Instrumentation for Isotachophoresis

 

The Single Column System

 

In general an isotachophoretic separation is carried out in a PTFE tube 0.2 to 0.4 mm internal diameter. In most instruments the system is fitted with both conductivity and UV absorption detection. A typical example of an isotachophoretic instrument is shown in figure 14.

 

Figure 14. Basic Isotachophoretic Apparatus

 

The apparatus consists of two electrode compartments carrying the leading and terminating electrolytes respectively in which are inserted the anode and cathode, which in turn, are connected to a constant current power supply. A PTFE tube, with inside and outside diameters of 0.2 and 0.3 mm respectively, has been found to be optimum for this type of separation. Tubes of these dimensions have very small convective contributions and minimal temperature affects. The sample is introduced by injection into the sample block.  As the zones are formed each one will assume its own physical characteristics such as temperature, conductance, pH, and potential gradient. In addition some zones may exhibit other characteristics such as radioactivity, UV absorption optical rotation, and fluorescence all of which, using appropriated detecting systems can aid in sample identification. One of the first detection methods was thermal measurement employing very thin constantan/copper thermocouples and although the signal is small it is one of the more sensitive detection devices. Potential gradient detection and conductivity detection is carried out using 25-micron diameter electrodes. The electrodes are cemented into the detector block and the sensor volume is a few nanolitres. The UV sources is normally a low pressure mercury vapour lamp and light is guided by an optical quality glass rod through the separation tube and then by another optical glass rod system onto a suitable photoelectric cell. The different parts of the apparatus are labelled in figure 14.

 

   The Coupled Column System

 

Unfortunately, samples can rarely be directly placed onto the isotachophoretic instrument for analysis, as most will invariably need some form of sample preparation. Sample preparation techniques can vary widely in complexity from simple dilution to a complex extraction followed by derivatization procedures. A frequent problem involving sample preparation arises when the substances of interest are included in mixtures containing high concentrations of other materials.  By employing a column coupling procedure a large amount of sample can be used without the need for sample pre-treatment such as extraction followed by concentration.  The coupled column system that will be described has a wide range of application as it can also be used to employ other techniques in tandem with isotachophoresis such as liquid chromatography or zone electrophoresis etc.

 

A diagram of a coupled column isotachophoresis instrument is shown in figure 15. The coupled column apparatus consists of three main parts, vis.,

 

1.      A pre-separation section

2.      A bifurcation section with a fitted detector

3.      A final separation section

 

In turn the bifurcation block has also three important components, viz.,

 

a  A relatively wide bore hole (0.8 mm) in which the detector sensor(s) is mounted and which is connected to the wide bore separation tube in the pre-separation section.-

 

b  A narrow bore hole (0.2 mm) in line with the wide bore hole (0.8 mm)  to which the PTFE narrow bore separation tube is connected.

C  A rectangular hole (0.05mm x 1.0 mm) at right-angles to the other holes which forms an electrical connection to the counter electrode compartment applied during the pre-preparation.

 

Figure 15. A Coupled Column Apparatus

 

A diagram of a commercially available isotachophoresis instrument manufactured by Flowspek is shown in figure 16.

 

The separation sections are in line with each other so that zone mixing is minimised during trapping. To select the chosen band with precision, the time interval between the detector sensing this band of interest and the junction to the separation section can be calculated from the value of dV/dt of the constant current power supply in a leading electrolyte/terminating electrolyte experiment. Thus, those zones requiring further separation can be selected even if they are not migrating sequentially.

 

 

 

 

Figure 16. A Commercial Isotachophoresis Instrument

 

The choice of the bands of interest can be programmed so that the selection is automatic and consequently highly reproducible. Using this technique, the load that can be used for analysis can be increased by an order of magnitude.