Table Of ContentAnalytical Methods
for Glycerol
M. R. F. Ashworth
Organische und Instrumentelle Analytik
Universitat des Saarlandes
66 Saarbriicken, Germany
Edited and with a final chapter by A. A. Newman
1 9 7 9
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Preface
Arguably, glycerol is one of the most important compounds in the list of
useful chemicals, and its world production is between 300,000 and 400,000
tonnes per annum. It is a major technical raw material with literally hundreds
of applications. Direct uses are, for example, as a humectant in tobacco;
a component of therapeutic and cosmetic preparations; a preservative for
foodstuffs; a solvent for dyes; a plasticiser for cellophane; an antifreeze
agent. It is also used to prepare esters, including: "monostearate", as an emulsi-
fying agent, employed with foodstuffs such as margarine and ice cream;
triacetate, "acetin", used as a solvent, e.g. for film; trinitrate, "nitro-
glycerine", used both medicinally and as an explosive; glycerol dichloro-
hydrins, as intermediates in the preparation of epichlorohydrin; glycero-
phosphate salts, used as tonics. Further, it plays an absolutely vital role in
the biochemistry of most living organisms, and is listed in the pharmacopoeas
of all nations.
It was felt that these varied and extensive uses justified an attempt to com-
pile in detail a work devoted to the detection, identification, determination
and separation of glycerol in analytical procedures. The chemical and physical
information is concentrated in Chapter 1. Analytical work on the important
naturally occurring and synthetic materials containing combined glycerol
is treated in Chapters 2, 3 and 4, always trying to keep the glycerol moiety
in mind. Chapter 5 deals with the analysis of glycerol samples, especially
from the point of view of pharmacopoiea specifications. The important
methods for enzymic determination of glycerol are given in Chapter 6. In
this virtually complete survey of the available analytical methods, it should
be possible to find a procedure best suited to any particular situation.
The literature cited in this volume covers the period up to the end of 1976.
M. R. F. Ashworth
A. A. Newman
1
Glycerol
1.1. O X I D A T I O N M E T H O D S
Glycerol can be oxidised to many products, e.g. glyceraldehyde, glyceric
acid, tartronic acid, dihydroxyacetone, mesoxalic acid, formic acid, and
formaldehyde, and of course carbon dioxide and water. Some of these
oxidations are stoichiometrically uniform, well-defined, and controllable
and are thus adaptable to analytical, especially quantitative, aims.
Analytical information is classified below in alphabetical order of reagent.
1.1.1. Br o m i n e
Bromine is a classical reagent, yielding a mixture of glyceraldehyde and
dihydroxyacetone, known as glycerose. Dihydroxyacetone can be dehydrated
with concentrated sulphuric acid to methylglyoxal:
C H 2 O H
I
CHOH
l
C H 2 O H CRO
C HOH ° > +
C H 2 O H CH2OH C H 3
' - H , 0 I
c=o — ^ c = o
I I
C H 2 O H CHO
These carbonyl compounds yield colours with various reagents, pre-
l
2 GLYCEROL 1.1
dominantly phenols, which are the bases of detection and quantitative
determinations.
Deniges (1909, 1910) tested for glycerol by heating 0-1 g or less of sample
with 10 ml of 0*3 % bromine water for 20 min, then boiling out excess bromine
and showing that the product(s) gave colours with reagents such as codeine,
salicylic acid, resorcinol, thymol, and (3-naphthol; the first two were the most
sensitive reagents. Others basing analytical methods on the blue colour
yielded with codeine have been: de Coquet (1928), for wine glycerol, who
also heated the sample for 20 min with bromine water on the boiling water
bath and then added 10% alcoholic codeine solution and finally concen-
trated sulphuric acid; Helweg-Mikkelsen (1948), with a similar procedure,
boiling off the excess of bromine for 5 min and then treating an aliquot with
a solution of 0-5 g of codeine in 10 ml of 96% alcohol and sulphuric acid;
and, in a later publication (1949), using bromide-bromate-2N-sulphuric
acid so as to give 2-5-4 mol of bromine per mol of glycerol. Ka (1940) tested
the method and claimed that a bromine oxidation period of 25 min and a
colour development of 20-25 min were best; excess of bromine water
appeared to make no difference.
Resorcinol has been a popular reagent for demonstrating the presence of
the carbonyl compounds derived from glycerol by reaction with bromine.
