The Chemistry of Sulphinic Acids, Esters and their Derivatives

The Chemistry of Sulphinic Acids, Esters and their Derivatives

Edited by S. Patai
Edited by S. Patai
4"01990
1990John Wiley&&Sons
JohnWiley SonsLtd
Ltd

CHAPTER 4

Analytical methods
M.R. F. ASHWORTH
Organische und lnstrumentelle Analytik, Universitat des Saarlandes, 0-6600 Saar-
brucken. FRG

1. INTRODUCTION . . . . . . . . . . . . . . . . . . . . 87
11. CHEMICAL METHODS . . . . . . . . . . . . . . . . . . 88
A. Oxidation to Sulphur(V1) . . . . . . . . . . . . . . . . . 88
B. Acid/Base Reactions of the Acids and their Salts . . . . . . . . . 95
C. Reactions with Metal-containing Reagents . . . . . . . . . . . 95
D. Miscellaneous Other Reactions . . . . . . . . . . . . . . . 96
111. PHYSICAL/INSTRUMENTAL METHODS . . . . . . . . . . . 99
A. Polarography . . . . . . . . . . . . . . . . . . . . . 99
B. Chromatography . . . . . . . . . . . . . . . . . . . . 100
C. Spectroscopy. . . . . . . . . . . . . . . . . . . . . . 103
IV. MICROBIOLOGICAL METHODS . . . . . . . . . . . . . . 103
V. REFERENCES. . . . . . . . . . . . . . . . . . . . . . 103

1. INTRODUCTION
Few general analytical procedures have been published for sulphinic acids and their
derivatives, such as salts, esters, acid amides, acid chlorides and anhydrides. Almost all of
these few are for the free acids and their alkali metal salts.
Interest has centred often on some special individual compounds, which may be listed
here:
(a) So-called 'Rongalite', HOCH2S02Na, variously termed sodium formaldehyde
sulphoxylate, sodium hydroxymethyl sulphite and sodium hydroxymethanesulphinate.
(b) Thiourea dioxide, with a tautomeric form of

termed also formamidinesulphuric acid or amino-imino-methanesulphinicacid.

(c) Some amino acids, such as cysteinesulphinic acid (3-sulphinoalanine)
HOOCCHCH2S02H
I
NH2
87
88 M. R. F. Ashworth
and hypotaurine(2-aminoethanesulphinic acid). NH,CH2CH2S02H. Considerable ana-
lytical work (especially chromatography) has been carried out on the amino acids. The
published examples include occasionally one or other of these sulphinyl-amino acids. It is
unreasonable to quote any publication unless the sulphinyl compounds form a significant
proportion of the whole, say about one quarter, or where these acids receive special
mention.
(d) Neoarsphenamine is a derivative ofan early chemotherapeutic agent, arsphenamine,
in which a -SO,Na group has been introduced in order to increase solubility in water.
The methods for assaying neoarsphenamine are usually based on determination of arsenic
or sulphur, or on biological tests of toxicity. It could be argued that the determination of
sulphur is indirectly a determination of the sulphinate group, since it is the only source of
sulphur in the molecule. However, it cannot be regarded as a determination of the
functional group, -SO,Na, and such examples of determination are therefore not
included here.
Methods have been given here for some compound classes containing sulphur(1V)
which, with some goodwill, can be considered as sufficiently closely related to the sulphinic
acids. These include:
R\ R,
sulphilimines or sulphidimines S-NR’ or S=NSO,Tos
R” R‘
sulphinylamines RN=S=O
The analytical methods have been divided into so-called chemical methods (i.e. those
based on chemical reaction of the compound or compounds to be detected or determined),
physical/instrumentaI methods (including polarography) and microbiological methods.

ii. CHEMICAL METHODS

The principal chemical methods are based on oxidation to the corresponding sulphonic
acid or to sulphate, i.e. to sulphur(V1). About a dozen oxidizing agents have found use. A
second group is that of acid/base reactions. These are usually direct titrations, under
suitable conditions, of the sulphinic acids with standard bases, or of the salts (mostly alkali
metal) of sulphinic acids with a standard acid. Further types of chemical methods are
reactions of precipitation or colour change with metal-containing reagents (cations or
anions), and a category of assorted procedures under the heading ‘miscellaneous’.
There is no reason to doubt that derivatives of sulphinic acids can be determined by
standard methods, for example: esters by hydrolysis using excess standard alkali and back
titration of the unused amount; amides by hydrolysis with alkali and determination of the
ammonia evolved; acid halides and anhydrides by the differential procedure using an
alcohol and water, or by reaction with excess primary or secondary amines and
determination of the unused amine. However, no published example could be found.

A. Oxidation to Sulphur(V1)
This oxidation proceeds comparatively easily so that many reagents have been used. In
fact, sulphinic acids are highly susceptible to atmospheric oxidation, which endangers the
accuracy of oxidative determinations. Most quantitative determinations have been
titrimetric. A convenient classification is according to frequency of use.

1 . Nitnte

-
The reaction of sulphinic acids with nitrous acid was used preparatively by Koenigs’ as
long ago as 1878. His reaction equation was
2 RS0,H + HNO, (RSO,),NOH + H,O
 4. Analytical methods 89
This reaction has been the basis of methods of titration. Thus Marvel and coworkers'
determined the purity of dodecanesulphinic acid by potentiometric titration in acetic acid
solution with sodium nitrite. In a study of the reactions of this sulphinic acid, Marvel and
Johnson3 titrated the magnesium salt in acetic acid/hydrochloric acid at 0 "C with sodium
nitrite to an external indicator ofstarch/iodide. They referred to a tendency to formation of
the product (RSO,),NO in the presence ofexcess nitrous acid. This should not affect direct
titration in a significant way. Further examples of titration to external starch/iodide can be
found in the work of Ponzini4, who titrated aromatic sulphinic acids in water cooled to
5 "C and containing hydrochloric acid, taking as end-point a colour persisting for at least
several minutes; Kice and Bowers' in a study ofdisproportionation of sulphinic acids; and
Danehy and Elia' for p-chlorobenzenesulphinicacid.
In his study of various methods for determining aromatic sulphinic acids and their salts,
Lindberg' titrated with sodium nitrite a solution of the sample in dilute sulphuric acid
containing potassium bromide and determined the end-point potentiometrically at a
platinum electrode. Fleszar', likewise in an investigation of various procedures, titrated an
acidified solution (hydrochloric acid) of sodium benzenesulphinate with sodium nitrite but
used amperometric end-point indication with two platinum electrodes. Matrka and
collaborators' also employed amperometric or potentiometric end-point determination
with platinum and calomel electrodes, or titrated biamperometrically with two platinum
electrodes; they titrated aromatic sulphinic acids in dilute hydrochloric acid at 20 C.
Marek I " detected some aromatic sulphinic acids and their derivatives on paper
chromatograms by exposure for 1 min to the vapours from a mixture ofsodium nitrite and
hydrochloric acid, followed by spraying on a solution of R-salt (2-naphthol-3,6-
disulphonic acid) and exposure to ammonia to yield yellow-green spots. Czerwicz and
Malata' ' visualized substituted phenylsulphinylamines Ph-N=S=O, on silica gel thin
layer chromatograms, by exposure for 30-60s to nitrous gases from a sodium
nitrite/hydrochloric acid mixture. After 24 h the compounds appeared as yellow. orange or
red-brown spots.

