The weighed substance is placed in a small flask. With solid bodies this is a very simple operation, but with liquids more difficult. Liquids which are not decomposed, on heating, should be evaporated in a thin glass dish which can be ground up and placed in the digestion flask with the desiccated sample. The strongest sulfuric acid is added in sufficient quantity to secure complete decomposition, not less than ten cubic centimeters in any case. The acid must be free of ammonia and be kept in such a way as not to absorb ammonia from the atmosphere of the laboratory. To guard against danger of error from such an impurity frequent control determinations should be made. In control experiments one or two grams of pure sugar should be used as the organic matter. If the acid employed contain traces of ammonia the necessary corrections should be made in each analysis. The flask having been charged is placed on a wire gauze over a small flame. The organic matter becomes black and tar-like and soon there is a rapid decomposition attended with the evolution of gaseous products among which sulfur dioxid is found. To avoid danger from spurting, the digestion flask should be placed in an oblique position. The flask should have, at least, a capacity of 100 cubic centimeters and a long neck and be made of a kind of glass capable of withstanding the action of the boiling acid. Particles of the carbonized organic matter left on the sides of the flask by the foaming of the mass at first are gradually dissolved by the vapors of the boiling acid as the digestion proceeds. The action of the sulfuric acid is not entirely finished when gases cease to be given off but the digestion should be continued until the liquid in the flask is clear and colorless or nearly so. Usually about two hours are required to secure this result. When aided by the means mentioned below the time of digestion can be very materially shortened. By adding some fuming sulfuric acid, or glacial phosphoric acid, the dilution caused by the formation of water in the combustion of the organic matter, can be avoided. For albuminoid bodies it is hardly necessary to continue the combustion until all carbonaceous matter is destroyed. The full complement of ammonia is usually obtained after an hour’s combustion even if the liquid be still black or brown, but with other nitrogenous bodies the case is different so that upon the whole it is safest to secure complete decoloration.
The temperature must be maintained at the boiling-point of the acid or near thereto since at a lower temperature, for instance from 100° to 150°, the formation of ammonia is incomplete. Since all organic substances of whatever kind are dissolved by the boiling acid the previous pulverization of the material need be carried only far enough to secure a fair sample. Many substances give up practically all their nitrogen as ammonium sulfate when heated with sulfuric acid as, for instance, urea, asparagin, and the glutens. In most of the other organic bodies fully ninety per cent of the nitrogen are likewise secured as the ammonium salt. In the aromatic compounds, or even in the form of amid in anilin salts, the nitrogen is more resistant to the action of sulfuric acid. In the alkaloids where the nitrogen is probably a real component of the benzol skeleton the formation of ammonia is very incomplete. But even in the cases where the conversion of the nitrogen into ammonia is practically perfect it is advisable to finish the process by completing the oxidation with potassium permanganate. The permanganate should be used in a dry powdered form and added little by little to the hot contents of the digestion flask, the latter being held in an upright position and removed meanwhile from the lamp. When carefully performed there is no danger of loss of ammonia although the oxidation is, at times, so vigorous as to be attended with evolution of light. The permanganate must always be added in excess and until a permanent green color is produced. The flask is then gently heated for from five to ten minutes over a small flame, but this is not important. The heating must not be too strong or else a strong evolution of oxygen will take place with a consequent reduction of the manganese compound. When this happens the liquid again becomes clear and there is a loss of ammonia.
