V10 Electrokinetic II Capillary Electrophoresis WS 23 PDF

V10 V10 Electrokinetic II Capillary Electrophoresis - CE - Lecture „Microfluidic Systems - Bio-MEMS“ - Capillary Electrophoresis Prof. Dr.-Ing. Uwe Schnakenberg | Institute of Materials in Electrical Engineering 1 | WS 23 1 Contents V10 Contents and Learning targets 10.1 Electrophoresis  Introducti...

V10 V10 Electrokinetic II Capillary Electrophoresis - CE - Lecture „Microfluidic Systems - Bio-MEMS“ - Capillary Electrophoresis Prof. Dr.-Ing. Uwe Schnakenberg | Institute of Materials in Electrical Engineering 1 | WS 23 1 Contents V10 Contents and Learning targets 10.1 Electrophoresis  Introduction 10.2 Capillary Electrophoresis  Setup  Influence of EDL  Peak broadening 10.3 Capillary Electrophoresis on a Chip 10.4 Capillary Isoelectric Focusing CIEF Lecture „Microfluidic Systems - Bio-MEMS“ - Capillary Electrophoresis Prof. Dr.-Ing. Uwe Schnakenberg | Institute of Materials in Electrical Engineering 1 | WS 23 2 V10 10.1 Introduction Lecture „Microfluidic Systems - Bio-MEMS“ - Capillary Electrophoresis Prof. Dr.-Ing. Uwe Schnakenberg | Institute of Materials in Electrical Engineering 1 | WS 23 3 10.1 Electrophoresis - Introduction Electrophoresis* V10 4 Electrophoresis is the transport of ions** through a carrier material in an electric field Friedrich Kohlrausch (1840-1910) https://de.wikipedia.org  Separation method * Mathematical description by Friedrich Kohlrausch: Ann. Phys. Chem. 62 14 (1897) 1948 Nobel price in chemistry to Arne Tiselius: Trans. Farad. Soc. 33 524 (1937), Adv. Prot. Chem. 8 461 (1953) ** Ions here includes also charged molecules Lecture „Microfluidic Systems - Bio-MEMS“ - Capillary Electrophoresis Prof. Dr.-Ing. Uwe Schnakenberg | Institute of Materials in Electrical Engineering 1 | WS 23 Arne Tiselius (1902-1971) 10.1 Electrophoresis - Introduction V10 Principle of EP - Capillary Wet paper strip Gel + Mixture of ions at t = 0 min Negative pole Small positive charged ions U Positive pole Neutral particles Teilchen Large negative Large positive charged ions charged ions Ionen Small negative charged ions Ionen Ionen Lecture „Microfluidic Systems - Bio-MEMS“ - Capillary Electrophoresis Prof. Dr.-Ing. Uwe Schnakenberg | Institute of Materials in Electrical Engineering 1 | WS 23 http://www.u-helmich.de 5 10.1 Electrophoresis - Introduction EP force Counter force V10 F  qE Pushing force F  6    r   v Drag force Friction force for spherical particle Stokes law* 6 In equilibrium vEP q   E   EP  E 6    r  Electrophoretic mobility μEP E… r… q... η… v… External electric field strength Ion radius Ion charge Viscosity of fluid Ion velocity, migration rate * The drag force cannot be predicted precisely without detailed molecular dynamics calculation, but the appoximation by Stokes law is valid within one order of magitude for most ions Lecture „Microfluidic Systems - Bio-MEMS“ - Capillary Electrophoresis Prof. Dr.-Ing. Uwe Schnakenberg | Institute of Materials in Electrical Engineering 1 | WS 23 10.1 Electrophoresis - Introduction Migration rate vEP,i for species i vEP ,i  V10 qi E 6    ri   Differences in charges and radii result in → different migration rates → separation  Ion charge can be adjusted through buffer solution (pH value) E … External electric field strength r … Ion radius q... Ion charge η… Viscosity of fluid v … Ion velocity, migration rate i …. Ion species Lecture „Microfluidic Systems - Bio-MEMS“ - Capillary Electrophoresis Prof. Dr.