High Performance Capillary Electrophoresis A Primer A Primer High Performance Capillary Electrophoresis Second completely revised edition by Henk H. Lauer and Gerard P. Rozing Foreword Electrophoresis was born at the beginning of nineteenth century, even earlier than chromatography. It has taken a long development path, which in the early days was framed by names like Kohlrausch, Tiselius and later Everaerts. At the beginning of eighties in the last century, Jorgenson cause a paradigm shift by executing electrophoresis in a very thin capillary with inner diameter less then 100 μm leading to capillary electrophoresis. Since then, capillary electrophoresis has been constantly improved and has become a routine technique. Instrument manufacturers like Agilent Technologies have developed instrumentation for capillary electrophoresis serving many analytical laboratories reliably for many years. After a decline at the beginning of this decade, the capillary electrophoresis market is recovering strongly. Especially, hyphenation with mass spectrometers has been very fruitful and brought a remarkable ability to unravel complex structures of biomolecules with invincible speed and sensitivity. And there are several modes of capillary electrophoresis, which make it useful for separation of really wide class of compounds, such as small ions, peptides, proteins, DNA fragments both sequencing and restriction ones, and even complete cells or uncharged molecules. When compared with liquid chromatography capillary electrophoresis has one big advantage: the separation process takes place in a rather simple environment – in homogeneous solutions or in a polymeric sieving network and in a separation space with simple geometry, like a cylindrical channel. The equations describing completely movement of matter in space and time can be than easily formulated and solved by advanced mathematical methods. Here several researchers have done a great job. The deep understanding of basic principles of the separation process allows fast development of instru- mentation, sophisticated simulation and prediction of separation, and easier development of new electrophoretic separation methods. This primer is an excellent entry for novices in the field. I am sure capillary electrophoresis will live long in the new instrumentation and will serve well for separation scientists. And will excite them with its elegancy and efficiency. Bohuslav Gaš Professor Faculty of Science, Charles University in Prague II Foreword From the early days of electrophoresis as separation technique, introduced by Tiselius, capillary electrophoresis has been applied for the separation of substances from many different origins. After the pioneering work of Hjertén, Everaerts and Jorgenson, the 1990s showed a real explosion of applications using capillary electrophoresis (CE). Both creative solutions to separation problems as well as validated methods from industry appeared in literature. After the dust of the application explosion settled at the start of this century, it became apparent that the CE techniques are an important part in the toolbox of analytical scientists. Some of the applications are easy to observe, such as general chapters in Pharmacopoeia, CE methods in pharmacopoeial monographs and new drug applications from pharmaceutical companies and CE methods replacing slab gel methods in the biotech industry. Many pharmaceutical com- panies use CE for chiral separations and small ions. There are many application kits available through several suppliers. But there are also “hidden” applications. Several instruments for specific applications such as DNA/RNA analyzers and protein analyzers use CE as the separation mechanism. Through further miniaturization, CE contributes to the development of lab-on-a-chip. Today’s challenges to strengthen the implementation of CE techniques in the pharmaceutical industry, are further improved equipment and training. After the big steps made towards good instrumentation in the late 1980s and 1990s lots of lessons have been learned from the use of CE in both R&D and quality control laboratories which should be translated in new generation instruments. For future instrumental improvements it is important to work from the fundamental concepts of electrophoresis. As for training, there is a strong need for skilled people. CE method development is very much a conceptual approach, that is thinking in mechanisms. Additionally it is important to train on “Good CE Practice”, since CE in its handling is neither liquid chromatography nor slab gel electrophoresis. The first primer from HP/Agilent was an important tool in acquainting and training people in the CE techniques. Hopefully this renewed primer will prove an as good, or even better, instrument