After cooling, the contents of the flask are diluted with water, the green color giving place to a brown with a rise of temperature. After cooling a second time the whole is brought into a distillation flask of about three-quarters of a liter capacity and attached to a condenser which ends in a vessel containing titrated sulfuric acid. About forty cubic centimeters of sodium hydroxid solution of one and three-tenths specific gravity are added and the stopper at once inserted to prevent any loss of ammonia. To prevent bumping during the distillation some zinc dust is added securing an evolution of hydrogen during the progress of the distillation. In this case the bumping is prevented until near the end of the operation when it begins anew, probably by reason of the separation of solid sodium sulfate. After the end of the distillation, the excess of acid remaining in the receiver is determined by a set alkali solution and thus the quantity of ammonia obtained easily calculated. Kjeldahl, however, preferred to titrate the solution after adding potassium iodate and iodid, a mixture which in the presence of a strong acid sets free a quantity of iodin equivalent to the free acid present. The iodin thus set free is titrated by a set solution of sodium thiosulfate using starch as an indicator. The merits of this method are sharpness of the end reaction and the possibility of using only a small quantity of the nitrogenous body for the combustion. The sulfuric acid used in the receiver is made of the same strength as the thiosulfate solution; viz., about one-twentieth normal. Thirty cubic centimeters of this were found to be the proper amount for use with substances taken in such quantities as to produce ammonia enough to neutralize about half of it. The titration is carried on as follows: A few crystals of potassium iodid are dissolved in the acid mixture obtained after the distillation is completed, then a few drops of the starch-paste and finally a few drops of a four per cent solution of potassium iodate. The iodin set free is then oxidized by the addition of the one-twentieth normal sodium thiosulfate solution until the blue color disappears.
| Example: Sulfuric acid used, | 30 | cc. | ||
| Equivalent to sodium thiosulfate, | 30 | cc. | ||
| Blank combustion required, | 29.8 | cc. | thiosulfate | solution. |
| Combustion of 0.645 gram of barley required, | 14.5 | cc. | “ | “ |
| Thiosulfate corresponding to barley, | 15.3 | cc. |
In the computation it is more simple to multiply the corresponding number of cubic centimeters of thiosulfate by seven, half the atomic weight of nitrogen, and divide the product by the weight of the substance taken, which will give the per cent of nitrogen therein.
| Then | 15.3 × 7 | × 100 = 1.66 = per cent of nitrogen in sample taken. |
| 0.645 |
A more detailed description of the method of making the titration follows: After the distillation is finished the condensing-tube is rinsed with a little water, after which the sulfuric acid unneutralized in the receiver is determined. It is advisable first to test the reaction of the distillate with litmus paper before going any further; for if at any time all the acid should be found neutralized it will be necessary to add a sufficient quantity of one-twentieth normal sulfuric acid before adding the potassium iodid, etc., otherwise the determination will be irreparably lost. Add to the contents of the flask ten cubic centimeters of the potassium iodid and two cubic centimeters of the potassium iodate solutions, described further on, and the sodium thiosulfate is then run in from a burette till the fluid, which is constantly kept agitated by shaking the flask, shows only a bare trace of yellow coloration from the iodin still present. Starch solution is then added, and the blue color obtained is at once removed by additional thiosulfate solution. When some experience has been gained, the eye is able to discern, with great certainty, even the slight coloration caused by only a small trace of free iodin.
In regard to the sensitiveness of the end reaction and the accuracy of the result, this method of titration leaves nothing to be wished for. The strength of the thiosulfate solution is determined in exactly the same manner, and with starch as an indicator. For this purpose, measure ten cubic centimeters of one-twentieth normal sulfuric acid into an erlenmeyer, add 120 cubic centimeters of ammonia-free water, ten cubic centimeters of potassium iodid solution, and two cubic centimeters of iodate solution; add thiosulfate solution till the fluid shows only the above-mentioned light yellow tint, then add starch, and finally thiosulfate. In this way the strength of the thiosulfate is ascertained, which of course must be occasionally re-determined, under exactly the same conditions as with the nitrogen determinations themselves, and every possible error is thereby excluded. That the solution once decolorized within a short time again assumes a deep blue color, is a matter of no concern, inasmuch as both solutions are added in such a manner that the end reaction lies exactly at the point when the starch iodid reaction distinctly disappears.
180. Theory of the Reactions.—As has been seen above the final product of heating a nitrogenous organic compound with sulfuric acid and an oxidizing body is ammonium sulfate. The various steps by which this is obtained have been traced by Dafert:[150]
(1) The sulfuric acid abstracts from the organic matter the elements of water: