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Tuatara: Volume 6, Issue 3, December 1957

Precautions in the use of Picric Acid in the Laboratory

page 96

Precautions in the use of Picric Acid in the Laboratory.

Each year usually brings to the writer of this article at least one student who, wishing to prepare a solution containing picric acid, is surprised and perturbed to find the stock bottle of this substance containing‘… a lot of watery liquid above the crystals’. Students are even aggrieved, thinking that some previous user has been careless, and are annoyed with the presumed prospect of having to dry out the wet mass of crystals.

Since there appears to be a steady stream of similar misconceptions, the following notes might profitably be drawn to the attention of all biologists, especially those engaged in histological work.

Picric acid (2 : 4 : 6 trinitro-phenol) is a common ingredient of fixatives and even staining solutions used in histological and cytological techniques in biological laboratories. Its use depends on several important properties including a strong precipitating action on cytoplasmic proteins without concomitant hardening (though it does promote shrinking); and penetration of chitin. It is worth while noting, however, that there are stringent precautions to be observed in handling this material which is better known in the field of explosives. ‘Picric acid if stored in bulk should, for safety, first be damped. Smaller quantities may be safely kept whilst dry, but should be stored in bottles having cork or rubber stoppers; glass stoppers should never be used for potentially explosive substances, because on replacing the stopper some of the material may be ground between the neck of the flask and the stopper, and so caused to explode.’ (Mann and Saunders.) The necessity for ensuring that non-rigid stoppers are used and that the material be maintained damp cannot be over-emphasised. Fortunately precautions can be readily observed in biological laboratories, since wide-mouthed containers with large corks can be employed for storage, and the concentrations of picric acid required are low — they can easily be derived starting from the saturated aqueous solution from the stock bottle. Thus, it is recommended that a stock bottle (say, 1 litre capacity), suitably stoppered with cork or rubber, be maintained with a layer of picric acid some 3 cm. thick and filled with distilled water. The picric acid dissolves slowly to form a saturated solution of which the concentration varies with temperature as follows (data from Seidell): page 97
Temperature ° C.grams picric acid/100 grams solution
Intermediate values, if desired, can be determined by plotting these points on a graph, when a curved relationship will be given. However, the variations within the range of room temperatures are sufficiently small to be ignored in preparing biological fixatives, etc., and the term ‘saturated solution in distilled water’ will suffice for all such purposes. Solution of the acid is slow, and if continual use is being made of the stock saturated solution, two or more similar bottles should be kept and used in rotation. It is essential that after removing the solution, the deficiency in volume be made up with distilled water, and that the bottles be clearly labelled to this effect.

Recipes for preparing fixatives incorporating picric acid are very conveniently given in terms of a dozen or so ‘basal fixatives’ of which volumetric quantities are combined variously to give a great range of standard fixatives. (For details see Gray or Bolles Lee.) Consequently, a saturated solution of picric acid being one of the basal solutions, it is never necessary to contemplate weighing out dry picric acid. Where picric acid in alcoholic solution is called for, this may conveniently be prepared by filtering the water-wet crysals, preferably through a Büchner funnel under suction, washing with alcohol and taking up the crystals from the filter paper in alcohol of the appropriate strength. One of the basal fixatives is 95% alcohol saturated with picric acid; this may therefore be obtained by allowing the acid to stand covered with 95% alcohol, and the supernatant solution withdrawn as required, replacement being made each time with 95% alcohol. Picric acid is several times more soluble in ethyl alcohol than in water, maximal solubility being achieved at about 80% alcohol.

Particular attention is to be drawn to the picrates. ‘Metallic salts of picric acid are much less stable than the free acid, and should always be stored damp.’ (Mann and Saunders.) Fortunately, very few fixatives in common use include both picric acid and cations of heavy metals (which produce the most dangerous picrates), but those which do should be handled with respect in the realisation that the metal picrates are even more explosive than is the acid itself. One of the standard tests for explosiveness is that of the ‘fall-hammer’ in which a 2-kilogram hammer is allowed to drop on to the substance under test. The following figures, taken from International Critical Tables, indicate the potential dangers involved: height of fall necessary to explode picric acid — 35-95 cm.; ammonium and sodium picrates — 80 cm.; zinc picrate — 60 cm.; copper, iron, lead and page 98 silver picrates — 5 cm. These are to be compared with compressed guncotton (15% water) — 85 cm.; nitroglycerine (dry or moist) — 4 cm.

Emphasis is laid on the fact that heavy metals are common impurities of other chemicals, especially where commercial grade chemicals are being used; traces of such metals, inadvertently and unconsciously introduced into picric acid solutions are hazardous. The cautions against allowing the solution to evaporate to dryness and against the use of glass stoppers are therefore strongly reiterated. They should be applied to all stock solutions and working solutions of picric acid, picrates, fixatives and staining solutions containing any of these materials, whether in aqueous or alcoholic solution.

Literature Cited

Bolles Lee, 1950.—The Microtomist's Vade-Mecum. 11th Ed., by J. Brontë Gatenby and H. W. Beams. London: Churchill.

Gray, P., 1933.— J. R. Micr. Soc., 53, 13-19; or 1954, The Microtomist's Formulary and Guide. London: Constable.

Mann, F.G., and Saunders, B. C., 1936.—et seq. Practical Organic Chemistry. London: Longmans.

Seidell, A., 1941.—Solubilities of Organic Compounds, 3rd Ed., Vol. II. New York: van Nostrand.