Electrophoresis techniques have been primarily developed for analytical purposes— to investigate the characteristics of the molecules in the sample of interest. The techniques can be modified to serve a preparative purpose, in which a large amount of a specific molecule can be obtained at the end of an electrophoresis run.
Preparative electrophoresis uses the same principles as analytical electrophoresis but contains the additional step of sample collection or fractionation. Most analytical electrophoresis formats can be modified to address issues related to the generation of excessive heat and used as a basis for preparative electrophoresis.
Electrophoresis For Preparatory Purposes
Electrophoresis is primarily conceived as an analytical technique, in which molecules in a liquid sample are electrically charged and separated by the characteristics of the charged molecules. At the end of an analytical electrophoresis run, the separation pattern is analyzed to determine the characteristics of the molecules in the sample.
Analytical electrophoresis can be upscaled and modified for the preparation of a specific molecule in a preparative electrophoresis. Unlike analytical electrophoresis, the characteristics of the desired molecules must be known before being subjected to preparative electrophoresis. At the end of the preparative run, the target molecules are collected for use in downstream processes.
Preparative electrophoresis uses the same principle as that of its analytical counterpart despite possessing a larger scale. As with analytical electrophoresis, molecules in the preparative electrophoresis system are also under the influence of an electric field, which ionizes the ionizable groups and propels the ionized molecules to migrate. The migration pace differs, depending on the mass and the net charge of each molecule in the sample. The difference in the migration pace is reflected in the time and distance and the direction each molecule moves from the starting point of electrophoresis.[1,2] Preparative electrophoresis is applicable to all molecules that can be separated by its analytical counterpart although it is more popular for the purification and large-scale preparation of a specific protein.
The Setup Of Preparative Electrophoresis Is More Vulnerable To Convection Currents
As a rule, all formats of analytical electrophoresis can be modified to accommodate preparative electrophoresis. Nevertheless, preparative electrophoresis is performed on a much larger scale, with a higher volume of the separating samples and electrophoretic running buffer than its analytical counterpart. The overall system is, therefore, more vulnerable to changes during the electrophoretic run than its analytic counterpart.
During electrophoresis, heat can be converted from electrical energy to thermal energy, also known as Joule heating or Ohmic heating. The electrical energy is generated when electric currents meet the resistance, which exists in the components used in the system such as the molecules of the electrophoretic running buffer, etc. If the generated heat is in excess, it can be transferred to samples and the electrophoretic system, resulting in convection currents. Consequently, excessive heat can cause the running buffer to be evaporated, leading to instability in the temperature and pH of the system or the mixing of samples, which can damage the samples and their migration rate and pattern.[1,2,4]
Most of the countermeasures to excessive heat and convection currents used in analytical electrophoresis can be applied to preparative electrophoresis. For example, the use of a stabilized power supply which can control the voltage or current applied to the electrophoresis system and the use of an anticonvective or support medium.[1,3] In a certain format of preparative electrophoresis, in which the use of an anticonvective medium is not compatible, preparative electrophoresis may be performed in a cold room (approximately 4-5°C) to maintain the temperature of the electrophoretic system during the electrophoretic run. Based on the same idea, newer preparative electrophoresis apparatuses have a cooling system incorporated to prevent the generation of convection currents.
Preparative Electrophoresis Involves The Collection Of Sample Fractions
Similar to analytical electrophoresis, samples used for the separation in preparative electrophoresis are in their liquid state. However, since the goal of preparative electrophoresis is to acquire a purified target molecule in large quantities, a collection of each species of molecules in the sample, also termed sample fraction, is necessary when electrophoretic separation is finished. To date, there are two approaches in sample fractionation in preparative electrophoresis[3,5]:
In electroelution or discontinuous elution, the separated target molecules are eluted or purified from a support matrix after the electrophoresis run is completed. This approach applies a compatible electric charge to the target molecule to release it from the support matrix. Once released, the target molecule can be captured using, for example, semipermeable membranes or dialysis tubing. After electroelution, the target molecule is collected and further processed in a post-electroelution process to remove other contaminants such as buffer salts or staining dyes before subjecting them to a downstream purification process.
