Hilic column for vitamine B complex

[centraing]

Hello,

Have anyone experience with Hilic columns for the determination of vitamine B complex (nicotinamin, pyridoxine, thiamine, riboflavine phosphate and dexpanthenol)?

Thursday I had an nice chromatogram for the separation of nicotinamin, pyridoxine, dexpanthenol and benzylalcohol. Thursday night I set up the HPLC with an mobile phase of 50% buffer (100mM ammoniumacetate pH5. and 50% ACN. IS this to much buffer?
Friday morning I can not recieve the same chromatogram as thursday. The peaks are now broad and they have a shoulder at the end. How can this hapen and how can I solve the problem? This weekend I set up the HPLC with 5% ACN and 95 % water. In an articel of hilic columns I have read that this is an regenerating method for hilic columns.

Thanks

Ing

[mjordan]

I'm curious to know what kind of HILIC column you're using. Do you have any more information regarding the specific column you have? Manufacturer? Model number? Sorbent lot number?

[tom jupille]

50% water (buffer) is toward the high end for HILIC (sort of the equivalent of running 100% organic in reversed phase), but should not hurt the column.

If you have multiple peaks all showing the same problem (e.g., shoulder), then there is probably a physical problem: something line a partially blocked inlet frit or a void space at the head of the column. If it's a frit problem, back-flushing the column may clear it up (check with the vendor first to make sure that the column can be back-flushed without doing damage!). If that doesn't clear it up, it's usually more cost-effective to simply replace the column rather than spending time to change the frit and/or repack the void ("your mileage may vary!").