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S. Kulka, B. Lendl:
"Capillary electrophoresis with on-line FTIR detection for the separation of proteins";
Poster: 3rd International Conference on Advanced Infrared and Raman Spectroscopy, Delavan, Wisconsin, USA; 14.08.2005 - 19.08.2005; in: "ICAVS 03", B. Lendl (Hrg.); (2005).

Here we present the on-line hyphenation between capillary electrophoresis and FT-IR [1] applied to the study of the secondary structure of proteins. FT-IR offers the advantage of real-time, non-destructive and molecule-specific detection as compared to UV, LIF or MS. Therefore FT-IR can be employed as a detection method complementary to the above mentioned techniques. The position of the C=O stretch vibration (the so-called amide I band) can be used as an indicator for the secondary structure of a protein. In helical structures the vibration is centered around 1650 cm-1, while in pleated structures it lies around 1635 cm-1.
The main problem when using FT-IR in aqueous solution was overcome by using heavy water (D2O) where the solvent absorption bands are shifted away from the amide I towards lower wavenumbers. The detection was performed in a micro-machined IR transparent flow-cell [1] and checked using a UV detector to ensure that the proteins were separated as detected in the IR spectrum. The proteins separated were myoglobin, a-lactoglobulin and b-lactalbumin (consisting of two isoforms).
The results of the IR analysis agree very well with ATR data taken in water and D2O. The secondary structure given by the spectral data was confirmed by data recorded using X-ray crystallography. Therefore the technique used can be employed to study proteins, interaction between proteins and other analytes and to study misfolding/refolding of proteins.

http://aleph.ub.tuwien.ac.at/F?base=tuw01&func=find-c&ccl_term=AC05937123

http://publik.tuwien.ac.at/files/pub-tch_4956.pdf