ITP Physics and Experimental Methodology Principal Investigators: Giancarlo Garcia-Schwarz, Anita Rogacs, Supreet S. Bahga, and Juan G. Santiago Isotachophoresis (ITP) is a robust electrokinetic separation and preconcentration technique with applications in toxin detection, sample preparation, fractionation, and nucleic acid analysis. We here discuss the physics of ITP and the methodology of applying this technique to two specific example applications: separation and detection of small molecules, and purification of nucleic acids from cell culture lysate. Physics of ITP ITP Fundamentals: ITP uses an applied current and two electrolyte mixtures (e.g., two buffers) to focus and separate sample ions. Sample ions focus within a moving sharp boundary separating these two mixtures. Here, we discuss ITP focusing of anionic samples in the ITP zone formed by the anions of a low-conductivity trailing (TE) and a high-conductivity leading electrolyte (LE). An applied electric field causes a strong electric field gradient between TE and LE. ITP Interface: The ITP interface is self-sharpening and established by the competing effects of diffusion (acting to broaden the interface) and the electric field gradient, which causes it to sharpen. The TE and LE anion mobilities are chosen to bracket the mobility of sample ions. Sample ions in the TE, overspeed neighboring anions and focus at the ITP interface. Sample ions in the LE are oversped by anions in the LE, but not by anions in the TE, and therefore also focus at the ITP interface. Peak Mode ITP: In this mode of ITP, sample ion concentrations are at all times much lower than those of LE and TE co-ions and so contribute negligibly to local conductivity. This has the useful effect of causing multiple sample ions to focus within the same narrow ITP interface region. Sample ions meeting the ITP focusing criteria focus as largely overlapping peaks. Plateau Mode ITP: In this mode of ITP, sample ions increase in concentration and saturate at a level governed by the properties of the initial LE mixture. As they focus, sample ions in plateau mode also displace each other and separate into adjoining pure zones. Example Applications Purifying DNA from cell lysate: ITP selectively focuses only ionic species whose mobility is bracketed by those of TE and LE co-ions. Our group has shown that this ability makes ITP ideal for purifying target species from complex biological samples including cell culture, urine, and blood lysates. In the video below, we will demonstrate purification of nucleic acids from cell lysate. We do this by selecting a trailing anion with an effective mobility magnitude lower than the target nucleic acid, but higher than co-ionic PCR inhibitors. Such inhibitors include proteins, anionic detergents, and organic solvents. Cationic PCR inhibitors, such as alkali metals, cationic proteins, or detergents, of course, do not focus. These latter molecules migrate in the opposite direction as the (anionic) ITP interface. Purification and detection of amino acids: In plateau mode, ITP can separate and focus sample ions into adjoining plateaus bracketed by the TE and LE. These zones can be detected and quantified using a variety of physicochemical properties (e.g., conductivity, UV absorbance, temperature, index of refraction). In the video below, we demonstrate detection of plateau zones using a non-focusing tracer (NFT) species. This NFT is a fluorescent species which is not focused by ITP but which migrates through all ITP zones and whose concentration adapts to local electric fields. VIDEO Reference G. Garcia-Schwarz, A. Rogacs, S.S. Bahga, and J.G. Santiago, “On-chip Isotachophoresis for Separation of Ions and Purification of Nucleic Acids,” Journal of Visualized Experiments 2012, 61, e3890.