Potential for Automation

Commercially available capillary electrophoresis systems are remarkably simple from an instrumentation point of view. The basic instrument consists of four parts: 1) a fused silica capillary holder, 2) a high voltage power supply, 3) a detector, and 4) a safety interlock. Most instruments also provide a mechanism to control the temperature in the capillary. This may be controlled either by air cooling, using a peltier device, or by enclosing the capillary in a cartridge, which is filled with a coolant that is maintained at a constant temperature. All components of the CE are monitored or controlled by computer software. This allows the end user to develop methods and then analyze the separated components, post-electrophoresis. In this respect current CE instruments compare favorably with automated HPLC systems used in the clinical laboratory. However, unlike the highly automated instruments found in a clinical laboratory, the operation of CE instruments requires an above average amount of technical expertise. In other words, these instruments are not black boxes that just require an operator to push a switch and then wait for a result to be generated. Most importantly, in order for CE to be used routinely in the highly automated clinical laboratory, primary sample tube sampling, the ability to read a bar code, and a bidirectional interface to Laboratory Information System are required. With the exception of Beckman Paragon CZE 2000®, no other CE system has this capability.

2.4. Accuracy, Sensitivity, and Precision

In CE, accuracy has never been an issue because the analyte is usually separated from interfering substances. However, the limit of detection (LOD) has been and still is the main problem with CE. Various manufacturers of commercial CE units have tried to address this problem by using modified capillaries (i.e., bubble-cell or z-shaped cells) or by using laser-induced fluorescence (LIF). Typical detection limits range from 1 x 10-6 M for UV detection to 1 x 10-9 M for LIF. Alternatively, sensitivity can also be enhanced by sample stacking. Chien et al. (6) has comprehensively reviewed sample stacking in capillary zone electrophoresis and its applications for routine analysis. Typically, sample stacking involves a movement of sample ions across a boundary, which divides regions into high (low-conductivity sample solution or water plug) and low (high-conductivity separation solution) electric fields inside the capillary. The sample is usually dissolved in a buffer having an ionic strength that is 1/10 that of the run buffer. As a result, the electric-field gradient across the sample zone is relatively high, causing the analyte ions to migrate rapidly until they reach the interface between the sample buffer and the run buffer. On reaching the interface, the ion mobility drops, causing stacking of sample at the boundary of two solutions giving a 5- to 10-fold increase in signal-to-noise ratio. Because sample stacking relies on the enhanced electrophoretic velocities of ionic species in the low-conductivity or high-electric field region, neutral analytes are not concentrated unless they are compartmentalized in an ionic micelle. Quirino et al. (7)

described an exceptional narrowing of neutral analytes zones in electro-kinetic chromatography that allowed a 5000-fold concentration of neutral analytes, such as steroids, racemic herbicides, and other biologically important compounds. This special preconcentration phenomenon, dubbed as sweeping, works for all charged and neutral compounds. Detection limits ranging from 1.7 to 9.6 ng/mL have been reported by these authors for various clinically important steroids. Although very promising, the technique needs to be applied to real patient samples. It may also require a prior sample clean up in order prevent the interference of unwanted proteins and other analytes present in the sample that also under go preconcentration.

The ability to measure precisely (analytical imprecision) an analyte is very important in a clinical laboratory. Coefficients of variation (CV) <5% are routinely observed for various analytes, such as electrolytes, enzymes, proteins, and so forth, on automated analyzers. The various factors that can affect both with in- and between-run imprecision in CE are listed here (a more in-depth discussion on these factors can be found in Chapter 2):

1. Capillary surface: The inner surface of the capillary, which participates in the separation process, can be a major factor in the cause of imprecision in CE. Not only does the sample, and therefore the various analytes, come in contact with the surface, but the analytes can also bind to the surface, changing the properties of the capillary. For the most part, it is best to try and eliminate any interaction of the analyte with the surface of the capillary. This can be done by deliberately changing the surface by treating with polymers, various detergents, or simply by changing the buffer or buffer concentration.

2. Current: When a CE instrument is "turned on" a current is generated. It is extremely important to control this current, because a variation in the current can cause a change in the temperature. A change in the temperature can result in a change in pH, which can then lead to a change in the current, the original variable that was being controlled. With the power sources available today, controlling the current is not an issue.

3. Capillary conditioning: In order to ensure good reproducibility in bare and coated fused silica capillaries, daily conditioning with a series of buffers is required before a capillary is used. In our experience, a daily rinse of 30 min for serum protein electrophoresis, 30 min for hemoglobin variants using capillary isoelectric focusing, 15 min for steroids using coated capillary, and 45 min on a bare fused silica capillary are very typical. Furthermore, the rinsing time is very dependent on the capillary surface and can change from one lot of capillaries to another. Most of the buffers used for capillary conditioning are quite stable if protected from the atmosphere and stored at 4°C. Hence, buffer stability is not much of an issue. For IEF, preconditioning the capillary with a 0.04% methylcellulose and ampholine solution is extremely important to ensure reproducible migration times. In our hands, the CV for migration time increased from <5 to >20% when the pre-rinsing time is shortened from

30 min to 10 min for the analysis of hemoglobin variants using capillary isoelectric focusing on a bare fused silica capillary.

