Capillary Electrophoresis

Transcription

1 Electrophoresis is a saration techniqe based on the differential transportation velocities of charged species in an electric field throgh a condctive medim. Capillary Electrophoresis Primary candidates for CE saration are ions. The basic instrmental set-p consists of a high voltage power spply (0 to 30 kv), a fsed silica (SiO 2 ) capillary filled with a (backgrond) bffer soltion, two bffer soltion reservoirs, two electrodes, and an on-colmn detector. Detector + Injection (analyte(s) plg) L t _ L d V The very high resoltion of CE is a conseqence of the techniqe s extremely high efficiency. B H A C A lower vale of H corresponds to a higher saration efficiency when the plate height is redced, more thretical plates (N ~ 50,000 to 500,000) can be packed into a given length along the saration axis. Resoltion is proportional to N. In CE, two terms in Van Deemter Eqation are zero; mltiple-path term, A, mass-transfer term, C, becase the saration is carried ot in a single phase of niformly flowing carrier liqid. Only sorce of band broadening nder ideal conditions is from the longitdinal diffsion term, B.

2 Electrophoretic Velocity: The velocity of a charged analyte nder the inflence of an electric field relative to the backgrond electrolyte. Electrophoretic Velocity F drag At steady state: 6 r F elect F 6 r zee 0 6 +z r zee zee (+) (-) 2r Electrophoretic velocity z = charge on ion r ~ size and shape zee ee z 6 r 6 r ze e z E 6 r 6 r Electrophoretic mobility E Electro-osmotic velocity The mobility de to a species being swt along in a flow arising from a bffer soltion s response to an applied electric field (electro-osmotic flow). In the case CE, negative charges on the silica capillary wall create a doble layer of charge. The layer next to srface is rich in mobile, solvated cations which move towards the cathode and drag along anions and netrals. Si silanol pk a 3-4 H H Si - ph>2 Strctre of the capillary srface in contact with a bffer soltion is electrically charged when in contact with bffer. In a basic bffer, for example the srface is negatively charged. Tightly bond layer and srface charges

3 Cross-sectional view Net charge = 0 no net charge region Diffse net charge + Diffsed Tightly held Tightly held net charge + Srface charges, tightly bond layer, diffsed layer and no net charge regions. Srface net charge - Bffer/blk soltion Soltion next to srface is net positively charged. Diffsed layer. Doble Layer Diffsed layer positively charged The blk soltion containing the bffer moves toward an electrode, here toward (-); electro-osmotic flow (EOF). E Doble Layer blk soltion app In general for ph>3 Positive ions, apparent velocity app Negative ions, apparent velocity Netral molecles, apparent velocity (ph<=2, srface netral; EOF=0) (ph>=11, highly charged)

4 Apparent mobilities app app app app Ld / t E V / L Ld / t V / L netral t t The electro-osmotic mobility and electrophoretic mobility can case anions, cations and netrals to have a net migration towards the cathode becase generally the blk soltion has a net positive charge. Dending on the charge and size, the molecles/ions move throgh at different speeds; saration is achieved. app app Electropherogram Example: Determine the charge of nmodified protein r ~ nearly same for all species. Highest positive z Lowest positive z Resoltion of (consective) peaks: fast slow t 0.589t R w w av 1/ 2av N R ( 1) 4 fast tslow t slow fast saration factor Protein charge ladder. Acetylated bovine carbonic anhydrase

5 Efficiency in CE The only contribtor to peak broadening, practically, is diffsion. A plg of analyte diffses ot to prodce a Gassian crve of distribtion of concentration. The of it, in this case is given by the Einstein s Law; 2 2Dt and H= L 2 L d app Ld N V 2Dt 2D L 2 d t Electro-osmotic flow as opposed to laminar flow decreases broadening. Controlling the natre of EOF: The direction of the EOF can be changed with a cationic srfactant bilayer. Hexadecyl-trimethyl-ammonim bromide N + Br N + Cl 1-Hexadecylpyridinim chloride Diffsed layer negatively charged Wall charge can be maniplated by derivatization of silanol grops on the wall srface, and also by adding modifiers to the (rnning) bffer soltion derivatization Sample Injection: psi 5 kv NH 3 + NH 3 + modifier Hydrodynamic Uses the pressre difference between capillary ends Electrolytic Electric field drives sample into capillary

6 Hydrodynamic injection is accomplished by the application of a pressre difference between the two ends of a capillary. The amont of sample injected can be calclated by the Poiseille eqation. Hydrodynamic injection volme: Volme 128 L 4 P d t P is the pressre difference between the ends of the capillary, d is the inner diameter of the capillary, t is the injection time, is the sample viscosity, and L t is the total length of the capillary. t Electro-kinetic injection is performed by simply trning on the voltage for a certain period of time. The moles of each analyte injected, determined by the apparent mobility of each analyte, app ; the injection time, t; and the ratio of condctivities of the saration bffer and sample, concentration of the analyte ion C. Electro-kinetic injection amont: b 2 Moles app E t r C s V/m mol/m 3 Concentrations (optimm): Sample bffer ~ 1/10 backgrond electrolyte (bffer) Sample ~1/500 backgrond electrolyte (bffer) Stacked/concentrated sample Becase each analyte has a different mobility, electro-kinetic injection is biased. For qalitative analysis, this is not sally a problem. For qantitative analysis, the concentration/composition of the injected sample can be different than that of the original sample. One of the main advantages of CE is its ability to inject extremely small volmes of sample. Typical injection volmes range from pico-liters to nanoliters. Band skewing: If the condctivities of the rn electrolyte (backgrond) and that of the analyte region are very different, distortions of the peaks occr. b a (+) ion

7 Jole heating. Jole heating is a conseqence of the resistance of the soltion to the flow of crrent. H=VIt (+) ion low E slow high E fast - diffsion If the heat is not sfficiently dissipated from the system the reslting temperatre and density gradients can redce the saration efficiency. The capillary walls sed in CE mst dissipate efficiently. Detectors UV/Visible absorption Florescence Radiometric (for radioactive sbstances) Mass Spec. LOD (mol) Micellar Electro-kinetic Chromatography Mobile phase = micelles

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