1Build your own CE workshop, Brno Oct 15-16, 2018
BUILD YOUR OWN CAPILLARY ELECTROPHORESISINSTRUMENT
Petr Kubáň
Department of Bioanalytical InstrumentationCentral Institute of Technology (CEITEC) – Masaryk University
Institute of Analytical Chemistry of the Czech Academy of SciencesVeveří 97, 602 00, Brno, Czech Republic
Email: [email protected]
2Build your own CE workshop, Brno Oct 15-16, 2018
LITERATURE (open source)
Tutorial: Capillary electrophoresishttp://www.esi.umontreal.ca/~badiaa/CE%20basics.pdf
Basic Principles and Modes of Capillary Electrophoresishttps://www.springer.com/cda/content/document/cda_downloaddocument/9780896036451-c2.pdf?SGWID=0-0-45-454204-p173727706
HP primer: Capillary electrophoresis: An Introductionhttp://www.colby.edu/chemistry/CH332/laboratory/Agilent%20CE%20Primer.pdf
Introduction to capillary electrophoresis (Beckman coulter)https://ls.beckmancoulter.co.jp/files/appli_note/CEPrimer1.pdf
Basics of Capillary Electrophoresis (Ronald E, Majors, HP)matematicas.udea.edu.co/~carlopez/cnq533/CE_Basics.pdf
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CAPILLARY ELECTROPHORESIS
separation technique (liquid phase)
narrow-bore fused silica capillaries (10–75 μm id)
small sample volume required (1 to 50 nl injected)
high voltages (10 to 30 kV) and high electric fields (100 to 1000 V/cm) are applied across the capillary
high resistance of the capillary limits current generation and internal heating (currents typically µA)
high efficiency (N>10.000 to 100.000)
short analysis time
4Build your own CE workshop, Brno Oct 15-16, 2018
sample sample
high voltage
separation electrolyte
capillary
+
+
++_
___
+ _
detector
CAPILLARY ELECTROPHORESIS SYSTEM
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PHOTO OF A SIMPLEST CE SYSTEM
HV supply
DAQ
capillary
VIAL + electrode
VIAL + electrode
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B
HV C4D
T
CPDAS
INJ + G
Agilent
Prince
Wynsep
EH systems
Home build CE systems
7Build your own CE workshop, Brno Oct 15-16, 2018
Basics of Capillary Electrophoresis(Ronald E, Majors, HP)matematicas.udea.edu.co/~carlopez/cnq533/CE_Basics.pdf
ELECTROOSMOTIC FLOW
Negatively charged
fused silica surface
(Si-O ¯ )
Hydrated cations
accumulate near
the surface
Bulk flow is towards
the cathode upon
application of the
electric field
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ELECTROPHORETIC MOBILITY IN CE, µA
A
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https://www.youtube.com/watch?v=wStV1rFjHOo
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EOF (CE) vs. Pressure flow (HPLC)
Basics of Capillary Electrophoresis(Ronald E, Majors, HP)matematicas.udea.edu.co/~carlopez/cnq533/CE_Basics.pdf
11Build your own CE workshop, Brno Oct 15-16, 2018
INJECTION IN CAPILLARY ELECTROPHORESIS
Basics of Capillary Electrophoresis (Ronald E, Majors, HP) matematicas.udea.edu.co/~carlopez/cnq533/CE_Basics.pdf
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BUFFER (BACKGROUND ELECTROLYTE) CONSIDERATIONS
Always need to consider co- and counter- ion
Good buffering capacity (pKa), pH influence separation
Co-ion mobility matching the analytes
Suitable solubility, no interaction with analytes/wall
Suitable optical properties/conductivity
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BGE
Asr
CONCENTRATION PROFILE ELECTROPHEROGRAM
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direct UV detection
indirect UV detection
µA > µBGE µA = µBGE µA < µBGE
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Conductivity detection
µA> µBGE µA = µBGE µA < µBGE
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20Build your own CE workshop, Brno Oct 15-16, 2018
FUSED SILICA CAPILLARIES
ID 10-75 um
Fused silica, PTFE, PEEK
Shape (circular, rectangular)
PRODUCERS
Polymicrotechnologies MOLEX
Microquartz
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HIGH VOLTAGE POWER SUPPLIES
Ready to use Modules
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PHOTO OF OUR IN-HOUSE BUILT HVPS
Based on EMCO DX250 module
Based on Spellmann UM8*4 module
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DETECTORS
TYPE Commercially available
DIY
UV-VIS absorbance YES YES
Contactless conductometric (C4D, CCD etc.) YES YES
Fluorescence (LIF, LED-LIF) YES YES
Amperometric NO YES
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DATA ACQUISITION DEVICES
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CapillaryCE SYSTEM CONSISTS OF 3 MAJOR PARTS
CONCLUSIONS
HV power suppplyDetector
CE is easy to build from ready modules ~10 minutes
CE is cheap – total cost: 500-3000 EUR
CE is easily made portable
CE is the best separation technique for the OPEN SOURCE HW