Lecture 2 - Tan.pptx

CH4306 Bioanalytical Techniques Assoc Prof TAN Meng How N1.2-B2-33 [email protected] 1 Lecture 2 – Electrophoresis 2 Lecture 2 Outline • Electrophoretic mobility • Electroosmotic flow • Gel electrophoresis • Capillary electrophoresis 3 What is electrophoresis? • Electrophoresis is the moveme...

CH4306 Bioanalytical Techniques Assoc Prof TAN Meng How N1.2-B2-33 [email protected] 1 Lecture 2 – Electrophoresis 2 Lecture 2 Outline • Electrophoretic mobility • Electroosmotic flow • Gel electrophoresis • Capillary electrophoresis 3 What is electrophoresis? • Electrophoresis is the movement of particles through a fluid due to the application of a spatially uniform electric field. • This phenomenon was observed for the first time in 1807 by Ferdinand Frederic Reuss (Moscow State University), who noticed that the application of a constant electric field caused clay particles dispersed in water to migrate. • It is ultimately caused by the presence of a charged interface between the particle surface and the surrounding fluid. • Electrophoresis is a bioanalytical tool used in fundamental research and diagnostics 4 What determines the speed of movement? • During electrophoresis, different molecules migrate at different speeds. • The determining factors include: (1) Magnitude of the charge (2) The molecular weight (size) (3) The structure or shape (4) The isoelectric point (for proteins) • Most biopolymers, such as proteins and nucleic acids, are charged and they can be separated and quantitated by electrophoretic methods. 5 A pioneer in electrophoresis • Swedish biochemist • President of the International Union of Pure and Applied Chemistry 1951-1955 • Vice President of the Nobel Foundation 1947-1960 • President of the Nobel Foundation 1960-1964 • Member of the Nobel Committee for Chemistry 1947-1971 • Chairman of the Nobel Committee for Chemistry 1965-1971 • Died of a heart attack in 1971 6 Principle of Electrophoresis • An electrophoretic separation occurs in an intervening (support) medium that separates two electrodes. • At one end of the medium is the positively charged anode, and at the other is the negatively charged cathode. • The intervening (support) medium may be as short as 10cm or as long as 1m. • Throughout this medium, positively charged species will migrate toward the cathode and negatively charged species will move toward the anode. • Differences in charge and size can lead to different mobilities and thus separation of different sample components. 7 Principle of Electrophoresis • Electrophoretic separations can be performed in: (1) Free solution - the separation of ions occurs due to differences in mobility (2) A solution containing a non-conductive matrix such as agarose or polyacrylamide gel - The separation of analytes in a gel is based not only on differences in mobility, but also on the sieving effect of the gel. Large compounds are retarded more than smaller compounds. So two compounds with same charge to size ratio can be separated as long as they are different in size. • The efficiency of an electrophoretic separation is governed by two main factors: (1) The electrophoretic mobility (μep) of the analytes (2) The electroosmotic flow (EOF) of bulk solution 8 Simple Analogy • Running against headwind • Running with tailwind 9 Electrophoretic mobility • Electrical mobility is the ability of charged particles (such as electrons or protons) to move through a medium in response to an electric field that is pulling them. • The separation of ions according to their mobility in gas phase is called ion mobility spectrometry, and in liquid phase is called electrophoresis. • When a charged particle in a gas or liquid is acted upon by a uniform electric field, it will be accelerated until it reaches a constant drift velocity according to the formula: vd = µepE where vd is the drift velocity E is the magnitude of the applied electric field µep is the mobility 10 What are the units of mobility? • Units of drift velocity, vd = m s-1 • For a constant electric field, E: where V is the applied voltage d is the distance between the electrodes • Hence, units of E = V m-1 • Since µep = vd/E, units of mobility = m2 V-1 s-1 11 Balance of forces Consider a particle with charge q moving at constant velocity through a medium: Electrostatic force, Fe = qE (Similar to Newton’s 2rd law: F=ma) Drag force, FD = fvd (f is the frictional coefficient) At constant velocity, net force = 0 FD = Fe fvd = qE f(µepE) = qE Hence, µep = q/f Electrical mobility is proportional to the net charge of the particle. 12 Stokes’ Law • The Stokes radius or Stokes-Einstein radius of a solute is the radius of a hard sphere that diffuses at the same rate as that solute. • According to Stokes’ law, a perfect sphere traveling through a viscous liquid feels a drag force proportional to the frictional coefficient: FD = fvd = (6  R)vd where is the liquid's viscosity R is the Stokes radius • Note that Stokes’ law is derived from the Navier-Stokes equations for fluid flow with low Reynolds number (i.e. Re << 1 or inertia force is much smaller than viscous force). 