THE OR Y & ME TITODS OF SPECTROPHO TO.VETRY. 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- 
photometry constants of oxy haemoglobin — (a) The theory. — Inte- 
resting and attractive though it undoubtedly is, the examination of an 
absorption-spectrum, or bhe 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 oxyhemoglobin bands. The first, less 
refrangible band (a), 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 Vierordt'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," Jowrn. Ohem. 
Soc, London, 185S, vol. x. p. 79. 
2 A. Rollett, "Physiologie desBlutes," Hermann's "Handbuch," Leipzig, 1880, Bd. iv. 
Th. 1, S. 50. 
3 <: Die Anwendung des Spektral-apparates zur Photometrie der Absorptionsspektren," 
Tubingen, 1873. 
