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Title: On Sulphonfluoresceïn and some of its Derivatives
Author: Hayes, C. Willard
Language: English
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            Presented for the Degree
               Doctor of Philosophy
                     at the
            Johns Hopkins University

                C. Willard Hayes,



     Introduction                                                     3.
  I  Ortho-sulpho-benzoic acid.
     Preparation from toluene and H_{2}SO_{4}                         7.
           ”       ”   para-nitro-toluene and H_{2}SO_{4}             8.
     Oxidation  of   toluene-o-sodium sulphonate                     12.
     Preparation of Sulphinide from ”    ”                           14.
          ”            ”        ”           by chlorsulphonic acid.  17.
     Conversion of sulphinide into o-sulphobenzoic acid.             25.
     Analysis of and crystallography of  ”    ”      ”               28.
  II Sulphonfluoresceïn--previous attempts                           29.
     Preparation and purification of s-fluoresceïn                   30.
     Analyses of                            ”                        34.
     Properties of                          ”                        35.
     Salts. Ba. and Ca. of                  ”                        37.
     Acetyl derivative of                   ”                        41.
     Br substitution products of            ”                        42.
     Action of H_{2}SO_{4} on               ”                        44.
     Action of HCl on                       ”                        45.
     Reduction of                           ”                        46.
     Conclusion.                                                     47.

On Sulphonfluoresceïn and some of its Derivatives.


The close analogy in composition and structure between phthalic acid
and ortho-sulpho-benzoic acid suggests the possibility of obtaining
from the latter, by its action on phenols, substances analogous to
the phthaleïns. If such compounds could be made they would afford
a favorable opportunity of studying the effects produced in the
properties of a compound by the replacement of CO by SO_{2}. It was
with a view to such study that the following discussed work was
undertaken at the suggestion of Prof. Remsen and carried on under his
constant guidance.

Some experiments previously performed by Remsen and Palmer (A.G.)
indicated the possibility of the formation of a fluorescent substance
by the action of ortho-sulpho-benzoic acid on resorcin but they did
not succeed in obtaining any definite crystallized compound from the

The chief obstacle to be overcome in the work is the difficulty in
obtaining the o-sulpho-benzoic acid and a large proportion of the
work here described was applied in that direction.

Ortho-sulpho-benzoic acid: Methods for its preparation.

1. From toluene and H_{2}SO_{4}.

A method employed by Remsen and Fahlberg (Am. Ch. Jour. Vol. 1.
p __ ) for getting the sulphonic acid group in the ortho position to
methyl was; (a) treat toluene with fuming H_{2}SO_{4} forming thus
ortho- and para- toluene sulphonic acids, (b) make the calcium salt
of the sulphonic acids thus formed and from this the potassium
salt. (c) treat this mixture of potassium toluene sulphonates
with phosphorous penta-chloride forming the corresponding
sulphonchlorides. One of these (para) being a solid and the other
(ortho) an oily liquid a nearly complete separation could be
effected. The difficulty with this method is however that the larger
part of the product is the para and only a comparatively small
proportion of the ortho compound is formed.

2. From p-nitro-toluene and H_{2}SO_{4}.

A second method employed consists in starting with p-nitro-toluene.
This when treated with H_{2}SO_{4} forms toluene p-nitro o-sulphonic
acid. If now a method could be obtained for removing the nitro group
the desired result would be attained.

The attempt was made by Remsen and Palmer (A.G.) to accomplish this
by (a) reducing the nitro compound to the amide, (b) making the diazo
compound and (c) boiling this with absolute alcohol. According to
generally accepted views this should effect the removal of the diazo
group and its replacement by hydrogen.

Experiments however showed that the replacement was made not by
hydrogen but by the ethoxy group -OC_{2}H_{5}. This method was
therefore impracticable.

A modification of this method was suggested by an observation of
Baeyer and Liebermann that if phenyl hydrazine be boiled with a
dilute solution of copper sulphate the hydrazine group is replaced
by hydrogen and benzene thus formed. Hence it was believed that if
the hydrazine compound should be made from diazo compound mentioned
above, the corresponding hydrocarbon, i.e. toluene o-sulphonic acid
could be obtained. The results of experiments showed that this
afforded a practicable method of preparing toluene ortho-sulphonic

After experimenting with various modifications of the method the
following was found to be the best adapted to the purpose.

