The small, direct-vision (hand) spectroscope suffices. A wavelength scale is a convenient attachment. Daylight or strong artificial light (such as the “daylite” lamp) is used. Have solution in a small test tube or, preferably, a flat cell with a thickness of about 1 cm. Before use, focus Frauenhofer’s lines sharply.
Reducing agents are employed, such as ammonium sulphide, or Stokes’ solution made up as follows: Dissolve 3 gm. FeSO4 in cold H2O; add cold, aqueous solution of 2 gm. tartaric acid; make up to 100 cc.; immediately before use, add strong NH4OH until precipitate first formed is dissolved. Both solutions must be freshly prepared, and the sulphide must be warmed to about 50°C.
Material that is uncontaminated, relatively fresh and in relatively concentrated aqueous solution may give any or all of the upper three spectra, a few drops of reducer changing the first to the second.
If the material is older, dissolve the suspected stain in 1-2 cc. of 10% NaOH, heat almost to boiling, cool, and add a few drops of reducer. Examination shows Spectrum 5.
It is better, however, especially with much contamination, to prepare an ethereal, acid extract. After having ground the material thoroughly with water, if it is not already in liquid form, shake it with an equal volume of neutral ether. Reject ether extract, and, to 10 cc. of residue, add 3 to 5 cc. of glacial acetic acid. Shake thoroughly with an equal volume of ether. If the ether does not separate readily, mix gently with a few drops of alcohol. Remove ethereal extract, and evaporate it to a small bulk for use in tests. Examination will show spectrum of acid haematin, which, however, in ethereal solution, resembles Spectrum 3 more than 4.
Donogány’s Method increases the delicacy of the spectroscopic test, and is also a color test. Dissolve the pigment with 20% NaOH, add fresh pyridin and, if necessary, fresh ammonium sulphide. Filter. The filtrate will be more or less orange-red according to blood content, and will show Spectrum 5.
Color Tests.—The reliability of these may be enhanced by the use of methods which involve the removal or destruction of interfering substances. In such a method, the original aqueous solution is boiled for 15 to 20 seconds, and the acid ethereal extract is prepared as previously described. This extract is dropped on filter-paper, the reagents being applied to the moistened spot. The delicacy of these several tests is variable, being greater with blood in aqueous solution than in biological fluids, but it may be given as approximately 1-25,000 for the guaiac and aloin, and 1-250,000 for the benzidine test.
(a) Treat moist spot with a few drops of freshly prepared 2% alcoholic solution of guaiac resin, and then a few drops of hydrogen peroxide. A blue color is “positive.”
(b) Treat moist spot with a few drops of 3% aloin in 70% alcohol, and then with ozonized turpentine (turpentine that has stood for a few days in an open vessel in sunlight). A purplish-red color within 10 minutes is “positive.”