-Ing. Uwe Schnakenberg | Institute of Materials in Electrical Engineering 1 | WS 23 7 10.1 Electrophoresis - Introduction V10 8 Comparison Electrophoretic velocity Electroosmotic velocity of ions of fluid vEP ,i qi   E   EP ,i  E 6    ri  vEOF    0    Ex   EO  E  Describes the ion motion in a fluid in Describes the fluid motion in regard to regard to an electric field applied to the ion an electric field applied to EDL Lecture „Microfluidic Systems - Bio-MEMS“ - Capillary Electrophoresis Prof. Dr.-Ing. Uwe Schnakenberg | Institute of Materials in Electrical Engineering 1 | WS 23 10.1 Electrophoresis – Capillary Electrophoresis vEP ,i V10 qi   E   EP ,i  E 6    ri  Electrophoresis Separation process Charged molecules are separated in an electric field with regard to the charge/size ratio Capillary electrophoresis Separation in a small capillary Important in analytical chemistry Lecture „Microfluidic Systems - Bio-MEMS“ - Capillary Electrophoresis Prof. Dr.-Ing. Uwe Schnakenberg | Institute of Materials in Electrical Engineering 1 | WS 23 9 10.2 Capillary Electrophoresis V10 10 10.2 Capillary Electrophoresis CE , CEP Lecture „Microfluidic Systems - Bio-MEMS“ - Capillary Electrophoresis Prof. Dr.-Ing. Uwe Schnakenberg | Institute of Materials in Electrical Engineering 1 | WS 23 10.2 Capillary Electrophoresis V10 11 Basic Setup for Capillary Electrophoresis Detector Capillary Capillary diameter 10 - 100 µm Capillary length 30 - 100 cm Electrode High voltage supply 5 - 30 kV* * Current in 100 µA range Inlet reservoir Lecture „Microfluidic Systems - Bio-MEMS“ - Capillary Electrophoresis Prof. Dr.-Ing. Uwe Schnakenberg | Institute of Materials in Electrical Engineering 1 | WS 23 Outlet reservoir 10.2 Capillary Electrophoresis Capillary  UV transparent materials  Fused silica  Borosilicate glass  Outer layer: polyimide (5 - 15 μm thick) Electrolyte  Water-based buffer solutions  Phosphate or citrate buffer for pH < 7  Borat or and TRIS* buffer for pH > 7 * TRIS …. Tris(hydroxymethyl)-aminomethane (THAM) Lecture „Microfluidic Systems - Bio-MEMS“ - Capillary Electrophoresis Prof. Dr.-Ing. Uwe Schnakenberg | Institute of Materials in Electrical Engineering 1 | WS 23 V10 12 10.2 Capillary Electrophoresis V10 13 Influence of EDL on Electrophoretic Flow (EPF) Glass capillaries are used  Exhibit negatively charged inner wall When EP is applied in a capillary with surface charge electroosmotic flow is superimposed V.M. Upaz: „Electrophoresis in Microfluidic Systems“, in Microfluidic Technologies for Miniaturized Analysis Systems“ Steffen Hardt and Friedhelm Schönfeld (eds.), Chap. 10, Springer Verlag, Berlin (2007) D. Wu et al.: J. Chromatography A 1184 542-559 (2008) Lecture „Microfluidic Systems - Bio-MEMS“ - Capillary Electrophoresis Prof. Dr.-Ing. Uwe Schnakenberg | Institute of Materials in Electrical Engineering 1 | WS 23 10.2 Capillary Electrophoresis Electrophoretic transport mechanism in detail 1. Solvent and solutes both migrate due to electroosmotic motion of the solvent 2. Additionally, charged molecules migrate driven by electrophoresis 3. Charged molecules are separated due to differences in their electrophoretic mobility (migration rates) D.J. Harrison et al.: Anal. Chem. 64 1926-1932 (1992) Lecture „Microfluidic Systems - Bio-MEMS“ - Capillary Electrophoresis Prof. Dr.-Ing. Uwe Schnakenberg | Institute of Materials in Electrical Engineering 1 | WS 23 V10 14 10.2 Capillary Electrophoresis Advantages of Electroosmotic Flow in Capillary Electrophoresis  Cations and anions can be analyzed in one run (which is not possible in, e.g., gel electrophoresis)  Large ions with low electrophoretic mobilities (low velocities) can enter the detector (without flow → too long migration times) J.W. Jorgenson et al.: Anal. Chem. 53 1298-1302 (1991) https://chem.libretexts.org/ Lecture „Microfluidic Systems - Bio-MEMS“ - Capillary Electrophoresis Prof. Dr.