From Clinical and Forensic Applications of Capillary Electrophoresis 2 Basic Principles and Modes of Capillary Electrophoresis Harry Whatley 1 BASIC PRINCIPLES OF CAPILLARY ELECTROPHORESIS 1 1 Fundamentals of Electrophoresis Capillary electrophoresis CE is a special technique that uses an electrical field in order to separate the components present in a mixture Electrophoresis in a capillary can be differentiated from other types of electrophoresis that it is done within the walls of a narrow tube To understand the functioning of molecules influenced by an electrical field inside a capillary it is important to know the phenomena that result from the geometry of a capillary 1 1 1 Basic Principles It has long been well known that molecules can possess positive or negative electrical charge When the quantity of negative and positive charges are equal the charges will cancel creating an uncharged neutral molecule If allowed to move charged particles will go for regions like an electrode having an opposite charge which means that opposites will attract Figure 1 demonstrates an electrophoresis example In this example mixture of ions is dissolved in such a suitable solvent like water Before applying an electrical field these ions are in a random motion state When the electrical field is applied charged species start moving A separation happens leading to a less random distribution of particles Cations positive ions move toward the cathode electrode of negative charge and anions negative ions move toward the anode electrode of positive charge Figure 1 also shows another point of electrophoresis in solution the effect of the mass charge ratio m z In figure 1 there are four types of ions large and small of positive charge and large and small of negative charge If each of the particles has only one single charge then will be the same absolute value of the force on each of the particles
The acceleration created by this force can be calculated by the equation Force acceleration mass F ma The separation medium viscosity opposes the acceleration with the result that a steady velocity is reached under constant conditions This means that the system is only able to separate particles of opposite charges but is also able to separate particles having the same charge when there are other differences between them The electrophoresis science is concerned with the creation of systems that utilize the differences between the molecules Antother way is that the analyst tries to make a system that creates differences between molecules Changing the pH of the separation method is an example At pH 10 0 acetic acid and glycine will have the same charge 1 At pH 7 0 glycine will have a very small net charge while acetic acid will have a charge of 1 Separation of these two molecules would differ at pH 7 0 and at pH 10 0 Many other factors other than pH affect the separations of electrophoretic These include the hydrodynamic radius of the molecules the temperature and the separation medium viscosity In real systems there are other forces in addition to the electrical field affecting the charged molecules e g the entire fluid mass may be moving relative to the vessel in which it is contained Some of these factors can affect the electrophoresis in a very complex manner for example the movement of current through a liquid can elevate the liquid temperature This temperature change can affect the electrical resistance of the system ie the current the viscosity and the velocity of the molecules moving in the field
These factors will be discussed later in the specific context of CE Janini and Issaq presented a detailed discussion of the theoretical underpinnings of these factors 1 Janini G M and Issaq H J 1993 The buffer in capillary zone electrophoresis in Capillary Electrophoresis Technology Guzman N A ed Marcel Dekker Inc New York pp Fig 1 Simple electrophoresis Principles and Modes of CE 23 1 1 2 Electrophoresis in a Capillary The electrophoretic process in a capillary has all of the properties previously described In addition the capillary has a small diameter and a large aspect ratio length width presenting additional factors What actually a capillary means For this article the discussion will be only about capillaries made of fused silica and having an inner diameter not more than 100 µm Usually the inner diameters is about 20 100 µm Normally the capillaries used in Capillary Electrophoresis are circular in cross section However capillaries with square cross sections have been produced These may provide some advantages in terms of temperature regulation and sensitivity of detection 2 Cifuentes A and Poppe H 1994 Rectangular capillary electrophoresis some theoretical considerations Chromatographia 39 391 404 1 1 2 1 ADVANTAGES OF THE CAPILLARY Introduction of capillaries into electrophoresis was as an anti convective and heat controlling innovation In wide tubes thermal gradients leads to band mixing and resolution loss The use of capillaries made of glass of 200 500 µm i d was reported by Virtanen 3 in 1969 Jorgensen s 4 introduction of 75 µm capillary tubes was the start of modern high performance CE Figure 2 shows the separation of a mixture of two components
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