2 Hypohahte (and chlorarnine T )

After Allen" had found low results in oxidative titrations (e.g. with permanganate) of
acidified solutions of sodium and magnesium alkanesulphinates, he proposed direct
titration with basic calcium hypochlorite [Ca(OCI)CI, 'bleaching powder'] in alkaline or
neutral solution and using an external indicator of starch/iodide. Ackermani3 titrated

-
sodium benzenesulphinate with standardized sodium hypochlorite, likewise using an
external starch/iodide indicator. He gave the reaction equation
2 PhSO'Na + NaOCl PhS0,Na + PhS0,CI
In order to determine halates, Atkini4 standardized hypochlorite solution by titrating
with sodium benzenesulphinate solution; he, too, used the starch/iodide external
indicator.
Coulometric titration with hypochlorite and hypobromite was performed by Liberti
and Lazzari' '. Their examples included sodium benzenesulphinate. They generated
chlorine electrolytically at pH 1.3, bromine likewise at pH 5.8. In each case the halogen was
led into a 0.6 M solution of hydrogen carbonate of pH 8.3 containing the sample They
used amperometric end-point indication with rotating platinum electrodes at t 0.2 V.
Hashmi and Ayazl' also utilized the reaction of benzenesulphinate (and other reducing
agents) with hypochlorite and chlorite, but their aim was the determination of inorganic
ions. They titrated the sulphinate with hypochlorite using the starch/iodide external
indicator. but also with tartrazine and Bordeaux as internal indicators if sodium hydrogen
carbonate and a little potassium bromide were added to the solution to improve the end-
point.
90 M. R. F. Ashworth
In an adaptation of the Ackerman hypochlorite procedure (see above) Uhlenhroek and
coworkers” used chloramine T for titrating benzenesulphinic acids containing various
substituents [e.g. o-acetylamino-, p-(2-hydroxy)ethoxy-]. They added sodium hydroxide
solution to the acid sample until methyl orange indicator just changed colour. Barium
chloride was then added and any precipitate filtered off. An aliquot of the filtrate was
treated with hydrochloric acid and a measured amount in excess of the chloramine T
reagent solution was added. After 2 min reaction time they added solid potassium iodide
and titrated with thiosulphate the iodine liberated by unused reagent.
Sumizu’8 determined hypotaurine (2-aminoethanesulphinic acid) by oxidation with
excess hypoiodite. Unreacted reagent was then decomposed with phosphoric acid to
iodine which was estimated by absorption at 590nm after treatment with starch.

3 lodme, iodme oxyacids and [odme monochloride

Iodine has been used to titrate sodium hydroxymethanesulphinate (‘Rongalite’)
according to the reaction
HOCH,SO, + I, + H,O - HOCH,SO, + 2HI
Examples are the work of: Salkin’”, who titrated with iodine but considered it to be less
satisfactory than titration with copper(l1) because the iodine reacted also with sodium
hydrogen sulphite and sodium thiosulphate; Furness2’, who titrated the Rongalite
in connection with a polarographic study; Badinand and Rondelet”, who deter-
mined the hydroxymethanesulphinate present as an antioxidant in p-aminosalicylate
by adding phosphoric acid before titrating with iodine: and Maros”, who determined it in
the presence of formaldehyde bisulphite, HOCH,SOJ, by adding formaldehyde and
acetate/acetic acid and titrating with iodine. Tsau and P ~ o l e commented
,~ on the difficult
end-point indication in conventional titration of antioxidants including the hydroxymeth-
anesulphinate. They proposed a method involving H PLC of reaction mixtures obtained
by adding consecutive amounts of titrant. They used a column of C 18 HL (Alltech) and
four mobile phases, monitoring spectrophotometrically.
Thiourea dioxide has also been determined by titration with iodine, e.g. by
Wojtasiewicz-Obrzutz4 and Shafran and [Link] compound is oxidized to urea

-
and sulphate:

NH *C-SGo
NH,’ ’ O H
+ 21, +6NaHC0,
NH,
‘C=O + 4 N a l + Na,S04 + 6C0, + 3H,O
NH,’

The former added excess standard iodine solution to an aqueous solution of the sample
containing sodium hydrogen carbonate. After 2 min he acidified with sulphuric acid and
back titrated with thiosulphate to starch indicator. This yielded a value for the thiourea
dioxide and any oxidizable impurities. These impurities were determined and corrected for
by an analogous procedure in which the sample in acid solution was treated with the excess
iodine, back titrating as before after 2 min. Shafran and coworkers also added excess
iodine reagent to a solution of the sample in water containing sodium hydrogen carbonate;
they, too, used 2 min reaction time before acidifying with sulphuric acid and back titrating
with thiosulphate to starch, while impurities were similarly determined in a blank.
Mahadevappa26used potassium iodate and periodate to titrate sodium hydroxymethane-
sulphinate, finding also that 4 equivalents of oxidizing agent were consumed.
Iodine monochloride was used by Krishnan Nambisan and Ramachandran Nair2’ to
 4. Analytical methods 91
determine some poorly soluble compounds, including Rongalite. They used excess reagent
in 5 M hydrochloric acid. After 10 min they added lW;<, potassium iodide solution and
titrated the liberated iodine with thiosulphate. Four equivalents took part in the
oxidation:
HOCH,SO, + 41C1+ 2H,O - HCHO + SO:- + 21, + 4C1- + 5H'
4 Bromine and brornidelbromate
Ramberg,' titrated ethanesulphinic acid in a hydrochloric acid medium with potassium
bromate, taking decolouration of added methyl orange as end-point. He admitted that
some atmospheric oxidation took place. Faster titration gave higher values and results
were better in the presence of potassium bromide. Fleszar' titrated sodium benzenesulphi-
nate with several reagents, including potassium bromate. He dissolved the sample in
water, strongly acidified with hydrochloric acid, and added potassium bromide. End-point
indication was potentiometric. An example of titrimetric determination of thiosulphinate
using bromide/bromate is the work of Ostermayer and [Link] dissolved the

-
sample in 80% acetic acid and took the first permanent appearance of bromine colour in
the solution as their end-point. The reaction equation is
CH,S=O + 4Br, + 3H,O 2CH,SO,Br + 6HBr
I
SCH,
(Siggia and Edsberg,' used similar conditions for titrating disulphides, which also yield
sulphonyl bromides)
Bromine has found some limited use for detecting organic sulphur compounds on
chromatograms. Thus Bayfield and collaborators3' studied the visualization of thiosul-
phinates and sulphinamides (also thiols and sulphides) by drawing the paper chromat-
ograms through 3% aniline in petrol ether, allowing the petrol ether to evaporate and then
exposing to bromine vapour. This gave blue or mauve spots within 30-60 s and was more
sensitive for thiosulphinates than for sulphinamides.