-Ing. Uwe Schnakenberg | Institute of Materials in Electrical Engineering 1 | WS 23 V10 15 10.2 Capillary Electrophoresis https://www.youtube.com/watch?v=-rDRU_qxwYA Alternative: https://www.youtube.com/watch?v=wStV1rFjHOo Lecture „Microfluidic Systems - Bio-MEMS“ - Capillary Electrophoresis Prof. Dr.-Ing. Uwe Schnakenberg | Institute of Materials in Electrical Engineering 1 | WS 23 V10 16 10.2 Capillary Electrophoresis Detection principle V10 17 Detector limit / (10-18 mol) (for 10 nL injection volume) Spectrometry Absorption 1 -1000 Fluorescence 1 - 0.01 Electrochemical Amperometry 100 Potentiometry 0.1 Mass spectrometry Lecture „Microfluidic Systems - Bio-MEMS“ - Capillary Electrophoresis Prof. Dr.-Ing. Uwe Schnakenberg | Institute of Materials in Electrical Engineering 1 | WS 23 1 - 0.01 10.2 Capillary Electrophoresis V10 18 Sample Injection by Electrophoresis vi  li   EP ,i  E ti High voltage Plug length li li  tinj  Einj   EP ,i supply 5 - 30 kV* Different plug length li for each ion i, Inlet reservoir due to their differences in mobility µEP,i K. Seiler et al.: Anal. Chem. 55 1481-1488 (1993) Lecture „Microfluidic Systems - Bio-MEMS“ - Capillary Electrophoresis Prof. Dr.-Ing. Uwe Schnakenberg | Institute of Materials in Electrical Engineering 1 | WS 23 tinj … Injection time Einj.. Electric field strength during injection µEP,i. Mobility of ion i 10.2 Capillary Electrophoresis V10 19 Separation Sequence Migration velocity vi for species i vi  µi  E  µi  Migration time tm,i for species i L L2   vi µi V t m ,i V L required for a zone to migrate over the distance L From tm,i the total mobility µ can be determined Total mobility µ Separation S of peak centers µi  µEO ,i  µEP ,i S  µEP  E  t J.W. Jorgenson et al.: Anal. Chem. 53 1298-1302 (1991) Lecture „Microfluidic Systems - Bio-MEMS“ - Capillary Electrophoresis Prof. Dr.-Ing. Uwe Schnakenberg | Institute of Materials in Electrical Engineering 1 | WS 23 µ …. Overall electrophoretic mobility µEO.. Electroosmotic mobility µEP.. Electrophoretic mobility E … Electric field strength V …. Applied voltage L …. Length of separation zone tm … Migration time v …. Migration velocity 10.2 Capillary Electrophoresis V10 20 After Separation Zone (Peak) Sharpness Depends on Effects Inside Separation Channel Outside Separation Channel  Diffusion  Injected volume (plug length)  Interaction of fluid with  Detector spot size inner capillary walls  Detector response time Lecture „Microfluidic Systems - Bio-MEMS“ - Capillary Electrophoresis Prof. Dr.-Ing. Uwe Schnakenberg | Institute of Materials in Electrical Engineering 1 | WS 23 10.2 Capillary Electrophoresis V10 21 Inside Separation Channel Peak Broadening due to Diffusion 2. Fick‘s Law for one dimension c  2c  D 2 t x c0  w0 c ( x, t )  e   4 D t B.J. Kirby: ISBN 978-0-521-11903-0  Distribution of single analyte with motion caused by electrophoresis and peak broadening owing to diffusion x2 4 Dt c0 ….. Concentration of species at peak center x=0 and t = 0 w0 ….. Width of peak at t= 0 c0 w0. Total number of species is constant D …. Diffusion coefficient t ……. Migration time Lecture „Microfluidic Systems - Bio-MEMS“ - Capillary Electrophoresis Prof. Dr.