13 More on the Stokes radius µep = q/f = q/(6  R) • Electrical mobility is also inversely proportional to the Stokes radius of the ion, which is the effective radius of the moving ion including any molecules of water or other solvent which move with it. • For different ions with the same charge (such as Li+, Na+, and K+), the electrical forces are equal, so that the drift speed and the mobility are inversely proportional to the Stokes radius R. • Measurements show that mobility increases from Li+ to Cs+ and therefore that Stokes radius decreases from Li+ to Cs+. This is opposite to the order of ionic radii for crystals because in solution, the smaller ions (Li+) are more extensively hydrated than the larger ions (Cs+). • A smaller ion with stronger hydration may have a greater Stokes radius than a larger ion with weaker hydration. The smaller ion drags more 14 water molecules with it as it moves through the solution. Electroosmotic flow • Electroosmotic flow (often abbreviated EOF) is the motion of liquid induced by an applied potential across any fluid conduit. • Electroosmotic flow was first reported in 1807 by Ferdinand Frederic Reuss (Moscow State University). He noticed that the application of a constant electric field caused clay particles dispersed in water to migrate. • In the same experiment, he also found that there was an opposite flow of water (electroosmosis) associated with the movement of the clay particles. Water flows through the narrow spaces between these particles. 15 Explanation for EOF • Clay is composed of closely packed particles of silica and other minerals. (Capillaries used for electrophoresis are also made of silicate.) • The silanol groups on the surface of silica are deprotonated at pH > 4 and are thus negatively charged. At lower pH values, they are protonated. • The negative charges on the surface can attract positive charges from the surrounding solution. 16 The electric double layer • The electric double layer consists of the fixed Stern layer right next to the silicate capillary wall and the diffuse layer extending into the bulk solution. • In this double layer, the counterions (ions with charge opposite the wall) are present at a higher concentration, while the coions (ions with charge of same sign as the wall) are present at a lower concentration. • The counterions in the fixed layer cannot move, while those in the diffuse layer are mobile. 17 Movement of solvent molecules • Upon application of an electric field, the counterions in the diffuse layer (positive charge) migrate towards the cathode and carry solvent molecules in the same direction. • This overall solvent movement is called electroosmotic flow. 18 Electroosmotic flow as a function of pH (silica walls) 19 Control of EOF The electroosmotic flow can be controlled in different ways: (1) Low pH (< 4) – surface charges are neutralized by protonating the silanol group (2) Chemical surface modification (e.g. coating of the capillary walls with a polymer layer) (3) Using additives to change the viscosity and the zeta potential  (e.g., the organic solvents methanol and acetonitrile are used to reduce and increase respectively the viscosity). 20 Zeta ( ) Potential Slipping Plane: distance from particle surface where ions move with the particle  -potential: potential (mV) at the slipping plane or potential at the boundary of the diffuse layer and the bulk solution (relative to a point in the bulk fluid far away from the interface) The potential drop inside the double layer is exponential. 21 EOF Equation • The velocity of the solvent flow depends on the electroosmotic mobility µeo, which in turn depends on the charge density on the capillary internal wall and the buffer characteristics. • The electroosmotic velocity veo is given by the following equation: where  is the dielectric constant of the buffer is the zeta potential of the capillary surface is the liquid's viscosity V is the applied voltage L is the distance between the electrodes 22 Apparent Mobility • The efficiency and resolution of an electrophoretic separation are influenced by the electrophoretic motion as well as the EOF. • The apparent mobility (µ) is the sum of the electrophoretic mobility and the electroosmotic mobility, i.e. µ = µep + µeo • Hence, the migration velocity of an analyte under an electric field E is determined by the intrinsic electrophoretic mobility of the analyte and the electroosmotic mobility of the buffer inside the capillary. • For most biomolecular separations, the analyte ions are negatively charged and will migrate towards the anode, whereas the EOF is directed to the cathode (opposite directions). 