5. Ceriurn(/V)
The stoichiometry of the cerium(1V) reaction with sulphinates is controversial. Forrest
and Ryan32 reported titrations with cerium(1V) sulphate of sodium benzenesulphinate,
using ferroin as indicator, in which they found a mole ratio of cerium to sulphinate of 1.8 to
1; they quoted diphenyl disulphone, PhSO,SO,Ph, as a reaction product. Gringras and
SjOstedt3, also titrated aromatic sulphinic acids with cerium(1V) sulphate, using
potentiometric end-point indication with platinum electrodes. They found varying mole
ratios, depending on the concentration (within the range 5 x lo-, to l o - ' M). One
equation given by them was
4ArSO; + 6Ce4+ + 2H,O
T ~ a i k o reported
v~~
- ArSO,SO,Ar + 2ArSO; + 6Ce3' + 4H'
titrations of benzene- and p-toluenesulphinic acids in sulphuric acid
solution at pH 1-2, using cerium(1V) sulphate (and also potassium dichromate) and
potentiometric end-point determination at a platinum electrode. Grossert and Langler35
used the 'cerium ammonium nitrate' reagent (ammonium hexanitratocerium),
(NH,),[Ce(NO,),], in nitric acid solution as a spray reagent for visualizing some organic
sulphur compounds on silica gel thin layers. These compounds included thiols, disulphides
and two thiosulphinate esters, the methyl esters of benzenesulphinic and chloromethane-
sulphinic acids. These esters appeared at room temperature as colourless zones on a yellow
background.
92 M. R. F. Ashworth
6. Perrnanganate
Permanganate has long been known as an oxidizing agent for sulphinates. Reu-
t e r ~ k i o l dtitrated
~ ~ sulphinic acids and sulphinoacetic acid, HOOCCH,SO,H, with this
reagent. As mentioned in Section II.A.2, Allen” used potassium permanganate to titrate
aliphatic sulphinate salts. He found low results in acid solution but gave two procedures
for the use of permanganate in non-acidic solution: (a) direct potentiometric titration in
alkaline or neutral solution; (b) the sample together with sodium hydroxide was left in
contact with a measured amount of permanganate reagent in excess. After 5 min he added
excess standard arsenite solution, some potassium iodate as catalyst, then concentrated
hydrochloric acid and finally titrated the unused arsenite with potassium permanganate.
Lindberg’ investigated comprehensively the determination of aromatic sulphinic acids
and their sodium salts. His procedures included direct potentiometric titration (platinum
indicator electrode) with potassium permanganate, and oxidation with excess permanga-
nate reagent in neutral solution, followed after 10min by adding acid and potassium
iodide and finally titrating the liberated iodine with thiosulphate to a dead-stop end-point.
Fleszar and coworkers3’ determined the 4,4’-disulphinic acid of diphenyl ether by
precipitation as zinc salt (Section II.C.5) and titrating this in acid solution with
permanganate. In an extensive study of the paper chromatography of many compounds
R e i detected
~ ~ ~ cysteinesulphinic acid (3-sulphinoalanine) with several reagents which
included permanganate.

7. Copper(//)
Copper(I1) was used as a titrant for hydroxymethanesulphinate in early work. Thus
Helwig3’ titrated this sulphinate (sodium salt) in aqueous solution with an ammoniacal
copper(1I)reagent in a stream of carbon dioxide until decolourization ceased. The solution
was then heated over a free flame and titrated further until a faint blue colour persisted for
10s at the boiling temperature. Salkin4’ also considered copper(I1) sulphate in concen-
trated ammonium hydroxide to be the best titrant for Rongalite, sodium
hydroxymethanesulphinate, superior to iodine which reacted with other compounds
possibly present, such as hydrogen sulphite and thiosulphate. Spitzer4’ determined the
sodium hydroxymethanesulphinate by adding excess concentrated copper(I1) sulphate
solution, acidified with sulphuric acid. After a short interval he added potassium iodide to
yield iodine with the unused copper(I1); this was titrated with thiosulphate.

8. Mercuric ion

-
Mercuric chloride has been used in analytical work with sulphinates. Thus Spitzer4’
determined Rongalite by reacting it with mercuric chloride:
HOCH,SOT + 2HgCI, + H,O HOCH,SO; + Hg,CI, + 2HCl
He filtered off the precipitated mercurous chloride, dissolved it in excess standard iodine
and back titrated the unused part.

-
Mercuric chloride has also been used to prepare solid crystalline derivatives of sulphinic
acids:
RSO,H(Na) + HgCI, RHgCl + H(Na)CI + SOz
This reaction was described as long ago as 1905 by Peters43. Marvel and coworkers’ were
the first to prepare such a derivative of an aliphatic sulphinic acid (dodecanesulphinic
acid).
A spot test for aromatic sulphinic acids depends on this reaction yielding sulphur
dioxide. Feigl and heated the sample to 80 “C with mercuric chloride and
 4. Analytical methods 93
tested for sulphur dioxide evolved through the blue colour given by Congo paper treated
with hydrogen peroxide and held in the issuing gases.

9. Chromium(Vl)
T ~ a i k o titrated
v ~ ~ benzene- and p-toluenesulphinic acids in sulphuric acid solution at
pH 1-2 with potassium dichromate, using potentiometric end-point indication with a
platinum electrode.
Langler45 visualized some sulphur-containing compound classes (including sulphinate
esters) on silica gel HF,,, thin layers with a chromium trioxide/acetic acid reagent. The
sulphinate esters tested (methyl esters of benzenesulphinic and chloromethanesulphinic
acids) appeared after 15-30 min as blue-green spots. This detection was considered
superior to that with cerium(1V) (see Section II.A.5).

10. 0- and p-Dinitrobenzene

Feigl and M a i r ~ b e r g e rdistinguished
~~ hydroxymethanesulphinate (Rongalite) and
dithionite through their reactions with o- or p-dinitrobenzene in the presence of alkaline
ethanol. Both reduce the reagent to violet or orange quinonoid compounds on heating but
only dithionite accomplishes this in the cold. Feigl and coworkers44 detected alkali metal
or calcium salts of aromatic sulphinic acids by reaction with o-dinitrobenzene to yield
nitrite, which is itself detected with the help of the Griess reagent of sulphanilic acid/a-
naphthylamine (the former is diazotized in acid solution by the nitrous acid and the

diazonium ion couples with the latter component). Feigl and coworkers tested this with
benzene-, p-toluene- and 3-acetamido-4-methoxybenzene-sulphinic acids.