For example, Ohl (1938) detected glycerol in textile sizing agents through
the blood-red colour yielded by heating an extract with bromine water until
colourless and then mixing the product with 5% alcoholic resorcinol and
concentrated sulphuric acid. Cunha (1939) employed a similar procedure to
estimate glycerol in wines. Jones (1947) detected glycerol in preservatives by
oxidising with bromine water, boiling off the excess of reagent, cooling, and
adding concentrated sulphuric acid and then 0-5% resorcinol to give a
wine-red colour. Weigel (1955) also based determinations of glycerol (as a
measure of the lipid fraction of neutral fats, lecithin, and cephalin) on the
bromine oxidation-resorcinol reaction, quoting, however, an orange to
violet product. Javicoli and Mattei (1956) detected and determined glycerol
in faeces according to the same principle.
Pyrocatechol as final component was used by Ghimicescu (1935) for
micro-colorimetric determination of glycerol in wine. He oxidised with
bromine water in a sealed tube, removed the excess of bromine with zinc,
and ultimately treated an aliquot of the resulting solution with 5% pyro-
catechol solution and concentrated sulphuric acid, heating for 5 min on the
water bath for colour development. Ghimicescu et al. (1963) evidently
slightly modified this procedure for micro-determination in the presence of
sugar and tartaric acid. After the bromination, Arreguine (1936) added
concentrated sulphuric acid and 1 % alcoholic veratrole (which presumably
functions like pyrocatechol, of which it is the dimethyl ether).
1.1 OXIDATION METHODS 3
Thomas and Micsa (1924) described tests for polyalcohols based on
heating with bromine water, then removing the excess of bromine in a
current of air and finally adding a solution of 2-hydroxynaphthalene-3,6-
disulphonic acid in concentrated sulphuric acid; glycerol gave a greenish-
blue colour with a yellow ring. Salzer and Weber (1950) gave this test among
others for polyalcohols; they also quoted the use of guaiacol as phenol
component.
Fiirst (1948a) used a reagent of 2,7-dihydroxynaphthalene in concentrated
sulphuric acid as phenol reagent, obtaining a reddish-violet colour; and
Bonino (1952) recorded a violet product using Bertrand's orcinol reagent
after bromine oxidation of glycerol.
de Prada (1934) employed carbazole-sulphuric acid as final reagent in a
colorimetric micro-determination of glycerol in beverages.
Conclusions other than observation or estimation of colour appear to be
extremely rare. Juhlin (1938) determined glycerol in aqueous solutions by
treating a sample containing 20-40 mg (neutralised to Methyl Orange) with
10 ml of 0-1 % bromine water for 15 min. He then determined the unreacted
bromine by adding 10ml of 10% potassium iodide and 50-100 ml of water
and titrating liberated iodine with thiosulphate. One molecule of bromine
evidently reacted to yield one molecule of dihydroxyacetone.
Bacila (1949) detected glycerol by oxidation with bromine water to
dihydroxyacetone, conversion of this into methylglyoxal with sulphuric acid,
distillation of the methylglyoxal, and reaction of it with iodine-alkali to give
iodoform:
C H 3 - C O — C H O + 3I 2 - • C I 3 — C O — C H O + 3HI( + OH~)
CI 3 — C O—CHO + O H " CHI3 + CHO—COO"
1.1.2. C e r i u m ( I V )
Cuthill and Atkins (1938) seem to have been the first to apply cerium(IV)
oxidation to glycerol determination. They refluxed the sample for 1 h with
eerie sulphate in acid solution and back-titrated unused reagent with ferrous
iron to Xylene Cyanol FF indicator. They stated that 1 mol of glycerine
reacted with 8 mol of cerium(IV) and presumed that tartronic acid was the
end-product of oxidation. Fulmer et al (1940) used this principle of eerie
sulphate oxidation for estimating glycerol in fermentation media in the
presence of dextrose, back-titrating with ferrous iron to erioglaucin or
phenanthroline, and determining the glucose through a separate titration
with copper(II). Mull (1943) likewise determined glycerol and pentoses
together by oxidation with excess eerie sulphate in sulphuric acid solution
(for 45 min on the boiling water bath) and back-titrating with Mohr salt to
4 GLYCEROL 1.1
o-phenanthroline, and determined pentose alone through copper(II) titra-
tion. More recently, Rao and Gopala Rao (1972) determined glycerol (also
ethylene glycol or mannitol) by 45 min heating at 50-60°C with a 1 -5-2-fold
excess of ammonium hexanitratocerate, ( N H 4 ) 2 [ C e ( N 0 3 ) 6 ] , in 0 5 N nitric
acid and back-titrating with iron(II) to ferroin after adding sulphuric acid.
