CrimpJiggler - 4-1-2012 at 01:15
So the silanol groups of the fused silica capillary tube are deprotonated at pHs above 3-4 which results in the interior wall of the capillary being
negatively charged. Cations present in the liquid inside the capillary tube are attracted to the walls and form a positively charged layer along the
wall. When an electrical field is applied across the capillary tube, the cations move towards the cathode and drag everything with them (the anions
and the solvent). What are these buffer reservoirs for? Are they to keep the liquid buffered at a pH above 4 to ensure that the silanol groups remain
ionised?
jwpa17 - 16-8-2013 at 19:05
For one thing, it's easier to apply the driving potential by simply immersing the electrodes in the buffer reservoirs, rather than threading
electrodes into the capillary itself. Also, in the early days (and maybe now - it's not my specialty) the buffer was changed every run to improve
reproducibility.
Protium1 - 17-8-2013 at 03:50
This is my First post here at ScienceMadness...YEAH!!!
While it is true that the at a pH < 3, the silanol groups along the wall of the column would be re-protonated, the main reason for the buffer
solution is to effect chromatographic resolution of differently charged analyte species.
Consider the separation of two amino acids, Asparagine(Asn) and Glycine(Gly). Let us make our buffer solution to be of pH=9.
Amino Acid______pKa COOH___pKa NH3+
Asparagine________2.02______8.80
Glycine___________2.34______9.60
At pH=9, both the Asparagine AND Glycine carboxylic acid groups are deprotonated, giving BOTH Asparagene and Glycine a -1 charge... The Amino groups
however differ...
For Asparagine, at pH=9, the amino group is deprotonated(neutral), since the pKa of the amino group is 8.80.
For Glycine, at pH=9, the amino group remains protonated(+1 charge), since the pKa of the amino groupt is 9.60
Amino Acid___Carboxylic___Amine_______Net Charge
Asparagine_____- 1______0 (neutral)_______-1
Glycine________- 1________+1_________0 (neutral)
Since Asparagine has a Net Negative(-) Charge, it will be attracted toward the Positive(+) Anode.
Glycine, having a Neutral Net Charge, is not attracted by either the anode nor cathode, and therefore only moves along the column with the speed of
the liquid mobile phase.
If, we assume the following:
The capillary Inlet is at a positively charged Anode,
The capillary Outlet is at a negatively charged Cathode,
Then Glycine will be Eluted Faster than Asparagine since both are drawn along by the movement of the liquid mobile phase, but Asparagine will be 'held
back' by the positive anode while Glycince is allowed to flow more or less freely long the column since it is not affected by the electrode charges.
NOTICE, it is the BUFFER that held these Net Charges of Asparagine and Glycine stable so that they be chromatographically resolved by their
differences in interaction with the electric field between the anode and cathode.