Buffer isotachophoresis

Composition of some common capillary isotachophoresis buffers... 

Capillary tube isotachophoresis using a potential gradient detector is another technique that has been applied to the analysis of alcohol sulfates, such as sodium and lithium alcohol sulfates [303]. The leading electrolyte solution is a mixture of methyl cyanate and aqueous histidine buffer containing calcium chloride. The terminating electrolyte solution is an aqueous solution of sodium octanoate. 

Isotachophoresis. In isotachophoresis (ITP), or displacement electrophoresis or multizonal electrophoresis, the sample is inserted between two different buffers (electrolytes) without electroosmotic flow. The electrolytes are chosen so that one (the leading electrolyte) has a higher mobility and the other (the trailing electrolyte) has a lower mobility than the sample ions. An electric field is applied and the ions start to migrate towards the anode (anions) or cathode (cations). The ions separate into zones (bands) determined by their mobilities, after which each band migrates at a steady-state velocity and steady-state stacking of bands is achieved. Note that in ITP, unlike ZE, there is no electroosmotic flow and cations and anions cannot be separated simultaneously. Reference 26 provides a recent example of capillary isotachophoresis/zone electrophoresis coupled with nanoflow ESI-MS. 

In practice isotachophoresis is usually performed in narrow tubes with electrodes at either end and is one form of capillary electrophoresis. For the separation of a particular type of ion, e.g. an anion, two buffered electrolyte solutions are selected that have different anions but a common cation with a buffering capacity. One of the anions (termed the leading electrolyte) should show a greater mobility than the other anion and occupies the anodic end of... 

And CITP is favorably utilized in the analysis of low-molecular-weight ionic species. A difficulty often arises with finding suitable buffer systems that provide leading and terminating ions and also form the appropriate buffer pH. One of the advantages is that the capillary can be loaded with sample up to 30-50% of its length, enabling the analysis of very dilute samples. Furthermore, the principle of predetermined solute concentrations in isotachophoresis is also used as preconcentration step for very dilute samples prior to CZE, MEKC, or CGE. 

Capillary isotachophoresis (CITP) is an electromigration technique, which is performed using a discontinnous buffer system, formed by a leading electrolyte (LE) and a terminating electrolyte... 

Kostelecka and Haller have determined procaine in mass-produced and extemporaneous pharmaceuticals by capillary isotachophoresis [152]. The method was carried out using pH 4.85 acetate buffer solution, and 0.01 M formic acid as leading and terminating electrolytes, respectively. 

Capillary isotachophoresis (CITP) — An electrophoretic separation technique (-> electrophoresis) in a discontinuous -> buffer system, in which the analytes migrate according to their -> electrophoretic mobilities, forming a chain of adjacent zones moving with equal velocity between two solutions, i.e., leading and terminating electrolyte, which bracket the mobility range of the analytes. Ref [i] Riekkola ML, Jonsson jA, Smith RM (2004) Pure Appl Chem 76 443... 

Figure 4-20. Schematic principles of various electrophoretic separation methods. In zonal electrophoresis (left) particles move according to their electrophoretic mobilities towards the oppositely charged electrode separation into discrete bands depends on mobility differences. In isotachophoresis (centre), the electrode and separation buffers are different. Particles in the sample form tight bands or zones, ordered according to electrophoretic mobility,...

II) Isotachophoresis K separation in a discontinuity between two buffer solutions (the leading and the terminating electrolyte). The sample components are introduced in small quantities in the discontinuity, a d.c. current is applied, and the various components start to move. Consecutive zones are formed and a steady state is attained where each zone moves with equal velocity, hence the name. 

Whereas in conventional zone electrophoresis most of the electrical current is carried by the buffer, in isotachophoresis the ions being separated carry most of the current. In isoelectric a... 

Capillary Isotachophoresis. In isotachophoresis, the capillary is first filled with a buffer of higher mobility than any of the solutes, then the sample, and, finally, a second buffer with lower mobility than any of the analytes. Separation occurs in the zone formed between the two electrolytes. 

Two kinds of conductivity detector are distinguished contact detectors and contactless detectors. Both types were originally developed for isotachophoresis in 0.2-0.5-mm-inner diameter (i.d.) PTFE tubes. Contactless detectors are based on the measurement of high-frequency cell resistance and, as such, inversely proportional to the conductivity. The advantage is that electrodes do not make contact with the buffer solution and are, therefore, outside the electric field. As these types of detectors are difficult to miniaturize down to the usual 50-75-jU.m capillar inner diameter, their actual application in capillary electrophoresis (CE) is limited. 

The main separation modes used in CE are capillary zone electrophoresis (CZE), micellar electrokinetic capillary chromatography (MEKC), capillary isotachophoresis, capillary gel electrophoresis, and capillary isoelectric focusing. CZE and MEKC are used most often. CE buffers are generally aqueous-based, though nonaqueous systems are exploited as well, particularly for analytes that are insoluble or sparingly soluble. 

Isotachophoresis is similar to isoelectric focusing but includes the use of additional ampholines or multiphasic buffer systems to act as spacers to improve the separation and resolution. Each of these techniques can be run using columns, tubes, thin layers, or slabs and are comprehensively dealt with by Andrews (1981). 

To get some idea what composition of buffers can be used for protein separation by isotachophoresis the following examples are presented. 

The advantage of isotachophoresis from the point of view of preparative scale operations is the increase in resolution with increasing load. A higher load increases the length of individual zones. Separation is carried out in an adapted LKB prepara-tive electrophoresis column (Uniphor) and the following buffer systems are recommended by Svendsen [293]. 

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