4. Buffer: Although the capillary wall can significantly affect the separation, the separation also takes place in the presence of a buffer. This buffer has multiple purposes, one of which is to control the pH. It is extremely important to control pH because analyte charge, EOF, and heat production all change with even small changes in pH. The actual buffer used can also have an affect on the separation process because of interaction between the buffer and the analyte. In addition some buffers, i.e., borate or zwitterionic buffers, carry less current than mono-functional buffers like phosphate. Buffer concentration is also important because higher buffer concentrations will lessen the interaction of the analyte with the capillary wall.

5. Temperature: Control of the temperature within the capillary is extremely important. However, temperature control is complicated by the generation of heat (Joule heating) when current passes through a buffered solution. As mentioned previously, multiple factors (temperature, pH, and current) are interrelated. In other words, what affects one will potentially affect the other. With current instruments, the temperature is controlled either by air cooling, a peltier device, or by enclosing the capillary in a cartridge, which is filled with a coolant maintained at a constant temperature. For most applications, the use of a coolant will control temperature better than air-cooling.

6. Sample Injection: In the past, sample injection was a major source of error for traditional CE systems. However, with the use of high-precision pressure valves, this problem has been significantly reduced.

CE is highly versatile, with numerous modes of operation, which are accessed, in many instances, by altering the buffer composition. The commonly used modes of CE include CZE, capillary gel electrophoresis (CGE), CIEF, and capillary isotachophoresis (CITP) (8).

2.5.1. Capillary Zone Electrophoresis (CZE)

CZE, the simplest form of CE, requires filling the capillary with only running buffer. In this form of CE, the ionic solutes migrate with different velocities (as determined by their charge-to-mass ratio) forming discrete zones in the running buffer. By using the electroosmotic flow (EOF), separation of many of the cationic and anionic solutes is possible. Neutral solutes, which move with the EOF, are not separated (8). Applications of CZE include analysis of amino acids (see Chapter 9), peptides (see Chapter 16), protein analysis (see Chapters 4, 5, 6, 7, and 8)—including screening of proteins variants and evaluating protein purity—and forensic applications (see Chapter 20).

2.5.2. Micellar Electrokinetic Chromatography (MEKC or MECC)

MEKC is a combination of electrophoresis and chromatography in which both neutral and charged solutes can be separated (9). This form of CE takes advantage of a property that when the concentration of some surfactants reach the critical micelle concentration, aggregates (micelles) are formed that help separate neutral species. These spherical micelles contain the hydrophobic tails of the surfactant molecules directed towards the center and charged heads directed toward the outside buffer. The micelles are thus charged and migrate, depending on their charge, after application of a potential field across the capillary. Solutes are partitioned between the micelles and liquid phase leading to differential retention and separation of the solutes (10). The physical nature of the micelle can be changed using different types of surfactant thus altering the selectivity of the micelle. Some applications of MEKC include separation of amino acids (see Chapter 9), heavy metals (see Chapter 18), nucleotides, vitamins, drugs (see Chapters 17 and 18).

2.5.3. Capillary Gel Electrophoresis (CGE)

"Gels" used in CGE are a polymer network of compounds such as bis-polyacrylamide, agarose, or methylcellulose, and separate high molecular weight compounds by the sieving effect of the polymer network. CGE can separate DNA and denatured proteins (11). CGE has been applied in the analysis of polymerase chain reaction (PCR) products (see Chapters 13 and 14), purity of oligonucleotides, sequencing of DNA, and so forth.

2.5.4. Capillary Isoelectric Focusing (CIEF)

CIEF is a technique that separates peptides and proteins on the basis of isoelectric point (pi) and can separate proteins with a pi difference of as little as 0.005 pi units (12). This technique is applied to the separation of hemoglobins and hemoglobin variants (see Chapter 8), protein isoforms, and immunoglobulins that are difficult to separate by other methods.

2.5.5. Capillary Isotachophoresis (CITP)

CITP is a moving boundary electrophoretic technique that uses a combination of two buffer systems to create a state where the separated zones move at the same velocity. These zones are sandwiched between leading and terminating electrolytes, making it possible to separate either anions or cations, but not both, in a single experiment. CITP can be used to concentrate the solutes before CZE, MEKC, or CGE (13). When used in this manner it is known as transient CITP.

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