23 Influence of EOF • The movement of the bulk solution can affect solute ions in the following ways: (1) Positively-charged solute ions move faster. (2) Uncharged solute ions move at the same velocity as the electroosmotic flow. All these uncharged ions remain unseparated. (3) Negatively-charged solute ions move slower. • Velocity of the solute, vtot = vep + veo = (µep + µeo)E 24 What is gel electrophoresis? • Gel electrophoresis is a method for separation and analysis of biomacromolecules (DNA, RNA and proteins) and their fragments using an electrically neutral (non-conducting) hydrogel medium in an electrolyte buffer. • The pores of the gel can act as a molecular sieve and retard the migrating molecules according to their size. • The gel can also act as an anti-convective support medium. It suppresses the thermal convection caused by application of the electric field, thereby reducing band broadening. • Additionally, gels can simply serve to maintain the finished separation, so that a post electrophoresis stain can be applied for visualization. • There is no electroosmotic flow. Only analytes with a net charge can be separated. Neutral compounds cannot be separated. 25 History of Gel Electrophoresis 1930s – first reports of the use of sucrose for gel electrophoresis 1955 – introduction of starch gels, mediocre separation 1959 – introduction of acrylamide gels; accurate control of parameters such as pore size 1966 – first use of agar gels 1969 – introduction of denaturing agents especially SDS for separation of proteins 1970 – Laemmli separated 28 components of T4 phage using a stacking gel and SDS 1972 – agarose gels with ethidium bromide stain 1975 – 2-dimensional gels; isoelectric focusing then SDS gel electrophoresis 1977 – sequencing gels 1983 – pulsed field gel electrophoresis enables separation of large DNA molecules 1983 – introduction of capillary electrophoresis 26 Choice of gel media • The most common gels today are made from agarose or polyacrylamide. • The gel pore size is an important parameter for electrophoresis separation. • In restrictive gels, the pores are small enough to act as molecular sieves • In non-restrictive gels, the pores are too large to impede the sample movement. • Polyacrylamide gels are usually used for proteins, and have very high resolving power for small fragments of DNA (5-500 bp). • Agarose gels on the other hand have lower resolving power for DNA but have greater range of separation, and are therefore used for DNA fragments of usually 50-20,000 bp in size 27 Agarose • Extracted from seaweed • Molecular weight of about 120,000 • Consists of alternating D-galactose and 3,6,-anhydro-L-galactopyranose • Pyruvate and sulfate are also found in small quantities • Pore diameter ranges from 50 nm to >200 nm depending on the concentration of agarose used • Pore sizes of agarose gels are much larger than polyacrylamide gels • On standing the agarose gels are prone to syneresis (extrusion of water through the gel surface) • The melting and gelling temperature of agarose can be modified by chemical modifications, most commonly by hydroxyethylation. With low melt agarose, one can make gels of even higher concentrations. 28 An overview of agarose gel electrophoresis Gel appears translucent after it solidifies. Remove comb before using gel. (DNA is negatively charged) Be careful of orientation when plugging in the power supply! (Black-toblack, red-to-red) 29 Casting and running an agarose gel • With a greater gel concentration, porosity and average pore size decrease. • When agarose has dissolved by heating the solution, add a reagent that allows you to readily visualize the DNA, swirl, and then pour into a casting mold with a comb. Allow the agarose gel to solidify. • Mix each DNA sample with DNA loading dye. The DNA loading dye contains glycerol, which weighs the sample down to the bottom of the well, as well as two different dyes (typically bromophenol blue and xylene cyanol) for visual tracking of DNA migration during electrophoresis. • A different DNA sample is loaded in each well. A known DNA ladder is also loaded. • You will see bubbles moving inside the gel box due to the current passing through. • Shorter DNA strands will move along the gel faster than the longer ones. 1% = 1g agarose in 100ml buffer (Low-concentration gels (0.1–0.2%) are fragile and hard to handle.) 30 DNA Visualization • Ethidium Bromide (EtBr) Ethidium bromide is an intercalating agent commonly used as a fluorescent tag (nucleic acid stain) in molecular biology laboratories for techniques such as agarose gel electrophoresis. Absorption maxima of EtBr in aqueous solution are at 210 nm and 285 nm, which correspond to ultraviolet (UV) light. Hence, to visualize DNA using EtBr stain, we need to expose the gel to UV light. Upon exposure to UV light, EtBr will fluoresce with an orange colour, intensifying almost 20-fold after binding to the DNA. EtBr intercalates between the bases of DNA. By moving into the hydrophobic environment found between the base pairs, the ethidium cation is forced to shed any water molecules that are associated with it. As water is a highly efficient fluorescent quencher, removal of these water molecules allows the ethidium to fluoresce. EtBr is a mutagen. Safer alternatives (such as SYBR Safe or GelRed), which are cell impermeable, have been developed. 