Chromatography Forum

My question is in regards to buffers and running gradients. For example, if I will prepare a 10mM phosphate buffer for reservoir A and want to run a gradient (5%-95%B), would I need to add phosphate to reservoir B, the organic (e.g. ACN/MeOH), and thereby make it 10mM as well? I assume not, but don't know.

If I don't add phosphate buffer to the organic reservoir (B), then what would one consider the optimal buffer concentration if peaks eluted ideally, for example, at 50% A(10mM) and 50% B? In other words, would an isocratic run to follow use a 5mM phosphate concentration for mobile phase (50:50)? Or would I keep the phosphate concentration at 10mM? This probably sounds elementary, but hope to get some feedback! Thanks alot!

Lastly, I heard one should not run gradients with IP reagents such as alkylsulfonates. Any thoughts?

Thanks,
Phil

I think it is a good question, which I heard the first time.
Here is my thinking:
1) usually we want enough buffer capacity instead of accurate. for example in your gradient

For example, if I will prepare a 10mM phosphate buffer for reservoir A and want to run a gradient (5%-95%B),

at 95% B MP your buffer concentration is 0.5mM. If 0.5mM is enough, the whole run has good buffer.

2) if you want to keep the buffer concentration constant through the gredient you can (if you are able to since you may not be able to dissolve the same amount of the salt into the organic). this may help a bit for the base line if you do though may not much.

the major reason preventing IP gradient run probably is that it takes a long time for the column to stablize in the IP MP. an unstabilized run gives rough base line.

Thank you for the feedback ym3142. I greatly appreciate the insight. Certainly, the question wasn't worded the best, but it sounds like you caught on to what I was attempting to communicate.

Basically, it would seem then that the optimal isocratic follow up would then be (potentially) the decreased mM concentration relative to the % organic ramped in... True, the salt solubility would not be the same as that of the organic, but sounds like it helped to get the string of ideas across.


Thanks alot for the feedback,

Phil

When I am doing development work, I often use ternary systems with organic in A, water in B, and buffer/IP/etc in C. I can always turn it into a binary system later.