INTRODUCTION AND METHODS 



a monochromator. Passages drilled in the carrier allow circulation of 

 water from a thermostat, and hence control of the temperature of the 

 solutions during measurement. 



In Fig. 1.9 the cell holder is shown in 'exploded' relationship to 

 the monochromator (which delivers light of any wavelength within a 

 certain range), and the Hght-measuring instrument. Since the visual 

 pigments are photosensitive it is essential to employ light of very 

 feeble intensity. The apparatus must accordingly be of high sensi- 

 tivity; a photocell of the multipHer type (e.g. RCA 931 A) in con- 

 junction with a galvanometer and scale is suitable. With such an 

 arrangement, optical densities can be measured with great precision 

 (e.g. with a standard deviation of 0-0006 for densities within the 

 range to 0-5). On the other hand, a manually controlled apparatus 

 is slow to operate, the time required to traverse the visible spectrum 

 at 10 mju intervals (each observation checked by a second one) being 

 40-50 mins. 



So far as speed is concerned the Hardy recording photoelectric 

 spectrophotometer has a great advantage. This costly instrument 

 draws the absorption spectrum on paper in about 2 min. It is thus of 

 particular value in studying the transient thermal reactions which 

 follow the photodecomposition of the visual pigments (wald, 1938, 

 1939). The instrument would also be useful for investigating the 

 homogeneity of visual pigment preparations (Chapter 6), a laborious 

 undertaking with a manual apparatus. 



DIFFERENCE SPECTRA 



The density spectrum of an unbleached visual pigment preparation 

 is not wholly characteristic, for it includes the contribution of Ught- 

 absorbing impurities. If the impurities are stable, and are not 

 affected by exposure to light, the density spectrum of the bleached 

 preparation includes the same contribution from impurities. Conse- 

 quently, by subtracting one density spectrum from the other, a 

 function which is independent of impurities is obtained. This 

 function, the difference spectrum, is simply the difference between 

 the density spectrum of the visual pigment and that of the product 

 into which it is changed by bleaching. 



The value of the difference spectrum for characterizing a visual 

 pigment in the presence of impurities is illustrated by examples in 

 Fig. 1.10. In the upper part of Fig. 1.10 are shown the density 

 spectra, before and after bleaching, of two extracts from the retinae 



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