-Ing. Uwe Schnakenberg | Institute of Materials in Electrical Engineering 1 | WS 23 10.2 Capillary Electrophoresis V10 22 Peak Broadening due to Diffusion Peak widths can be expressed in terms of their full width of half maximum (FWHM), which can be related to the standard deviation σdiff Standard deviation Variance  diff FWHM  2 2 ln 2 2  diff  2  D  tm J.W. Jorgenson et al.: Anal. Chem. 53 1298-1302 (1991) Lecture „Microfluidic Systems - Bio-MEMS“ - Capillary Electrophoresis Prof. Dr.-Ing. Uwe Schnakenberg | Institute of Materials in Electrical Engineering 1 | WS 23 D...... Diffusion coefficient tm ….. Migration time 10.2 Capillary Electrophoresis V10 23 Peak Broadening due to Diffusion 2  diff  2  D  tm L L2 Substitution of tm   v µ V 2  diff J.W. Jorgenson et al.: Anal. Chem. 53 1298-1302 (1991) 2  D  L2  µ V σdiff2..Spatial variance of a zone by diffusion D... Diffusion coefficient of solute in the zone tm … Migration time µ … Overall mobility µEOF + µEP V … Applied voltage L … Length of capillary Lecture „Microfluidic Systems - Bio-MEMS“ - Capillary Electrophoresis Prof. Dr.-Ing. Uwe Schnakenberg | Institute of Materials in Electrical Engineering 1 | WS 23 10.2 Capillary Electrophoresis V10 24 Driving Force to Enable Electrophoresis is the Voltage ! V v  µE  µ L 2  diff 2  D  L2  µ V Higher voltage Higher voltage Higher velocity Narrower peak Lecture „Microfluidic Systems - Bio-MEMS“ - Capillary Electrophoresis Prof. Dr.-Ing. Uwe Schnakenberg | Institute of Materials in Electrical Engineering 1 | WS 23 10.2 Capillary Electrophoresis V10 25 Principal Challenge High voltages (electric field strength) generate serious Joule heat with power Q V  Q    E2       L 2 J.W. Jorgenson et al.: Anal. Chem. 53 1298-1302 (1991) D. Wu et al.: J. Chromatography A 1184 542-559 (2008) Lecture „Microfluidic Systems - Bio-MEMS“ - Capillary Electrophoresis Prof. Dr.-Ing. Uwe Schnakenberg | Institute of Materials in Electrical Engineering 1 | WS 23 Q... Power ρ … Fluid conductivity E … Electric field strength V … Voltage L … Capillary length Temperature Gradient Across the Capillary  Fluid is warmer in center and colder V10 26 Parabolic profile Inner diameter r1 at the wall  Results in zone broadening of solute through increase of mobility µ (2 %/°C) Outer diameter r2 http://www2.tci.uni-hannover.de/pdf/Kapillarelektrophorese_Kasper_2009.pdf 10.2 Capillary Electrophoresis Q... Power x … Radius distance from the center of capillary  Solute in the warmer center of the r1 … Radius of microchannel/capillary lumen r2 … Radius of capillary capillary migrates faster k … Thermal conductivity of capillary wall D. Wu et al.: J. Chromatography A 1184 542-559 (2008) kB.. Boltzmann constant X. Xuan et al.: J. Chromatography A 1064 227-237 (2005) h … Heat transfer coefficient to surroundings Lecture „Microfluidic Systems - Bio-MEMS“ - Capillary Electrophoresis Prof. Dr.-Ing. Uwe Schnakenberg | Institute of Materials in Electrical Engineering 1 | WS 23 10.2 Capillary Electrophoresis V10 27 Influence of Applied Voltage on Axial Temperature in Capillary Measured by change of fluorescence intensity of rhodamine B dye 15 s after voltage application Inlet Outlet X. Xuan et al.: Electrophoresis 298 33-43 (2008) und Lab Chip 4 230-236 (2004) Lecture „Microfluidic Systems - Bio-MEMS“ - Capillary Electrophoresis Prof. Dr.-Ing. Uwe Schnakenberg | Institute of Materials in Electrical Engineering 1 | WS 23 10.2 Capillary Electrophoresis V10 28 Inside Separation Channel Zone Broadening due to Joule Heating Electropherogram with heating without heating Zone Under Heat  Moves faster → shorter throughput time (higher mobility)  Dispersed faster → larger half width of full maximum → decrease of peak maximum → decrease in resolution X. Xuan et al.: J. Chromatography A 1064 227-237 (2005) Lecture „Microfluidic Systems - Bio-MEMS“ - Capillary Electrophoresis Prof. Dr.