need help?

[moon704520]

hi:
I meet a strange phenomenon!have you ever met before?
we have a 50mmID*250mmL column here,when we test the column efficency with
mobile phase: 50%ACN:50water
Detector was 254nm,
flowrate:55ml/min
silica:C4
testing probe:paraben
everything is ok!but when I change the mobile phase to 50%MeOH:50water,the column efficency will decrease a lot ,and the T-factor will encreased a lot !
I just want to know why is so ?and do you have some recommendation on testing the column efficiency(50mmID*250mmL,C4)
thanks !

[sassman]

Methanol and acetonitrile are not the same. Changing from one to the other can dramatically affect selectivity, retention, and peak shape depending on the compound. You calculate column efficiency using the Van Deemter equation. Test column efficiency under the conditions that you plan to use for your analysis.

[mbulentcakar]

Hi,
To substitute %50 ACN, I would suggest %62.5 MeOH to obtain similar retention.
As a general rule, you can substitute ACN with MeOH but with proper ratios. 50% MeOH has a roughly equal solvent strength with 40% ACN.
But do not forget that this is not the only thing, Sassman already explained selectivity and peak shape issues.
For testing column efficiency: a column will have different efficiencies for different methods. There may be a lot of ways, virtually all of them previously discussed at the forum.
If you have a good correlation of paraben efficiency and your effective column life (for your method), then stick to your paraben injecting method.
Good luck,
Bulent

[Mark Tracy]

The viscosity of the mobile phase has a big effect on the efficiency of the separation. 50% MeOH is about three times as viscous as 50% MeCN.

[Consumer Products Guy]

Agree with above posters. You'll also get more reproducible results with parabens if you add a little acidity to the mobile phase (e.g. acetic or phosphoric).