CE instrument

The concentration mode is used when the extract from the SEE module is not concentrated enough to be directly analyzed by the CE instrument, and thus requires a concentration step, which is carried out in the concentrator device. In this mode, during the concentration step the extract from the SEE cell enters the first valve (positioned in the concentration mode (Eigure 6.11(a)), and is then directed to and adsorbed into the sample concentrator which contains an SPE cartridge. While the... 

The direct mode is used when the concentration of the SEE extract is enough for direct analysis in the CE instrument without the need for a pre-concentration step. In this case, the sample concentrator is by-passed and the SEE extract goes directly to the CE instrument. The extract is collected in a CE vial containing an appropriate solvent and is thus ready for the CE analysis (Eigure 6.12). 

The only other group to have performed comprehensive multidimensional reverse-phase HPLC-CZE separations is at Hewlett-Packard. In 1996, a two-dimensional LC-CE instrument was described at the Erederick Conference on Capillary Electrophoresis by Vonda K. Smith (21). The possibility for a commercial multidimensional instrument may have been explored at that time. 

Capillary electrophoresis offers several useful methods for (i) fast, highly efficient separations of ionic species (ii) fast separations of macromolecules (biopolymers) and (iii) development of small volume separations-based sensors. The very low-solvent flow (l-10nL min-1) CE technique, which is capable of providing exceptional separation efficiencies, places great demands on injection, detection and the other processes involved. The total volume of the capillaries typically used in CE is a few microlitres. CE instrumentation must deliver nL volumes reproducibly every time. The peak width of an analyte obtained from an electropherogram depends not only on the bandwidth of the analyte in the capillary but also on the migration rate of the analyte. 

With the advancing automatization and computerization of CE instruments, the application of micromachining techniques, and the improvement of the devices for coupling CE with CL detection, it is hoped that both techniques may be incorporated in the future as suitable methodology in routine laboratories, being complementary to classical techniques such as HPLC and offering new alternatives to the analytical chemist. 

The first commercial CE instruments were introduced in 1988 by Applied Biosystems (Foster City, CA) and Beckman Coulter (Fullerton, CA). The main challenge during these days was to find out how to overcome poor reproducibility and improve separation efficiency. In the early 1990s, many instrument-building companies introduced CE systems (e.g. Isco (Lincoln, NE), Bio-Rad (Hercules, CA), Waters (Milford, MA), Applied Biosystems, ThermoQuest (Santa Fe, NM) and Dionex (Sunnyvale, CA)), but at the end of the decade only Beckman Coulter... 

This section provides a brief discussion of the basic theoretical concepts of CE (including separation mechanisms), a description of CE instrumentation, and some guidelines in selecting conditions for a CE separation. Readers interested in more detailed presentations of CE theory and practice may consult References 1 to 8. Several general reviews of CE have been published,911 as well as specific reviews of protein analysis by CE.12-16... 

The core components of a CE instrument are a power supply, a detector, and devices that allow for temperature control of the capillary and sample compartment. A wide variety of commercial CE instruments are available, from simple modular systems to fully integrated automated systems under computer control. 

One of the major advantages of CE as a separation technique is the wide variety of separation modes available. Analytes can be separated on the basis of charge, molecular size or shape, pi, or hydrophobicity. The same CE instrument can be used for zone electrophoresis, IEF, sieving separations, isotachophoresis, and chromatographic techniques such as MEKC and capillary electrokinetic chromatography. This section provides a brief description of each separation mode. Zone electrophoresis, IEF, and sieving are the primary modes used for protein separations, and these will be discussed in detail in the following sections. 

For all CE instruments that use on-line detection at a fixed point along the capillary, CIEF must include a means of transporting the focused zones past the... 

Capillary Electrophoresis (CE) The CE instrument consists of a source/ sample vial, a destination vial and a small capillary filled with electrolyte joining the two vials. A voltage is applied and separates the sample according to size and charge, which is detected by UV absorbance. 

CE instrumentation is quite simple (see Chapter 3). A core instrument utilizes a high-voltage power supply (capable of voltages in excess of 30,000 V), capillaries (approximately 25—lOOpm I.D.), buffers to complete the circuit (e.g., citrate, phosphate, or acetate), and a detector (e.g., UV-visible). CE provides simplicity of method development, reliability, speed, and versatility. It is a valuable technique because it can separate compounds that have traditionally been difficult to handle by HPLC. Furthermore, it can be automated for quantitative analysis. CE can play an important role in process analytical technology (PAT). For example, an on-line CE system can completely automate the sampling, sample preparation, and analysis of proteins or other species that can be separated by CE. 