The potassium salt of toluene p-nitro-o-sulphonic acid is easily
obtained, as already stated, by heating p-nitro toluene on the water
bath with three times its weight of fuming H_{2}SO_{4}, neutralizing
with chalk and to the solution of calcium salt thus obtained adding
a slight excess of K_{2}CO_{3}. On filtering from. the precipitated
CaCO_{3} and evaporating slightly, the salt is obtained in long
needle shaped crystals of a pale straw yellow color. This is

    C_{6}H_{3}┤SO_{2}OH  (o)
              │NO_{2}    (p).

The reduction of the nitro group is best effected by means of tin and
HCl, in the proportion, salt 5 parts, tin 6 parts and concentrated
HCl 30 parts.

The amido acid forms a compound with tin which crystallizes from the
HCl together with stannous chloride. This compound may be broken up
and the tin removed by continued boiling with water.

A better method of removing the tin is by dissolving the compound in
Na_{2}CO_{3}. This forms a salt with the amido acid and throws down
the tin as Sn(OH)_{2}, a white flocculent precipitate. On filtering
and adding to the solution conc. HCl, the free amido acid is
deposited in characteristic colorless, rhombic crystals, having the

    C_{6}H_{3}┤SO_{2}OH    (o)
              │NH_{2}      (p)

The method at first employed for preparing the hydrazine compound
consisted in treating the amido acid, suspended in HCl, with
potassium nitrite and then with stannous chloride. The tin was then
removed from the solution by the addition of sodium carbonate and the
hydrazine compound thrown down with HCl. This method however gave
poor results the yield being only about 50% of the theoretical.

Another method was accordingly substituted for the above, namely that
of Strecker and Römer (Ber. IV. s 784.) By this the diazo compound
is made first and isolated. This is done by suspending the finely
powdered acid in absolute alcohol, cooling and passing a current of
the oxides of nitrogen through in the ordinary way. The acid changes
in appearance, becoming more crystalline and slightly darker and
settles quickly on being shaken. The reaction here may be expressed

               ┌─                                ┌─
               │ CH_{3}                          │ CH_{3}
    C_{6}H_{3} ┤ SO_{2}OH + HNO_{2} = C_{6}H_{3} ┤ SO_{3} + 2H_{2}O
               │ NH_{2}                          │   \
               └─                                │ N=N

When the reaction is completed as shown by the appearance of the
suspended powder it is filtered and while still fresh is added to a
solution of acid sodium sulphite as long as it continues to dissolve

To this solution there is added a quantity of solution of acid
sodium sulphite equivalent to that already used and the solution is
then boiled. It has at first a deep red color but in a few moments
becomes light reddish yellow. The reaction of HNaSO_{3} on the diazo
compound may be represented in two stages, the first portion forming
an addition product and the second acting as a reducing agent. Thus,

                  ┌─                               ┌─
                  │ CH_{3}                         │ CH_{3}
   1.  C_{6}H_{3} ┤ SO_{3} + HNaSO_{3} = C_{6}H_{3}┤ SO_{2}ONa
                  │  \                             │
                  │ N═N                            │ N═NSO_{3}H
                  └─                               └─
                  │ CH_{3}
   2.  C_{6}H_{3} ┤ SO_{2}ONa  + HNaSO_{3} + H_{2}O
                  │ N═NSO_{3}H
                  └─                       ┌─
                                           │ CH_{3}
                              = C_{6}H_{3} ┤ SO_{2}ONa    + HNaSO_{4}.
                                           │ NH-NHSO_{3}H

To the hot solution an excess of conc. HCl is added when the
hydrazine compound separates in a few moments in lustrous yellow
scales which completely fill the solution. On the addition of the HCl
a large amount of SO_{2} is given off from the excess of HNaSO_{3} and
the solution becomes deep red. When the hydrazine has separated the
mother liquor is again yellow.

The reaction is represented as follows:

    C_{6}H_{6}┤SO_{2}ONa    + HCl + H_{2}O
                            = C_{6}H_{3}┤SO_{2}OH  + H_{2}SO_{4} + NaCl

The yield of hydrazine when both the diazo and the NaHSO_{3} are
freshly prepared is practically quantitative.