-Ing. Uwe Schnakenberg | Institute of Materials in Electrical Engineering 1 | WS 23 10.2 Capillary Electrophoresis V10 29 Solving the Heat Problem Decrease of capillary diameter  Heat dissipation is more efficient (increased surface/volume ratio)  ΔT from the center to the capillary wall  r12  x 2 Q... Power x … Radius distance from the center of capillary r1 … Radius of microchannel/capillary lumen r2 … Radius of capillary k … Thermal conductivity of capillary wall kB.. Boltzmann constant J.W. Jorgenson et al.: Anal. Chem. 53 1298-1302 (1991) h … Heat transfer coefficient to surroundings Lecture „Microfluidic Systems - Bio-MEMS“ - Capillary Electrophoresis Prof. Dr.-Ing. Uwe Schnakenberg | Institute of Materials in Electrical Engineering 1 | WS 23 10.2 Capillary Electrophoresis V10 30 Five Factors Contribute to Total Peak Broadening 2  Injection plug length 𝜎𝑖𝑛𝑗 2  Molecular diffusion 𝜎𝑑𝑖𝑓𝑓 2  Joule heating 𝜎𝑗𝑜𝑢𝑙 2  Dimensions of the detector spot 𝜎𝑑𝑒𝑡 2  Interaction of fluid with inner capillary walls 𝜎𝑎𝑑𝑠 2 2 2 2 2  tot   inj   det   diff   2joul   ads 2  inj 2 linj  12  2 det 2 ldet  12 2  diff  2  D  tm D. Wu et al.: J. Chromat. A 1184 542-559 (2008) D.J. Harrison et al.: Anal. Chem. 64 1926-1932 (1992) X. Huang et al.: J. Chromat. 480 95-110 (1989) Lecture „Microfluidic Systems - Bio-MEMS“ - Capillary Electrophoresis Prof. Dr.-Ing. Uwe Schnakenberg | Institute of Materials in Electrical Engineering 1 | WS 23 linj … Length of initial sample plug ldet.. Length of detector window σ2 …. Variance V10 31 10.3 Capillary Electrophoresis on a Chip Lecture „Microfluidic Systems - Bio-MEMS“ - Capillary Electrophoresis Prof. Dr.-Ing. Uwe Schnakenberg | Institute of Materials in Electrical Engineering 1 | WS 23 10.3 Capillary Electrophoresis on a Chip V10 32 Principle of Capillary Electrophoresis on a Chip Sample inlet Detector Outlet Buffer inlet Channel Inlets and outlets with electrodes Lecture „Microfluidic Systems - Bio-MEMS“ - Capillary Electrophoresis Prof. Dr.-Ing. Uwe Schnakenberg | Institute of Materials in Electrical Engineering 1 | WS 23 Glass substrate 10.3 Capillary Electrophoresis on a Chip V10 33 Sample inlet Stack Injection Sample is continuously stacked into Buffer the separation channel during injection time Sample inlet Size of injection zone is determined by  Injection time Buffer  Electrophoretic mobility of ions in sample A.T. Woolley et al.: Proc. Natl. Acad. Sci. 91 11348-11352 (1994) Lecture „Microfluidic Systems - Bio-MEMS“ - Capillary Electrophoresis Prof. Dr.-Ing. Uwe Schnakenberg | Institute of Materials in Electrical Engineering 1 | WS 23 10.3 Capillary Electrophoresis on a Chip V10 34 Sample inlet Plug Injection Sample is stacked into the inlet channel Buffer Size of injection zone is determined by  geometry of channel intersection Sample inlet Buffer With plug injection, the detection signal is lower than for the stack injection, but resolution is superior A.T. Woolley et al.: Proc. Natl. Acad. Sci. 91 11348-11352 (1994) Lecture „Microfluidic Systems - Bio-MEMS“ - Capillary Electrophoresis Prof. Dr.-Ing. Uwe Schnakenberg | Institute of Materials in Electrical Engineering 1 | WS 23 10.3 Capillary Electrophoresis on a Chip V10 35 Sample Injection Electrokinetic injection Is preferred for sample injection, since the flow rate of solvent is controlled by electrokinetic effects independently of capillary dimensions Pressure driven injection Conventional pumps not preferred, because of extremely high fluid resistance Rfl in the small capillaries - not suitable for injection A.T. Woolley et al.: Proc. Natl. Acad. Sci. 91 11348-11352 (1994) Lecture „Microfluidic Systems - Bio-MEMS“ - Capillary Electrophoresis Prof. Dr.