Table Of ContentThis book is dedicated to
PROFESSOR M. A. MaCCONAILL, M.A., M.B., D.SC, M.R.I.Α.,
of University College, Cork
Anatomist, Chemist, Mathematician, Linguist, Soldier and Poet—
but, above all, a dear friend
SYNTHETIC DYES
in
Biology, Medicine and Chemistry
EDWARD GURR
1971
®
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PREFACE
This book treats of dyes as coloured organic chemical reagents, not as mere
agents for imparting colour. It is intended as a guide in the selection of dyes
for special purposes in biology, medicine, chemistry and allied fields. The
information contained herein is of the kind which, in my experience as a
consultant, is most frequently required by histologists and cytologists and
other workers in the field of biology and medicine as well as chemists and
certain other scientists, whether they be engaged in industry or in the academic
sphere.
The dyes described in this book are arranged in 17 different classes according
to the nature of their salt-forming sidechains ("Colligators"). Each class is
arranged with its members placed in order of ionic or molecular weight.
Colligators, ionic and/or molecular weights of dyes are important factors to
be taken into consideration in the selection of dyes for special purposes in
biology, medicine, chemistry and even in certain industrial processes.
The known uses of each dye are stated, together with references to original
papers. The spectral curve, together with structural and empirical formulae,
ionic and/or molecular weight of each individual dye, is given as well as its
relative solubilities in 15 different solvents.
This book is not intended for the textile dyer. Its emphasis is mainly on
biology, although it is hoped that it will be equally useful as a guide and a work
of reference in a number of other spheres of science and industry.
This work was begun in April 1965. As in the case of all my previous works,
the idea presented itself to me as a challenge. This book, I felt, was needed.
Also, like all its predecessors, it began as a pleasure and ended as an obsession.
I am grateful to my wife, Mrs. F. P. Gurr, Β.Sc., for her patience and for-
bearance; for constructive criticism, moral support and co-operation.
Professor M. A. MacConaill, M.R.I.Α., inspired the "chemical" classifica-
tion of dyes used in this book. I am grateful to him and count it an honour
to be a member of his academic staff.
I consider myself fortunate to have Col. T. Ryczel, O.B.E., as my research
assistant. He has been a source of considerable assistance to me in the com-
pilation of statistics.
Acknowledgements are also due to Mr. J. E. Jones, Mr. B. F. White,
Mrs. Pegler, Miss Lynda Eastwood and Mrs. D. Gray for relieving me of
ν
vi PREFACE
day-to-day duties and responsibilities, thereby making it possible for me to
find the time to carry out the work involved in producing this book.
Finally it should be mentioned that the original aim of this book, since
extended, was to supplement three previous works of mine, namely: Encyclo-
paedia of Microscopic Stains (1960), Staining, Practical & Theoretical
(1962) and Rational Use of Dyes in Biology (1965). I believe that biologists
will be interested in all the dyes described herein, though many of these are
at present unknown in the field of micro-technique.
Last but not least, I wish to place on record my appreciation of the speed
and efficiency with which the publishers have produced this book. Together
with the printer they are to be congratulated for their accurate and helpful
interpretation of my MS, which Was by no means a model of neatness.
EDWARD GURR
Michrome Laboratories
East Sheen, London, England
November 1970
PART 1
Introduction
HISTORICAL
The first artificial dye, picric acid, was prepared by Woulfe (1771) from the
natural pigment, indigo. It was not, however, produced commercially until
nearly one hundred years later following the work of Schmitt and Glutz who
synthesized it from phenol sulphonic acid.
The second artificial dye, rosolic acid, was discovered by Runge (1834).
It was not, however, produced commercially until around 1860 following
the work of Kolbe and Schmitt (1859).
Meanwhile a young English chemist, William Henry Perkin, discovered the
third artificial dye, mauveine, in 1856 and founded the synthetic dye-making
industry. A specimen of the actual sample synthesized by Perkin himself in
that year is still in existence today in the Science Museum, South Kensington,
London. The writer was recently given the opportunity of examining a
minute sample of this historic dye for the purpose of recording its spectral
curve which is reproduced in this book (pp. 123-124).
As Venkataraman (1952) states, it was during the course of attempts to
synthesize quinine by the addition of oxygen to allyltoluidine that Perkin
studied the action of potassium dichromate on aniline sulphate, and from the
very unpromising-looking black product he isolated a small amount of
mauve colouring matter which dyed silk directly. He had the vision to foresee
immediately the tremendous importance of his discovery and, overcoming
all obstacles that stood in his way, he built in 1857 a factory for the manu-
facture of this dye. His essential achievements were, in the first place, the
transformation of a casual laboratory observation into manufacturing prac-
tice and secondly his success in demonstrating the value of his product to the
dyer and printer.
For a detailed account of the early history of the synthetic dyestuifs in-
dustry readers are referred to Welham (1963a, b, c, d).
ANATOMY OF DYE MOLECULES
With few exceptions all synthetic dyes are aromatic organic compounds.
They may be divided into three main groups : non-ionic, anionic and cationic.
