Sciencemadness Discussion Board
Not logged in [Login ]
Go To Bottom

Printable Version  
Author: Subject: Capillary Electrophoreses - What is the buffer for?
Hazard to Self

Posts: 75
Registered: 1-9-2011
Member Is Offline

Mood: No Mood

[*] posted on 4-1-2012 at 01:15
Capillary Electrophoreses - What is the buffer for?

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?
View user's profile View All Posts By User

Posts: 45
Registered: 28-5-2013
Member Is Offline

Mood: No Mood

[*] posted on 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.
View user's profile View All Posts By User

Posts: 6
Registered: 17-8-2013
Member Is Offline

Mood: No Mood

[*] posted on 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+

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.
View user's profile View All Posts By User

  Go To Top