1 1 . Elementary sulphur
Scandurra and Most4’ reported an unusual method of determining sulphinates. They
refluxed the sample for 10 min with a solution of elementary sulphur in 95% ethanol. plus
some drops of concentrated ammonium hydroxide. After evaporating to dryness they
dissolved the residue in water and removed unused sulphur with chloroform. The aqueous
phase was then heated with ammonium hydroxide and potassium cyanide on the water
bath for 30 min. After cooling they added a reagent of ferric nitrate in nitric acid, yielding a
red colour, evaluated at 470 nm after 10 min. Evidently the sulphinate is first converted by
the sulphur into thiosulphinate, ArS(=O)SH, which then reacts with the cyanide to give
thiocyanate. This in turn reacts with the ferric ion to give the characteristic red product.

12. Vanadatelferrocyanide
Sodium hydroxymethanesulphinate (also hydrosulphite and stannous chloride) but
neither sulphite, hydrogen sulphite nor formaldehyde, respond positively to the test of
Gentil and Miranda48. They used a reagent of potassium ferrocyanide and vanadate
which together yield a green precipitate. This is reduced by the sulphinate to a red product.
94 M. R. F. Ashworth
13. Photochemical oxidation
An unusual example of oxidation of sulphinate to sulphonate come from the work of
Kataoka and colleagues49 on hypotaurine. They treated it first with i-butyl chloroformate,
then photooxidized this product through irradiation with a 300 W tungsten lamp for 5 min
at 20cm distance. The product was then converted into the sulphonyl chloride with
thionyl chloride and the sulphonyl chloride finally reacted with dibutylamine to yield the
end-product of 2-(isobutoxycarbonylamino)-N,N'-dibutylethanesulphonamide.
CICOOHu-I Photooxlddtlon
H,NCH,CH,SO,H ~-BuOCONHCH,CH,SO,H +

- -
+

S0('Iz HulNH
i-BuOCONHCH,CH,SO,H i-BuOCONHCH,SO,CI
i-BuOCONHCH ,CH ,SO,NH Bu

B. AcldlBase Reactions of the Acids and their Salts

7. Determination and identification of free acids w/th bases

Free sulphinic acids can, of course, be directly titrated with bases. They are several
powers of ten weaker than the corresponding sulphonic acids so that a differentiating
titration of the two, e.g. potentiometrically, is possible. An early reference to an acidimetric
determination is that of Krishna and Bhagwan Das3'. They used the potassium

-
iodide/potassium iodate reagent at 0 ' C from which the acid liberated iodine according to
the usual reaction:
KIO, + 5KI + 6 H + 31, + 3H,O
They did not determine the iodine by the customary titration with thiosulphate or arsenite.
Instead, the reaction mixture was allowed to warm up to room temperature and then
treated with alkaline hydrogen peroxide. This yielded oxygen which they determined gas-
volumetricall y.
Wetzel and Meloan' ' titrated several aromatic sulphinic acids (with methyl, ethyl, butyl
and other nuclear substituents) in non-aqueous solution. They tested many solvents, using
quaternary ammonium hydroxides in benzene-methanol as titrant. End-point indication
was potentiometric. They were able to titrate the sulphinic acids in the presence of the
corresponding sulphonic acids in benzene-methanol, t-butanol, D M F -DMSO, tetra-
hydrofuran and pyridine solution.
The usual organic bases can serve for preparing sulphinate salts for identification of the
free sulphinic acids. An early example is the work of Halssig5, who prepared salts with
phenylhydrazine. Solid derivatives for identification through melting point are, however,
generally prepared from alkali metal salts of the acids (see Section II.D.2b below).

2 DetermmWon of sulphinate salts with a o d s

As salts of strong bases and comparatively weak acids, the alkali metal sulphinates can
be titrated with strong acids. End-points may not be very sharp in aqueous solution
because the sulphinic acids have pK values between 2 and 3 (methanesulphinic acid,
ca. 2.3; benzenesulphinic acid, ca. 2.8). Titration is non-aqueous solution is, however,
successful. Examples of this are the extensive investigations of Lindberg' and Fleszar'.
Among other procedures, each titrated with perchloric acid. Lindberg titrated p-methoxy-
benzenesulphinate in acetone-methanol or methanol-i-butyl acetate. using perchloric
 4. Analytical methods 95

acid in dioxan, and some other substituted benzenesulphinic acids (p-methyl, p-chloro)
in acetic acid using perchloric acid in this solvent also. End-point determination
was potentiometric with a platinum indicator electrode. Fleszar titrated sodium
benzenesulphinate potentiometrically in acetic acid-dioxan ( 1 + 1) using glass and silver
chloride electrodes.

C. Reactions with Metal-containing Reagents

Reaction with metal-containing cations or anions has been used for quantitative
determination of sulphinates via precipitation or colour change, and also for detection and
identification.

1 . Ferric ion
The earliest reference to the sensibly quantitative reaction of sulphinates with ferric ion
appears to be that of Thomas”. He obtained orange-yellow ferric salts of aromatic
sulphinic acids in practically theoretical yield by adding ferric chloride to the strongly
acidified sulphinic acid solution. Krishna and Singh54 determined a wide range of
aromatic sulphinic acids by addition to acidified ferric chloride in excess. After filtration
they reduced unused ferric ion in the filtrate with stannous chloride in concentrated
hydrochloric acid or zinc dust and sulphuric acid, and titrated the resulting ferrous ion
with dichromate to an external indicator of potassium ferrocyanide. Forrest and Rjan”
undertook a comprehensive study of the reactions between a wide range of metal cations
and several aromatic sulphinic acids (benzene-, p-toluene-, naphthalene-2-, thiophene-2-
and benzyl-). The work was aimed more at detection, identification and determination of
the metals. They stated that ferric ion gave at least two products with benzenesulphinic
acid. The ferric trisulphinates were soluble in many organic solvents, a property which
does not appear to have been exploited analytically. Alimarin and Kuznetsovs6 tested a
new reagent for ferric ion, o-hydroxybenzenesulphinic acid. At pH values between 1.9 and
7.53 it yielded a violet complex with the ferric ion, evidently in the ratio of 1 : I . This could
be used for colorimetric estimation of ferric ion. Presumably the method could be used in
the reverse sense, to determine the sulphinic acid. However, the colour is probably due to
the o-hydroxyl group (like salicylic acid).
Ferric chloride has been used for chromatographic visualization of sulphinic acids and
related compounds. Thus Mondovi and coworkerss7 used it in the paper electrophoresis
of some sulphur-containing compounds of biological interest, inclusing cysteinesulphinic
acids and hypotaurine. Fondarai and Richert’” also used ferric chloride to reveal
cysteinesulphinic acid on paper chromatograms. An example of its use in thin-layer
chromatography comes from the work of Westley and Westley’”, who visualized organic
thiosulphinates (also sulphonates and thiosulphonates) on silica gel layers using ferric
chloride in acetone.