Smith and Duke (1941) studied the reaction and its stoichiometry, suggest-
ing a procedure with excess ammonium hexanitratocerate in the presence of
perchloric acid. They heated for 15 min at 50°C (not more than 60°C) and
back-titrated with standard sodium oxalate to nitroferroin. In the presence
of sulphuric acid instead of perchloric acid, temperatures of 90-100°C were
necessary. They reported also a 1:8 reaction stoichiometry with formic
acid as end-product, according to:
C H 2 O H
I
4 + + 3 +
CHOH + 3 H 2 0 + 8 C e - > 3 H C O O H + 8H + 8Ce
I
C H 2 O H
In later work (1943) they discussed reaction mechanisms for the cerium(IV)
oxidation of glycerol and other polyols, comparing with periodate oxidation.
Silverman (1947) determined glycerol in soap, employing oxidation for
12-13 min at 50°C with cerium(IV) in perchloric acid solution (18% in the
final solution) and also back-titrating with oxalate to nitroferroin. Guardia
(1950) reviewed the use of eerie perchlorate in determinations of many
materials, including crude glycerol, based on the work of Smith and co-
workers.
Other investigations or reviews have been made by Michalski and Stapor
(1966) who compared cerium(IV) oxidation of alcohols (methanol, ethanol,
ethylene glycol, glycerol, etc.) with permanganate, dichromate, and periodate
oxidations, and by.Misantone (1966) who recommended back titration with
ferrous iron to o-phenanthroline and found cerium(IV) to be superior to
permanganate for glycerol and other materials.
Guilbault and McCurdy (1961) studied catalysis by silver(I)-manganese(II)
of the oxidation of polyols with cerium(IV) and were able to suggest a much
faster procedure. They heated a 3-25 mg sample with reagent in 2 0 - 6 5 %
excess + catalyst + 72% perchloric acid for 3-5 min at 95°C until the
solution was red (from permanganate). The solution was then cooled
immediately, 6F sulphuric acid added, and they back-titrated with standard
iron(II) to ferroin.
Gordon (1951) determined unused cerium(IV) by estimating the residual
amount of an oxidisable triphenylmethane dye which it destroyed. He
1.1 OXIDATION METHODS 5
accomplished this by measuring the diminution in light absorbance of the
dye (e.g. at 628 nm for Fast Green FCF). His procedure was empirical,
requiring standard conditions because of the competition between two
reactions, the one quoted above and another with only four cerium(lV)
molecules:
C H 2 O H
4 + 3 + +
CHOH + H 2 0 + 4 C e - ^ 2 H C H O + HCOOH + 4 C e + 4H
I
C H 2 O H
For glycerol, Gordon oxidised 1-10 jig amounts for 15 min at 165°C.
Sharma and Mehrotra (1955) analysed glycerol-ethylene glycol mixtures
by using two oxidation procedures with cerium(IV). In one, formic acid was
the end-product (30 min heating), and in the other, carbon dioxide and
water (70 min, in presence of additional sulphuric acid and a drop of 1 %
chromium(III) sulphate catalyst). Khan and Bose (1969) also touch on the
subject of formic acid decomposition by more drastic treatment. They
carried out two oxidations with ammonium hexanitratocerate in perchloric
acid at 10°C in sun or ultraviolet light for 3h or 90 min, respectively, with
reagent excesses of 100 and 1000%. They determined unused reagent
iodometrically. This enabled them to determine compounds that yield
formic acid on oxidation in the dark (e.g. glycerol, also ethylene glycol and
methanol) in the presence of those that do not (e.g. ethanol or benzyl
alcohol) by employing two sets of oxidation conditions, one with and one
without photochemical decomposition.
Sand and Huber (1967) were able to titrate directly 0-5-5 mg amounts of
glycerol in aqueous solution 2F in perchloric acid, using ammonium
hexanitratocerate. They employed constant-current (100 |iA) potentiometry
at 80°C (70°C was too low).
Knappe et al (1964) detected glycerol and other polyols in TLC by
spraying with ammonium hexanitratocerate reagent in nitric acid, +
Is ,iV-dimethylphenylenediamine reagent (1 + 10) or + N,N,N',AT-tetra-
methyl-4,4'-diaminodiphenylmethane (tetrabase) reagent (then 1 +1), and
heating at 105°C for 10 or 5 min respectively. This gave white or pale blue
zones on a blue background of oxidation products of the organic bases.
