Method of using the Micro-Spectroscope.
A beginner with the micro-spectroscope should first make himself fully acquainted with the spectroscope by holding it up to the sky and noting the effects of opening and regulating the slit, by rotating the screw C, Figs. 195 and 197. The lines will be well seen on closing down the opening. This screw diminishes the length of the slit, when the spectrum is seen as a narrow ribbon of prismatic colours. The screw E regulates the admission of light through the aperture above D. The better objects with which to commence the study of the absorption bands are, aniline dye, much diluted, madder, permanganate of potash, and blood. As each colour varies in refrangibility, the focus must be adjusted by the screw E. When it is desired to view the spectrum of a very minute object, the prisms should be removed by withdrawing the tube containing them, the slit set open, and the object brought into the centre of the field; the vertical and horizontal slits must then be partially closed up, and the prisms replaced, when a suitable objective is employed to examine the spectrum. For ordinary observations a magnifying power of an inch and a half or two inches will be suitable, but for small quantities of material a higher power must be employed, when a single blood corpuscle can be made to show its characteristic absorption band. After having obtained the best image of any object on stage, throw it slightly out of focus, and substitute the micro-spectroscopic eye-piece for the Huyghenian. Opaque objects should be examined by reflected light, by means of the bull’s-eye condenser, or side reflector. Mr. Sorby uses a binocular microscope, which enables him to regulate the focussing and throwing out of focus of the object.
In examining crystals or other small objects, a small cardboard diaphragm should be placed beneath them; and when examining the spectra of liquids in cells, slip a small cap with a perforation of 1⁄10-inch in diameter over the tube containing the ½-inch or 2-inch objective. Substances which give absorption bands or lines in the red are best seen by artificial light, while those which show bands in the violet are better seen by daylight. By following rules of the kind we are less likely to mix the bands of the absorption spectrum with the Fräunhofer lines. For example, if the edge of a band happens to coincide with a Fräunhofer line, the observer is apt to imagine that the band is better defined and more abruptly shaded on one side than it really is.
Standard Spectrum Scale.
Cells for use with Spectroscope.
Fig. 198.
Cells and Tubes.—These are either supplied ready-made by the optician, or can be formed out of small pieces of barometer tubing, with the edges ground down and cemented on ordinary glass slips. In [Fig. 198] is seen the several kinds of cells and tubes usually employed, while the little flat tubes commonly in use as bouquet holders will be found of use, with the side stage reflecting spectrum as comparison tubes; being of different diameters they allow of two or more depths of colour in the fluid intended for examination.
In the case of many other fluids the sloping form of cell ([Fig. 198]) will be useful, as different shades of fluids can be examined without removal from the stage of the microscope. The deeper cells are cut from a piece of barometer tubing of about half to an inch long, one end being cemented to a piece of flatted glass, and the other covered over temporarily or permanently with a thin piece of glass on the top, held in its place by capillary attraction, thus admitting of the tube being turned upside down.
Re-agents required.—A diluted solution of ammonia, citric acid, double tartrate of potash and soda (the last being used to prevent the precipitation of oxide of iron), and the double sulphate of the protoxide of iron and ammonia (employed to deoxidise blood, etc.). In some special cases, dilute hydrochloric acid, purified boric acid, and sulphate of soda are required.
The character of stains of blood varies with age and with the nature of the substance with which it happens to be combined. This is important to remember in connection with Jurisprudence, when the micro-spectroscope is brought into use for the detection of blood stains. The spectrum used in important cases of the kind should have a compound prism, with enough, but not too great dispersive power, otherwise the bands become, as it were, diluted, and less distinct.
If the blood stain is quite recent, the colouring matter will be hæmoglobin only. This easily dissolves out in water, and when sufficiently diluted gives the spectrum of oxy-hæmoglobin, which on the addition of ammonia, together with a small quantity of the double tartrate, a small piece of ferrous salt, and stirring carefully without the admission of air, changes the spectrum of reduced hæmoglobin. When stirred again, so as to expose the solution as much as possible to air, the two bands reappear; on gradually adding citric acid in small quantities the colour begins to change, and the bands are seen to gradually fade away; if there should have been much blood present, a band appears in the red; the further addition of ammonia makes all clear again, but does not restore the original bands, because the hæmoglobin has been permanently changed into hæmatin. This reaction alone distinguishes blood from most other colouring matters, since other substances after being changed by acids are restored by alkalies to their original state. There are many other curious facts connected with the spectroscopic analysis of blood, which are fully explained and illustrated by Dr. Maemunn in his book on “The Use of the Spectroscope in Medicine,” and also in Dr. Thudicum’s[36] reports and charts, which are the most complete. Sir George Stokes, F.R.S., was one of the first to show the essential value of the spectral phenomena of hematine, and who proved, after Hoppe had first drawn attention to the fact, that this colouring matter is capable of existing in two states of oxidation, and that a very different spectrum is produced according as the substance, which he termed cruorine, is in a more or less oxidised condition. The chart appended to his paper[37] affords an imperfect representation of the changes seen in the spectrum.
