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

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

Historical notes Initially developed by Arne Tiselius in the 1930 s Separated serum proteins Slab gel electrophoresis: developed in the 1950 s Capillary electrophoresis: developed in the 1980 s Narrow bore tubes used Applications of electrophoresis: Separation of proteins and nucleic acids (single nucleotide differentiation capability!) The human genome Project Human DNA: ~three billion nucleotides B. Principle and Theory of Electrophoresis Basic requirements Conducting medium (aqueous buffer/ electrolyte/ run buffer) Applied Electric Field Positively charged species move to the cathode (-) Negatively charged species move to the anode (+) Two Basic Techniques 1. Free solution: narrow capillary bore (Instrumental) 2. Non-conducting matrix (Agarose, Polyacrylamide gel/page) a. Joule heating is minimal b. Sieving effect of gel allows separation of species with same charge to size ratio if they have different sizes. 2

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

Electrophoretic Mobility F F ef fr = q E = 6 π η r v ep η : vis cos ity r : radius v : electropho retic migration ep velocity F ef = F fr v ep = q E 6 π η r µ ep = v E ep = q 6 π η r = const q r µ : electropho retic mobility ep B-2 Joule Heating Ohmic/Joule heating heating occurs as charged particles move within the conducting buffer upon application of an electrical field. Temperature gradient are generated leading to convective flows within the electrolyte Convective flow leads to band broadening Increase in temperature can also damage macromolecules. Method for decreasing joule heating: Decrease applied voltage- leads to long analysis time Dissipate heat- use thin gel or small diameter capillaries which have large surface-to-volume ratio. Their electrical resistance is high, thus current flow is reduced for a given voltage. 4

B-3 ElectoOsmotic Flow (EOF) EOF refers to migration of the bulk liquid toward the cathode Origin: formation of a double layer at the wall of the capillary. Glass, fused silica, agarose exibit surface charges Silanol groups are deprotonated at ph higher than 4. Potential difference is established Positive ions in solution migrate to the wall. A double layer is developed at the wall of the capillary. Stern layer (SL): layer of positive charge that compensate for the negative charge on the surface Diffuse layer (DL): layer adjacent to the Stern layer: layer of mobile cations SL Incomplete neutralization B-3 ElectroOsmotic Flow (EOF) Upon application of the electrical field, cations within the double layer are attracted towards the cathode and drag the bulk solvent with them. EOF is in opposite direction to analyte electrophoretic flow (generally) EOF is practically equal across the capillary, thus band broadening is minimal EOF are not reproducible thus leading to irreproducible separation efficiencies EOF in GE is minimal or practically inexistent v dielectric cons t ς ε : tan : zeta potential( SL DL) µ v EOF EOF EOF ε ς E = 4 π η v = E = µ EOF EOF E EOF HPLC flow 5

Use of EOF in Capillary Electrophoresis If EOF is more important that electrophoretic mobility, all analytes are dragged by the bulk solvent towards the negative electrode (Mobile Phase??!!) Electropherogram The order of elution is: fastest cation, slower cations, neutrals (single zone), slowest anion, faster anions ++ + - -- ++ + _ - -- Control of EOF in Capillary Electrophoresis Work at low ph Dynamic coating of the channel walls With Polyethylene glycol (PEG) added to the buffer Polymer layer masks charges and suppress EOF Chemical modification With trimethylchlorosilane-tmcs bonds to the surface and reduces the number of silanol groups- low EOF Sulfonic acid- high EOF Problem: long term stability Use additives to change the viscosity and zeta potential Hydroxy ethyl cellulose or polyvinyl alcohol increase viscosity of the run buffer and thus reduce v EOF. Organic solvents: 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

B-4 Separation Efficiency and Resolution Efficiency and resolution depend on both the electrophoretic flow and the EOF Apparent mobility is given by: µ app = µ ep + µ EOF If EOF dominates, all analytes move towards the cathode (-) Efficiency and Resolution If EOF is more important that electrophoretic mobility, all analytes are dragged by the bulk solvent towards the negative electrode (Mobile Phase??!!) Electropherogram The order of elution is: 1. fastest cation 2. slower cations 3. neutrals (single zone), 4. slowest anion 5. faster anions v = t ( µ + µ ) ep EOF 2 L L = = v µ V app E = µ app t : migration time L : length of capillary V L 7

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 voltages are on the order of 10 6. In practice: joule heating, sample injection and adsorption of analyte to matrix decrease efficiency σ 2 = 2 D t = 2 L N = σ 2 µ app V = 2 D 2 D L µ V app 2 Resolution Resolution depends on the difference in electrophoretic mobility What is the equivalent in LC? Resolution depends on the applied voltage V, the apparent electrophoretic mobility and the diffusion coefficient D. What are the equivalents of these terms in LC? Optimizing resolution: R s = µ ep V 1 µ app 4 1 2 D 8

C. GEL ELECTROPHORESIS Matrix: electrically non-conductive hydrogel containing buffer Agarose Polyacrylamide Advantages Porous gel acts as a sieve Gel limits diffusion of sample molecules EOF is suppressed Joule heating is suppressed Disadvantages Slow, labor intensive and not readily automated Techniques Native gel electrophoresis: analyte separated according to differences in apparent mobility Sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE): analyte separated according to size Isoelectric Focussing (IEF) Two dimensional gel electrophoresis (2D-GE) C-1 Instrumentation for Gel Electrophoresis Power Supply 200-500 V 400 µa -100 ma Electrophoresis Chamber with Buffer Reservoir Electrodes in buffer reservoir Mini Gel 8 cm x 8 cm Larger gel 40 x 20 cm 9

