Separation methods based completely or largely on charge differences are those using [1]:
Among the latter, conventional macroscopic methods are confined to an analytical or small scale preparative range. However, with the advent of microchips a more flexible approach to microbiological measurements has emerged. Microlaboratories are introduced on chips that present significant advantages in terms of speed, reagent usage and, of course, integration.
The basic principles of separation techniques involving charge differences are presented below.
Electrophoresis is a technique for separating the components of a mixture of charged molecules in an electric field within a gel or other support (called a buffer). The movement of the electrically charged molecules under the influence of the electric field results in their separation. The technique can be summarized as follows [2]: The driving force qE (q electric charge, E electric field) on the particle is opposed by the frictional resistance of the medium which depends on the viscosity of the medium, gel density and particle size. As a result, the distance d travelled by the particle in time t is proportional to the electrophoretic mobility m and the product tE. Comparison between experiments is possible only if tE is constant for different experiments. An experiment run at half time and double E results in the same d. The time is only approximate because of other effects such as the extra heat generated by an increasing current.
The mobility of the charged particle depends on the ionic strength of the buffer. The higher the ionic strength, the greater its conductivity and the greater the amount of heat generated. With increasing temperature there is an increased rate of diffusion and therefore an increased mobility of the particle. Also, with increasing temperature, the viscosity of the medium is decreased which decreases the electrical resistance. At constant voltage there is increased current, therefore increased heat output. The choice of buffer strength is crucial since it determines the amount of electrical power that can be applied.
If the power input is too high, excessive heat generation leads to unacceptable rate of evaporation solvent from the medium, resulting in convection currents and mixing of separated zones. It can also lead to denaturation of the specimen.
Too low power input overcomes the heating problem but leads to poor separation because of a higher diffusion amount due to a long running time.
Removal of heat leads to temperature gradients between different cooled parts. This in turn leads to distortion of bands due to variations in rates of migration.
Other factors that influence mobility and sharpness of separation:
In conclusion, the basic concept of electrophoresis is very simple but there is a variety of factors that influence the technique. These factors can be exploited for separation.
Isotachophoresis [3] is a related technique, also called displacement electrophoresis. The underlying theory and separation process are the same as those ocurring during electrophoresis. Its designation as an independent method relies upon operational considerations. The method is applicable where other methods are difficult to use such as for small molecules. Isotachophoresis can be used in capillary tubes, gel rods or slabs.
Isoelectric Focusing [4] is a technique that employs an important concept, the isoelectric point. Substances which contain acidic and basic groups in their molecules have a so-called isoelectric point pI which is the pH value at which they have no net charge. In solution, when their pH is equal to their pI they do not migrate when placed in an electric field. At higher pH they become negatively charged so that they migrate toward the anode if an electric field is applied. Their net charge is positive at lower pH and therefore they will migrate toward the cathode.
The basic principle of isoelectric focusing is that a buffer gradient is used such that the pH in the separation chamber increases from one side to the other, the lowest pH being obtained at the anode and the highest pH at the cathode. When a mixture of substances is introduced into the system, all substances will acquire different net charges according to their pI values, and hence will have different mobilities. When an electric field is applied, each substance will migrate toward that position where the pH is equal to its pI value. At this position its net charge is zero and its velocity decreases to zero. Using the technique of isoelectric focusing, all substances in the sample will be concentrated in narrow zones. Because pI values are characteristic of specific substances, the latter can be separated by this means.
On a micro-level: Capillary electrophoresis (CE) has been implemented on fused-silica capillary and is currently under study for use on glass microchips. Microchannels are built on a glass chip using a technique called photolithography which allows a high density of channels. Work performed on DNA [5] has demonstrated that there is no loss of electrophoretic performance in microchannels built on glass as compared to a fused-silica capillary with similar cross-sectional area.
In conclusion: A system that integrates microbiology protocols has the advantage that the device simply implements the protocol after the user applies the sample.
Bibliography:
[1] Haugland, R.P. Handbook of Fluorescent Probes and Research Chemicals, 6th ed. Molecular Probes, Eugene, OR 1996
[2] Kilar, F.; Hjerten, S. Electrophoresis 1989
[3] Weinberger, R. Practical Capillary Electrophoresis Academic Press: Boston 1993
[4] Righetti, P.G. Isoelectric Focusing: Theory, Methodology and Applications Elsevier Amsterdam 1983
[5] Backhouse, C.; Caamano, M.; Oaks, F.; Nordman, E.; Carrillo, A.; Johnson, B.; Bay, S. Electrophoresis 21 (2000) 150-156