Chromatography Forum

(Continue from my previous post)
If two compounds are too similar, extremely, if their molecule retention are the same and their ion retentions are the same, too, they still can be separated providing their pKs have a little bit difference. Then you adjust your mobile phase pH to ½ (pK1+pK2), you can get maximum separation. Even if the difference is as small as 0.5pK unit or even less, you still can get significant separation.

But how can we know the compound's pKs in applied mobile Phase? Adjust mobile phase to different pHs (at least 3 points) and get related retentions. Then, do regression. Because this is a three-dimension regression, we have to use matrix calculation. I already created the matrix regression Excels from 3 points to 7 points. If you have more points, I can create for more. You just simply fill in the pHs and related retentions (RT). You can get pK, correlation coefficient, molecule retention and ion retention. But I don’t know how to post my Excel on Forum without losing its functions.

But I don’t know how to post my Excel on Forum without losing its functions.

There is no way to "post" a spreadsheet in the Forum. The easiest approach would be to simply give an e-mail address for inquiries and then attach the Excel file to your replies. An alternative would be to save a copy in a web-accessible location and then put the URL in a posting here on the Forum.

"simply fill in the pHs". Sounds simple but where do these pH values come from? Once an orgainic solvent is added to an aqueous buffer the buffer pH changes and so does the pKa of what ever is being separated. So the optimum pH for the separation is a function of the relevant pKa as well as where in the gradient the separation occurs. And then to further confuse the theoretical situation we can toss in effects of ion pairing equilibria if the spearation is done at high organic content. I think the situation is too complex to model.

If you don't mind, I would appreciate if you send your spreadsheet to my e-mail address.

Thanks
Uwe

[quote="Bill Tindall"]"simply fill in the pHs". Sounds simple but where do these pH values come from? Once an orgainic solvent is added to an aqueous buffer the buffer pH changes and so does the pKa of what ever is being separated. So the optimum pH for the separation is a function of the relevant pKa as well as where in the gradient the separation occurs. And then to further confuse the theoretical situation we can toss in effects of ion pairing equilibria if the spearation is done at high organic content. I think the situation is too complex to model.[/quote]

pH can be obtained by pH meter. That means in isocratic, first you use mobile phase with pH 3, for example, run sample and get a retention, then just change mobile phase pH to 5, run the same sample and get the second retention. So on, keep mobile phase all other components the same, just change pH, and run the same sample to get different retentions. Definitely, this is apparent pH, and practical pH, also.

[quote="Uwe Neue"]If you don't mind, I would appreciate if you send your spreadsheet to my e-mail address.

Thanks
Uwe[/quote]

Sure, but I can't find your email address. Could you give me your email address?

All you need to do is click on the e-mail button. In case that you can't get it to work:

uwe.neue@prodigy.net

OK. if the separation is isocratic and pH measured in the actual mobile phase, which you made clear, then I have no fault with the approach. In practice it may even work to get a useful estimate in gradients.

But I wonder what equations you use with what approximations/assumptions to get within about 0.5pKa units based on retention time and pH. I am also having a considerable problem with that max separation at 1/2 the sum of pKa.

[quote="HW Mueller"]But I wonder what equations you use with what approximations/assumptions to get within about 0.5pKa units based on retention time and pH. I am also having a considerable problem with that max separation at 1/2 the sum of pKa.[/quote]

I have a presentation to give the whole theoretical understanding, explanation and calculation in detail. If there is a suitable chance, I like to present it.

Given that the three factors for column resolution are
based upon selectivity, efficiencey, and retention - one
can increase resolution by increasing the efficiency of
the column being used.

Our Cadenza and Unison (3 micron) RP columns can get
200,000 plates/m. Please see below for column efficiency
and pressure data.

http://www.imtakt.com/TecInfo/TI064E.pdf

http://www.imtakt.com/TecInfo/TI147E.pdf

Ok, Bryan, I'll play "agent provocateur" here.

200,000 plates sounds great. How soon can you deliver a 1-m long column?

Ok, Bryan, I'll play "agent provocateur" here.

200,000 plates sounds great. How soon can you deliver a 1-m long column?

200,000 plates will probably take a while, but we can send you
a 100,000 plate column immediately!

Bryan,

Well I'm still interested. Please let us know the dimensions of your 100,000 plate column and the flow rate necessary to realize this figure.

sunht, why don't you publish in Journal of Chromatography?