10 February / March 2019
[10,11,12] . In their original work, a short, 4 cm, glass separation capillary, was used and like CIEF was connected to the anolyte and catholyte vial respectively. In a later implementation, the separation capillary was connected to an inlet and outlet capillary by a hollow fibre membrane which allows the passage of protons and hydroxyl anions into the separation capillary. This method was commercialised by Convergent Biosciences (now Protein Simple) who entered the market in 2000 with the ICE280 system, followed by Advanced Electrophoresis Solutions with their CEInfinite system in 2016. A typical sketch of this system is given in Figure 5.
Figure 4: Principle of chemical mobilisation using acetic acid. The pH gradient is disrupted by the acid and the analytes are positively charged and move by electrophoretic force towards the cathode
As mentioned before though, all analytes are focussed before the PoD and need to be mobilised past the P0D.
The preferred mobilisation approach is to replace the catholyte vial with a vial containing acetic acid and reapply the voltage. All ampholyte molecules regain a positive charge and start to move toward the outlet vial by electrophoretic mobility as illustrated in Figure 4 (the capillary part past the detector is not shown here). The zones are detected with the built-in spectrophotometric detector (UV-DAD) at 280 nm. Since the high pI zones are closest to the PoD they are detected first. This chemical mobilisation is preferred over the pressure-driven displacement of the sample train in the capillary since significant zone broadening will occur with hydraulic flow.
The fundamental work by Vigh et al. has been commercialised by both Beckman Coulter (now Sciex Separations) and Agilent Technologies who both offer complete
solutions for CIEF which has allowed the technique to become a standard methodology in the biopharmaceutical industry.
Imaged Capillary Iso- Electric Focusing (iCIEF)
The CIEF method described above has three shortcomings;
• the focussing process occurs undetected
• the focussing time is subjectively established based on the current as an indirect measurement
• the mobilisation step adds additional time to the overall run (see Figure 3).
Pawliszyn and Wu proposed to use the whole separation capillary for continuous monitoring of the focusing process which allowed visual control of the focusing process and on-column detection of the zones once focusing has been completed
The essential elements of this system are the narrow, 5 cm wide deep UV LED light source, the illuminated separation capillary, a collection lens, and a detector (camera) by which the focusing process in the separation capillary is continuously monitored. A sample mixture, as described before, fills the separation capillary completely. The sample transfer and outlet capillary are connected to the separation capillary (50 x 0.1 or 50 x 0.2 mm) by a porous hollow fibre membrane (cut-off typically 9000 Dalton). These connections are in the anolyte and catholyte vial and allow protons and hydroxyl ions to enter the separation capillary. The frames in Figure 6 clearly illustrate the focusing process. A simple sample consisting of 5 peptide pI markers are used in this example.
The first frame in Figure 6 shows a baseline of the mixture of ampholytes and 5 pI markers (peptides). Once the voltage is switched on, the marker peptides and ampholytes which are close to the anolyte vial become positively charged by protons flowing in and start to move towards the cathode. Simultaneously, the marker peptides and ampholytes close to the cathode become negatively charged by hydroxyl ions moving in and move towards the anode. Each peptide marker forms two zones which are detected as peaks moving in from the anolyte and catholyte side. It must be realised that simultaneously the ampholytes behave the same and establish the ‘pH gradient’ but are not detected at the wavelength (280 nm) used (frame 2 and 3).
In the 4th frame, 10 peaks from the 5 peptide markers are discerned. The small ‘peaks’ on the low pI side are the front of the zones of the anionic form of the marker while the high ‘peaks’ are the front of the zone of the cationic form of the same marker. Vice versa at the high pI side.
Figure 5: Schematic of an iCIEF set-up (Picture courtesy of Dr Hanno Stutz, Salzburg University). Frame 5 shows the focussing is coming
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