-Ing. Uwe Schnakenberg | Institute of Materials in Electrical Engineering 1 | WS 23 10.3 Capillary Electrophoresis on a Chip V10 36 One of the First Examples for CE on a Glass Chip  Channels  Inlets  Outlet 14.8 cm / Buffer 1 mm 3.9 cm Detector 30 µm D.J. Harrison et al.: Anal. Chem. 64 1926-1932 (1992) K. Seiler et al.: Anal. Chem. 55 1481-1488 (1993) Lecture „Microfluidic Systems - Bio-MEMS“ - Capillary Electrophoresis Prof. Dr.-Ing. Uwe Schnakenberg | Institute of Materials in Electrical Engineering 1 | WS 23 10.3 Capillary Electrophoresis on a Chip High Voltage Supply D.J. Harrison et al.: Anal. Chem. 64 1926-1932 (1992) K. Seiler et al.: Anal. Chem. 55 1481-1488 (1993) Lecture „Microfluidic Systems - Bio-MEMS“ - Capillary Electrophoresis Prof. Dr.-Ing. Uwe Schnakenberg | Institute of Materials in Electrical Engineering 1 | WS 23 V10 37 10.3 Capillary Electrophoresis on a Chip V10 38 Stack Injection  250 V between  and  for 30 s / Buffer draw sample into channel   3000 V between  and  drive the injection zone along channel  affecting Detector electrophoretic separation   Calcein  D.J. Harrison et al.: Anal. Chem. 64 1926-1932 (1992) K. Seiler et al.: Anal. Chem. 55 1481-1488 (1993) Lecture „Microfluidic Systems - Bio-MEMS“ - Capillary Electrophoresis Prof. Dr.-Ing. Uwe Schnakenberg | Institute of Materials in Electrical Engineering 1 | WS 23 Fluorescein 10.3 Capillary Electrophoresis on a Chip Influence of Electric Field Strength During Stack Injection K. Seiler et al.: Anal. Chem. 55 1481-1488 (1993) Lecture „Microfluidic Systems - Bio-MEMS“ - Capillary Electrophoresis Prof. Dr.-Ing. Uwe Schnakenberg | Institute of Materials in Electrical Engineering 1 | WS 23 V10 39 10.3 Capillary Electrophoresis on a Chip V10 40 Agilent 2100 Bioanalyzer www.lokmis.lt https://www.ksre.k-state.edul C.-Y. Lu et al.: Clinica Chimica Acta 318 97-105 (2002) Lecture „Microfluidic Systems - Bio-MEMS“ - Capillary Electrophoresis Prof. Dr.-Ing. Uwe Schnakenberg | Institute of Materials in Electrical Engineering 1 | WS 23 www.my.ilabsolutions.com 10.3 Capillary Electrophoresis on a Chip Agilent 2100 Bioanalyzer http://biochem.cores.ucla.edu/ Lecture „Microfluidic Systems - Bio-MEMS“ - Capillary Electrophoresis Prof. Dr.-Ing. Uwe Schnakenberg | Institute of Materials in Electrical Engineering 1 | WS 23 V10 41 10.3 Capillary Electrophoresis on a Chip V10 42 Agilent Bio-Rad Perkin Elmer 4200 TapeStation system Expersion System LabChip GXII Touch HT allows analysis of up to 96 samples www.agilent.com www.bio-rad.com Lecture „Microfluidic Systems - Bio-MEMS“ - Capillary Electrophoresis Prof. Dr.-Ing. Uwe Schnakenberg | Institute of Materials in Electrical Engineering 1 | WS 23 www.perkinelmer.com 10.4 Capillary Isoelectric Focusing (cIEF) 10.4 Capillary Isoelectric Focusing (CIEF) https://www.agilent.com/cs/library/primers/public/5991-1660EN.pdf https://www.youtube.com/watch?v=ph7JtxVNF5A Lecture „Microfluidic Systems - Bio-MEMS“ - Capillary Electrophoresis Prof. Dr.-Ing. Uwe Schnakenberg | Institute of Materials in Electrical Engineering 1 | WS 23 V10 43 10.4 Capillary Isoelectric Focusing (CIEF) In electrophoresis  Separation of charged molecules/proteins according to q/r-ratio vEP ,i  qi E 6    ri  Also separation by EP with respect to charge only is possible Capillary Isoelectric Focusing (CIEF) Lecture „Microfluidic Systems - Bio-MEMS“ - Capillary Electrophoresis Prof. Dr.