2 P/atinurn(/V)fhexachloro, hexa/odo-p/at/nate)
These reagents with a metal-containing anion have been used in analytical procedures
depending on colour change. Thus Winegard and collaborators6’ visualized sulphur-
containing acids, including cysteinesulphinic acid, on paper chromatograms by spraying
with a hexachloroplatinate-potassium iodide reagent. They then suspended the paper strip
in hydrogen chloride fumes, which revealed the amino acids as uncoloured zones on a pink
background. Fondarai and RichertS8 also visualized cysteinesulphinic acid and other
amino acids with this iodoplatinate reagent, while De Marco and coworkers“ likewise
applied this method of detection to hypotaurine and homohypotaurine (and correspond-
96 M. R. F. Ashworth
ing selenium compounds) on paper chromatograms. Jolles-Bergeret6' profited from the
fact that cysteine- and homocysteinesulphinic acids decolourize hexachloroplatinic acid in
acetic acid solution. Quantitative determination was possible, based on the decrease in
light absorption at 500nm.

3. Palladium(//)(tetrachloropalladate)
Akerfeldt and L ~ e v g r e nreported
~~ that various sulphur-containing compound classes
(thiols, sulphides, disulphides and sulphinic acids) yielded coloured complexes with
palladium(I1). They treated the sample with ammonium tetrachloropalladate,
(NH,),[PdCI,], in hydrochloric acid. Colour developed within 5 min with the thiols and
sulphinic acids and spectrophotometric determination was possible at wavelengths
between 350 and 415 nm.

4. Thallium(///)
Gilman and Abbott" characterized the sodium salt of p-toluenesulphinic acid (and
many sulphonic acids) by conversion to a derivative of the formula p-TolSOTICI,. This
was accomplished by mixing aqueous solutions of the salt(s) and thallium(l1I) chloride.
Other sulphinate salts should react similarly.

5. Ztnc(l/)
Fleszar and coworkers3' precipitated the 4,4'-disulphinic acid of diphenyl ether (as
disodium salt) with excess zinc sulphate, centrifuged the product, acidified it and titrated
with permanganate (Section II.A.6).

D. Mlscellaneous Other Reactions

1. Reduction
Methods of reduction apply only in special cases because the sulphinic acids are not
normally oxidizing agents. Three examples of reagent are given below:

a. Iodide. Compounds with oxidizing properties can convert iodide to the easily
detectable and determinable iodine. Thus Bretschneider and Klotzer6s treated thiosulphi-
nate esters with iodide in sulphuric acid solution:
RS(=O)SR + 21- + 2H+ RS-SR + 1, + HZO
They then titrated the iodine. Barnard and Cole66found low results with this method and
developed their own procedure for alkyl (ethyl, butyl) esters of aromatic thiosulphinic
acids (benzene-, p-methoxybenzene-). They dissolved the sample in glacial acetic acid,
added aqueous potassium iodide solution in an oxygen-free atmosphere (stream of carbon
dioxide) and titrated the iodine yielded with thiosulphate. They also visualized thiosulphi-
nate esters on paper chromatograms as blue-violet spots by exposure for 10 s to hydrogen
chloride and then spraying with a starch-iodide reagent. A similar method of detection was
used also by Fondarai and Richert" for cysteinesulphinic acid on paper chromatograms
and by Freytag and Ney6' for aliphatic sulphilimines on thin-layer chromatograms of
silica gel by spraying first with potassium iodide-dilute hydrochloric acid, heating the plate
for 8 min at 120°C and then spraying with starch solution to give violet to brown-violet
zones. Bayfield and coworkers" visualized aromatic sulphinamides (and sulphenamides)
as blue-mauve spots on paper and thin-layer chromatograms by exposure to hydrogen
 4. Analytical methods 97
chloride or trichloroacetic acid fumes and then spraying with starch-iodide. De Marco and
collaborators6' visualized hypotaurine and homohypotaurine (and the selenium-
containing acids) by spraying with potassium iodide-hydrochloric acid.

b. Methyl violet. Yakovleva and coworkers69 determined thiourea dioxide and hydro-
gen peroxide through reaction with methyl violet for 10 min at 100 "C and pH 8.5 to 9. The
diminution in absorbance of the dye at 590nm was proportional to the amount of the
oxidizing agents [hydrogen peroxide alone was estimated through a colour reaction with
titanium(IV), enabling the thiourea dioxide to be determined by the difference].

c. Reduction with metals. Feig17' gave methods for detecting benzenesulphinic acid by
reduction. Zinc-hydrochloric acid or Devarda's alloy reduced it to thiophenol, detected in
the vapours through the blackening of paper saturated with lead acetate solution held
there. Raney alloy (nickel-aluminium) with some drops of hydrochloric or sulphuric acid
reduces the sulphur to hydrogen sulphide, which can be detected in the same way. This test
must be applicable to other sulphinic acids but probably also to other sulphur-containing
organic compounds.

2. introduction of characterizing organic groups

-
a. Diazonium coupling. Babbs and Gale" determined sulphinic acids by reaction with
the diazonium salt Fast Blue BB to yield a coloured azo compound:
+
RS(=O)OH + R'N-N RS(=O)ON=NR'
They extracted the azo compound into toluene-butanol, stabilized this solution with
pyridine and evaluated colorimetrically through the light absorption at 420nm. It is
surprising that no further examples have been found of such coupling procedures. They
must be reasonably specific, easy to carry out and sensitive, and there is a wide choice of
diazonium salts.

b. Reaction with active halides. Jayson and coworkers7' visualized oxidation products
of cysteamine by reaction for 45 min at 100 "C with l-fluoro-2,4-dinitrobenzenein the
presence of sodium hydrogen carbonate. The amino group of the amino acid products
reacted with the fluoro compound, but it is of interest that the sulphinic acid group of
hypotaurine also reacted, to yield a di-substituted product:

The sulphonic acid group of taurine does not react. This could enable sulphinic acids to be
determined in the presence of sulphonic acids, although this idea does not appear to have
been tested.
Derivatives for identification of sulphinic acids have been prepared by reaction of their

ArS0,Na -
alkali metal salts with suitable halides. Thus Marvel and Johnson3 used chloroacetic acid:
+ CICH,COOH ArSO,CH,COOH + NaCl

