Sulphonfluoresceïn.
Several attempts had already been made to obtain from the action of o-sulphobenzoic acid and resorcin a substance analogous to the fluoresceïn obtained by Baeyer[1] from phthalic anhydride and resorcin but while a strongly fluorescent substance was easily obtained, no definite compound could be separated from it. Thus Palmer obtained, by heating together the above named substances to 160°(?) a solid mass, part soluble in water and part insoluble as a dark brown amorphous powder. Both parts gave a strong fluorescence with alkalis. He was unable however to obtain the substance itself or any derivative in a crystalline form.
The first experiments in this series gave the same negative results. The mixture of acid and resorcin was heated in a sulphuric acid both for several hours to 150°-170°, and as it showed no sign of solidification the temperature was raised to 200° and then to 235° where it was kept several hours longer. The black viscous mass obtained in this way became vitreous on cooling, and in all respects resembled that described by Palmer. This however is not the normal course of the reaction as shown later but is probably due to a decomposition of the normal product produced by too high heating.
An experiment was made with the acid salt of ortho-sulphobenzoic acid.
| COOH | ||
| ╱ | ||
| C6H4 | ||
| ╲ | ||
| SO2OK |
resorcin and H2SO4 heating the mixture to 150°-170°. A solid black mass was obtained strongly fluorescent in alkaline solution and in all other respects like the substance obtained above.
Preparation of Sulphonfluoresceïn.
As the result of a number of experiments the following method of preparing and purifying the sulphonfluoresceïn was found to give the best results.
The free acid is mixed with resorcin in the proportion of 1 part acid to 1.2 parts resorcin, or a slight excess over two molecules of the latter to one of the former. The mixture is placed in a deep vessel, a test tube or beaker, which is placed in a sulphuric acid bath, a thermometer being suspended in the mixture. The bath is heated and at about 100° the resorcin melts and the acid slowly dissolves in the liquid. No action appears to take place till the temperature reaches 175° where water begins to be given off and the liquid slowly assumes a darker color. White crystals of resorcin collect on the sides of the vessel. After the heating has been continued for about seven hours at 178°-185° the liquid has a clear deep red color but shows no signs of becoming viscous. At length lustrous yellow crystalline plates appear in the liquid and soon the whole mass becomes a thick nearly solid yellow paste. Continuous heating at the same temperature causes no further apparent change. This mass, which on cooling is made up of yellow crystals imbedded in a red vitreous matrix, is then dissolved in hot water forming a clear red solution or at most one containing but a small quantity of a brown flocculent precipitate. This solution is filtered if necessary and evaporated to a small volume from which the substance separates on cooling in reddish yellow radial crystals. These are filtered and washed with ether till the washings are perfectly colorless. The substance on repeated crystallization from water has a pale straw yellow color and when deposited slowly forms transparent crystal blocks from 2·6 mm long, arranged in radial groups.
A considerable amount of resorcin is lost by sublimation during the reaction especially if the operation is carried on in a beaker so that some excess should be added. But even when the resorcin is present in excess at the end of the reaction some free acid is always left which may be obtained from the mother liquid in the characteristic colorless orthorhombic crystals.
The water of crystallization and sulphur were determined in the new compound. In estimating the water, the substance was heated to 106°-123° for about ten hours till it attained a constant weight. On standing in the air it quickly regains its original weight. When heated to 130°-140° for some time it turns slightly reddish and loses over 10% of its weight which is not regained by standing in the air.
Estimation No. I. was made by fusion with KOH and KNO3. Nos. II and III were made by Mr. Mindileff by Morse’s method, oxidizing with KMnO4 in KOH solution.
·3882 gr. heated to 106°-123° lost ·0302 gr. = 8·5%
Calculation for 2H2O - C19H12O6S + 2H2O = 8·9% water.
| I. | ·2007 | gr sub. gave | ·1286 | gr BaSO4 | = 8·77% S. |
| II. | gr ” ” | ” | gr ” | = 7·91” ” | |
| III. | gr ” ” | ” | gr ” | = 7·89” ” |
Calculation for C19H12O6S + 2H2O = 7·92% S.
These analyses show with but little doubt that the substance has the composition indicated above i.e. C19H12O6S + 2H2O. The reaction therefore which takes place between ortho-sulpho benzoic acid and resorcin from its analogy to that taking place between phthalic anhydride and resorcin may be represented thus, as shown by Baeyer in his second paper (Ann. 202. S. 43)
Representing the formation of the anhydride as the first action.
| COOH | CO | |||||
| ╱ | ╱ | ╲ | ||||
| C6H4 | = C6H4 | O + H2O | ||||
| ╲ | ╲ | ╱ | ||||
| SO2OH (o) | SO2 |
and the action of resorcin on this anhydride thus.
| O | ||||||||||
| ╱ | ╲ | |||||||||
| (HO)H4C6 | C6H4(OH) | |||||||||
| ╲ | ╱ | |||||||||
| CO | OH | C | ||||||||
| ╱ | ╲ | ╱ | ╱ | ╲ | ||||||
| C6H4 | O + C6H4 | = | C6H4 | O + 2H2O. | ||||||
| ╲ | ╱ | ╲ | ╲ | ╱ | ||||||
| SO2 | OH (m) | SO2 |
The substance thus formed would naturally receive the name Sulphonfluoresceïn from its analogy with Fluoresceïn.
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| Sulphonfluoresceïn. | Fluoresceïn. | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Properties of S.fluoresceïn.