-Ing. Uwe Schnakenberg | Institute of Materials in Electrical Engineering 1 | WS 23 V10 44 10.4 Capillary Isoelectric Focusing (CIEF) Isoelectric Focusing (IEF) - Electrophoretic Separation by Charge  Net charge of molecules depends on pH value of solute  Net charge of a molecule is zero at a distinct pH value, the isoelectric point (IEP, pI)  The IEP is the pH where the net charge of molecule is zero  Separation of charged molecules with respect to IEP Lecture „Microfluidic Systems - Bio-MEMS“ - Capillary Electrophoresis Prof. Dr.-Ing. Uwe Schnakenberg | Institute of Materials in Electrical Engineering 1 | WS 23 V10 45 10.4 Capillary Isoelectric Focusing (CIEF) V10 46 Principle of Isoelectric Focusing When a Charged Molecule Approximates the IEP  Stop of movement  Collection / Concentration of particles of the same charge at one IEP  Focusing of molecules of same charge Lecture „Microfluidic Systems - Bio-MEMS“ - Capillary Electrophoresis Prof. Dr.-Ing. Uwe Schnakenberg | Institute of Materials in Electrical Engineering 1 | WS 23 en.wikipedia.org 10.4 Capillary Isoelectric Focusing (CIEF) V10 47 How to Form a Stable pH Gradient inside a Capillary? - Stable pH Gradient is Formed by Ampholytes - Ampholytes are molecules that  contain ionizable acidic and alkaline groups depending on pH of molecule’s environment  become neutral at the pH where positive and negative charges in the molecule are balanced, at the IEP Structural formula Lecture „Microfluidic Systems - Bio-MEMS“ - Capillary Electrophoresis Prof. Dr.-Ing. Uwe Schnakenberg | Institute of Materials in Electrical Engineering 1 | WS 23 10.4 Capillary Isoelectric Focusing (CIEF) Principle of IEF http://www.youtube.com/watch?v=9jW8n1Ailic Lecture „Microfluidic Systems - Bio-MEMS“ - Capillary Electrophoresis Prof. Dr.-Ing. Uwe Schnakenberg | Institute of Materials in Electrical Engineering 1 | WS 23 V10 48 10.4 Capillary Isoelectric Focusing (CIEF) V10 49 Preparation of Capillary for CIEF 1. Loading the fused silica capillary by pressure in essence with premixed  Sample proteins  Ampholytes 2. Connecting one end of the filled capillary with low pH electrolyte (anolyte) 3. Connecting the other end of the filled capillary with high pH electrolyte (catholyte) 4. Applying an electric field Anode Cathode + Anolyte H3PO4 https://www.agilent.com/cs/library/primers/public/5991-1660EN.pdf Lecture „Microfluidic Systems - Bio-MEMS“ - Capillary Electrophoresis Prof. Dr.-Ing. Uwe Schnakenberg | Institute of Materials in Electrical Engineering 1 | WS 23 Catholyte NaOH 10.4 Capillary Isoelectric Focusing (CIEF) V10 50 The following three steps occur simultaneously Step 1 of 3  Hydronium ions from the anolyte vial will move fast towards the cathode  Hydroxide ions will move fast from the cathode to the anode  Establishment of a non-stable pH gradient H3O+ Anode OH- + Anolyte H3PO4 https://www.agilent.com/cs/library/primers/public/5991-1660EN.pdf Lecture „Microfluidic Systems - Bio-MEMS“ - Capillary Electrophoresis Prof. Dr.-Ing. Uwe Schnakenberg | Institute of Materials in Electrical Engineering 1 | WS 23 Cathode Catholyte NaOH 10.4 Capillary Isoelectric Focusing (CIEF) V10 51 Step 2 of 3  Carrier ampholytes will automatically arrange themselves in the order of their pls so that those with the lowest pls will end up at the anode and those with the highest pls will be found at the cathode  When the pH gradient is fully established, the net charges of all the carrier ampholytes will be zero and the gradient becomes stationary  Formation of a stable pH gradient Anode Cathode Stable pH gradient + 3 4 5 6 7 8 9 10 Anolyte H3PO4 https://www.agilent.com/cs/library/primers/public/5991-1660EN.pdf Lecture „Microfluidic Systems - Bio-MEMS“ - Capillary Electrophoresis Prof. Dr.