ELECTROPHORESIS SLAB (THIN LAYER GEL) AND CAPILLARY METHODS. A. General Introduction

Transcription

1 ELECTROPHORESIS SLAB (THIN LAYER GEL) AND CAPILLARY METHODS A. General Introduction Electrophoresis: a saration method based on differential rate of migration of charged species in an applied dc electric field Rate of migration Dends on charge and size Saration based on differences in charge-to-size ratios High efficiency and resolution 1

2 Historical notes Initially deeloped by Arne Tiselius in the 1930 s Sarated serum proteins Slab gel electrophoresis: deelopped in the 1950 s Capillary electrophoresis: deelopped in the 1980 s Narrow bore tubes used Applications of electrophoresis: B. Principle and Theory of Electrophoresis Basic requirements Conductie medium (aqueous buffer/ electrolyte/ run buffer) Applied Electric Field Positiely charged species moe to the cathode (-) Negatiely charged species moe to the anode (+) Two Basic Techniques 1. Free solution: narrow capillary bore (Instrumental). Non-conducting matrix (Agarose, Polyacrylamide gel/page) a. Joule heating is minimal b. Sieing effect of gel allows saration of species with same charge to size ratio if they hae different sizes.

3 Efficiency dends on: Electrophoretic mobility (µ ) Electroosmotic flow of the bulk solution () Joule heating B-1 Electrophoretic Mobility Ion placed in an electrical field (E) experiences force F ef that is proportional to field strength of the charge (q) on the ion As the ion moes, a frictional force (f fr ) opposes the forward moement of the ion In a constant electrical field the elocity of particle is constant and dends on the balance between the two forces Electrophoretic mobility is the fundamental parameter which determines the efficiency of saration based on charge to size ratio (q/r) Change in ph effectiely alters the charge on the ions and their electrophoretic mobility. 3

4 Electrophoretic Mobility F F ef fr q E 6 π η r η : is cos ity r : radius : electropho retic migration elocity F ef F fr q E 6 π η r µ E q 6 π η r const q r µ : electropho retic mobility B- Joule Heating Ohmic/Joule heating heating occurs as charged particles moe within the conducting buffer upon application of an electrical field. Temperature gradient are generated leading to conectie flows within the electrolyte Conectie flow leads to band broadening Increase in temperature can also damage macromolecules. Method for decreasing joule heating: Decrease applied oltage- leads to long analysis time Dissipate heat- use thin gel or small diameter capillaries which hae large surface-to-olume ratio. Their electrical resistance is high, thus current flow is reduced for a gien oltage. 4

5 B-3 ElectoOsmotic Flow () refers to migration of the bulk liquid toward the cathode Origin: formation of a double layer at the wall of the capillary. Silanol groups are drotonated at ph higher than 4. Potential difference is established Positie ions in solution migrate to the wall. A double layer is deeloped at the wall of the capillary. Stern layer (SL): layer of positie charge that compensate for the negatie charge on the surface Diffuse layer (DL): layer adjacent to the Stern layer: layer of mobile cation B-3 ElectroOsmotic Flow () Upon application of the electrical field, cations within the double layer are attracted towards the cathode and drag the bulk solent with them. is in opposite direction to analyte electrophoretic flow is practically equal across the capillary, thus band broadening is minimal are not rroducible thus leading to irrroducible saration efficiencies in GE is minimal or practically inexistent dielectric cons t ς ε : tan : zeta potential( SL DL) µ ε ς E 4 π η E µ E 5

6 Use of in Capillary Electrophoresis If is more important that electrophoretic mobility, all analytes are dragged by the bulk solent towards the negatie electrode (Mobile Phase??!!) Electropherogram The order of elution is: fastest cation, slower cations, neutrals (single zone), slowest anion, faster anions ( µ e + µ eo ) E ll tm ( µ e + µ eo ) V l : lengt of ector L : length of det capillary Control of in Capillary Electrophoresis Work at low ph Dynamic coating of the channel walls With Polyethylene glycol (PEG) added to the buffer Polymer layer masks chages and suppress Chemical modification With trimethylchlorosilane-tmcs bonds to the surface and reduces the number of silanol groups- low Sulfonic acid- high Problem: long term stability Use addities to change the iscosity and zeta potential Hydroxy ethyl cellulose or polyinyl alcohol increase iscosity of the run buffer and thus reduce. Organic solents: methanol (reduce) and acetonitrile (increase) Cationic surfactants such as dodecyl trimethyl ammonium bromide (DoTAB) adsorb onto the capillary walls and thus change the surface charge 6

7 B-4 Saration Efficiency and Resolution Efficiency and resolution dend on both the electrophoretic flow and the Apparent mobility is gien by: µ app µ + µ If dominates, all analytes moe towards the cathode Efficiency and Resolution If is more important that electrophoretic mobility, all analytes are dragged by the bulk solent towards the negatie electrode (Mobile Phase??!!) Electropherogram The order of elution is: 1. fastest cation. slower cations 3. neutrals (single zone), 4. slowest anion 5. faster anions t ( µ + µ ) E ( µ + µ ) L L µ V app l : lengt of ector L length of det : capillary V L 7

8 Band Broadening Main contribution to band broadening: longitudinal diffusion Peak dispersion is proportional to the diffusion coefficient, D and the migration time of the analyte In theory: efficiency superior to LC. Calculated N at operating oltages are on the oreder of In practice: joule heating, sample injection and adsorption of analyte to matrix decrease efficiency σ D t L N σ µ app V D D L µ V app Resolution Resolution dends on the difference in electrophoretic mobility What is the equialent in LC? Resolution dends on the applied oltage V, the apparent electrophoretic mobility and the diffusion coefficient D. What are the equialents of these terms in LC? R s µ V 1 µ app 4 1 D 8

9 C. GEL ELECTROPHORESIS Matrix: electrically non-conductie hydrogel containing buffer Agarose Polyacrylamide Adantages Porous gel acts as a siee Gel limits diffusion of sample molecules is suppressed Joule heating is suppressed Disadantages Slow, labor intensie and not readily automated Techniques Natie gel electrophoresis: analyte sarated according to differences in apparent mobility Sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE): analyte sarated according to size Isoelectric Focussing (IEF) Two dimensional gel electrophoresis (D-GE) 9