THEOR Y 6- METHODS OF SPECTRO PHOTO ME TRY. 213 



the slit being perpendicular to the edge of the wedge, the accuracy of 

 the diagram can be realised objectively, each section of the slit forming 

 a spectrum corresponding with a given thickness of stratum, which 

 increases in a continuous manner from the edge towards the base of the 

 wedge. This method of examination was first employed by J. H. Glad- 

 stone. 1 



The theory and methods of spectrophotometry. The spectro- 

 photometric constants of oxyhsemoglobin (a) The theory. Inte- 

 resting and attractive though it undoubtedly is, the examination of an 

 absorption-spectrum, or the comparison of allied absorption-spectra, by 

 the unaided sense of sight, may be singularly deceptive. 



The impression which the unaided eye enables us to form of the 

 boundaries, the breadth, the intensity of an absorption-band, or of the 

 extent and depth of a less defined general absorption, is often very 

 fallacious. When, for instance, the absorption of a definite region of the 

 spectrum commences and ceases abruptly, the band appears to the eye 

 more intense than when the absorption commences and ceases more 

 gradually. 2 The most striking illustration of the truth of these remarks 

 is indeed furnished by the two oxyhaemoglobin bands. The first, less 

 refrangible band (), has always been described as much more intense 

 than the second, which is broader and less sharply defined, and un- 

 questionably this is the impression which we form by ordinary methods 

 of examination. Vierordt 3 has, however, shown that, in opposition to 

 the visual impression, a greater percentage of light is absorbed in the 

 spectral region which corresponds to the second band than in that 

 corresponding to the first band. Measuring, spectrophotometrically, the 

 percentage of light remaining unabsorbed, after traversing a stratum 1 cm. 

 broad of a solution containing 1 per cent, of defibrinated mammalian 

 blood, he found that in the region of the first, apparently more intense 

 band, 87 per cent, of the light was absorbed and 13 per cent, trans- 

 mitted ; whilst in the region of the second, apparently less intense band, 

 90 per cent, of the light was absorbed, and only 10 per cent, transmitted. 



This result at once suggests the necessity of a method of determin- 

 ing quantitatively the amount of light absorbed by any medium whose 

 absorption-spectrum forms the subject of investigation, instead of trusting 

 to our unaided sense of sight. When, however, we are made acquainted 

 with the remarkable and far-reaching conclusions which can be legiti- 

 mately drawn from an accurate determination of the percentage of light 

 of a definite wave length, absorbed by colouring matters existing in 

 solution, the beauty and the importance of the method of spectrophoto- 

 metry become apparent. Until Yierordt's discovery, those coloured 

 bodies whose visible spectrum presented no definite absorption-bands, 

 were held to be beyond the scope of spectroscopic research. Now, how- 

 ever, we know that a photometric study of the spectrum affords us not 

 only the means of identifying them, but supplies us with a method 

 for the quantitative analysis of colouring matters, surpassing all others 

 in accuracy, and permitting, in certain cases, of the accurate determination 

 of data not to be ascertained in any other way. 



1 J. H. Gladstone, "On the Use of the Prism in Qualitative Analysis," Journ. Chem. 

 Soc., London, 1858, vol. x. p. 79. 



2 A. Rollett, "PhysiologiedesBlutes," Hermann's "Handbuch," Leipzig, 1880, Bd. iv. 

 Th. 1, S. 50. 



3 "Die Anwendung des Spektral-apparates zur Photometric der Absorptionsspektren," 

 Tubingen, 1873. 