-Ing. Uwe Schnakenberg | Institute of Materials in Electrical Engineering 1 | WS 23 Catholyte NaOH 10.4 Capillary Isoelectric Focusing (CIEF) V10 52 Step 3 of 3  Sample particles/proteins become sorted by electrophoretic migration from low pl to high pl in a charge-free medium  Sorting stops when the molecule is electroneutral Anode Cathode + 3 4 5 6 7 8 9 10 Anolyte H3PO4 Catholyte NaOH Protein pl 5 Protein pl 7 Protein pl 9 de.wikipedia.org https://www.agilent.com/cs/library/primers/public/5991-1660EN.pdf Lecture „Microfluidic Systems - Bio-MEMS“ - Capillary Electrophoresis Prof. Dr.-Ing. Uwe Schnakenberg | Institute of Materials in Electrical Engineering 1 | WS 23 10.4 Capillary Isoelectric Focusing (CIEF) Characterization of Focusing Processes  At the beginning high current is observed, which continuously decreases of several minutes until it reaches a minimum (10 % of original value)  Current is related to formation of pH gradient  The analytes and ampholytes initially posses a net charge, which decreases upon neutralization by H3O+ and OHduring the focusing process Lecture „Microfluidic Systems - Bio-MEMS“ - Capillary Electrophoresis Prof. Dr.-Ing. Uwe Schnakenberg | Institute of Materials in Electrical Engineering 1 | WS 23 V10 53 10.4 Capillary Isoelectric Focusing (CIEF) V10 54 Detection  EOF takes place simultaneously to sorting  Analytes move towards the detector while being focused  EOF must be low so that the entire train of proteins can reach equilibrium before emerging at the detector → Conditioning of inner wall surface (hydrophobic polymer, hydroxymethyl cellulose) to neutralize EDL and Detector decrease EOF Anode Cathode + 3 4 5 6 7 Anolyte H3PO4 Lecture „Microfluidic Systems - Bio-MEMS“ - Capillary Electrophoresis Prof. Dr.-Ing. Uwe Schnakenberg | Institute of Materials in Electrical Engineering 1 | WS 23 8 9 10 Catholyte NaOH 10.4 Capillary Isoelectric Focusing (CIEF) V10 55 Capillary Electrophoresis Disadvantages Advantages  Small sample volume (μL - nL)  Not applicable for insoluble molecules  Fast separation and detection  High voltages needed  Measuring time 2-10 min  Inefficient for continuous monitoring  High resolution  More expensive than standard gel  Use of sensitive detector electrophoresis  Automation  Anoins and cations detectable Lecture „Microfluidic Systems - Bio-MEMS“ - Capillary Electrophoresis Prof. Dr.-Ing. Uwe Schnakenberg | Institute of Materials in Electrical Engineering 1 | WS 23 Conclusion V10 56 Capillary Electrophoresis (CE)  Well established in analytical chemistry Conclusion Capillary Electrophoresis on Chip  Commercialized  Competition to gel electrophoresis  Advantage: Very small sample volumes Capillary Isoelectric Focusing (CIEF)  Can be applied in standard CE equipment  Mainly used for determination of pl of purified proteins and/or assessment of the charge heterogeneity of the particular proteins Lecture „Microfluidic Systems - Bio-MEMS“ - Capillary Electrophoresis Prof. Dr.-Ing. Uwe Schnakenberg | Institute of Materials in Electrical Engineering 1 | WS 23 V10 57 One Minute Paper 1. What was the most important topic you understood? 2. What was the topic you didn‘t catch? Lecture „Microfluidic Systems - Bio-MEMS“ - Capillary Electrophoresis Prof. Dr.-Ing. Uwe Schnakenberg | Institute of Materials in Electrical Engineering 1 | WS 23