31 Types of Buffer • TAE Buffer - Contains(i) Tris buffer (ii) Acetic acid (iii) EDTA - Most commonly used for routine DNA agarose gel electrophoresis • TBE Buffer - Contains(i) Tris buffer (ii) Boric acid (iii) EDTA - Also commonly used • SB Buffer - Contains(i) Sodium borate (ii) Boric acid - Has low conductivity and allows for less heat buildup and thus higher voltage and faster runs 32 Joule Heating Joule heating, also known as ohmic heating and resistive heating, is the process by which the passage of a current through the conductive buffer releases heat. The amount of heat released is given by: When the charged particles making up the current collide with ions in the buffer, the particles are scattered and so their motion becomes random and therefore thermal, increasing the temperature of the system. Due to heat transfer, a temperature gradient is formed across the gel cross-section, resulting in band broadening and loss of separation resolution. There are several ways to minimize Joule heating: - apply a low electric field - decrease the conductivity of the buffer - improve the dissipation of heat by using a thin gel - run experiment in a temperature controlled environment 33 Video: agarose gel electrophoresis 34 Pulsed-field gel electrophoresis Developed in 1983 at Columbia University (USA) Pulsed-field gel electrophoresis (PFGE) is a technique used for the separation of large DNA molecules by applying to a gel matrix an electric field that periodically changes direction. Small DNA fragments can find their way through the gel matrix more easily than larger DNA fragments. Additionally, above a threshold length of about 30-50kb, all large fragments will run at the same rate and appear in the gel as a single diffuse band. However, with periodic changing of field direction, various lengths of DNA react to the change at different rates. Hence, over the course of time with consistent changing of electric field directions, the DNA fragments will begin to separate more and more even for the very large ones. This procedure takes longer than normal gel electrophoresis due to the size of the fragments being resolved and the fact that the DNA does not move in a straight line through the gel. 35 Varying electric fields The pulse times are equal for each direction, resulting in a net forward migration of the DNA. 36 37 What is SDS-PAGE? • The separation principle of SDS-PAGE is based solely on the difference in protein size (molecular weight). • Proteins are denatured in the presence of the anionic detergent SDS (sodium dodecyl sulfate) with binding ratio of 1.4g SDS to 1g protein. (The detergent solubilizes or “dissolves” hydrophobic molecules.) • Since each protein chain will be coated by many SDS molecules, the large negative charges of SDS will mask the intrinsic charges of the protein. • Hence, separation depends entirely on the molecular sieving effect of the gel. The larger the molecular weight, the slower the protein migrates. 38 Effect of SDS on proteins A protein sample is boiled in SDS and β-mercaptoethanol. The β-mercaptoethanol reduces disulfide bonds. The end result has two important features: (1) All proteins contain only primary structure (are linear) (2) All proteins have a large negative charge, which mean they will all migrate towards the positive pole when placed in an electric field. 39 How to prepare a gel for SDS-PAGE • Demonstration: https://www.youtube.com/watch?v=pnBZeL8nFEo • In the reaction mix, there are: (1) Acrylamide (gel matrix) (2) Bisacrylamide (cross-linker) (3) SDS (maintains denaturation and negative charge of proteins) (4) Ammonium persulfate (5)TEMED (Tetramethylethylenediamine) • TEMED, which is always added last, catalyses the decomposition of the persulfate ion to give a free radical, which is required to initiate the polymerization of acrylamide into chains (with bisacrylamide linking the chains together into a network). 40 Acrylamide • Acrylamide is a potent neurotoxin, so handle with care! • Porosity is controlled by the proportions of acrylamide and bisacrylamide. 41 Why are there two layers of gel in SDS-PAGE? Sample well Stacking gel Resolving or separating gel • In order to accurately size fractionate, the proteins all have to be at the starting line at the same time. • The stacking gel has large sized pores that allow the proteins to migrate freely and be lined up in a row at the interface of the two layers (we want all the proteins to start migrating from the same level). • In the stacking gel, the migrating proteins are sandwiched tightly between the negatively charged glycinate ions (from the buffer) and chloride ions (in the gel), which are migrating towards the anode. This causes the proteins to get aligned in a sharp band. • The resolving gel is the actual track where proteins run according to 42 their molecular weight. Visualization of proteins • Once protein bands have been separated by electrophoresis, they can be visualized using different methods of in-gel detection. • Demands for improved sensitivity for small sample sizes and compatibility with downstream applications have driven development of visualization methods. • Typically, the gel is suspended in a tray filled with the necessary reagents. • Most protein staining methods involve the same general incubation steps: (1) A