On-line capillary electrophoresis FTIR detection for the separation and characterization of proteins

Capillary electrophoresis (CE) with on-line FTIR spectroscopic detection was used to separate a mixture of three model proteins and to provide information on the dominant secondary structures of the separated proteins. On-line FTIR transmission measurements were achieved using a micro-machined flow cell made out of CaF2 and SU-8 polymer which was attached to the fused silica capillaries using commercial O-rings. The IR beam was focused on the detection window using an off-axis parabolic mirror attached to an external optical port of the spectrometer. As the absorption of water heavily overlaps the so-called amide I band of the proteins the separations were carried out in heavy water (D2O). The model proteins studied were myoglobin, α-lactalbumin and β-lactoglobulin having different secondary structures. It could be shown that the performance of the separation was comparable to conventional UV–vis detection and that the IR-peak positions of the proteins studied were the same as obtained in reference measurements using the ATR technique.