2 SYNTHETIC DYES
The molecules of ionic (anionic or cationic) dyes are composed of two
main parts, one of which is a complex aryl radicle. This is the colour-
imparting ion. If the balance of the charge on the latter is negative then the
dye is classed as anionic. On the other hand, if the balance of the charge on
this ion is positive then the dye is classed as cationic.
The second part of an ionic dye molecule is an inorganic ion (or an aliphatic
organic ion or, in a few cases, an aryl ion) of opposite charge to that of the
colour-imparting aryl ion. The former is sometimes called the gegen-ion, and
the latter the dye-ion.
The function of the gegen-ion is to balance the charge on the dye-ion and
to render the dye soluble in water.
With few exceptions anionic dyes are manufactured as metallic salts. The
vast majority of anionic dyes are sodium salts of sulphonic acids. Some, how-
ever, are sodium salts of carboxylic acids, uranine being a notable example.
In the case of cationic dyes, the gegen-ion is usually the chloride ion.
In the vast majority of cases the gegen-ion could be replaced by another of
the same charge without seriously affecting the functioning of the dye. There
might, however, be differences in solubility. For example, a calcium salt
might be less soluble in water than a sodium salt. Similarly a sulphate might
be less soluble in water than a chloride. And obviously, when dyes are used as
reagents for the detection of certain ions, due consideration has to be given
to the gegen-ion of the dyes. However, unless the gegen-ion itself is coloured,
it has no influence whatever upon the colour of the dye in aqueous solution.
For example, pararosaniline chloride or sulphate or acetate exhibit exactly
the same colour in solution.
On the other hand, the dye-ion cannot be replaced by another without
profound changes in the nature and functioning of the dye-molecule. The
individual chemical, physical and tinctorial characteristics of a dye are due
to its dye-ions.
The simple gegen-ion of a dye could be replaced by another dye-ion of the
same charge as the original gegen-ion and, of course, of opposite charge to
that of the first dye-ion. This would result in the formation of a polychrome
(if the interacting ions were of contrasting colour) compound dye. Such dyes
are prepared for special purposes. Their molecules consist of an electro-
positively charged dye-ion and a negatively charged dye-ion. Dyes of this
kind are insoluble in water but soluble in absolute and aqueous alcohol.
Partial dissociation takes place in the latter. Such dyes are used in biological
microtechnique for the differential staining of acidic and basic elements of
cells and tissues (see Gurr, 1962, 1965).
The Colour Index gives the structures of about 2100 anionic dyes and
classifies them variously as "acid", "basic", "direct" and "mordant" accord-
ing to their manner of usage in the textile-dyeing industry.
INTRODUCTION 3
About 1400 of these anionic dyes are amino or imino amino acids. The
remainder are wholly acid in that they have no basic side-chains.
The term "acid" dye has a special meaning in the textile-dyeing industry.
By definition an "acid" dye is one which dyes wool from a dyebath which
contains acid. Such dyes now, however, find their main application not only
for wool but for silk, polyamide, acrylic and regenerated protein fibres. They
are applied from dyebaths containing sulphuric or formic or acetic acid or
ammonium sulphate; sometimes from a neutral and occasionally from a
slightly alkaline bath. The pH of the dyebaths may vary from 2-0 to 8-0.
The "acid" dyes are also used in textile printing. They are not necessarily
acid in reaction. Some basic (cationic) dyes are classified as "acid" by the
textile dyer on account of the fact that he sometimes uses them in acid
solution for wool. However, all but a few dyes classed as "acid" by the textile-
dyer could also be classed under that heading by the chemist since their
dye-ions are negatively charged. From a standpoint of chemical constitution
it is more rational to refer to these as "anionic" rather than as "acid" dyes.
The textile-dyer is understandably more concerned with the manner of usage
of the dye for the aesthetic colouring of fibres and fabrics than he is with its
molecular structure and chemical constitution. On the other hand, the
research chemist, the biologist and other scientists and technologists are
more often than not just as interested and concerned with the structural and
chemical constitution of the dye as he is in the object and the manner of its
use.
It is well known, of course, that dyes are used in chromatography and
electrophoresis for the identification of proteins. There is apparently a need
for a greater variety of dyes for this purpose. The ideal dyes are apparently
those which will stain proteins but not cellulose. It is not possible to find
such dyes except by trial and error, which can be a time-consuming process.
Time can be saved, however, by restricting the experimental work in this
connexion to those dyes known to the textile-dyer as "acid" dyes. In the
following pages dyes of this class have been noted for the benefit of readers
interested in the selection of new dyes as protein stains in chromatography
and electrophoresis. The information regarding the relative affinities of these
dyes for protein and cellulose material has been gleaned by the writer from
the Colour Index. The relative affinities mentioned are those which obtain
when mixtures of protein, cellulose and other fibres are immersed together in
the same dyebath, each class of fibre being in competition with the other
classes for the dye. These conditions differ to some extent from those obtain-
ing in chromatography and electrophoresis. Nevertheless the results serve as
a guide in the selection of the more promising dyes for trial as protein stains
in chromatography, electrophoresis and biological microtechnique.