initial wash (e.g. with water) to remove electrophoresis buffers and residual SDS from the gel matrix (2) An acid- or alcohol-wash to condition or fix the gel to limit diffusion of protein bands from the matrix (3) Treatment with the stain reagent to allow the dye or chemical to diffuse into the gel and bind (or react with) the proteins (4) Washing to remove excess dye from the background gel matrix 43 Coomassie Blue • Coomassie blue dyes are a family of dyes commonly used to stain proteins in SDS-PAGE gels. The gels are soaked in dye, and excess stain is then eluted with a solvent. This treatment allows the visualization of proteins as blue bands on a clear background. • In acidic buffer conditions, coomassie dye binds to basic and hydrophobic residues of proteins • Coomassie dye reagents detect some proteins better than others due to differences in protein composition. Thus, coomassie dye reagents can detect as few as 8-10 nanograms for some proteins but more typically 25-100 nanograms for most proteins. • Coomassie dye staining does not permanently chemically modify the target proteins. Because no chemical modification occurs, excised protein bands can be completely destained and the proteins recovered for analysis by mass spectrometry or sequencing. 44 Silver staining • Silver staining is the most sensitive colorimetric method for detecting total protein. • Silver ions (from silver nitrate in the stain reagent) interact and bind with certain protein functional groups. Strongest interactions occur with carboxylic acid groups (Asp and Glu), imidazole (His), sulfhydryls (Cys), and amines (Lys). • The bound silver ions are reduced to metallic silver, resulting in brown-black color. The development process is essentially the same as for photographic film. • Silver staining can detect less than 0.5 nanograms of protein in typical gels. • In some protocols, glutaraldehyde or formaldehyde is used (as a reducing agent). These aldehydes can cause chemical crosslinking of the proteins in the gel matrix, limiting compatibility with downstream analysis by mass spectrometry. 45 Amino acids are zwitterionic • Isoelectric point (pI) is the pH where the amino acid exhibits no net charge. • Basic proteins have a higher pI, while acidic proteins have a lower pI. • At the isoelectric point, the amino acid remains stationary under an applied electric field. Isoelectric focusing • Isoelectric point (pI) is the pH value, where the overall protein charge equals to zero. • This can be obtained by creating a pH gradient in the gel where protein is loaded and applying an electric current. • Proteins migrate towards the cathode or anode according to their total charge up to the point where the gel pH equals pI of a given protein. • Isoelectric focusing has a high resolution. Bands as narrow as 0.001 pH units can be obtained. 47 How to obtain a gel with a pH gradient? • Isoelectric focusing has been successfully used in research, clinical, and agricultural fields to separate proteins in (for example) blood, muscle extracts, and seed extracts. • In isoelectric focusing, a stable pH gradient with constant conductivity is very important. • This can be achieved by: (1) Carrier ampholytes (2) Immobilized pH gradients 48 What are carrier ampholytes? An amphoteric compound is a molecule or ion that can react both as an acid as well as a base. Carrier ampholytes are amphoteric molecules that contain both acidic and basic groups and will exist mostly as zwitterions with a high buffering capacity near their pI. Commercial carrier ampholyte mixtures comprise hundreds of individual polymeric species with pIs spanning a specific pH range. When a voltage is applied across a carrier ampholyte mixture, the carrier ampholyte with the lowest pI (and the most negative charge) migrates towards the anode (+). The carrier ampholyte with the highest pI (and the most positive charge) migrates towards the cathode (-). The other carrier ampholytes align themselves between the extremes, according to ther pIs, and buffer their environment to the corresponding pH. 49 Illustration of carrier ampholytes Each ampholyte reaches an equilibrium position along the separation medium. A pH gradient with increasing pH over the gel length formed by a mixture of hundreds of ampholytes each with a different pI. The concentration of each ampholyte is the same to ensure a homogeneous conductivity. 50 Limitations of carrier ampholytes • Since the carrier ampholyte-generated gradient is dependent on an electric field, it breaks down when the field is removed. • The pH gradient is susceptible to drift towards the ends of the gel, especially towards the cathode (-). Over time, this leads to a plateau in the middle of the gradient with gaps in the conductivity. Hence, we need to restrict the focusing time of the experiment. • There can be significant batch-to-batch and company-to-company variations in the properties of carrier ampholytes, which limits the reproducibility of focusing experiments. • Carrier ampholytes have a tendency to bind to the sample proteins, which may incorrectly alter the migration of the proteins. 