12 February / March 2019


broadening of the protein zone with time, the diffusion coefficient and hydrodynamic radius can be calculated.


Alternatively, iso-electric focusing of a sample containing the protein of interest and a low molecular weight LMW ligand, one will initially see two focused zones viz the protein-ligand complex and the free protein. While continuing the focusing, one will observe that the complex zone will decrease, and the protein zone increases (see Figure 10), thus allowing KD to be calculated [14].


iCIEF/CIEF MS-Coupling


Figure 9: Effect of using smaller pI range. Sample, acidic mAB, top trace Pharmalyte 3-10, 6 mins focussing time; lower trace Servalyte 4-7, 12 mins focussing time. Anolyte 80 mM phosphoric acid, catholyte 100 mM sodium hydroxide. (Figure courtesy of Tiemin Huang, Advanced Electrophoresis Solutions)


capillary) is 5-6 times shorter than for an analysis by CIEF. Furthermore, in CIEF the result of the focussing step is only seen after mobilisation has finished whereas with iCIEF the separation is monitored as it develops. Any aberrance that may have occurred during CIEF focusing goes by unnoticed and becomes visible while mobilising. In addition, during focusing in CIEF, peaks are observed (cathodic peaks) which may raise questions about the composition of the sample under investigation.


A high level of practical competence in CE is a necessary requirement to successfully apply CIEF, whereas iCIEF is easier to use, as in most cases a standard protocol for injecting and observing the data can be followed.


The typical LODs in CIEF are 2-3x better than those obtained by iCIEF [9] since the spectrograph in CE equipment for UV-VIS detection has a stable, optimal focused optical system and highly sensitive photodiode array. The introduction of the CMOS camera as a standard detector in iCIEF has improved sensitivity, but still has some way to compete with the CIEF. Currently commercial CE and iCIEF instrumentation is rugged and robust.


There are two major providers of CE equipment (Sciex Separations and Agilent Technologies) committed to serve the market and support CIEF with consumables and application notes. For iCIEF there is one dominant manufacturer on the market (Protein Simple), although the recent


introduction of the CEInfinite iCIEF system by Advanced Electrophoresis Solutions Inc. has created a more competitive landscape which will be beneficial for analytical scientists who should see improvements in the technology as the competition between the companies grows.


New developments of iCIEF


Given the ability of an iCIEF system to continuously monitor dynamic changes in the separation capillary, kinetic measurements are feasible. Eg. iCIEF can be used for determination of diffusion coefficient and hydrodynamic radius of proteins by Taylor dispersion measurement. A protein mixture is separated by IEF after which a hydrodynamic pressure is applied to move the focussed protein zone along the imaged separation channel after the high voltage is stopped. By observing the


Table 1: Comparison CIEF and iCIEF


CAPILLARY ISO-ELECTRIC FOCUSSING IMAGED CAPILLARY ISO-ELECTRIC FOCUSSING


Standard open platform CE Equipment: multiple purpose CE methods


Long cycle time (sample introduction, focusing and mobilisation)


Long method development time


Competence and experience required especially for troubleshooting and diagnosis


Protein concentration > 0.05 mg/mL


Dedicated, CE system: lean system, optimised and targeted for CIEF


Fast analysis time


Short method development Easy to use (‘black box’)


Protein concentration >0.1 mg/mL, LOQ 1 µM [13]


The superior resolution of CIEF and the 50-60 x concentration factor in the focussing process makes CIEF very attractive for the separation and quantitation of complex protein samples. Unfortunately, the UV detection does not allow for identity or structural information, so soon after CE-MS ESI interfacing became available, many groups have investigated coupling CIEF with MS.


In coupling CIEF/iCIEF with ESI-MS, several hurdles can be readily identified.


The zones that are mobilised from the capillary to ESI-sprayer are in nanolitre volume (4-20 nL) range, which due to the necessity of using a transfer capillary result in a loss of resolution when compared to the UV detector.


Besides the separated protein zones, the CA and other additives will become transferred to the ESI interface. This is undesirable while the ionisation process will become interfered and/or ion suppression will occur and contaminate the MS inlet capillary.


The two high voltage sources on the capillaries viz. voltage from CE and the ESI voltage need to be decoupled electrically so that there is no interference in both processes. MS systems from Agilent and Bruker have an advantage here since the ES


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