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- Posts: 22
- Joined: Wed Sep 29, 2004 6:33 am
Dear Colleagues,
I found a listing of buffers to be used in certain pH's, and one option seemed to be potassium hydrogen phthalate in the range of 4.1-5.9. Does anyone have any expeciences of it? What is the UV cut-off?
I'm trying to optimize a method RP (C18 column) at the pH5 (the unionized form of API dominates having lambda max 215nm) that now has a acetate buffer. The main peak elutes nicely, but because of some late eluting peaks, I have tried several ways to make method faster.
In the current method the column lenght is 250mm for the best resolution of API and related substances. Shortening the column makes the early eluting peaks to co-elute. For the best resolution of early eluting peaks, less organic would help, but having a lower concentration of organic modifier (acetonitrile) in the mobile phase makes the late eluting peaks to elute even later. I also tried other option, like changing to monolithic column in order to have flow-gradient for faster method, but that was not that successful because of slightly worse retention power of monolithic (100mm long).
So best thing I can figure out is to have a gradient. For pH=5 it is hard to find a buffer other than acetate. I've tried having a gradient (even very easy), but acetate gives downward drifting baseline making the integration difficult. Citrate is out of question because of high UV cut-off.
I could maybe have a slightly higher pH of mobile phase (pH=6) so possible buffers might be carbonate or phosphate. But the buffering capacity of these is still at minimum at pH=6. Furthermore carbonate (CO2 volatile, possible pH changes of MP to alkaline) might give problems, so final option seems to be phosphate. Then I found this phthalate, but I have no experiences. Any advices or comments?
Thank you in advance.
I found a listing of buffers to be used in certain pH's, and one option seemed to be potassium hydrogen phthalate in the range of 4.1-5.9. Does anyone have any expeciences of it? What is the UV cut-off?
I'm trying to optimize a method RP (C18 column) at the pH5 (the unionized form of API dominates having lambda max 215nm) that now has a acetate buffer. The main peak elutes nicely, but because of some late eluting peaks, I have tried several ways to make method faster.
In the current method the column lenght is 250mm for the best resolution of API and related substances. Shortening the column makes the early eluting peaks to co-elute. For the best resolution of early eluting peaks, less organic would help, but having a lower concentration of organic modifier (acetonitrile) in the mobile phase makes the late eluting peaks to elute even later. I also tried other option, like changing to monolithic column in order to have flow-gradient for faster method, but that was not that successful because of slightly worse retention power of monolithic (100mm long).
So best thing I can figure out is to have a gradient. For pH=5 it is hard to find a buffer other than acetate. I've tried having a gradient (even very easy), but acetate gives downward drifting baseline making the integration difficult. Citrate is out of question because of high UV cut-off.
I could maybe have a slightly higher pH of mobile phase (pH=6) so possible buffers might be carbonate or phosphate. But the buffering capacity of these is still at minimum at pH=6. Furthermore carbonate (CO2 volatile, possible pH changes of MP to alkaline) might give problems, so final option seems to be phosphate. Then I found this phthalate, but I have no experiences. Any advices or comments?
Thank you in advance.