51 Immobilized pH gradients (IPG) • An immobiline is a weak acid or base that can be incorporated into an acrylamide gel matrix for isoelectric focusing. • Immobilines are not zwitterionic; they are either acidic or basic. • Gels with immobilized pH gradients (IPG) are made using a cassette system and a gradient maker to mix two kinds of acrylamide solutions, one with immobiline having acidic buffering property and the other with basic buffering property. • The immobilines co-polymerize with the acrylamide gel matrix. • The pH depends on the ratio of the two immobilines. • The gels can be dried and stored. To use them, a simple rehydration step is needed. (A is a weakly acidic or basic buffering group) 52 Advantages of IPG • The protein sample can be applied immediately (no prefocusing is needed). • The pH gradient is stable and does not drift in an electric field because the buffers that form the pH gradient are immobilized within the gel matrix. • Isoelectric focusing experiments using IPG are more reproducible. • The resolution possible with immobilized pH gradient gels is 10100 times greater than that obtained with carrier ampholytes. 53 What is 2D Gel Electrophoresis? • Two modes of electrophoresis are combined in a single gel. • Usually, proteins are separated by isoelectric focusing in 1 dimension, based on pI. • This is followed by SDS-PAGE in a perpendicular direction, based on size. • Mixtures of thousands of proteins can be separated with high resolution. • The result can be compared to electronic databases. 54 Application of 2D Gel Electrophoresis The resulting "maps" of proteins can be compared for example between the experimental and control sample or among samples from patients with specific disease and their healthy controls and thus identify differentially expressed proteins that can be linked with the pathogenesis of the studied disease. (to identify differentially produced proteins) 55 Capillary Electrophoresis • Separation method carried out in a buffer-filled capillary tube. • The tube extends between two buffer reservoirs. • The sample is introduced into one end of the tube. • A dc potential is applied between the two electrodes throughout the separation. • Charged analytes migrate at different rates in the presence of the electric field. • The separated analytes are observed by a detector at the opposite end of the capillary. 56 Sample Injection - Hydrodynamic • Hydrodynamic injection can be achieved by using pressure to force the sample into the capillary. • Alternatively, it can be achieved by natural gravity flow. In gravity flow injection (also known as siphoning injection), the inlet end of the capillary is raised so that the liquid level in the sample vial is at a height h above the level of the cathodic buffer, and is held in this position for a fixed time t. 57 Sample Injection - Electrokinetic Electrokinetic injection involves drawing sample ions into the capillary interior with an applied potential. A high voltage is applied over the capillary between the sample vial and the destination vial for a given time. This causes the sample to move into the capillary according to its apparent mobility, µ. 58 DNA Capillary Electrophoresis • DNA sequencers separate strands by size (or length) using capillary electrophoresis. • The fluorescently labeled products of the cycle sequencing reaction are injected electrokinetically into capillaries filled with polymer. High voltage is applied so that the negatively charged DNA fragments move through the polymer in the capillaries towards the positive electrode. • In capillary sequencing of DNA, the sieving polymer (typically polydimethylacrylamide) suppresses electroosmotic flow to very low levels. 59 Micellar Electrokinetic Chromatography (MEKC) Micellar electrokinetic chromatography (MEKC) can be thought of as a hybrid of electrophoresis and chromatography. The solution contains a surfactant (most commonly SDS) at a concentration that is greater than the critical micelle concentration. MEKC is performed under alkaline conditions to generate a strong electroosmotic flow. Since SDS is negatively charged, the micelles have an electrophoretic mobility that is counter to the electroosmotic flow. Hence, the micelles migrate quite slowly, though their net movement is still toward the cathode. 60 Migration of analytes in MEKC • During a MEKC separation, analytes distribute themselves between the hydrophobic interior of the micelle and hydrophilic buffer solution. • The analyte migration velocity depends on the partition coefficient between the micelle and the aqueous buffer: (1) Uncharged analytes that are insoluble in the interior of micelles should migrate at the electroosmotic flow velocity. (2) Analytes that solubilize completely within the micelles (analytes that are highly hydrophobic) should migrate at the micelle velocity. (3) For electrically charged solutes, the migration velocity depends not only on the partition coefficient and the electroosmotic flow, but also on the electrophoretic mobility, µep, of the solute in the absence of the micelle. 61