for that. Dr. Cari Sänger – van de Griend Pim Muijselaar Senior Analytical Scientists, Solvay Pharmaceuticals BV New Chemical Entities Development NCED, Weesp, The Netherlands III Table of Content 1. Principles of capillary 1.1. Historical background, current status and applications of CE .......2 1.2. Basic components ........................................4 electrophoresis 1.3.Theory ................................................5 1.3.1. Electrophoresis ....................................5 1.3.2. Electroosmotic flow (EOF) and control of EOF ............7 1.3.3. Mobility and migration time .........................12 1.3.4. Zone broadening and efficiency ......................14 1.3.4.1. Longitudinal diffusion .......................14 1.3.4.2. Joule heating .............................17 1.3.4.3. Injection plug length ........................22 1.3.4.4. Solute-surface interactions ...................23 1.3.4.5. Electrodispersion ...........................25 1.3.4.6. Unleveled buffer reservoirs ...................29 1.3.4.7. Detector .................................29 1.3.5. Resolution .......................................30 1.4. Characteristics of capillary electrophoresis ....................31 2. Modes of operation 2.1. Capillary Zone Electrophoresis (CZE) ........................34 2.2. Micellar Elektrokinetic Chromatography (MEKC) ...............37 2.3. Capillary Electrochromatography (CEC) ......................43 2.4. Capillary Gel Electrophoresis (CGE) .........................49 2.5. Capillary Isoelectric Focusing (CIEF) .........................53 2.6. Capillary Isotachophoresis (CITP) ...........................55 3. Instrumentation 3.1. High voltage power supply ................................58 3.2. Sample introduction .....................................59 3.2.1. Hydrodynamic injection .............................60 3.2.2. Electrokinetic injection .............................64 3.2.3. On-capillary sample concentration ....................65 3.2.3.1. Field amplified sample stacking (FASS) and field amplified sample injection (FASI) .......65 3.2.3.2. Isotachophoretic sample stacking ..............68 3.2.3.3.High-saltstacking..........................72 3.3. Temperature control .....................................73 3.4. Detection .............................................73 3.4.1. UV-vis spectrophotometric detection ..................74 3.4.1.1. Noise, sensitivity, linear detection range ........75 3.4.1.2. Extended path length flow cells ...............77 3.4.1.3. Application of spectral data ..................81 3.4.1.4. Indirect photometric detection ................87 3.4.2. Laser Induced Fluorescence (LIF) .....................90 3.4.3. Contactless Conductivity Detection (CCD) ..............96 IV Table of Content 3.5. Fraction collection .....................................101 3.6. Capillary electrophoresis coupled with mass spectrometric detection and ICP-MS ..................107 3.6.1. Fundamentals of CE-MS interfacing ..................108 3.6.2. Electrical interfacing ..............................109 3.6.3. Hydraulic interfacing ..............................110 3.6.3.1. Sheath flow interface for CE-MS .............111 3.6.3.2. Sheathless interface for CE-MS ..............113 3.6.4. Other ionization methods ..........................115 4. Practical operation 4.1. Separation capillary ....................................118 and method 4.1.1. Bare fused silica .................................118 4.1.2. Wall-coated capillaries ............................121 development 4.1.2.1. Permanent coating capillaries ...............122 4.1.2.2. Dynamically coated capillaries ...............123 4.2. Electrolyte replenishment & capillary conditioning .............129 4.2.1. Buffer replenishment ..............................129 4.2.2. Capillary conditioning .............................131 4.2.3. Common buffer systems ...........................132 4.3. Troubleshooting .......................................133 4.4. Method development ...................................137 4.4.1.Howtostart? ...................................139 4.4.2. Capillary Zone Electrophoresis (CZE) .................141 4.4.2.1. Simulations with PeakMaster ................145 4.4.3. Micellar Electrokinetic Chromatography (MEKC) ........147 4.4.3.1. Separation of enantiomers ..................149 4.4.4. Capillary Electrochromatography (CEC) ...............152 4.4.5.CE-MS.........................................153 4.4.5.1. Hydraulic interfacing and electrical connections .153 4.4.5.2. Setting nebulizing gas pressure ..............155 4.4.5.3. Delivering sheath solvent, composition of sheath solvent ...............155 4.4.5.4.BufferchoiceforCE-MS....................156 4.4.6. Capillary Gel Electrophoresis