This column gave large retention of these analytes (look mobile phase and temperature).
My chromatographic conditions:
Mobile phase: ACN: 0.3% formic acid 90:10 (isocratic)
Flow rate: 0.25 ml/min
Column Oven temp: 55 C
Detection: MS/MS
Retention times:
DEX: 4.5 min
BETA: 5.5 min

I hope my explanations are clear

[ym3142]

Dr. pyrex,
your separation impressed me too.
I am using luna C18(2) 3u 150x4.6, no temp control, buffer 20mM KH2PO4 pH 2.2 with 35% ACN isocratic. Rs1.5 is good enough for me since it is a related compound but the back pressure was too high so I do not like it when I try to use this column for my assay.

Thank you for quick reply.

[ym3142]

actually I found a Synergi Max RP can give Rs 1.2; Symmetry C8 Rs1.3-1.4.