Capillary electrophoresis Flashcards
State the effect of crosslinking on an ion exchange column
It gives decreased swelling, increased exchange capacity and selectivity but longer equilibrium time
Consider a negativly charged protein adsorbed on an anion exchange gel at pH8. How will gradient of eluent pH (from pH8 to some lower pH) be useful for eluting the protein? Assume that the ionic strenght of the eluent is kept constant.
As pH is lowered the anionic protein becomes protonated so the magnitude of the negative charge decreases. The protein becomes less strongly retained by anion exhcange gel.
Consider a negativly charged protein adsorbed on an anion exchange gel at pH8. How would a gradient of ionic strenght (at constant pH) be useful for eluting the protein?
As the ionic strenght of eluent is increased the protein will be displaced from the gel by the increasing concentration of anions in the eluent.
How can size exclusion chromatography be used to measure the molecular mass of a protein?
There is a range in which retention volume is logaritmhycly related to molecular mass. The unknown is compared to standards of a known molecular mass.
What is electroosmosis?
It’s the bulk flow of fluid in capillary caused by migration of the dominant ion in the diffuse part of the double layer toward the anode and the cathode.
State three different methods to reduce electroosmotic flow. Why does the direction of electroosmotic flow change when silica capillaru is coated with a catiomic sufactant?
It can be reduced by lowering the pH so the charge of the capillary wall is reduced, add ions that adhere to the capillary wall and effectivly neutrilize it’s charge, and covantly attatching silanes with neutral hydrophilic substiuents to the Si-O groups of the wall. A cationic surfactant layer that effectivly reverses the charge of the wall.
Explain how neutral molecules can be separated by micellar electrokinetic chromatography. Why is this a form of chromatography? Why are micelles called pseudostationary phase?
In abscence of micelles, neutral molecules are all swept throught the capillary at the electpopsmotic velocity. Negativly charged micelles swim upstream with some elecrophoretic velocity, so they take longer tan neutral molecules to reach the detector. A neutral molecule spends some time free in solution and some time dissolved in the micelles. Therefore the net velocity of the neutral molecule is reduced from the electroosmotic velocity. Because different neutral molecules have different partition coefficients between the solution and the micelles, each type of neutral molecule has it’s own net migration speed. We say that micellar electrokinetig chromatogarpht is a form of chromatographt because the micelles behave as station phase in capillary because their concentration is uniform throughout the capillary. Analyte partitions between the mobile phasde and the micelle as the analyte travels thriugh the capillary. Micelles are called a speudostationary phase because they are not stationary and their concentrationis constant in the capillary because they are åart of the run buffer.
