Generally, electrophoresis is an analytical technique used in the separation of charged species by means of an electric field. Electrostatic attraction pushes positively charged ions toward the negative electrode (cathode) and negatively charged ions toward the positive electrode (anode). The velocity (v) of an ion can be described mathematically by the product of the electrophoretic mobility (µe) and the applied electric field (E):

Considering that the movement of an ion is defined by Stoke’s law, the electrophoretic mobility can be described as follows:

Where z is the charge of the ion, r is the radius of the ion, and η  is the viscosity of the solvent. The equations show above allow us to reach the conclusion that the mobility of the ion increases with the increase of the charge but tends to decrease with the increase in radius and viscosity of the solvent. Very important is the pH of the medium, which can act on the degree of protonation and consequently on the charge of the ions involved in the separation processes.

Capillary electrophoresis includes several techniques with different characteristics, such as: capillary zone electrophoresis, isoelectric focusing, capillary gel electrophoresis, isotachophoresis, and micellar electrokinetic capillary chromatography. In this article, capillary zone electrophoresis (CZE) will be discussed.

The instrumentation for capillary electrophoresis is relatively simple. As the name suggests, it is made up, in most cases, of narrow silica capillaries, with an internal diameter of 50-75 µm and lengths of 30-70 cm.  The silanol groups that characterize silica tend to deprotonate when the pH is higher than about 4. This gives the internal surface a negative charge (increase in the range 4-9) which is balanced by the cations used to prepare the buffer solution. When a potential difference is applied (typically 10–30 kV), the cations of the electric double layer, being mobile, migrate towards the cathode dragging the hydration cloud with them, determining the migration of all species, regardless of their charge to migrate towards the cathode. This phenomenon is called electroosmotic flow. Different detectors can be used (e.g. fluorescence, amperometry, conductivity, mass spectrometry) but UV detectors are generally used, with devices exiting the column to increase sensitivity.

Capillary electrophoresis is an extremely versatile technique and offers a wide range of analysis possibilities. Proteins and peptides, monoclonal antibodies, numerous active ingredients, inorganic compounds, carbohydrates and much more can be analyzed. Analyzes for the separation of chiral compounds can also be performed.

Capillary electrophoresis is a technique that is developing a lot in the analysis of pharmaceutical products, in particular due to its high selectivity, short analysis times, its versatility in the analysis of small and large molecules, and for its use of a few quantities of solvents.

References

  • Finja Krebs, Holger Zagst, Matthias Stein, Ratih Ratih, Robert Minkner, Mais Olabi, Sophie Hartung, Christin Scheller, Blanca H. Lapizco-Encinas, Cari Sänger-van de Griend, Carlos D. García, Hermann Wätzig. Strategies for capillary electrophoresis: Method development and validation for pharmaceutical and biological applications—Updated and completely revised edition, Electrophoresis, 2023.
  • Steen Hansen, Stig Pedersen-Bjergaard, Knut Rasmussen. Introduction to Pharmaceutical Chemical Analysis, 2012.