The hydrazine thus prepared was treated with a hot 10% solution of
copper sulphate till a permanent blue color was obtained in the
solution. Nitrogen is evolved and the copper sulphate is reduced to
cuprous oxide which is precipitated as a red powder. The reaction is
as follows.

    C_{6}H_{3}┤SO_{2}OH  + 2CuSO_{4} + H_{2}O
                 = C_{6}H_{4}┤CH_{3}   + Cu_{2}O + N_{2} + 2H_{2}SO_{4}

Chalk was added to the solution to precipitate the H_{2}SO_{4} and
form a calcium salt of toluene-o-sulphonic acid. From this the
sodium salt was made by adding a slight excess of Na_{2}SO_{3} and
evaporating to dryness. The salt is very soluble being deliquescent
in the air while the corresponding potassium salt is not. From 1538
gr. of para-nitro-toluene, 655 gr. of toluene ortho-sodium sulphonate
were obtained.

Having thus obtained the toluene ortho-sulphonic acid the next
step in the problem was to find a convenient method for converting
this into ortho-sulph-benzoic acid. Two ways present themselves
for accomplishing this end. (1) direct oxidation of this salt and
(2) conversion into benzoic sulphinide from which the acid may be
obtained. Both of these methods were tried.

Oxidation of toluene-o-sodium sulphonate.


The sodium salt of toluene-o-sulphonic acid is oxidized to
ortho-sulphobenzoic acid with considerable difficulty by KMnO_{4} in
neutral solution.

Thus two experiments showed that the oxidation was not complete after
24 hours boiling with excess of permanganate. If the solution be made
alkaline however, the oxidation is completed in a few hours, yet the
greatest difficulty still remains in the separation of the free acid
from the products of oxidation in the solution. If HCl be added to
the solution the acid salt


is formed and this has nearly the same solubility as the KCl also
present. A better method therefore is to add a slight excess of
H_{2}SO_{4} and evaporate nearly to dryness. In this way are formed
sulphates and the free acid presumably. The mixture is heated with
alcohol (95%) which extracts the acid leaving the greater part of
the manganese salts. This extract is evaporated and reextracted
with alcohol. To this solution BaCO_{3} is added to precipitate the
H_{2}SO_{4} and form the Barium salt of the o-sulphobenzoic acid. The
solution is filtered from the BaSO_{4} and just enough H_{2}SO_{4}
is added to exactly precipitate the barium. The solution should
thus contain only the free acid sought, which crystallizes out on
evaporating to a small volume. While the method is theoretically
possible it presents so many difficulties that it is practically
useless. The yield is extremely small; only enough acid being
obtained in this way to show that it was possible.

Formation of Sulphinide from toluene-o-sodium sulphonate.

The second method for obtaining free o-sulphobenzoic acid from
toluene-ortho-sulphonic acid is by the conversion of the latter first
into benzoic sulphinide and then into the free acid. The sulphinide
was made essentially as described by Remsen (Am. Ch. Jour. Vol. I.
p. 428) with a few changes in the details as follows.

The salt C_{6}H_{4}┤CH_{3}     finely pulverized and in portions

of from 10 to 50 gr. was placed in a Florence flask; an equivalent
quantity of PCl_{5} added; An inverted condenser was then attached
and the flask shaken. The action takes place at once and involves
sufficient heat to distill off the oxychloride formed in the
reaction. This being returned to the flask by the condenser furnishes
a liquid medium in which the reaction takes place more readily and
completely than when it is not present. It is best to cool the flask
at first and afterwards heat gently on the water bath. The reaction
which takes place may be represented as follows.

           ┌─                               ┌─
           │CH_{3}                          │CH_{3}
 C_{6}H_{4}┤SO_{2}ONa + PCl_{5} = C_{6}H_{4}┤SO_{2}Cl + POCl_{3} + NaCl.
           └─                               └─

On the addition of water the chloride separates as a light yellow
oil. This is washed with water and concentrated aqueous ammonia
added, which forms toluene-o-sulphonamide thus.

