Analytical 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 ACADEMIC PRESS London New York San Francisco A Subsidiary of Harcourt Brace Jovanovich, Publishers ACADEMIC PRESS INC. ( LONDO N ) LTD 24/28 Oval Road London NW1 United States edition published by ACADEMIC PRESS INC. I l l Fifth Avenue New York, New York 10003 Copyright © 1979 by ACADEMIC PRESS INC. (LONDON) LTD All Rights Reserved N o part of this book may be reproduced in any form by photostat, microfilm, or any other means, without written permission from the publishers Library of Congress Catalog Card Number: 78-68218 ISBN: 0-12-065050-9 Printed in Great Britain by Page Bros (Norwich) Ltd, Mile Cross Lane, Norwich 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.