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.