-
Douglas and collaborator^^^ prepared a derivative of ethanesulphinic acid by reaction
with benzyl chloride:
EtSO; + PhCH,CI EtSO,CH,Ph + CI
98 M. R. F. Ashworth
The well-known reagent benzylthiouronium chloride can be used for characterization, as
with carboxylic and sulphonic acids:
RSO;Na+ + [PhCH,SC-NH,It
I
CI- - [PhCH,SC=NH,]+
I
[RSO,]

NH, NH2
c. Reaction with N-ethylmaleirnide. This reaction, which has been used to determine
thiosulphinic acids, is not considered to be addition of the acid to the double bond of the
reagent but the formation of an enolic compound of the corresponding d i k e t ~ n eit~ has ~;
an absorption maximum at 515nm. Carson and Wong15 mixed the sample and N-ethyl-
maleimide, both in isopropanol, then added potassium hydroxide; they measured
the light absorption at 515nm after a reaction time of 10 to 18min from the moment of
addition of alkali. They tested the procedure with thiosulphinates containing phenyl,
p-tolyl, ethyl, propyl and 2-propenyl groups. They used the same colour reaction to
visualize thiosulphinates on paper chromatograms. The paper was dipped first into the
N-ethylmaleimide solution, dried for 5- 10 min, and then dipped into the potassium
hydroxide. Nakata and based their determination on the same principle,
mixing sample and reagent in isopropanol with potassium hydroxide but then adding
ascorbic acid to stabilize the colour before finally evaluating also at 515 min after 10 min
reaction time. According to Watanabe and K ~ m a d a who ~ ~ ,worked with the S-propyl
ester of propanethiosulphinic acid and the S-ally1 ester of 2-propenethiosulphinic acid
('allicin'), the light absorption of the product can be measured also in the ultraviolet region
at 355 nm after 20-30 min reaction time.

d. Pyrolysis. A spot test of Feigl and Costa Neta78 to distinguish dithionite and

-
hydroxymethanesulphinate (Rongalite) depends on heating the sample to 200-300 "C.
The latter decomposes according to
2HOCH,SO,Na 2 H C H O + N a 2 S 0 4 + H,S
The hydrogen sulphide is detected by the black stain formed on lead acetate paper held in
the vapours.
Only in rare cases can sulphinic acids be decomposed with loss of sulphur dioxide:
RS0,H -SO, + RH
There is thus no example parallel to decarboxylation for quantitative determination.

e. Miscellaneous colour reactions. Most colour reactions for sulphinic acids given in the
literature are carried out under rather drastic conditions and are therefore probably not
specific. Thus L i m p r i ~ h stated
t ~ ~ that aromatic sulphinic acids dissolve in concentrated
sulphuric acid without colour formation, but the addition of some phenol then yields a
blue to deep blue colour. Smiles and Le Rossigno18' reported a characteristic blue colour
given by aromatic sulphinic acids (e.g. p-ethoxybenzenesulphinicacid) with anisole o r
phenetole in concentrated sulphuric acid. The formation of ArS(=O)SC,H,Et (or Me)
was postulated. Further phenetole led to the disappearance of the colour, ascribed to
formation of diphenetyl phenylsulphonium sulphate. Bazlen and Scholz" detected
sodium and potassium salts of aliphatic and aromatic sulphinic acids through their ability
to decolourize indigo solutions in glycerol at 180°C. Probably many other sulphur-
containing compounds are also capable of this.

[Link] to double bonds. Sulphinic acids add comparatively readily to double

bonds, usually in a 1,4-addition, e.g. to r,P-unsaturated carbonyl compounds or quinones.
Addition to the last named appears to have been of special interest to Russian teams. Thus
 4. Analytical methods 99
Obtemperanskaya and Zlobin" studied the reaction of anthraquinone with thiourea
dioxide, which yielded anthrahydroquinone, the absorbance of which at 417 nm was used
to determine the anthraquinone in anthracene. Stadnik and coworkersR3investigated the
addition of benzenesulphinic acid to benzoquinone, substituted benzoquinones and
1,4-naphthaquinone; at pH 1-2 this yielded coloured products in a 1: 1 ratio. Stom
and collaboratorsR4 reported the chromatographic and spectroscopic determination of
quinones in aqueous solution through reaction with benzenesulphinic acid. In all this
work the interest has centred on determination of the quinones. The reaction does not
appear to have been the basis of determination of a sulphinic acid.

111. PHYSlCALllNSTRUMENTAL METHODS

Included under this heading are polarographic methods (these concern both reduction
and anodic oxidation but it was considered to be more convenient to deal with them in the
same section), chromatographic methods of separation and some spectroscopic proce-
dures, of which only a few have been published.

A. Polarography
Polarographic anodic waves (for oxidized sulphinates) and cathodic waves (for
thiosulphinates and thiourea dioxide) have been reported permitting, in some cases,
quantitative determination.
The earliest reference to polarographic determination of a sulphinic acid is evidently
that of Furness2', in work on dithionites, hydroxymethane- and hydroxyethane-
sulphinates. Horner and Nickels5 worked on the polarographic reduction of sulphinate
esters in 757{ dioxan, but their work was not expressly analytical. Several authors have
published results from the polarographic determination of sodium hydroxymethane-
sulphinate (Rongalite). They include Kolthoff and Tamberg", who obtained anodic

--
oxidation waves in alkaline solution (pH 9) showing a linear relation between wave height
and concentration. Their equations were
HOCH,SO; + 2 0 H - HOCH,SO; + H 2 0 + 2e
HOCH,SO; + O H - +
H C H O SO:- H20 +
Sokolov and Leonova8' determined the compound in latex by coagulating with nitric
acid-hydrochloric acid, bringing it to pH 1.2-1.5, deaerating and then submitting to
polarography in which wave height was proportional to sulphinate Concentration.
Fernandez-Martin and coworkers" found single anodic waves at pH values between 6.92
and 11.6, showing above pH 9 a linear relation between diffusion current and
concentration. Edgar" studied the polarography of the hydroxymethanesulphinate in a
McIlvaine citrate-monohydrogen phosphate buffer of pH 4, using a rotating platinum
electrode and obtaining a linear relation between the height of the anodic wave at 0.935 V
and concentration in the range from 0.02 to 0.2 mM (the influence of hydrogen sulphite
could be eliminated by adding formaldehyde).
Ruff and Kucsman" determined polarographically sulphimides, RR'S=NSO,Ar, in
deaerated Britton-Robinson buffer of pH 4.5, as part of a study of the reaction of organic
sulphides with chloramine T.
Grundler and Choschzick" made an alternating-current polarographic study of
sodium salts of aromatic sulphinic acids (components of photographic emulsions) in
borate buffer of pH 6.5 + potassium chloride. They were able to separate the polaro-
graphic waves from those of aryl thiosulphinates present (their attention was devoted to
these last named). In the cathodic region wave height was proportional to concentration.
Czerwicz and Bogaczeky2polarographed phenylsulphinylamine, PhN=S=O and the
100 M. R. F. Ashworth
0-,m- and p-substituted (methyl group) compounds in dimethylformamide or benzene-
dimethylformamide, using tetrabutylammonium iodide as supporting electrolyte. They
obtained two or three reduction waves, the second wave showing a linear relationship
between diffusion current and concentration.
Probably classifiable here is the linear sweep voltammetric method of Kirchnerova and
~ ~ , a vitreous carbon working electrode which they applied to thiourea and
P ~ r d y using
thiourea dioxide. They obtained a rectilinear graph up to 0.35 mV. The pH was below 5 in
a medium of mineral acid plus alkali metal or ammonium salt. Their sweep rate was 2 mV
per second.