                CH_{3}                          CH_{3}
              ╱                              ╱
    C_{6}H_{4}          + NH_{3} = C_{6}H_{4}               + HCl.
              ╲                              ╲
                SO_{2}Cl                        SO_{2}NH_{2}

The reaction is accompanied by a slight evolution of heat and the
formation, apparently, of an intermediate product having a yellowish
color, which passes over on longer standing into the white amide.
After standing several hours the excess of ammonia was driven off
by very gentle heating on the water bath. If the heat is too high
a large amount of a tarry product is formed and the yield of amide
is correspondingly small. In any case some of this tarry product is
formed. When nearly all the ammonia had been driven off the mass was
boiled with water which dissolves everything except the tar. The
hot solution was filtered through charcoal and on cooling the amide
separated in white feathery crystals which melt at 155°-156°.

The amide thus obtained was oxidized as described by Remsen (loc. cit.)
with potassium permanganate in neutral solution. The proportions are
10 gr amide. 40 gr KMnO_{4} and 1 L. water. The oxidation was usually
effected in from four to six hours.

To obtain the sulphinide from this solution after oxidation, the
latter, after filtration from the precipitated oxides of manganese,
was slightly acidified with HCl and evaporated to about one fourth
its original volume. On the addition of concentrated HCl to this
solution, the sulphinide separated out in white or slightly yellowish
feather shaped crystals melting at 212° and having the characteristic
intensely sweet taste.

Formation of Sulphinide from Toluene by means
of the chlorsulphonic acid reaction.

Before passing on to the methods used for converting the sulphinide
into free acid another method should be described by which
the former was obtained in larger quantities and much more easily
than by the one above described.

Beckurts and Otto (Ber. XI. 2061) found that by treatment of toluene
with sulphuryl hydroxy-chloride or chlorsulphonic acid, ClSO_{2}OH,
both o- and p- and as they supposed also m-toluene sulphonchlorides
were formed together with the corresponding sulphonic acid.

Claesson and Wallin (Ber. XII. p. 1848) repeated the work reaching
practically the same results and finally Noyes (Am. Ch. Jour. Vol.
VIII. p. 176) employed the reaction as a convenient method for
obtaining toluene o-sulphon-chloride.

Chlorsulphonic acid is made by passing dry HCl over solid sulphuric
acid so long as it continues to be absorbed. Since no solid sulphuric
acid was at hand, ordinary fuming Nordhausen acid was taken and
from one of two equal portions the SO_{3} was driven over into
the other. HCl was passed into the latter and the resulting
chlorsulphonic acid distilled off at about 156°.

This was placed in a flask, provided with a drop funnel and exit
tube, in portions of 150 gr. and to each portion 60 gr. of toluene
was added, very slowly, with constant shaking, the temperature
being kept near 10°. The action is violent and if any toluene is
allowed to collect on the surface of the liquid it is apt to produce
disastrous results. Large quantities of HCl are given off and the
liquid in the flask assumes a brown color. When all the toluene has
been added, it is poured into a large quantity of ice water, when
the sulphon-chlorides separate out, the ortho- as a heavy oil and
the para- as a white crystalline solid. After allowing to stand some
time in order that as much of the para-chloride might crystallize as
possible the ortho- was drawn off and subjected to a freezing
temperature for several hours. By this means more of the p-chloride
was removed and the operation was replicated as long as any crystals
continued to form, generally two or three times. In this way the
greater part of the para- may be removed, though some still remains
dissolved in the liquid chloride, which cannot be removed by repeated

The chloride thus obtained was treated with strong aqueous ammonia.
The conversion to the sulphamide does not take place so readily as
in case of the pure o-chloride obtained from the sulphonic acid and
phosphorus pentachloride.

After standing about two days the whole of the oily chloride had
solidified to a yellowish brown mass. The excess of ammonia was
driven off by gentle heating on the water bath and the mass then
boiled with water. Not enough water was added at first for complete
solution but when the first portion was saturated it was poured
off through a filter and from it the amide separated in yellowish
feathery crystals which melted at 105°-125° and consisted therefore
as shown by Fahlberg (Am. Ch. Jour. Vol. I. p. 170) of a mixture
of o- and p-sulphonamides. It was recrystallized and from it was
obtained a portion melting at 153°-5° and one at 108°-20°.

Since this mixture cannot be completely separated by
recrystallization another method was suggested. Remsen has shown that
K_{2}Cr_{2}O_{7} in acid solution does not oxidize the methyl group in

                SO_{2}NH_{2}   (o)

but does oxidize that in

                SO_{2}NH_{2}   (p).