This compound shows a marked similarity to the fluoresceïn described by Baeyer as would naturally be expected from its great similarity of composition and constitution, but it also shows decided differences which may be attributed to the replacement of CO by SO2.
Dissolved in water it shows a weak green fluorescence which in alkaline solution becomes much deeper but not by any means so strong as that of fluoresceïn. The dilute alkaline solution by transmitted light is almost perfectly colorless and by reflected light a clear green. Unlike fluoresceïn it is extremely soluble in water, about one part in two or three of hot and five or six of cold water. It is also soluble in absolute alcohol forming a yellow solution with weak fluorescence. It is soluble with difficulty in ether but when in solution is deposited only on evaporating to a small volume.
It does not melt at 250° but if held at a lower temperature for a long time becomes red undergoing some decomposition. If quickly heated somewhat above 300° it melts to a deep red liquid and then solidifies. If the mass is treated with water it partly dissolves leaving a dark brown flocculent precipitate which dissolves on the addition of an alkali, the solution having an intense fluorescence, nearly if not quite equaling that of fluoresceïn. This change produced by heating was not further studied.
The crystals are very thin blades, apparently monoclinic, showing the clinopinacoid αΡὰ and a very narrow prism αΡ and clinodome Ρὰ. The angle β = 75° and the extinction angle against the ϲ axis = 20°. The axial ratio could not be accurately determined.
Salts of sulphonfluoresceïn.
The influence of the SO2 group is shown by the fact that the substance acts as an acid decomposing carbonates and forming salts which is not the case with fluoresceïn.
Barium salt.
The substance was boiled with an excess of carefully purified BaCO3 for two hours. The filtrate from the BaCO3 evaporated to a small volume deposited yellow crystals resembling the original substance but in shorter and thicker prisms. These were twice recrystallized and had then a light straw yellow color.
A determination of the Ba gave the following results. The salt was dried in the air.
Transcriber's Note:
The following table was crossed out on the original. A note on the previous page beside the table was:
“All these calculations are wrong. J.R.”
| I | ·1078 | gr salt gave | ·0304 | gr BaSO4 | = 15·73% Ba. |
| II | ·1641 | ” ” ” | ·0457 | ” ” ” | = 15·53” ” |
| III | ·2425 | ” ” ” | ·0680 | ” ” ” | = 15·65” ” |
| IV | ·2860 | ” ” ” | ·0798 | ” ” ” | = 15·54” ” |
| V | ·1843 | ” ” ” | ·0498 | ” ” ” | = 15·08” ” |
| VI | ·2620 | ” ” ” | ·0708 | ” ” ” | = 15·08” ” |
| VII | ·3230 | ” ” ” | ·0906 | ” ” ” | = 15·65” ” |
| VIII | ·2875 | ” ” ” | ·0807 | ” ” ” | = 15·66” ” |
Calculated for C19H13O7SBa | = 15·10% Ba. | ||||
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.
| ⎧ | OH | ⎧ | OH | ||||||
| │ | ╱ | │ | ╱ | ||||||
| │ | C6H3 | │ | C6H3 | ||||||
| │ | ╲ | │ | ╲ | ||||||
| │ | O | │ | O | ||||||
| │ | ╱ | │ | ╱ | ||||||
| C | ⎨ | C6H3 | + 2H2O = | C | ⎨ | C6H3 | |||
| │ | ╲ | │ | ╲ | ||||||
| │ | OH | │ | OH | ||||||
| │ | C6H4SO2 | │ | C6H4SO2OH | ||||||
| │ | ╱ | │ | |||||||
| ⎩ | O | ⎩ | OH | ||||||
| ⎧ | OH | ⎧ | OH | ||||||||||
| │ | ╱ | │ | ╱ | ||||||||||
| │ | C6H3 | │ | C6H3 | ||||||||||
| │ | ╲ | │ | ╲ | ||||||||||
| │ | O | │ | O | ||||||||||
| │ | ╱ | │ | ╱ | ||||||||||
| 2 | C | ⎨ | C6H3 | + BaCO3 = | C | ⎨ | C6H3 | + Ba | |||||
| │ | ╲ | │ | ╲ | ||||||||||
| │ | OH | │ | OH | ||||||||||
| │ | C6H4SO2OH | │ | C6H4SO2OH | ||||||||||
| │ | │ | ||||||||||||
| ⎩ | OH | ⎩ | OH | 2 | |||||||||
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.
It was especially interesting to see what influence the SO2 group would exert upon the introduction of Bromine into the compound. In the case of fluoresceïn four Bromine atoms enter easily and special precautions are necessary to obtain a product containing a smaller number. The case however is different with 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.
| I. | ·2345 | gr sub. gave | ·1718 | gr AgBr | = 31·17% Br. |
| II. | ·2786 | ” ” ” | ·1815 | ” ” ” | = 27·72% Br. |
Calculated for C19H10Br2O6S | = 30·42% Br. | ||||
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.
Hydrochloric acid does not dissolve S.fluoresceïn but converts it into a light yellow granular powder. When recrystallized from water in which it is quite easily soluble it melts partially at 130° apparently with some decomposition. This compound was not further studied.
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—
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| Annalin. | No. | 183. | S. 1. No. 202. S. 36 & S. 153. |
| Berichte. | No. | IV. | S. 457. 555. 658. 662. |
| ” | ” | VIII. | S. 66. 146. |
Transcriber's Notes:
Illustration has been moved so it does not break up the paragraph.
As this was a hand-written thesis, the spelling, punctuation and hyphenation is very inconsistent.
The original spelling, hyphenation and punctuation have been left unchanged.