B. Chromatography

1. Paper Chromatography
Barnard and Cole66 separated alkyl and aryl thiosulphinate esters on Whatman No. 1
paper, impregnated with phenoxyethanol, and using heptane as developing solvent. They
visualized with hydrochloric acid-iodide (Section [Link]). Gringras and S j O ~ t e d t ~ ~
carried out ascending paper chromatography of some aliphatic and (mostly) aromatic
sulphinic acids and their salts. They used Whatman No. 1 paper, developing for 16h
at 20 "C with the solvent mixture butanol-propanol-water (1 + 1 + 1) and visualized
with tetrazotized o-dianisidine (Echtblau B Salt), stabilized with zinc chloride which
gave canary yellow dye products, insoluble in water. They were able to determine
p-toluenesulphinic acid quantitatively via spot size. Fondarai and Richert5' conducted
paper chromatography of sulphur-containing amino acids, using as mobile phase 95%
ethanol-chloroform-water (6 + 3 + 1); cysteinesulphinic acid was among the compounds
and was visualized with iodoplatinate, ferric chloride and hydrochloric acid-iodide
(Sections II.C.1, II.C.2 and [Link]). Bayfield and coworkers6' used conditions similar to
those of Barnard and Cole for paper chromatographic separation of some aromatic
sulphinamides (and sulphenamides), i.e. with Whatman No. 1 paper, impregnated with
phenoxyethanol, and heptane as developing solvent in descending chromatography. They
also visualized with acid-iodide (Section [Link]). De Marco and collaborators6'
separated sulphur- and selenium-containing amino acids, including hypotaurine and
homohypotaurine and their selenium analogues, on Whatman No. 1 paper using three
solvents: water-saturated phenol in the presence of ammonia vapour, the upper phase of
butanol-acetic acid-water (4 + 1 + 5) and water-saturated 2,4,6-trimethylpyridine-2,6-
dimethylpyridine. They visualized with hydrochloric acid-iodide and iodoplatinate
(Section II.C.2 and [Link]). Marek" separated mixtures of sulphinic acids (benzene-,
p-toluene-, p-acetamidobenzene- and m-nitrobenzene-) at 24 "C on Whatman No. 1 paper
using various solvent systems made up of propanol + ammonium hydroxide and/or
butanol and/or water. He visualized by exposure to nitrous oxides, then spraying with R
salt solution and finally exposing to ammonia to give yellow-green zones (Section II.A.1).

2. Thin-layer chromatography
In addition to separating some aromatic sulphinamides and sulphenamides by paper
chromatography, Bayfield and colleagues68 also tried thin-layer chromatography on
kieselguhr G and cellulose layers, using methanol of various concentrations, of which 65%
proved to be the best; however, they found separation to be less satisfactory than with
paper chromatography. Detection was carried out with the help of an acid-iodide reagent
(Section [Link]). Freytag and Ney6' carried out thin-layer chromatography of S,S-
dialkyl-N-(p-toluenesulphonyl) sulphilimines (alkyl groups being methyl, ethyl, propyl,
butyl and pentyl) on silica gel G layers. Their mobile phase was diethyl ether-ethanol
 4. Analytical methods 101
(4+ 1 for lower alkyl groups, 20+ 1 for higher). Acid-iodide was used for visualization
(Section [Link]). Czerwicz and Malata" used thin-layer chromatography on silica gel
layers to separate isomeric sulphinylamines, PhN=S=O, with methyl substituents in the
2,3-,2,4-,2,5-.2.6-,3,4- and 3,5-positions. As solvent they used binary and ternary
mixtures of hexane, benzene, chloroform, carbon tetrachloride, amyl alcohol, diethyl
ether, acetone and ethyl acetate (also benzene alone). They visualized with nitrous fumes
(Section III.A.1). Westley and Westleys9 performed thin-layer chromatography of alipha-
tic and aromatic thiosulphonates, sulphonates and sulphinates, on silica gel with various
mobile phases: isopropanol-0.2 M ammonium hydroxide (3 + l), ethyl acetate-
methanol-water (4+ 1 + 1) and acetone-butanol-water (2+ 2 + 1). They visualized also
with ferric chloride (Section II.C.l). The R values were in the sequence: RS0,S- > RSO;
> RSO;.

3. Gas-liquid chromatography
Block and 0 C o n n o r 9 ' subjected alkyl thiosulphinates (also deuteriated compounds) to
gas-liquid chromatography on 10% silicone rubber UCW-98 on 80-100 mesh Chromo-
sorb W at 70-75°C and using flame ionization detection. The work was primarily to
obtain mass spectrometric data. Czerwicz and M a r k ~ w s k icarried ~~ out gas-liquid
chromatography of aromatic sulphinylamines on Chromosorb P + 20% Rheoplex at
170 "C, using hydrogen as carrier gas and a heat conductivity detector. Their samples were
phenylsulphinylamine, PhN=S=O, also with 0-,m- and p-methyl and chloro substitu-
ents. The same authors9' later used the same principle to separate the six isomeric
dimethyl-substituted (2,3-,2,4-,2,5-,2,6-,3,4-and 3,5-)phenylsulphinylamines,injecting
10% solutions of the samples in benzene and using hydrogen as carrier gas and flame
ionization detection. Their best results were obtained using Apiezon N on 80-100 mesh
Chromosorb G AW-DMCS at 200°C. Good results were also obtained with the
impregnation polyphenyl ether 0s 138, also at 200°C. Other packings were less
satisfactory. Silar IOC on 80-100 mesh Chromosorb G AW-DMCS yielded a different
elution sequence of the isomers. Czerwicz9* separated the six dichlorophenyl-
sulphinylamines (substituents in the same positions as in the dimethyl compounds
mentioned above) by gas-liquid chromatography, the best column packing being
polyphenyl ether 0s 138 on Chromosorb G at 180 "C;with hydrogen carrier gas and flame
ionization detector.
MacKenzie and F i n l a y ~ o ngas ~ ~chromatographed cysteic and cysteinesulphinic acids
as their N-heptafluorobutyryl isobutyl ester derivatives. Their column was 3% SE-30 on
100-120mesh Chromosorb W HP. It was kept for 2 min at IOO'C, then warmed to 250°C
at 4"/min. Carrier gases were hydrogen, nitrogen and air with detection by flame
ionization.
Gas-liquid chromatography of methyl alkanesulphinates, alkanesulphonates and
dialkyl disulphides was carried out by Toro'" using a column of 10% Ucon on 80- 100
mesh Chromosorb H P. Column temperature was programmed, helium was carrier gas
and detection was by flame photometry.
Kataoka and colleagues'o' determined cysteinesulphinic acid and hypotaurine in
animal tissues by extraction and centrifuging followed by gas-liquid chromatography on
a glass column treated with dichlorosilane and packed with 1% silicone OV-17 + 0.2%
FFAP at 210-250 "C (programmed at 4'jmin) with flame ionization detection.