It was thought that in a mixture of the two the former might be left
unchanged while the latter was oxidized to p-sulphamine benzoic acid.

To test this 15 gr of the mixture, melting at 105°-125°, was heated
with 40 gr K_{2}Cr_{2}O_{7}, 55 gr H_{2}SO_{4} and 2 vols. of water
for about two hours. It was then tested and shown to be still a
mixture of p-& o-amides, since it was again heated for several
hours with half the original quantity of oxidizing mixture, then
diluted, filtered and washed. The white crystalline residue was
treated with sodium carbonate to dissolve the benzoic para-sulphamide
and the residue was found to be pure toluene o-sulphonamide melting
at 153°-155°. The small quantity remaining, 3 gr., indicated that
part of the o-amide had been completely broken down by the strong
oxidizing agent, though the proportion of o-& p-amides in the
original mixtures was known only approximately. The evolution of gas
during the oxidation would point to the same conclusion.

Although this effects a complete separation it is hardly economical
since it will be shown later that a separation can be conveniently
effected after the oxidation with KMnO_{4} so that the o-amide
contained in the mixture need not be lost.

The original mass was treated with successive portions of water
till nothing remained but a black tarry substance. The amide which
separated from these extracts was perfectly white and melted at
153-5°. It was therefore regarded as practically pure o-amide. The
yield in amide melting above 153° was a little over one sixth the
weight of toluene used.

The amide obtained in this way was oxidized in the manner already
described. It was found however that there was always some benzoic
p-sulphamide in the solution of the oxidation, due to the slight
admixture of p- with the o-amide used. This is thrown down with
the sulphinide on acidifying the solution and may be removed by
re-crystallization since it is somewhat less soluble in hot and cold
water than sulphinide.

A better way to effect the separation, however, was found to be
the following. After having evaporated the solution containing the
products of oxidation, nearly neutralized with HCl, to about one
fifth its original volume, it is made very slightly acid and allowed
to cool. In this way very nearly all the benzoic p-sulphamide is
separated from the solution and none of the sulphinide. After
filtering, strong HCl is added and the sulphinide then separates
in its characteristic form. This indicates that sulphinide forms
an alkaline salt which is not decomposed by diluted HCl while the
p-sulphamide does not.

The mixture of amides meeting at 105°-120° was oxidized and the
products separated in this way gave about equal quantities of
sulphinide and benzoic p-sulphamide.

When toluene is treated with chlorsulphonic acid there are formed
besides the ortho- and para- chlorides also ortho and para sulphonic
acids. These of course are in solution in the water from which the
chlorides separated. In order to recover the ortho-acid, the solution
was neutralized with chalk forming the calcium salt: this converted
into the potassium salt which by evaporating the solution to dryness
was obtained as a white crystalline powder. When treated with PCl_{5}
in the manner already described this gave a mixture of ortho and para
sulphonchlorides consisting of about ⅓ ortho and ⅔ para.

Formation of Orthosulphobenzoic acid from Sulphinide.

Benzoic sulphinide may be converted into a sulpho-benzoic
acid (1) by boiling with Ba(OH)_{2}, (2) by heating in a closed tube
with conc. HCl or (3) by evaporating on the water bath with HCl.

1. Three gramms of sulphinide were boiled in a flask connected
with an inverted condenser for about two days with an excess of
Ba(OH)_{2}. There was formed in the flask a hard mineral-like mass
which was insoluble in water and cold diluted HCl but dissolved in
hot HCl with effervesence. This was a Barium salt, probably basic (?)
of ortho sulphobenzoic acid. There was also formed an easily soluble
barium salt of that acid. The former was dissolved in H_{2}SO_{4}
and treated with BaCO_{3}; the filtrate from the BaSO_{4} which
contained a soluble barium salt was added to that above mentioned and
the barium exactly precipitated with H_{2}SO_{4} and the filtrate
evaporated to dryness giving the free acid but not in a perfectly
pure condition.

