# Background

### Fundamentals

There are two types of transport in free zone capillary electrophoresis. The first is electrophoretic transport, which is based on the charge attraction or repulsion of the analyte to a cathode (or anode). This is affected by frictional drag, so electrophoretic transport is related to the size-to-charge ratio of the analyte. Electrophoretic velocity, νeph, is the product of electrophoretic mobility, μeph, applied voltage, V, and capillary length, L, (equation 1.1). The second type of transport in electrophoresis is a bulk flow, termed electroosmotic flow. The negative surface charge on the surface of the separation capillary leads to a double layer. When the high voltage is applied to the capillary, the electrolyte moves towards the cathode and induces bulk liquid flow with a plug profile. Electroosmotic velocity, νeof, is the product of the electroosmotic mobility, μeof, applied voltage, V, and capillary length, L (equation 1.2).

$ν_{eph} = \dfrac{μ_{eph} \mathrm{V}}{\mathrm{L}} \tag{equation 1.1}$

$ν_{eof} = \dfrac{μ_{eof}\mathrm{V}}{\mathrm{L}} \tag{equation 1.2}$

The entire is occurs in the presence of the applied electric field and charged surface sites on the wall of the fused silica capillary. In a typical capillary electrophoresis configuration the anode is located at injection side of capillary and the cathode at the detection end of the capillary. In such a configuration, the electroosmotic flow moves from the injection to detection end of the capillary (anode to cathode). These two transport mechanisms are combined for net flow. The electrophoretic mobility of cations drives them towards the detection end of the capillary. Small highly charged cations have a faster electrophoretic velocity than large highly charged cations, and all cations are transported by the sum of electroosmotic and electrophoretic transport. Neutral compounds have no electrophoretic mobility and are transported solely by electroosmotic flow. Thus, a neutral analyte is not separated from other neutral compounds. The electrophoretic mobility of anions drives them towards the injection end of the capillary (against the electroosmotic flow). Small highly charged anions have a faster electrophoretic velocity than large highly charged anions, and all anions are transported by the difference of electroosmotic and electrophoretic transport. The net effect of these transport mechanisms is visually represented in Figure 1.1.

Figure 1.1 visually depicts analyte migration in a typical capillary electrophoresis system (anode at injection end, cathode at detection end). Cation migration is a result of the sum of electrophoretic and electroosmotic velocities. Neutral compounds migrate after cation. Anion migration is a result of the difference of electroosmotic flow toward the detector and electrophoretic transport away from the detector.

### The Instrument

A capillary electrophoresis instrument can be described in terms of five components: the injector, the capillary, the voltage source, the detector, the digital-to-analog converter. The purpose of the injection mechanism is to introduce discrete plugs of sample into the separation capillary. This may be accomplished using siphoning, pressure or voltage. No matter which of these injection methods is used, to obtain reproducible data injection parameters, such as the capillary height, applied pressure, or applied voltage, must be well-defined. In addition, each mode requires the duration of the injection is well-defined. Thus, a timer or timing device is often necessary.

The second component of a capillary electrophoresis system is the separation capillary. These capillaries are typically constructed of fused silica, although they have also fabricated from other materials such as borosilicate glass or Teflon [1-6]. The surface chargeof the capillary affects the electroosmotic flow. Furthermore, the surface may be covalently modified to display different functional groups, or additives to the running buffer itself may effect the surface charge [7-13]. The inner diameter generally ranges from 10 to 100 microns, although capillaries of < 1 micron inner diameter have been successfully used [14-16]. Fused silica capillary used in capillary electrophoresis is typically coated with polyimide. This polymer imparts flexibility to the capillary making it more practical for use. Both ends of the separation capillary are immersed in vials of ~2-5 mL volume and the vials and capillary are filled with a solution capable of conducting current. Usually, this is an aqueous solution buffered to a certain pH value using a good buffer producing separation currents less than 100 microamperes. Low separation current is desirable in a capillary electrophoresis separation because of resistive heating. If heat is not adequately dissipated from the separation capillary, convective flow will degrade the separation efficiency. At the extreme, solvent will boil, and current flow will cease due to bubble formation within the capillary. Smaller diameter capillaries have lower separation currents, and therefore generate lower resistive heating. In addition, smaller capillaries more efficiently dissipate heat generated in the separation. The drawback to small inner diameter capillaries is that they plug more frequently than larger diameter capillaries. Like inner diameter, the length of the separation capillary may also vary.