4. High performance liquid chromatography (HPLC)

Some work has been done recently on separating diastereoisomers with the help of
HPLC. Thus Szokan and colleagues'02 studied diastereoisomeric sulphoxides and
102 M. R. F. Ashworth
sulphilimines of the general formula

R’CHS=NSO~TOS
I \

R4
where R’ was hexyl or ethyl and R 2 was COOH or H. They used a column of either
Hypersil (Shandon GB) or Chromsfer-Sil (Budapest Labor-MIM), eluting with various
mixtures of diethyl ether, pentane, methanol and acetic acid. The best eluent for the
Chromsfer column proved to be diethyl ether- pentane (80 + 20) and, for the Hypersil
column, diethyl ether plus a few percent methanol. Detection was in the ultraviolet at
254 nm. In later work, Jalsovszky and coworkerslo3 investigated diastereoisomers of
cyclic sulphilimines, e.g.

containing alkyl group substituents in the nucleus. They used Hypersil columns, eluting
with mixtures of diethyl ether, pentane, methanol, 952, ethanol and 2-octanol and
detecting also at 254 nm. Wainer and colleagues’04 used HPLC to separate several classes
of compound, including sulphinamides, R’SONHR’ and sulphilimines, R3MeS=NR4
where R’ wasp-tolyl or phenyl, RZwas methyl or 2-pyridy1, R3 was phenyl or butyl and R4
was -COPh or -Tos, respectively. [They also worked with sulphoximines, but these
contain S(V1) and d o not qualify for inclusion here.] Their chiral stationary phase was a
cellulose triphenyl carbamate coated onto a macroporous silanized silica gel (ca 20-237,
by weight). The mobile phase was hexane modified with an alcohol, or an alcohol
+ acetonitrile; detection was again at 254 nm.
5. / o n exchange Chromatography
De Marco and coworkers’o5 separated sulphur-containing amino acids, e.g. cysteic and
cysteinesulphinic acids, taurine and hypotaurine, on the ion exchange resin 150A, the
elution agent being citric acid-sodium chloride. Identification was through light
absorption at 440 or 570nm after colour reaction with ninhydrin. Lombardini and
colleagueslo6 separated cysteinesulphinic acid from other enzymatic oxidation products
of L-cysteine on Dowex-50 at pH 2, ultimately forming and assaying the coloured product
with ninhydrin. Purdie and Hanafi’” separated sulphinic and sulphonic acid derivatives
of cysteine and glutathione on Dowex 1x8 anion exchange resin, improved by adding
Sephadex G 10 in the ratio 8:5, with the help of a monochloroacetate gradient.
Another example of ion exchange chromatography is the work of Williams’08, who
separated some organic sulphur-containing compounds, including aromatic sulphinates.
His stationary phase was a monolayer ofaminated latex beads, agglomerated to a styrene-
divinylbenzene (S/DVB) resin. The eluent system was an ordinary hydrogen
carbonate/carbonate one, especially satisfactory being 0.001 M sodium hydrogen carbo-
nate. Detection was by conductivity or, with aromatic compounds, also light absorption in
 4. Analytical methods 103
the ultraviolet. He was able to separate benzene- and p-toluenesulphinic acids. Ida and
KuriyamaLo9determined cysteic and cysteinesulphinic acids in rat brain on the strongly
basic anion exchanger ISA-O7/S 504. They detected by reacting with o-phthalaldehqde in
the presence of 2-mercaptoethanol to give fluorescent products.

C. Spectroscopy
Spectroscopic determination or detection of sulphinic acids or their derivatives have not
generally been the subject of special publications. Mostly they have been a tool in a
particular study. The best example of this is the monitoring of light absorption in the
ultraviolet.
t ~ ~ UV measurements to determine thiourea dioxide and
W o j t a s i e ~ i c z - O b r z u used
thiourea at 269 and 239 nm, where each has its respective absorption maximum. Mori and
Ueda' l o recorded the infrared spectra of 25 aromatic sulphinamides in chloroform
solution and in potassium bromide discs. They found characteristic bands at 1070 cm - '
(solution) and 1056cm-' (disc), both of which have undoubtedly found unpublished use
for identification. Freeman and McBreen" determined thiosulphinate in onions by
extraction of the juice plus water with hexane and measuring the ultraviolet absorption at
254nm. In a study of the reaction of organic sulphides with chloramine T, Ruff and
Kucsmango determined sulphimides (and sulphoxides) through their absorption at
286 nm.

IV. MICROBIOLOGICAL METHODS

Some microbiological assays of amino acids containing the sulphinic acid group may be
mentioned briefly. Leinweber and Monty' I determined cysteinesulphinic acid by
reaction with z-ketoglutarate in the presence of highly purified glutamic-oxaloacetic acid
transaminase. This gave sulphite, SO:-, which they determined colorimetrically with the
Schiff reaction using fuchsine (rosaniline).
Lombardini and coworkers106 studied the enzymatic oxygenation of L-cysteine to
L-cysteinesulphinic acid. They assayed this acid in two ways: (a) separation through ion
exchange chromatography (Section III.B.5) followed by colour reaction with ninhydrin
and colorimetric evaluation; (b) by transamination with a-ketoglutarate, followed by
desulphination using lactate dehydrogenase, yielding pyruvate which they determined
spectrophotometrically.
Baba and colleagues' l 3 assayed cysteinesulphinic acid by enzymatic conversion to
lactate, ultimately transforming NAD into NADH, the light absorption of which was
found to be proportional to the concentration of the cysteinesulphinic acid.
Soda and coworkers' l 4 degraded hypotaurine with an aminotransferase, leading to
sulphino-acetaldehyde which decomposed spontaneously into acetaldehyde and sulphur
dioxide. The former was assayed via aldehyde dehydrogenase, and the latter, more
relevant to a sulphinic acid determination, through the Schiff reaction.

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