For the pharmaceutical scientist, understanding the theory and application of the equipment is usually not sufficient there is the matter of compliance. The qualification of CE is similar to that of other instruments. Installation qualification (IQ), operational qualification (OQ), and performance qualification (PQ) apply in much the same way as high-performance liquid chromatography (HPEC). This chapter details the different parts of the modern CE instrument, equipment-related issues and troubleshooting, instrument qualification, and the future of the CE instrument. 

FIGURE I Schematic of a CE instrument. Drawing is courtesy of Kevin Daniel O Flaherty. 

After optimization of the correct capillary parameters (ID, OD, Lj), detection at the microscale level became the next major challenge for the survival of CE. Despite the challenges, many of the common HPLC detectors have a CE complement, e.g., absorbance, fluorescence, conductivity, photodiode array, and mass spectroscopy. Small dimensions mean universal detectors such as refractive index cannot be used. A sample of detectors will be discussed. The technical aspects of each detector will not be covered except in relation to the CE instrument. Readers are advised to consult an instrumentation textbook for more details on theory of operation. 

FIGURE 7 Schematic of a whole capillary-imaged CE instrument. Drawing courtesy of Kevin Daniel O Flaherty and Christopher James O Flaherty (adapted from the Convergent Bioscience Web site, www.convergentbiosci.com). 

CE has suffered from an assortment of common operator errors, which in turn have characterized the technique as not being robust. Like any analytical piece of equipment, there can be hardware and chemistry/operator issues. CE requires a keen background and user knowledge of the technique so as to avoid common problems that may initially be diagnosed as instrumental issues. For the early user of CE, the table below lists some common problems followed by their root causes and corrective actions. Following the suggested corrective actions should help the beginner get the maximum performance out of the CE instrumentation. 

Design qualification (DQ) is the process used to determine a system that will function within the intended purpose. It can be compared to a user s requirements document for a piece of software. For example, if a CE is being purchased to run DNA sequencing samples, then the system purchased will need to include a fluorescence detector. The main vendors for CEs have similar options for their CE instruments reducing the utility of DQ protocols. Their main utility is to define the specific equipment needed for the purchase order. This only needs to be performed before the system is purchased initially. If desired, this can also be done when additional features need to be purchased (i.e., new detectors, etc.). 

Another important part of IQ is the generation and approval of an instrument-operating procedure, if applicable. This procedure defines the use and care for a CE instrument. This generic procedure is useful to train new analysts on the technique, define the requirements for routine and corrective maintenance, and other relevant information. 

Early on in CE, the literature was flooded with applications that drove the technology beyond graduate school curiosity. Since Microphoretics introduced the first commercial CE instrument in 1988, equipment suppliers have come and gone. Many of the large equipment suppliers entered the CE market and learned the market cannot support too many instrument... 

CE instruments are thermostated to dissipate excessive Joule heat. Generally that covers only the main part of the capillary, and not, e.g., the autosampler with the buffer and sample vials. In some instruments, it is difficult to control the autosampler temperature due to the near presence of extraneous heating sources such as the detector lamp. Also, some labs... 

Apart from the qualification dossiers provided by vendors there seems, at present, to be very little information published on the performance of an operational qualification for capillary electrophoresis (CE) instruments other than a chapter in Analytical Method Validation and Instrument Performance. The chapter, written by Nichole E. Baryla of Eli Lilly Canada, Inc, discusses the various functions (injection, separation, and detection) within the instrument and provides guidance on the type of tests, including suggested acceptance criteria, that may be performed to ensure the correct working of the instrument. These include injection reproducibility and linearity, temperature and voltage stability, detector accuracy, linearity, and noise. 

To this end the following guideline describes a general approach toward risk management for a laboratory instrument and then applies it to the performance of the operational qualification of a CE instrument. Tests then need to be devised in order to determine the suitability of the instrument for its intended use and to fulfill those user requirements that have been defined by the operator. 

However for an equipment such as a CE instrument the utilization of a systematic technique as opposed to an empirical approach is advisable in order to ensure that all of the functionality of the instrument are correctly assessed. 

There are a number of techniques that can be employed as detection mechanisms within a CE instrument. These include absorbance of ultraviolet (UV) light, laser-induced fluorescence, electrochemical and mass spectrometry. UV absorbance is, at present, the most commonly used technique. 

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