Hi Everyone,
I work in a lab for a pharmaceutical company. The brand new column we purchased can only last 16 injections (a week) shown by lowered peak separation index and badly shaped peaks.
Here I list the parameters and any help will be greatly appreciated!
A polar compound is being tested so 100% aqueous mobile phase is used. Temperature 30C, Flow rate 1.0ml/min, pH 6.6, UV detection 210nm. Pressure 115 bar. Solvent A and B： 0.2% potassium dihydrogen phosphate and 0.1% TBAH (tetrabutylammonium hydroxide solution). Equipment: Agilent 1260 infinity. Column used: Thermo Hypersil Gold C18 AQ 4.6mmX250mm 5um.
Question: Anything we can change to lengthen the lifetime of the column? Are there other column recommended for TBAH which is believed to be a damaging agent?
If anyone had similar experience and happened to fix this please offer your insight. Thank you so much!
Simon
It is likely you have a non-polar compound in your sample matrix which is being completely retained by your column. I would reverse and wash the column with 100% IPA in order to extend the column's lifetime.
You might want to add a column wash step at the end of an injection sequence.
Although using a "AQ" type column, is it possible you are still seeing "phase collapse"? The buffer makes the solvent system more polar than plain water. Try washing with plain water (to keep the phosphate buffer from precipitating in the next step), then wash with methanol or acetonitrile. Wash with water again, and try another run.
Also, what pH are you running? If the column you are using is silica-based (I'm almost certain that it is), and the pH is above 7.5 or 8, the silica underlying the C18 will dissolve and you will see bad peak shape and possibly higher back pressure.
It is likely you have a non-polar compound in your sample matrix which is being completely retained by your column. I would reverse and wash the column with 100% IPA in order to extend the column's lifetime.
You might want to add a column wash step at the end of an injection sequence.
Wash column with deionized water after use directly ( if time of analysis is short) then wash with storage column solution (recommended from the manufacture).
if time of analysis is long, try to wash the column with deionized water every day or when the column back pressure is high.
I think others have covered how to deal with de-wetting of the column due to high water mobile phase composition well. Washing out salts and flushing with acetonitrile or other less polar organic solvent.
There are also other options as far as the type of column to be employed for very polar compounds that may fit better if there is flexibility in what method is used. HILIC columns and chromatography or other stationary phases (such as Polar-RP and family of similar columns from Phenomenex or others). This might offer you a more ideal solution. In HILIC the more polar compounds are retained longer, so may be worth a try.
Best of luck.
Randy
I agree with the idea of using HILIC columns for polar compounds. However, I have done a fair amount of work with the Phenomenex Gemini C18 column which tolerates wide range of pH (1-12) and 100% aqueous solutions. Seems almost indestructible.
- Badges
- Science method