A very excellent general account of the spectrophotometer and its applications in physiological chemistry is given by Lambling in a paper, which the reader is advised to consult. The method consists essentially in measuring the diminution in intensity which a beam of light undergoes in its passage through a coloured solution, and in deducing the concentration of the solution from such a measurement. Given two rays of equal initial intensity, one of which is perceived directly by the observer, and the other after its passage through a coloured solution, what one has to do is to measure the relative intensity of the two rays. But such observations must be made not with white light, which is mixed light, but with homogeneous light; in other words, the rays from a particular part of the spectrum; hence the term spectrophotometry. The amount of absorption varies for the same substance and the same region of the spectrum with the concentration and thickness of the layer of liquid examined. A double layer of the liquid would produce the same effect in absorbing light as a single layer of a liquid twice as concentrated. 1 The quantity of light absorbed, however, does not increase directly with the thickness or concentration of the coloured liquid. Suppose a luminous ray of intensity equal to 1 passing through a layer of coloured liquid of one unit's thickness, its intensity is reduced to ; when, however, this ray of diminished intensity passes through another similar layer, its intensity is diminished by - X - = and after passing through m similar layers to 1 1 The co-efficient of extinction (Bunsen) € of a coloured solution is the inverse of a number expressing the thickness of the layer of that solution which is necessary to reduce the intensity of the light to one tenth of its initial intensity. We have already seen if I'= final intensity, 1 = initial intensity, and m = thickness of layer, But by the definition of the co-efficient of extinction: And if munity, € = -log I'. In other words, the co-efficient of extinction is obtained by taking the negative logarithm of the fraction which represents the final intensity of the light. Suppose, for instance, that a solution of oxyhæmoglobin observed in a layer 1 centimetre thick reduced the luminous intensity in the region of the 8 band to -log 0.225 0.225 of the original, then € = 0.64782 Suppose that C, C', C" = series of solutions, and e, e', e'' C C' C" extinction, then : represent the respective concentrations of a the corresponding co-efficients of = A. This constant A can be easily measured in a solution of known strength: it is called the absorptive power: A = In other words, the concentration of the solution (number of Therefore C=Ae. grams in 1 c.c. of solution) can be ascertained by multiplying the co-efficient of extinction by the constant A. This method can also be applied to mixtures of two colouring matters in solution-e.g. hæmoglobin and oxyhæmoglobin-provided that the constant A is known for both substances in two regions of the spectrum. The co-efficient of extinction in the same two regions is then determined by observation. The formula is somewhat complicated, and the memoir already referred to must be consulted for this matter, as well as for other interesting suggestions relating to the examination of other animal pigments, as of the bile, urine, &c., by means of the spectrophotometric method. The forms of spectrophotometer that have been invented for the determinaI select for description one tion of co-efficients of extinction are very numerous. invented by Glazebrook, and described by Dr. Sheridan Lea in the Journal of Physiology." In principle it is the same as Hüfner's, but differs from it, in that the light from both sources is polarised, whereas in Hüfner's instrument the light from only one source is polarised. The light, then, from each of two sources is polarised by a nicol's prism, and then allowed to pass through a direct vision prism, whereby two adjacent superposed spectra are obtained, and these are observed through an eye-piece, in which is an analysing nicol. This eye-piece can be rotated on its axis, the amount of rotation being measured by a pointer which moves over a circle divided into degrees. The spectra are further observed through a narrow slit in the eye-piece, so that only a small piece of the spectrum is seen, the part, in fact, for which one is making the determination. Set the pointer of the eye-piece at 0°, and then rotate one of the polarising nicols until the spectrum formed by the light passing through it is eclipsed: now set the pointer at 90°, and rotate the other polarising nicol till the second spectrum is eclipsed. Then set the pointer in some intermediate position in which the two spectra are of equal brightness. Now let the solution of the pigment of unknown 1 Vol. v. p. 239. concentration C be introduced on the path of the light which forms one of the spectra. In order to produce equality of the spectra the pointer of the eye-piece must be rotated into a fresh position. Now if be the angle through which the eye-piece was rotated from 0° in order to produce the original equality of the spectra, and e' be the angle of rotation required to produce equality when the absorbing substance is interposed, our formula C=Ae becomes C=2Alog Suppose, now, tan @ tan that the same be done with a solution of known concentration, C, and that " be the angle of rotation required to produce equality when this solution is interposed in the path of the light from one source, then C'=2A log C log tan e-log tan o' Clog tan -log tan e" tan @ tan lated. Hence, = from which equation C can be calcu THE SPECTRO-POLARIMETER1 This instrument is one, in which a spectroscope and polarising apparatus are combined for the purpose of determining the concentration of solutions of substances which rotate the plane of polarised light. It was invented by E. v. Fleischl, for the estimation of diabetic sugar in urine. Its chief advantage is, that no difficulty arises of forming a judgment, as to the identity of two coloured surfaces, as in Soleil's saccharimeter, or of two shades of the same colour, as in Laurent's instrument. The light enters at the right-hand end of the instrument, is polarised by the nicol's prism b, and then passes through two quartz plates, ce, placed horizontally over one another. One of these plates is dextro-, the other lævorotatory, and they are of such a thickness (7.75 mm.) that the green rays between the E and b lines of the spectrum are circularly polarised through an angle of 90°, the one set passing off through the upper quartz to the left, the The following account of this instrument is taken from Dr. McKendrick's Physiology, vol. i. P. 154. other through the lower to the right. The light then continues through a long tube, ff, which contains 15 c.c. of the solution under examination. It then passes through an analysing nicol, d, and finally through a direct vision spectroscope, e. On looking through the instrument, the tube ff being empty, or filled with water or some other optically inert substance, two spectra are seen, one over the other, but each shows a dark band between E and b owing to the extinction of these rays by the circular polarisation, produced by the quartz. The analyser can be rotated: a vernier, g, is attached to, and moves with it, round a circular disc (seen in section at h) graduated in degrees. The two bands in the spectra coincide when the zeros of vernier and scale correspond. If now the tube ƒ is filled with an optically active substance like sugar, the bands are shifted, one to the right, the other to the left, according to the direction of rotation of the substance in ƒ. The rotation is corrected by rotating the analyser into such a position that the two bands exactly coincide once more as to vertical position. The number of degrees through which it is thus necessary to move the analyser, measures the amount of rotation produced by the substance in ƒ, and is a measure of the concentration of the solution. The degrees marked on the circular scale are not degrees of a circle, but an arbitrary degree of such a length that each corresponds to 1 per cent. of sugar in the given length of the column of fluid in ff (177-2 mm.). |