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capillary. When the voltage is switched on the situation changes as depicted in Figure 2.
Figure 1: Depiction of the capillary composition at the beginning of a CE run. The proteins to be separated have pI 5 and 7. Carrier ampholytes, pI markers and additives are homogenously distributed over the capillary and not visualised here. (picture with permission of Agilent Technologies)
When the focussing process starts, highly mobile hydrogen ions move towards the cathode, whilst hydroxyl ions move to the anode. At the same time low pI molecules which are initially positively charged move towards the cathode and negatively charged high pI molecules move to the anode. Upon migration to the electrode of opposite charge, an amphoteric molecule is protonated/deprotonated and focuses in a small zone. This is a dynamic process, with the amount of protonation and deprotonation depending on the pKa and pKb of the ionisable groups in each analyte molecule.
Figure 2: Schematic of initial steps of the focussing process. (Picture with permission of Agilent Technologies)
CIEF run, the whole capillary is filled with a mixture containing, carrier ampholytes, covering a specific pI range e.g. 3-8, blocking agents, the protein or peptide sample to be analysed, and pI markers. Additives may be added like methylcellulose to suppress residual EOF or urea, to improve the solubility of the focused molecules. The absence of EOF is essential in the execution of CIEF since the focussing process is disturbed by flow in the capillary.
All substances are distributed homogeneously over the whole capillary at the start. The overall pH of the sample
solution is determined by the concentration and dissociation state of all substances present. The inlet vial is filled with a low pH solution (anolyte, H3
PO4 ) and a positive
voltage is applied here, resulting in this electrode becoming the anode. The outlet vial contains a high pH (catholyte, NaOH) solution, is grounded and the electrode at this point becomes the cathode.
This is illustrated in Figures 1 and 2.
Before the focussing process commences molecules with a low pI will have a positive charge while molecules with high pI will have a negative charge at any location in the
The completion of the focusing process in a CE instrument is monitored by the current. When all molecules are neutralised and focused, the current will have dropped significantly to a minimum level. This normally occurs after approx. 20-30 minutes. At this time, it is believed that all proteins are arranged along the capillary according to their pI.
Figure 2 shows the problem with this approach. The PoD is remote (7-10 cm depending on the CE-instrument) from the end of the capillary. Therefore, high pI proteins may become focused outside of the PoD and will be lost at the start of the mobilisation.
In a collaboration with scientists from Beckman Coulter, Vigh developed a solution [7]. So-called sacrificing ampholytes or blocking agents, which have a pI lower or higher than the pI of all sample proteins and pI markers, are introduced into the sample mixture. Iminodiacetic acid, pI 2.2 and arginine, pI 10.7 are examples. Now it must be realised that the length of a zone occupied by any ampholyte in the capillary depends on its pI, its amount in the capillary as well as the pI values and amounts of all other ampholytes in the capillary. When the concentration of the blocker is chosen properly, the focussing of analyte molecules and pI markers occurs before the point of detection. The high pI blocking agent will focus beyond the PoD. An illustration is given in Figure 3 taken from a technical note [9].
Figure 3: cIEF separation of peptide pI markers (Table 3). Sample: 200 µL of cIEF gel, 12.0 µL of Pharmalyte 3-10, 2.0 µL of each pI marker (1.25 mM), 18.0 µL of cathodic stabiliser (0.5 M Arg.) and 4.0 µL of anodic sta- biliser (0.2 M IDA). Method: focusing: 15 min at 25 kV; chemical mobilisation: 25 min at 30 kV. Anolyte: 200 mM phosphoric acid. Catholyte: 300 mM sodium hydroxide. Chemical mobilising solution: 350 mM acetic acid. (Figure with permission from Sciex Separations)
The cathodic peaks are caused by the movement of high pI sample components from the cathodic side of the capillary which passes the PoD during the focusing phase towards the anodic side. They focus before the PoD.
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