In the above determinations the salt analysed was taken from specimens made at three different times and purified in slightly different ways, Nos 1, 2, & 3 being washed with absolute alcohol. Nos V and V were made by precipitating the Ba with H₂SO₄ from a solution of the salt.

The water was determined by heating at 110° till constant weight was reached. Part only of the weight lost was regained on standing in the air.

      ·3943 gr salt lost at 110° ·0286 gr = 7.25%
   Water calculated for C₁₉H₁₃O₇SBa+2H₂O = 7.35%

Although these analyses show a per cent. of Ba somewhat above that required by a compound having the formula C₁₉H₁₃O₇SBa still this appears to be the most probable formula which can be assigned to the substance. If this is the true composition of the salt, then in sulphonfluoresceïn the anhydride condition must be broken up by boiling with BaCO₃ forming the salt thus.

By treating the salt with H₂SO₄ the original substance is reformed.

Calcium Salt.

Attempts were made to prepare the calcium salt but without success. The S-fluoresceïn was boiled several hours with very finely powdered calcite, and some salt was formed as shown by the CO₂ evolved but on evaporating the solution and recrystallizing the substance deposited it was found to be the unchanged S-fluoresceïn. Some Ca. salt was in the mother liquors but its extreme solubility prevented a separation being made.

Acetyl derivative of S.fluoresceïn.

S.fluoresceïn was boiled with an excess of acetic anhydride for about three hours. The solution became quite dark and when evaporated on the water bath left a black tarry residue. This was treated with water which dissolved a part leaving a dark flocculent precipitate. The solution was boiled with animal charcoal and evaporated nearly to dryness. On cooling there separated a light yellow flocculent precipitate very soluble in hot water and but slightly less so in cold. This was dissolved in a small quantity of alcohol from which it separated on evaporation in small radial crystals having a light lavender color & satiny luster. They also have a peculiar odor resembling slippery elm which is not removed by recrystallization. They show a tendency to decompose, becoming yellow on exposure to the air. The substance does not melt or change in appearance under 245°. With alkalis it gives a slight greenish fluorescence. From the method of its formation this was taken to be an acetyl derivative of S.fluoresceïn but whether the mono-or di-acetyl could not be determined without analysis for which the substance did not suffice.

Bromine substitution products of S-fluoresceïn.

The latter was dissolved in glacial acetic acid in which it is soluble with some difficulty and to the solution was added a 20% solution of bromine in acetic acid, in sufficient quantity to make eight atoms of bromine to one molecule of S.fluoresceïn. This solution was evaporated on the water bath and while still having a considerable volume, small, red, sharply defined crystals began to separate. The solution was evaporated to a small volume and allowed to cool but nothing further separated. These crystals are difficultly soluble in water, alcohol and ether. The alkaline solution shows a green fluorescence and slight red color by transmitted light. These crystals were dissolved in a large quantity of alcohol which on evaporation gradually deposited very small yellow crystals, which were dried in the air and taken for analysis. The Br. was determined by Carius method.

These results, though not conclusive, indicate that under the given conditions it is the di-bromsulphonfluoresceïn which is formed. Whether this is due to the presence in the compound of the SO₂ group or simply to the greater insolubility of the di-than of the tetra-brom product cannot be definitely stated. When the original acetic acid mother liquor was evaporated to dryness, a red non-crystalline substance remained which more closely resembled rosin than the crystals. The concentrated alkaline solution had a deep red color without fluorescence and acted as a red dye stuff. The dilute alkaline solution showed the characteristic delicate pink of rosin.

Action of H₂SO₄ on S.fluoresceïn.

A test tube in which S.fluoresceïn was being made just at the end of the reaction broke and allowed the contents to run out into the sulphuric acid bath, which had a temperature of 175°. On standing several days the solution deposited a heavy precipitate which was separated by filtering through glass wool. When dry it formed a light yellow powder extremely soluble in water, alcohol and ether.

The alkaline solution had an intense green fluorescence with delicate shades of pink by transmitted light. On account of its great solubility it was impossible to purify it by crystallization, hence the Ba salt was made. The substance decomposed BaCO₃ with great ease forming an easily soluble salt. When it was attempted to evaporate the solution of this salt to crystallization the latter came out in a hard insoluble granular form and on continuous boiling of the solution turned brown. To avoid this undesirable form it was converted into the Ca. salt by treatment with H₂SO₄ and then CaCO₃. This also formed a hard granular insoluble mass on boiling but did not change in color. As there was no guarantee as to its purity and only a small quantity was obtained it was not analyzed.

Action of HCl on S.fluoresceïn.

Reduction of S.fluoresceïn.

When treated with zinc dust in a strong alkaline solution sulphonfluoresceïn is reduced to a colorless substance probably analogous to fluoresceïn which is formed in the same manner. On account of its great solubility it could not be obtained in the free state. It is quickly oxidized to s.fluoresceïn by oxidizing agents as KMnO₄ and HNO₃ and passes back spontaneously on standing in the air. The latter action is however much slower than in case of fluoresceïn.

Conclusion.

The principal results relating to s.fluoresceïn which have been reached in this work may be briefly summarized as follows. Orthosulphobenzoic acid acts on resorcin at a temperature of about 180° giving off water and forming a substance analogous to fluoresceïn but having the CO group replaced by SO₂. This substance sulphonfluoresceïn crystallizes from water in light yellow monoclinic crystals having the composition C₁₉H₁₂O₆S + 2H₂O. It is very soluble in alcohol and water and with difficulty in ether. It does not melt under 250° but above 300° melts with decomposition. It shows in alkaline solution a clear green fluorescence. It acts as an acid, decomposing carbonates and forming salts, the Ba salt having the composition C₁₉H₁₃O₇SBa. It forms an acetyl compound when boiled with acetic anhydride. It forms substitution products with Br, probably the dibrom-product most easily. It forms a compound with H₂SO₄, probably a substitution product, whose composition was not determined. It is reduced by zinc dust and KOH to a colorless substance analogous to fluoresceïn.

Finally in terms of the prevalent theory the substance itself may be represented thus—