Chromatography Forum

Continue from the same topic posted on Mar 25,2006(in page 33 now)


Based on (1):

B + H <-> BH

And (2):
B•H/BH=K

We can get a general equation (3):

Tr = (Tb•K+Tbh•H)/(K+H)

Where:

Tb is theoretical parameter, base (B) retention
Tbh is BH retention

Change equation (3) to (4):

Tb•K+Tbh•H-Tr•K=Tr•H

This is the calculation and regression equation from known Tr and pH by testing the compound in three or more different pH mobile phases. Then we’ll know Tb, Tbh and pK (from K).

Then we can calculate out the compound’s retention (Tr) in any pH of mobile phase by equation (3) and plot out the “pH—Tr “curve that just like a titration curve.

If two compounds their Tbs are almost the same and their Tbhs are almost the same, too, you can find out the maximum separation in certain pH. You can also put more compounds together to find the best separation.

All above if you can use Excel, it’s not only simple and even enjoyable.

Mobile phase is simple. Different pH buffers add the same ratio of organic to adjust the Tr in an acceptable range for tested pHs, that’s all.

I hope I can discuss with interested people. If you need my Excel spread sheet for your references, I can give you.

If you have any questions, you are welcome to post it and discuss it.

Sorry, it's posted on Mar 12, 2006(on p.33)

The key is the formula (1)

B+H<—>BH

B and BH are the two statuses of one species. They change to each other very quick.
This is a dynamic balance:

(B•H)/BH=K or B/BH=K/H

K is the balance constant.

That mean:

I. Any moment, in mobile phase, B/BH=K/H

II. But different moment, the particles are different. That mean, in moment A, may be particles a, b, c, d in B status and particles e, f, g, h in BH status. But in moment B, may be particles e, f, g, h in B status and particles a, b, c, d in BH status, though they still meet B/BH=K/H.

III. Finally, every particle undergoes two status, just timing different. And their time in B status / time in BH status are still =K/H

IV. Based on above analysis, every particle has the same retention time and the Tr is the Tb and Tbh weighted average.

That is Tr=Tb•(K/(K+H))+Tbh•(H/(K+H)), after rearrange:

Tr=(Tb•K+Tbh•H)/(K+H)

That is why two kinds of particles only show one peak. But this is only when particles average switch period between two status<T0 (void time). If period <<T0, the peak is very sharp. Peak becomes wider and wider along with longer and longer period. When period>T0, peak begin split. Peaks become away from each other along with the longer and longer period. Finally they become two independent peaks (two species)