B1 Analysis on PLRP-S column

Discussions about HPLC, CE, TLC, SFC, and other "liquid phase" separation techniques.

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Hi,
I am trying to come up with a gradient for this column. My two mobiles phases (pH=7) are 90/10 na2h2po4/meoh and 30/70 na2h2po4/meoh. I have ran this from 0%b to 100%b over 20min. After injecting 15uL of sample (pH=13) on a 2.1x50mm, 3um PLRP-s column (flow:0.3 mL/min) my two analytes (TMP and B1) eluted around 3min for TMP (very board mound, almost splitting) and 14.9min for B1 ( very sharp). What would be the best approach from here and why is the TMP not sharp like the B1? Is it due to the pH difference between MP and sample?
As a general rule, you should have the composition of your sample as close as possible to the starting composition of your mobile phase. So that's where I'd start.
Another possible cause might be the temperature of your mobile phase as it enters the column. If you have a preheater make sure it's switched on and set to the appropriate temperature.
2 posts Page 1 of 1

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