DISCOVER THE CAPILLARY ELECTROPHORESIS
In CE, a low volume of sample is loaded into the capillary in order to maintain a high efficiency.
Quantitative sample injection can be accomplished by two different modes :
- Hydrodynamic injection : it can be accomplished by application of pressure at the injection end of the capillary (or vacuum at the exit end of the capillary. With hydrodynamic injection mode, the injected quantity of sample is directly related to the injection time and the pressure value.
- Electrokinetic injection : It is performed by applying a voltage (some kilovolts) at both ends of the capillary. With this voltage, analyte enters the capillary by migration and by action of the electroosmotic flow. The injected quantity of sample is directly related to the injection time, the voltage value, the electrophoretic mobility of the compound and the electroosmotic mobility.
TRANSPORT PROCESSES IN CE
the electroosmotic flow and the electrophoretic migration
Electroosmotic flow (EOF)
Under main aqueous conditions (pH > 2.5), silica narrow bore capillary surface possesses an excess of negative charges resulting from the ionization of silanol groups. Counterions (cations, in most cases) ; which build up near the surface to maintain charge balance, form a double-layer and create a potential (zeta potential) difference close to the capillary wall. With the voltage application, the cation forming the double-layer are attracted toward the cathode and the resulting flow has a flat profile. This electroosmotic flow can be modified, inversed or deleted by covalent or dynamic capillary wall modifications using surfactants or neutral or ionized polymers.
In CE, separation is related to the differential migration of compounds in a applied Electric field. The Electrophoretic Migration velocity is directly related to the electrophoretic mobility. The electrophoretic mobility depends on pH buffer, ionic strength, buffer composition and viscosity.
Separation in CE
Today, CE appears as a performant and interesting analytical technique with fast, efficient and highly resolutive separations, low solvant and sample consumption, and low-running costs. CE is a valuable alternative to chromatographic techniques and can solve analytical problems.
The versatility of CE is partially derived from its numerous mode of separation. The separation mechanisms of each mode are different and can offer orthogonal and complementary information.
- a stationary phase of chromatography (this is called capillary electrochromatography)
- an ampholytic mixture creating an increasing pH gradient from the anode to the cathode (isoelectric focusing, IEF)
- several buffers of variable conductivities (capillary isotachophoresis, ITP).
Micellar electrokinetic chromatography (MEKC), is one of the most widely used CE modes. This mode can be used for the separation of neutral compounds as well as charged ones. The separation is accomplished by the used of surfactants in the running buffer. At concentration above the critical micelle concentration, micelles are formed in the capillary. During migration, micelles can interact with neutral compounds in a chromatographic manner through both hydrophobic and electrostatic interactions. The more the neutral compounds interacts with the micelle the longer is its migration time.
Capillary Gel Electrophoresis (CGE) allows the separation of molecules according to their sizes. The size separation is currently used for proteins analysis and is carried out with a capillary filled by a Gel Buffer which limits heating and acts as a molecular sieve.
Finally, CE can be used with a capillary filled with :
– A chromatographic stationnary phase (Capillary ElectroChromatography, CEC)
– A mixture of ampholytes creating a pH gradient in the capillary (Capillary isoelectric focusing, CIEF)
– Several running buffers with different conductivities (Capillary isotachophoresis, ITP)
UV-Visible absorption is the most widely used detection method. It was carried out accross the capillary with creation of a detection window by burning the external polyimide layer. To increase sensibility (which is low due to a short path length), Z-cell or bubble cell can be used.
Contactless Conductivity Detector (C4D) is a valuable alternative to UV detection for the analysis of ionized compounds. This detection method is universal and is based on conductivity measures between two electrodes placed on each side of the capillary.
For organic compounds, amperometric or fluorescence detections can also be implemented.
Detection by Mass Spectrometry are grown significantly for CE separations since 90s. Two interfaces are available : interface with or without sheath liquid.
Ionization process as ESI, APCI, APPI, ICP or MALDI are widely used as ionization modes for CE.