2. 2.75 gr. of sulphinide was sealed up in a tube with pure conc. HCl
and heated two hours to 150°. On cooling nothing separated; the liquid
was evaporated to dryness giving 3.2 gr of acid and ammonium
chloride. The reaction taking place here may be represented thus:

             CO                                    COOH
          ╱    ╲                                ╱
C_{6}H_{4}        NH + 2H_{2}O + HCl = C_{6}H_{4}          + NH_{4}Cl.
         ╲      ╱                               ╲
           SO_{2}                                  SO_{2}OH

3. A more convenient method for obtaining the acid than either of
the above, is to heat the sulphinide with conc. HCl on the water
bath for two days. Then evaporate to dryness and dissolve the
residue in a small quantity of water. If the sulphinide contained
any para-sulphamide, as is usually the case, this will be left
undissolved and most of the NH_{4}Cl will crystallize on standing.
This solution by slow evaporation deposits large colorless crystals
of the free acid.

This acid is soluble in about two parts of cold water, very
difficultly soluble in absolute alcohol and almost completely
insoluble in ether. It does not melt under 250° but considerably
above that it melts, at first apparently without change and then
with slight sublimation of a very deliquescent substance, probably
the anhydride.

Two determinations of the S. made by Mr. A. F. Linn, gave the
following results.

I ·1358 gr substance gave ·1855 gr BaSO_{4} representing 15·72% S.

II   ”          ”      ”             ”            ”         ”

Calculated for the formula C_{6}H_{4}           =  15·84% S.


A crystallographic examination of the acid showed it to belong to the
orthorhombic system. Axial ratio: a: b: c = ·8507: 1: ·8121.
Planes. Ρ and αΡὰ.

                   ┌─       │ Edge X = 131°  8'
                   │ Ρ ^ Ρ ─┤   ”  Y =  82° 18'
                   │        │   ”  Z = 118° 40'
  Angles measured ─┤        └─
                   │ Ρ ^ αΡὰ = 114° 38'

The crystals are up to 8 mm in length. The pyramidal faces are
generally etched so that the image is poor.


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.

[1] Annalin.  No. 183. S. 1. No. 202. S. 36 & S. 153.
    Berichte. No.   IV. S. 457. 555. 658. 662.
       ”      ”   VIII. S. 66. 146.

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.


resorcin and H_{2}SO_{4} 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 KNO_{3}. Nos. II
and III were made by Mr. Mindileff by Morse’s method, oxidizing with
KMnO_{4} in KOH solution.

    ·3882 gr. heated to 106°-123° lost ·0302 gr.        =   8·5%

 Calculation for 2H_{2}O - C_{19}H_{12}O_{6}S+2H_{2}O   =   8·9% water.

  I. ·2007 gr sub. gave ·1286 gr BaSO_{4}   =  8·77%  S.
 II.       gr  ”    ”     ”   gr   ”        =  7·91”  ”
III.       gr  ”    ”     ”   gr   ”        =  7·89”  ”

 Calculation for C_{19}H_{12}O_{6}S+2H_{2}O =  7·92%  S.

These analyses show with but little doubt that the substance has the
composition indicated above i.e. C_{19}H_{12}O_{6}S+2H_{2}O. 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
              ╱                            ╱    ╲
    C_{6}H_{4}                =  C_{6}H_{4}         O + H_{2}O
              \                            ╲      ╱
               SO_{2}OH(o)                   SO_{2}

and the action of resorcin on this anhydride thus.

                 CO                   OH
              ╱    ╲               ╱
    C_{6}H_{4}        O + 2C_{6}H_{4}
              ╲     ╱              ╲
                SO_{2}                 OH  (m)

                                      ╱  ╲
                         (HO)H_{4}C_{6}     C_{6}H_{4}(OH)
                                      ╲  ╱
                    =                   C                   + 2H_{2}O
                                      ╱  ╲
                              C_{6}H_{4}   O
                                      ╲  ╱

The substance thus formed would naturally receive the name
Sulphonfluoresceïn from its analogy with Fluoresceïn.

     │             OH
     │           ╱
     │ C_{6}H_{3}
     │           ╲
     │             O
     │           ╱
  C ─┤ C_{6}H_{3}
     │           ╲
     │             OH
     │ C_{6}H_{4}SO_{2}
     │          ╱
     │         O

     │             OH
     │           ╱
     │ C_{6}H_{3}
     │           ╲
     │             O
     │           ╱
  C ─┤ C_{6}H_{3}
     │           ╲
     │             OH
     │ C_{6}H_{4}CO
     │          ╱
     │        O

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 SO_{2}.

