Imaged cIEF

The CFR 21 part 11 compliant software newly released by Convergent Bioscience makes imaged cIEF a viable technique for use in QC environments for release testing of therapeutic proteins or antibody molecules. i-cIEF has also been used heavily for characterization of protein modifications such as deamidation and isomerization, as well as oligosaccharide structure analysis. ... 

Convergent Bioscience has an interesting approach to detection in CE by using a CCD (charge-coupled device) camera to image the entire capillary (Figure 7). A UV-transparent capillary is needed and the entire capillary is excited with a xenon lamp. This is useful in cIEF... 

Figure 3 Instrument setup of whole-column imaging detection for CIEF. Absorption or refractive index gradient mode with camera (I) placed in the direction of illuminated light fluorescence mode with camera (II) placed vertically to the direction of illuminated light. (Reproduced from Mao, Q., Pawliszyn, J J. Biochem. Bio-phys. Meth. 39, 1999, 93-110, with permission of Elsevier Science Publishers.)...

Peptides are not as commonly analyzed by CIEF as are proteins one reason is their lower resolution, another their lower (or lack of) detectability at 280 nm (the wavelength mostly used). The separation of tryptic digests (peptide mapping) of proteins have been performed by using absorption detection at 280 nm and refractive index gradient imaging detection no exact correlations were observed between measured and calculated p7 values [1,5]. Refractive index detection is a universal detection method (i.e., independent of chromophores like tyrosine and tryptophan) but suffers from low sensitivity. Assays of trypsin activity have also been performed with laser-induced fluorescence detection for enhanced sensitivity, with detectability down to picomolal concentrations [5,6]. 

Wu J, Wu XZ, Huang T, Pawliszyn J. Analysis of proteins by CE, CIEF, and microfluidic devices with whole-colunm-imaging detection. Methods Mol Biol 2004 276 229-52. 

In CIEF, the total time elapsed between sample introduction and detection depends on the focusing time and on the time for a band to reach the detection window during mobilization. These two effects can take place either simultaneously or consecutively depending on whether the process is a one-step or two-steps, respectively. So, the term migration time is not strictly correct in CIEF but it is used in this chapter for simplicity. It is important to note that the need for mobilization through the detector window can be eliminated in systems where whole-capillary imaging detection is used [100]. 

Although CE instruments are not well designed for CIEF, many variants can be separated better by this technique. In addition to the common variants, G Philadelphia, A2, and Bart s can all be separated by CIEF [46-48]. The separation by CIEF compares well with gel isoelectric focusing and with HPLC. The variants have also been analyzed by both CE and CIEF equipped with special absorption imaging detectors. These types of detection devices eliminate the extra steps needed to move the peaks, after the focusing step, to the detector and can simultaneously detect several capillaries with better precision and faster results than CE instruments. HB A2, which is increased in P-thal, is better quantified by CE compared with AG electrophoresis [44]. Hemoglobin analysis by CE has been reviewed recently [49-51]. 

A principal difference between lEF in a gel and in a capillary is that, in the latter, mobilization of the focused proteins past the detector has to be carried out if an online imaging detection system is not being used. Mainly three techniques are used chemical and hydrodynamic flow mobilization (in coated capillaries) and mobilization utilizing the EOF (in uncoated or partially coated capillaries). The last approach is troublesome, though, since the transit times of the focused zones change severely from run to run thus, it is preferable to perform cIEF in well-coated capillaries, where EOF is completely suppressed. 

Adaptation of CIEF to the microchip format can provide higher performance compared to conventional capillaries. Whole channel imaging can be performed, thereby eliminating the need for mobilization. The chip can be interfaced to the mass spectrometer while maintaining the short length of the separation channel which is a distinct advantage compared to the conventional capillary. 

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