Electroelution is considered to be a conventional sample fractionation approach due to its simplicity in the set-up. Many commercial electroeluter devices have been developed and are available in the market, allowing workers to perform the process in a semi-automatic manner. However, in electroelution, the target molecule must be first identified on the support matrix, which could lead to either a decrease in the yield of the target molecule or potential inaccurate identification. Another downside is the setup of electroelution which must be customized to suit the biochemical characteristics of each target molecule, which can be laborious if many molecules possessing different characteristics are targeted.[3,5]
2. Continuous Elution
In contrast to electroelution, continuous elution is a sample fractionation approach that occurs in succession to electrophoresis. This approach can be applied to electrophoresis with and without the use of a support matrix. In continuous elution, the sample is loaded into the system and subjected to electrophoretic separation as in analytical electrophoresis. When the target molecule is separated, it is either immediately captured using a collector incorporated into the electrophoresis apparatus, or a buffer stream that brings the target molecule to a fraction collector. After the target molecule has been collected, it can be further processed and purified for downstream applications.[3,5]
Continuous elution occurs consecutively after electrophoresis and does not require the customization of the setup to accommodate the characteristics of each targeting molecule. Thus, the approach is more applicable to high-capacity routine work, in which each sample is relatively similar and can be electrophoresed under a fixed condition. Nevertheless, the lack of customization also makes continuous elution less appropriate to non-characterized or less well-known target molecules. The use of a buffer stream can lead to unintentional mixing of samples and demands for the adjustment in the flowrate and the composition of the buffer, which could affect the downstream purification process. Another major disadvantage of the approach is the requirement of a sophisticated and specialized apparatus.
Types Of Preparative Electrophoresis
1. Preparative Free-Flowing Electrophoresis
Free-flowing electrophoresis is the electrophoresis of samples in a free solution, without the use of an anticonvective medium or a support matrix. Samples for electrophoresis are introduced as a continuous stream to the system, and electrophoretic separation occurs in the presence of an electric field, which allows molecules in the samples to be separated between the two electrodes, perpendicularly to the buffer flow. After separation, the target molecules are collected in a designated fraction collector incorporated into the electrophoresis apparatus.[5,6] This type of preparative electrophoresis is applicable to many formats of electrophoresis, including moving-boundary electrophoresis, isotachophoresis and isoelectric focusing electrophoresis.[4,6]
The generation of convection current during electrophoresis is a major disadvantage of free-flowing electrophoresis due to the lack of an antivective medium or a support matrix.[3,4] This calls for the need to reduce the heat generated during electrophoresis and to maintain the stability of the system. Apart from performing free-flowing electrophoresis in a cold environment, modern free-flowing electrophoresis apparatus has a cooling device or a cooling mechanism incorporated. For example, an electrophoresis chamber for free-flowing electrophoresis has capillary tubes filled with a coolant solution embedded to provide temperature and pH stability to the running buffer during the electrophoretic run.[3,7]
2. Preparative Electrophoresis On A Support Media
Opposite to free-flowing electrophoresis, preparative electrophoresis on a support media uses a support matrix to suppress excessive heat and convection currents. Analogous to zone electrophoresis, several support matrices are available, including filter paper and gel of various types. Apart from the decrease of heat generated from convection currents, the use of a support matrix also restricts the samples being separated to a certain area in the electrophoresis system, allowing the stirring or the flowing of the electrophoresis buffer during the run to ascertain that the temperature, pH, and the concentration of the running buffer are uniform throughout the system. After electrophoretic separation, the targeted molecules can be located on the support media, excised and collected using electroelution. Alternatively, the targeted molecules can be recovered using continuous elution if the necessary components are incorporated into the electrophoresis apparatus.[3,5]
Gel electrophoresis is the most popular form of non-free-flowing preparative electrophoresis to date. This is due to the availability of the types of gels, the simplicity in customization of the system and the molecular sieving property of the gel, which also adds to the resolving power of electrophoresis. The separation of a zone where electrophoretic separation occurs also enables the development of an all-in-one continuous elution-preparative gel electrophoresis apparatus, in which electrophoretic separation and collection of the sample occur consecutively in one device.[5,8]
All in all, preparative electrophoresis is the large-scale version of analytical electrophoresis, aimed to acquire large quantities of target molecules. The same concepts and theories related to analytical electrophoresis also apply to preparative electrophoresis. Nevertheless, the issues relating to convection currents are more pronounced, and the collection of the separated target molecules is necessary in preparative electrophoresis, which are circumvented in the design of preparative electrophoresis apparatuses.
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