1.1.3. Chloramine T
Afanas'ev (1949) reported the ready reactivity of polyols with chloramine T
at 80-90°C in dilute sulphuric acid, enabling a titrimetric determination to
B
6 GLYCEROL 1.1
be carried out. Glycerol reacted with 7 mol of reagent. Balwant Singh et al
(1953b) published work on determinations of about a dozen compounds,
including glycerol, by oxidation with excess chloramine T in acid solution to
give carbon dioxide. They estimated the unused reagent by adding potassium
iodide and titrating the liberated iodine with thiosulphate:
C H 2 O H
I
CHOH + / 7 A r S 0 2 N C l + 3 H 2 0 - * 3 C 0 2 + 7 A r S 0 2 N H 2 + 7CT
I
CH 2 O H
1.1.4. C h r o m i u m ( V I )
Chromium(VI) as dichromate is one of the great standard oxidising agents
and has been used extensively in quantitative methods for many oxidisable
organic compounds, including glycerol. The comparative ease of use is
neutralised by this rather too ready reaction. Oxidation of glycerol is
generally to carbon dioxide, according to:
C H 2 O H
I
2 + 3 +
3 CHOH + 7 C r 2 0 . ~ + 5 6 H - * 9 C 0 2 + 4 0 H 2 O + 14Cr
I
C H 2 O H
A vast amount of older literature describes the influence of factors such
as the concentrations of the dichromate, sample, and acid (usually sulphuric)
components of the medium, reaction temperature, and time and even order
of mixing of the reaction partners. Summing up, it can be said that it is not
difficult to obtain reliable results on fairly pure aqueous solutions of glycerol
but that impurities and other components of glycerol-containing samples
interfere through their own susceptibility to oxidation. It is not possible here
to do more than give a very brief summary of the basic methods used.
Fairly concentrated sulphuric acid medium is customary. Tortelli and
Ceccherelli (1913, 1914) used a 50% acid reagent, and Kellner (1922, 1924)
stated that the minimum sulphuric acid concentration for complete oxidation
of glycerol was about 32 % (density 1 -23). Reaction periods used to be 2-3 h
with the older macro-methods but more recently 5-20 min appear more
usual.
The earliest investigators, e.g. Legler (1885) and Cross and Bevan (1887),
gravimetrically determined the carbon dioxide product through the increase
in weight of an absorbent. This procedure was used by some later workers,
1.1 OXIDATION METHODS 7
e.g. Fachini (1923) and Pramme (1931) for glycerol in greases after saponifi-
cation. Neale (1926) also determined glycerol in sized cotton materials via
the carbon dioxide but used a gas volumetric method.
In the classical procedure of Hehner (1887, 1889), oxidising agent was used
in measured excess, and the unused was determined after complete reaction.
Hehner oxidised for 2 h on the boiling water bath and then back-titrated with
ferrous ammonium sulphate (Mohr salt) to an external indicator of ferri-
cyanide, responding to the first excess of titrant. Others who back-titrated
with a ferrous reagent include: Richardson and Jaffe (1898); Ferre and Bourges
(1928) and Semichon and Flanzy (1930) for wine glycerol; Fuchs (1942) on
technical products. (These investigators determined unused dichromate by
adding excess ferrous reagent and completing the titration with permanga-
nate); Randa (1937) on soaps and spent lyes; Procter and Gamble Co. (1937);
Launer and Tomimatsu (1953) who made use of the heat of dilution of
sulphuric acid; Karpov (1960) for drugs; Damyanov et al (1970) for spent
lyes, lard, and crude technical glycerol, back-titrating coulometrically with
ferrous iron.
Thivoile and Raveux (1941, 1942) (also Raveux, 1943) oxidised in con-
centrated nitric acid and back-titrated with the less usual ferrocyanide.
Erdey et al. (1955) back-titrated with ascorbic acid to Variamine Blue in their
dichromate oxidation procedure for numerous compounds, including
glycerol.
The other standard procedure for determining unused dichromate is to
add potassium iodide and titrate the liberated iodine with thiosulphate.
This was used by, for example, Steinfels (1910, 1915); Hoyt and Pemberton
(1922) in the presence of sugar; Bennett (1924); and Tschirch (1951) in a
semimicro adaptation of the Steinfels method.
Colorimetric and spectrophotometric procedures were introduced later.
These are based on the colours and light absorbance of unused yellow
dichromate and of green chromium(III) product. Johnson and Ladyn (1944)
determined glycerol in kettle soap by boiling for 2 min with excess dichromate
and 50% sulphuric acid and comparing the colour of the solution with those
of standards from known amounts of glycerol. Englis and Wollerman (1952)
oxidised for 20 min on the boiling water bath, then determined unreacted
dichromate through the light absorbance at 350 nm of the 50-fold diluted
solution, or alternatively, determined the chromium(III) through absorbance
measurements at 587 nm of the solution 4-4N in sulphuric acid. Almost at
the same time, Cardone and Compton (1952) also carried out absorbance
measurements of unused dichromate from oxidations, including that of
glycerol, at 349 nm and studied the effect of chromium(III) and sulphuric and
phosphoric acids on the values. For glycerol, they tested oxidation periods
at 100°C of 30, 60, and 120 min, finding that 30 min gave complete recovery.
