Electrophoresis: Instrumentation

Instrumentation

The basic instrumentation for capillary electrophoresis is shown in Figure 12.41 and includes a power supply for applying the electric field, anode and cathode compart- ments containing reservoirs of the buffer solution, a sample vial containing the sample, the capillary tube, and a detector. Each part of the instrument receives fur- ther consideration in this section.

Capillary Tubes 

Figure 12.42 shows a cross section of a typical capillary tube. Most capillary tubes are made from fused silica coated with a 20–35-μm layer of poly- imide to give it mechanical strength. The inner diameter is typically 25–75 μm, which is smaller than that for a capillary GC column, with an outer diameter of200–375 μm.

The narrow bore of the capillary column and the relative thickness of the capil- lary’s walls are important. When an electric field is applied to a capillary containing a conductive medium, such as a buffer solution, current flows through the capillary. This current leads to Joule heating, the extent of which is proportional to the capil- lary’s radius and the magnitude of the electric field. Joule heating is a problem be- cause it changes the buffer solution’s viscosity, with the solution at the center of the capillary being less viscous than that near the capillary walls. Since the solute’s elec- trophoretic mobility depends on the buffer’s viscosity (see equation 12.36), solutes in the center of the capillary migrate at a faster rate than solutes near the capillary walls. The result is additional band broadening that degrades the separation. Capil- laries with smaller inner diameters generate less Joule heating, and those with larger outer diameters are more effective at dissipating the heat. Capillary tubes may be placed inside a thermostated jacket to control heating, in which case smaller outer diameters allow a more rapid dissipation of thermal energy.

Injecting the Sample 

The mechanism by which samples are introduced in capil- lary electrophoresis is quite different from that used in GC or HPLC. Two types of injection are commonly used: hydrodynamic injection and electrokinetic injec- tion. In both cases the capillary tube is filled with buffer solution. One end of the capillary tube is placed in the destination reservoir, and the other is placed in the sample vial.

Hydrodynamic injection uses pressure to force a small portion of the sample into the capillary tubing. To inject a sample hydrodynamically a difference in pres- sure is applied across the capillary by either pressurizing the sample vial or by ap- plying a vacuum to the destination reservoir. The volume of sample injected, in liters, is given by the following equation

where ∆P is the pressure difference across the capillary in pascals, d is the capillary’s inner diameter in meters, t is the amount of time that the pressure is applied in sec- onds, μ is the buffer solution’s viscosity in kilograms per meter per second (kg m–1 s–1), and L is the length of the capillary tubing in meters. The factor of 103 changes the units from cubic meters to liters.

Electrokinetic injections are made by placing both the capillary and the anode into the sample vial and briefly applying an electric field. The moles of solute in- jected into the capillary, n_inj, are determined using

where C is the solute’s concentration in the sample, t is the amount of time that the electric field is applied, r is the capillary’s radius, μ_ep is the solute’s electrophoretic mobility, μ_eof is the electroosmotic mobility, E is the applied electric field, and κ_buf and κ_samp are the conductivities of the buffer solution and sample, respectively. An important consequence of equation 12.45 is that it is inherently biased toward sampling solutes with larger electrophoretic mobilities. Those solutes with the largest electrophoretic mobilities (smaller, more positively charged ions) are injected in greater numbers than those with the smallest electrophoretic mobilities (smaller, more negatively charged ions).

When a solute’s concentration in the sample is too small to reliably analyze, it may be possible to inject the solute in a manner that increases its concentration in the capillary tube. This method of injection is called stacking. Stacking is accom- plished by placing the sample in a solution whose ionic strength is significantly less than that of the buffering solution. Because the sample plug has a lower concentra- tion of ions than the buffering solution, its resistance is greater. Since the electric current passing through the capillary is fixed, we know from Ohm’s  law

E = iR

that the electric field in the sample plug is greater than that in the buffering solu- tion. Electrophoretic velocity is directly proportional to the electric field (see equation 12.35); thus, ions in the sample plug migrate with a greater velocity. When the solutes reach the boundary between the sample plug and the buffering solution, the electric field decreases and their electrophoretic velocity slows down, “stacking” to- gether in a smaller sampling zone (Figure 12.43).

Applying the Electric Field 

Migration in electrophoresis occurs in response to the applied electric field. The ability to apply a large electric field is important because higher voltages lead to shorter analysis times (see equation 12.41), more efficient separations (see equation 12.42), and better resolution (see equation 12.43). Be- cause narrow-bore capillary tubes dissipate Joule heating so efficiently, voltages of up to 40 kV can be applied.

Detectors 

Most of the detectors used in HPLC also find use in capillary elec- trophoresis. Among the more common detectors are those based on the absorption of UV/Vis radiation, fluorescence, conductivity, amperometry, and mass spectrom- etry. Whenever possible, detection is done “on-column” before the solutes elute from the capillary tube and additional band broadening occurs.

UV/Vis detectors are among the most popular. Because absorbance is directly proportional to path length, the capillary tubing’s small diameter leads to signals that are smaller than those obtained in HPLC. Several approaches have been used to increase the path length, including a Z-shaped sample cell or multiple reflections (Figure 12.44). Detection limits are about 10–7 M.

Better detection limits are obtained using fluorescence, particularly when using a laser as an excitation source. When using fluorescence detection, a small portion of the capillary’s protective coating is removed and the laser beam is focused on the inner portion of the capillary tubing. Emission is measured at an angle of 90° to the laser. Because the laser provides an intense source of radiation that can be focused to a narrow spot, detection limits are as low as 10–16 M.

Solutes that do not absorb UV/Vis radiation or undergo fluorescence can be detected by other detectors. Table 12.8 provides a list of detectors used in capillary electrophoresis along with some of their important characteristics.