No. 1.—Arterial Blood, Scarlet Cruorine.
No. 2.—Venous Blood, Purple Cruorine.
No. 3.—Blood treated with Acetic Acid.
No. 4.—Solution of Hæmatin.
Fig. 199.—Sir George Stokes’ Chart of the Absorption Bands of Blood.
Proto-sulphate of iron, or proto-chloride of tin, causes the reduction of the colouring-matter, but, on exposure to air, oxygen is absorbed, and the solution again exhibits the spectrum characteristic of the more oxidised state. The different substances obtained from blood colouring-matter produce different bands. Thus, hæmatin gives rise to a band in the red spectrum D; hæmato-globulin produces two bands, the second twice the breadth of the first in the yellow portion of the spectrum between the lines D and E, No. 1. The absorption-bands differ according to the strength of the solution employed, and the medium in which the blood-salt is dissolved; but an exceedingly minute proportion dissolved in water is sufficient to bring out very distinct bands. B represents the red end of the spectrum and G the green as it approaches the violet end.
Mapping the Spectra.—In the sectional view given of the micro-spectroscope ([Fig. 196]), the internal construction of the instrument is shown, and the arrangement made for throwing a bright point on to the surface of the upper prism is clearly seen. The mapping out is accomplished by means of a photographic scale fixed as a standard spectrum ([Fig. 198]), in the position of A A, illuminated by the small mirror at R, and focussed by a small lens at C, so that on looking into the instrument one can see the spectrum accurately divided into one hundred equal parts, and scale readings can be made at once; the only precaution needed is to be sure the D (or the sodium line, if D cannot be got) always stands at the same number on the scale. To map absorption spectra on this scale we have to lay down a line, as many millimetres long as there are divisions in the scale, and mark the position of the bands on this line. Mr. Browning supplies scales printed off ready for use. But the mapping out of spectra, as Mr. Sorby pointed out, requires some consideration; since the number of divisions depends on the thickness of the interference-plate, it becomes necessary to decide what number should be adopted. Ten it was thought would be most suitable; but, on trial, it appeared to be too few for practical work. Twenty is too many, since it then becomes extremely difficult to count them. Twelve is as many as can well be counted; it is a number easily remembered, is sufficiently accurate, and has other practical advantages. With twelve divisions the sodium-line 0 comes very accurately at 3½; thus, by adjusting the plate so that a bright sodium-light is brought into the centre of the band, when the Nicol’s prisms are also crossed accurately at 3½, parallelism is secured, together with a wider field of observation. The general character of the scale will be best understood from the following figure, in which the bands are numbered, and given below the principal Fräunhofer lines. The centre of the bands is black, and they are shaded off gradually at each side, so that the shaded part is about equal to the intermediate bright spaces. Taking, then, the centres of the black bands as 1, 2, 3, &c., the centres of the spaces are 1½, 2½, 3½, &c., the lower edges of each ¾, 1¾, &c., and the upper 1¼, 2¼, &c., we can easily divide these quarters into eighths by the eye: and this is as near as is required in the subject before us, and corresponds as nearly as possible to 1⁄100th part of the whole spectrum, visible under ordinary circumstances by gaslight and daylight. Absorption-bands at the red end are best seen by lamp-light, and those at the blue end by daylight.
(Red end.)
(Bue end.)
Fig. 200.—
On this scale the position of some of the principal lines of the solar spectrum is about as follows:—
| A | ¾ | B | 1½ | C | 23⁄8 | D | 3½ |
| E | 511⁄16 | b | 63⁄16 | F | 7½ | G | 105⁄8 |
At first plates of selenite, which are easily prepared, were used, because they can be split to nearly the requisite thickness with parallel faces; but their depolarising power varied much with temperature. Even the ordinary atmospheric changes alter the position of the bands. However, quartz cut parallel to the principal axis of the crystal is but slightly affected, and is not open to the same objection; but this is prepared with some difficulty. The sides should be perfectly parallel, the thickness about ·043-inch, and gradually polished down with rouge until the sodium-line is seen in its proper place. This must be done with care, since a difference of 1⁄10000-inch in thickness would make it almost worthless.
The two Nicol’s prisms and the intervening plate are mounted in a tube, and attached to a piece of brass in such a manner that the centre of the aperture exactly corresponds to the centre of any of the cells used in the experiments, and must be made to correspond with equal care, so that any of them, or this apparatus in particular, may be placed on the stage and in proper position without further adjustment, whereby both time and trouble are saved.