A 2 Experimental Considerations

Fourier transform infrared spectroscopy employs an interferometer to collect an entire spectrum over the full range of energies simultaneously. This provides a more rapid collection of spectra than scanning through the required energy range, as is done by dispersive spectrophotometers. Thus, the FT-IR spectrometer collects many spectra in a short time, improving the signal to noise ratio by an amount proportional to the square root of the number of scans [11], After the interferometric spectra are collected and averaged, the inverse Fourier transform is used to compute a vibrational spectrum, usually in the form of transmittance or absorbance vs. frequency in wavenumbers, from the interferogram. In modern spectrometers, this transformation is done automatically with software provided along with the FT-IR instrument.

FT-IR spectra can be obtained on many types of protein samples, such as solutions, gels, films, or colloidal dispersions. FT-IR spectra can be collected from microliters of protein solutions in the concentration range of 20-50 mg mL"1. In the past, the infrared (IR) spectra of proteins were obtained in D20, but FT-IR spectrometers make it possible to obtain the spectra in H20. This offers considerable advantages, because both amide I and II bands appear in H20, providing a larger information content enabling a more complete analysis of the secondary structure.

Careful subtraction of the water spectrum from the spectrum of the protein solution is required when experiments are conducted in water. This subtraction should yield a horizontal line in the 1800-2000 cm"1 region, where only water and not protein absorbs IR radiation. The spectrometer must be purged thoroughly with dry nitrogen or argon to remove water vapor and carbon dioxide before collection of the spectrum.

The following experimental conditions were used in the examples cited in this chapter [9, 10], Aqueous solutions were prepared as 4% protein in compatible buffers. All samples were introduced into a demountable cell with CaF2 windows separated by a 12 /¿m Teflon spacer. Spectra were obtained using a Nicolet 740 FT-IR spectrometer equipped with the Nicolet 660 data system. Nitrogen purging of the sample chamber was used to reduce water vapor and carbon dioxide to a minimum. Each spectrum consisted of 4096 interferograms, coadded, phase-corrected, apodized with the Happ-Genzel function, and inverse Fourier transformed. Nominal instrumental resolution was 2 cnr1, with one data point every 1 cm"1. Water vapor absorption was routinely subtracted from all spectra.

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