Physical and chemical research laboratories haxe expanded the use of infrared spectroscopy to such an extent that it has become indispensable for many branches of science within a relatively short period of time. Progress in biochemical application of infrared spectroscopy, on the other hand, has been rather slow. Some of the reasons for this were apparent when we considered utilization of infrared spectroscopy as an adjunct technique for tissue analysis. The 4 main difficulties which had to be evaluated and which greatly influenced development of the procedure were: 1. The infrared energy emitted by all commercial instruments is so small that loss of energy as may occur in examination of very small structures may make qualitative observations inaccurate and quantitative studies futile. The area which is necessary for satisfactory resolution with the present microspectrometer is of the order of about 25 u. by 200u. The necessity of using areas of this size make it impossible to obtain spectra from single cells except perhaps in very special instances, muscle. 2. Water contained in all living tissues is a strong absorbant of infrared energy. Weaker absorption bands, especially in the important 3 u. and 6 u. regions, may be obliterated by the very intense absorption of water. 3. Scattering of energy affects not only measurement of densities of absorption bands, but also ratios of densities of absorption bands. Since a complete correction for scattering is frequently close to impossible, the material used for spectroscopy has to be prepared in such a way that scattering is kept at a minimum. 4. Assignments in mixtures of large molecules as contained in tissues are empirical.
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