Posted: Thu Jan 26, 2006 2:51 am
by Uwe Neue
We find the pH manipulation very useful, especially in exploratory schemes. You can get very drastic differences in elution pattern going from pH 3 to pH 10. Then you can finetune the separation with the more conservative tools.
Posted: Thu Feb 23, 2006 4:20 pm
by sunht
[quote="Uwe Neue"]I do not agree that it is important to stay by 2 pH units away from the pK of your analytes. If you control the pH well (see the discussions on the other posts), you will get reproducible results in the interim range as well. In addition, the pK of your analyte and the pK of your buffer will cahnge as you add organic, therefore this rule can rarely be followed properly.[/quote]
That's right. It depends on your buffer capacity. If well buffered, for example: K2HPO4:NaH2PO4(they are very soluble in water) from 1:10 to 10:1 and in 20mM to 50mM, you even can work at less than 0.5 pH unit away from the pK. But you have to prepare buffer correctly and the ratio deviation should be less than 5% to keep the retention shift in tolerable range. Knowing this is very useful in separation of very similar compounds. See my later post.