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 SO_{2} 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
BaCO_{3} for two hours. The filtrate from the BaCO_{3} 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.

     I  ·1078 gr salt gave  ·0304 gr BaSO_{4} = 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 C_{19}H_{13}O_{7}SBa   = 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 H_{2}SO_{4} 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

                  ·3943 gr salt lost at 110° ·0286 gr = 7.25%

    Water calculated for C_{19}H_{13}O_{7}SBa+2H_{2}O = 7.35%

Although these analyses show a per cent. of Ba somewhat above that
required by a compound having the formula C_{19}H_{13}O_{7}SBa 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 BaCO_{3} forming the salt thus.

   ┌──                  ──┐            ┌──                    ──┐
   │                      │            │                        │
   │   ┌─            OH   │            │   ┌─           OH      │
   │   │           /      │            │   │           /        │
   │   │ C_{6}H_{3}       │            │   │ C_{6}H_{3}         │
   │   │           \      │            │   │           \        │
   │   │            O     │            │   │            O       │
   │   │           /      │            │   │           /        │
   │ C ┤ C_{6}H_{3}       │            │   │ C_{6}H_{3}         │
   │   │           \      │ + H_{2}O = │ C ┤           \        │
   │   │            OH    │            │   │            OH      │
   │   │                  │            │   │                    │
   │   │ C_{6}H_{4}SO_{2} │            │   │ C_{6}H_{4}SO_{2}OH │
   │   │           /      │            │   │                    │
   │   └─         O       │            │   └─ OH                │
   │                      │            │                        │
   └──                  ──┘            └──                    ──┘

   ┌──                   ──┐              ┌──                   ──┐
   │                       │              │                       │
   │   ┌            OH     │              │   ┌            OH     │
   │   │           /       │              │   │           /       │
   │   │ C_{6}H_{3}        │              │   │ C_{6}H_{3}        │
   │   │           \       │              │   │           \       │
   │   │            O      │              │   │            O      │
   │   │           /       │              │   │           /       │
   │   │ C_{6}H_{3}        │              │   │ C_{6}H_{3}        │
 2 │ C ┤           \       │ + BaCO_{3} = │ C ┤           \       │ Ba.
   │   │            OH     │              │   │            OH     │
   │   │                   │              │   │                   │
   │   │C_{6}H_{4}SO_{2}OH │              │   │ C_{6}H_{4}SO_{2}O │
   │   │                   │              │   │                   │
   │   └ OH                │              │   └ OH                │
   │                       │              │                       │
   └──                   ──┘              └──                   ──┘_{2}

By treating the salt with H_{2}SO_{4} 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 CO_{2} 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 SO_{2} 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 gr  ”    ”    ·1815 gr  ”      =  27·72% Br.

     Calculated for C_{19}H_{10}Br_{2}O_{6}S = 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
SO_{2} 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 H_{2}SO_{4} 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 BaCO_{3} 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 H_{2}SO_{4} and then
CaCO_{3}. 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

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 KMnO_{4} and HNO_{3} and passes back spontaneously on standing in
the air. The latter action is however much slower than in case of


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 SO_{2}.
This substance sulphonfluoresceïn crystallizes from water
in light yellow monoclinic crystals having the composition
C_{19}H_{12}O_{6}S+2H_{2}O. 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
C_{19}H_{13}O_{7}SBa. 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
H_{2}SO_{4}, 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--

            H             H                ┌─
            C      O      C                │
          ╱   ╲ ╱  ╲  ╱  ╲             │            OH
     (HO)C      C      C      C(HO)        │          ╱
         │      │      │      │            │ C_{6}H_{3}
        HC      C      C      CH           │          ╲
          ╲   ╱  ╲  ╱ ╲  ╱             │            O
             C      C      C        =  C ──┤          ╱
             H     ╱ ╲     H              │ C_{6}H_{3}
          HC     ╱     ╲                  │          ╲
        ╱   ╲ ╱         ╲                │           OH
      HC      C            O               │
       │      │          ╱                 │  C_{6}H_{4}SO_{2}
      HC      C        ╱                   │           ╱
        ╲  ╱   ╲   ╱                     │          O
          C       SO_{2}                    │
          H                                 └─

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