According to the Colour Index "direct" dyes were originally designed and
4 SYNTHETIC DYES
marketed for the primary purpose of dyeing cellulosic fibres, such dyes having
been defined as "Anionic dyes having affinity for cellulosic fibres when
applied from an aqueous dyebath containing an electrolyte". Direct dyes
provide the simplest means of colouring cellulosic materials as they are nor-
mally applied, in the textile-dyeing industry, from a neutral or slightly alka-
line dyebath, at or near boiling point, to which sodium chloride or sodium
sulphate is added.
All direct dyes are anionic. Whilst a few are wholly acidic the majority
may be regarded as amphoteric since they possess side-chains which are
basic in reaction. Wholly acid direct dyes are classed in this book as be-
longing to Group 3, Sub-group 1, denoted (3 : 1 : 1 to 3 : 1 : 6). Similarly,
amphoteric direct dyes are classed as belonging to Group 3, Sub-group 3,
denoted (3 : 3 : 1 to 3 : 3 : 3). Direct dyes of either type can readily be picked
out by reference to the tables (pp. 713-744).
It is among the direct dyes of Group (3 : 3) that the biologist is most likely
to find single dyes which will impart differential coloration to acidic and
basic elements of cells and tissues. This class of dyes also presents a pro-
mising source for chemists interested in finding new acid-base indicators.
The biochemist interested in finding new protein stains for use in chroma-
tography and electrophoresis, however, would be wasting his time by testing
dyes of this class.
Venkataraman describes "mordant" dyes as having the property of com-
bining with metallic oxides and salts to form metallic complexes. Dyes of this
class are, however, very difficult to define. The Colour Index states that the
make-up of this class is not a matter of strict definition but of a convention
which has grown during the years and which has no logical basis. The
majority of these dyes are used with the aid of mordants, in the textile
industry, mainly for dyeing wool, the secondary application being for the
dyeing of silk and nylon and for printing on cellulosic, silk and wool
fabrics.
It is amongst the mordant dyes that the chemist is most likely to find new
colorimetric reagents for the detection of polyvalent metals. He is also
likely to find temperature indicators and new acid-base indicators.
The Colour Index defines a basic dye as a cationic dye characterized by its
substantivity for tannin-mordanted cotton. Some basic dyes, as already
mentioned, could be classed as "acid" (in the textile-dyer's terminology)
since they are used in an acid bath for dyeing wool and silk (protein) fibres.
In this book basic dyes are classed as "cationic" since their dye ions are
positively charged. Amongst this class the chemist is likely to find new acid-
base indicators. This class is also likely to provide new protein stains for use
in chromatography and electrophoresis since some of its members will stain
protein fibres, but not cellulose, from an acid bath.
INTRODUCTION 5
Dyes of the "acid", "mordant", "direct", "basic" and other usage classes
are noted in the following pages for the convenience of the scientist and
technologist in his selections of dyes for trial as tools of research and pro-
duction.
A cationic dye is one in which the balance of the charge on the dye-ion is
positive. Such dyes can exist as free aromatic organic bases which are used
mainly for colouring lipid substances such as fats, oils and waxes although
they have other industrial and scientific applications. However, cationic
dyes are usually produced and marketed as salts of these aryl bases. Although
cationic dyes are described as "basic", many of them, dissolved in distilled or
deionized water, produce solutions which are very acid in reaction. For
example a 1% solution of methylene blue in deionized or distilled water
(previously adjusted to pH 7-0 with alkali) has a pH of 2-5 at 20°C. On the
other hand a 1 % solution of methylene violet under the same conditions has
a pH of 9*25; whilst another cationic dye, acriflavine, has a pH of 1-35. In-
cidentally ordinary distilled or deionized water as used in most laboratories
has a pH of around 5-4-6-0 (at normal room temperatures) not pH 7-0 as at
one time commonly believed: the effect of heat is to raise the pH, whilst the
withdrawal of heat results in a lowering of the pH (Gurr, 1962; 1965). Many
dyes are sensitive to slight changes of pH and readily undergo very pro-
nounced colour changes in dilute solution as a consequence.
Up to the present time it appears that only about two hundred cationic
dyes have been produced commercially and less than one hundred are still
commercially available. However, some newly invented cationic dyes have
recently become commercially available, although their structural formulae
have not yet been disclosed. Of the cationic dyes whose structures are given
in the Colour Index all but about six are wholly basic in character. That is to
say, they are positively charged and have no side-chains which might be
regarded as acidic in character.
A non-ionic dye molecule consists of three main parts, namely : the chro-
mogen, the chromophore(s) and the auxochrome(s).
A chromogen is an uncharged aryl substance which is coloured by virtue
of the fact that it contains a chromophore as part of its structure.
A chromophore is a configuration which has a group containing one or
more multiple bonds. The chromophore(s) is responsible for the chromogen
being coloured. It does not, however, determine the particular shade of
colour. This is evident from the fact that members of various groups of
dyes having identical chromogen/chromophore configurations are by no
means all of the same colour or shade of colour. A few chromogen/chromo-
phoric configurations do, however, appear to determine within certain narrow
limits the colour of the substances containing them. The acridine group of
dyes, for example, are all either yellow or orange. Members of this group