Gel Media Agarose 1g in 50 ml (2%) Dissolve, heat, cool, pour in casting stand Large pore size (e.g. 150 nm for 1%): no sieving Charged surfaces: EOF present Polyacrylamide Gel Polyacrylamide Copolymerization of acrylamide and N,N - methylene-bisacrylamide Initiator: TEMED (N,N,N,N - Tetramethylethylenedia mine) and Ammonium Persulfate(NH 4 ) 2 S 2 O 8 ) Sieving effect present: pore size depends on total gel concentration (%T: 5-20%) and degree of cross linking SDS for denaturinf and coating proteins Mercaptoethanol for reducing dissulfide bonds 10

Sample Preparation Buffer: Tris (ph 8), Tris-glycine (ph 9.1) 50-100 mm Amount of sample: µg Sample volume: depends on size of well ( µl to ml) Denaturing agent: SDS β-mercaptomethanol: to reduce disulfide bonds Visualization and detection Staining Coomasie brillant blue (alchoholic solution of dye) Silver nitrate Fluorescent reagents Ethidium Bromide for DNA 11

SDS PAGE Proteins are totally denatured and coated SDS charge (negative): rod-like shaped Charge on protein depend on its size: constant net charge per unit mass: same electrophoretic mobility Separation based on size/ molecular weight Sample preparation: heating (90) in the presence of SDS and β-mercaptoethanol Used to determine MW of proteins Molecular weight standards used Isoelectric Focussing (IEF) Separation based on pi Agarose gel used ph gradient from cathode to anode Several ampholytes with different pi used (same concentration to maintain conductivity constant) Gel immersed in medium ph mixture (1 2 % carrier ampholyte) Most ampholytes are charged Anode compartment: low ph buffer in (lower than the lowest pi) High ph buffer in cathode compartment (higher the highest pi) Apply field: Negatively charged amphoyte will towards the anode (lowest pi at end). Positively charged ampholytes will move towards the cathode (highest pi at end). Cathodic side becomes more acidic Anodic side becomes more basic ph range determined by choice of ampholytes 12

2D GEL First dimension separation in single lane: IEF Second dimension separation: SDS-PAGE 1000 to 2000 proteins can be separated Capillary Electrophoresis Instrumentation Capillary Fused silica Diameter: 20 to 100µm Length 10-100 cm Typical voltages: 10-30 kv Current 300µA Temperature control to control EOF and Joule heating Buffer (10 100 mm): ph and conductivity control Detection: UV, fluorescence, refractive index, etc. 13

CE: Performance Characteristics EOF present Convective flows due to joule heating Excellent for both large and small biomolecules (amino acids, peptides) Short separation time Small sample: nl Solvent consumption low: few ml Neutral analytes can be separated using Micellar ElectroKinetic Chromatography (MEKC). Automatic pressure or electrokinetic sample injection - Built-in capillary rinsing system - Convenient access to the vials and capillary - Possibility of visual check of the position of the vials and capillary rinsing - Liquid cooling system in CAPEL103 and in CAPEL-105, CAPEL-105M - Built-in monochromator (190-380 nm) in CAPEL-105 and CAPEL-105M 14

Capillary Zone Electrophoresis (CZE) Controlled electroosmotic flow used Separates anions as well as cations Separation of cations: no coating of walls Positively charged mobile phase moves towards cathode Separation of anions: treat walls with an alkyl ammonium salt: reverse osmotic flow Negatively charged mobile phase moves towards anode Apparent mobility µ + app = µ ep µ EOF Separation of 30 anions including glutamate, galactarate, (2 10 ppm) 15

CZE of Anti-inflammatory drugs (1)naporexen (2)ibuprofen (3) tolmetin CZE separation of protein mixture 1. Cytochrome c 12,400, pi = 10.7 2. Lysozyme, 14,100, pi = 11.1 3. Trypsin, 24,000, pi = 10.1 4. Trypsinogen, 23,700, pi=8.7 5. Trypsin inhibitor, 20, 100, pi= 4.5 ph = 2.7 16

Capillary Isoelectric Focussing (CIEF) Isoelectric focusing Capillary filled with ampholyte mixture Electric field applied: ph gradient develops within capillary Sample can be introduced with ampholyte mixture Capillary must be coated to suppress EOF Detection at a fixed point Mobilization of the focused bands is necessary for detection Mobilization Methods Hydrodynamic mobilization After focussing Capillary attached to a pressure or vaccum pump Electrophoretic (salt) mobilization After focussing Addition of salt to cathode buffer (NaCl): disrupts ph gradient (proteins dragged towards the cathode) CIEF of Proteins 17

Micellar Electrokinetic Chromatography (EKC)/CE and LC Micelles included in run buffer SDS - (CMC: 8 mm) (25 150 mm used) Partitioning of analyte between core of micelles (hydrophobic) and run buffer Both micelles and buffer are moved by applied electrical field Two mobile phases?! Neutral analytes are separated according to their hydrophobicity Elution in SDS Micelle is negatively charged: low apparent mobility due to electrophoretic mobility towards the anode Hydrophilic substances elute with the buffer (t 0 ) Very Hydrophobic substances which are completely solubilized by the micelle elute with the micelles (t mc ) Others elute between the two Applications: separation of amino acids, oligopeptides, nucleic acids, fatty acids, steroids and pharmaceutical drugs Capillary Gel Electrophoresis Gel anti-convective medium sieving medium Separation of analytes with similar mobilities Advantages Speed Efficiency Fully automated No casting of gels, no staining, no scanning!!! NO WORK!? Disadvantages No multisample capability No 2D capability Applications Separation of ssdna, dsdna, RNA Sizing of DNA fragments 18

CGE separation of SDS denatured proteins in polyethylene glycol column (1) lactalbumin (2) Soybean trypsin inhibitor (4) Ovalbumin (5) Bovine serum albumin (3)Carbonic anhydrase (6) Phosphorylase 19