The third component of a capillary electrophoresis system is the high voltage power supply. This is used to apply voltage to either the anodic or cathodic reservoir, via a platinum electrode in contact with the background electrolyte in the buffer reservoir. Voltage (either positive or negative) is applied to one reservoir, while the other reservoir is grounded. Platinum electrodes are used as a means to connect the high voltage to the capillary electrophoresis running buffer because platinum is relatively inert. It is important to remember that electrochemical side reactions will undoubtedly occur. For this reason, the running buffer volume is at least 1 mL and buffer is used to control the pH of the solution of background electrolyte. For an in-depth investigation of how capillary electrophoresis running buffer can be modified by the application of separation voltage see [17-21].

The fourth component is the detector. Capillary electrophoresis has been coupled to a number of different detection devices and can be made compatible with different detection strategies. The most common modes of detection in capillary electrophoresis are UV-visible absorbance detection, laser induced fluorescence, mass spectrometry, or electrochemical detection. Both UV-visible and fluorescence detection are usually performed on-column by removing a small portion of the polyimide coating to make an optically transparent window. Electrochemical and mass spectrometric detection are performed at the end of the capillary. The work outlined in this experiment is based on UV-visible absorbance detection coupled on-column with the electrophoresis capillary. Absorbance detection is a nearly universal technique, since most analyte absorbs radiation in the UV or visible region. The absorbance of incident radiation is linearly related to concentration (Beer’s Law). UV-visible absorbance detection can provide quantitative and qualitative information using standards, or spectral analysis.

The fifth component of a capillary electrophoresis instrument is the equipment that enables conversion of the analog data output by the detector to digital format for software analysis. The custom-built instrument outlined in these materials incorporates a computer with a data card that performs analog-to-digital conversion. Any card must be addressed using code or software. The instructions we have provided for building a capillary electrophoresis system invoke commercially available software that drives the analog-to-digital conversion card and provides a convenient means to fit the resulting data and return quantitative information (peak moment, height, width). The Igor driver and software we have proposed for use process the information collected by the card. We have included some startup tips for using software from this vendor. A commercial instrument will also incorporate a means of reporting data in digital form as well as software to allow data analysis. If you are using a commercial instrument, refer to the instrument manual for information regarding data collection and analysis.

### Basic Protocol

Successful application of a capillary electrophoresis instrument requires knowledge of the performance, standard operating procedures and a working knowledge of the figures of merit that will be necessary for analysis. Most users learn what is important through experience. To streamline this process, we have posed questions in a dry-lab format that will assist new researchers in documenting strategies for use and operation. As a result, this learning module requires the user to devise and document operating procedures for recording data in a laboratory notebook, preparing the separation capillary, and making chemical standards and running buffer. Each lab will have unique protocol based on practices and available equipment. Most standard operating procedures undergo multiple revisions as laboratory users become more experienced and focus on particular applications. Thus, we expect the protocol outlined for Learning Module I will undergo continual revision.

### Application

Capillary electrophoresis is useful for rapid efficient analyses of a variety of compounds including ions, small molecules, drugs, amino acids, peptides, proteins, DNA, RNA, oligonucleotides, lipids, other polymers, carbohydrates, and other compounds. The method may be applied for qualitative or quantitative analysis. In either case, the user must have formulated the experimental procedures necessary to implement these analyses. To emphasize fundamental concepts, Learning Module I requires the user to anticipate migration order in free zone capillary electrophoresis for anionic, neutral and cationic compounds (tolmetin, mesityl oxide, atenolol). Following completion of the dry-lab, the user will implement the protocol she/he has devised by completing a single capillary electrophoresis separation of these compounds.