Determination of Reactive Hydrogen in Organic Compounds

V O L U M E 2 4 , NO. 9, S E P T E M B E R 1 9 5 2

1425

LITERATURE CITED

Lewis, W. R., Quackenbush, F. M., and DeVriea, T.,

( 1 ) Alison, B., “Polarographic Studies of Lead in Methanol Solution,” unpublished M.S. thesis, Oklahoma A . and M. College, 1950. (2) Bachman, G . B.. and Astle, 11.J., J . Am. Chcm. SOC.,64, 1303 (1442). ~ - _-, - . (3) Ibid., p. 2177. (4) Black, H., “Polarographic Studies in Organic Solvents,” unpublished M.S. thesis, Oklahoma A. and hI. Collerre. - 1948. (5) Gentry, C. H. R., Aroture, 157,479 (1946). (6) Hall, M. E., ANAL. CHEX,23, 1382 (1951). (7) Kolthoff, I. M., and Lingane, J. J., “Polarography,” p. 111, New York, Interscience Publishers. 1941.

.bi.41,.

CHEW,2 1 , 7 6 2 (1949).

Parks, T. D., and Hansen, J. A., Ibid., 22, 1268 (1950). Vitek, V., ColEeetion Csechoslov. Chem. Commum., 7 , 537 11935). Weissberger, A., and Proskauer, E., “Organic S o l v e n t 3 , ” p. 143, London, Oxford Press, 1935. RECEIVED for review February 21, 1952. Accepted Jurtr. 113, 19.52. D a t a taken from a thesis presented b y Harold Lyons in partial fulfillment of t h e requirements for t h e P h . D . degree at Oklahoma A. and M. College, 1931. P a r t s of this material were presented before the Division of Analytical Chemistry a t t h e Seventh Southwest Regional Meeting, Aimtin, rex

Determination of Reactive Hydrogen in Organic Compounds A Review ELIZABETH D. OLLEIIZAN’ Coal Research Laboratory, Carnegie Institute of Technology, Pittsburgh, Pa. Methods for quantitatively determining reactive hydrogen in organic compounds have been reviewed to find the most suitable procedures for estimating the reactive structural units in coal degradation products. In the past, reaction with methylmagnesium iodide or lithium aluminum hydride has been used to determine total reactive hydrogen in organic compounds with an accuracy o f 1 3 to 5%. The types of compounds which have been determined are: alcohols, phenols, enols, mercaptans, acids. amines, amides, and water. Acylation methods. including acetylation, phthaloylation, benzoylation, ETHODS for quantitatively determiningreactive hydrogen in organic compounds-Le., hydrogen attached tonitrogen. oxygen, or sulfur and exceptionally reactive carbon-hydrogen structures-have been reviewed to find the most suitable procedures for estimating the reactive oxygen- and nitrogen-containing structures in coal degradation products. The chemical literature from 1927 to 1949, inclusive, has been thoroughly searched, and some earlier and more recent references have been consulted The literature previous to 1931 was reviewed by Meyer (93). Methyl Grignard reagents and lithium aluminum hydride will react quantitatively R ith reactive hydrogen atoms in organic compounds t o form methane and hydrogen, respectively. The gaa formed in each case is a memure of the “artive hydrogen.” Other reagents have been reported for the determination of active hydrogen but are generally less satisfactory. Some of the active hydrogen atoms can also be replaced b i a(-ylgroups, and compounds containing such hydrogen atoms are said to contain “acylatable hydrogen” or usually “acetylatable hydrogrn.” ““wylatable hydrogen” and ‘ acetylatable hydrogen” are not strictly correct t e i m ~ inaqmuch , as the hydrogen is not acylated but rather replaced by an acyl group. However, these are convenient ternis which distinctly differentiate methods which involve acylation of reactive groups from the methods IT hich determine total “active hydrogen.” Different reagents and conditions cause quantitative acylation of different functional groups, and consequently the terms “acylatable hydrogen” and “acetylatahle hydrogen” have little meaning unless the reagent and conditions are specified. 1

Pa

Present addreas, Verona Research Center Koppers Co , Inc , Verona,

formylation, and stearylation, vary greatly among themselves and give results depending on reagents and conditions. Variations of the most common method-i.e., acetylation using acetic anhydride and pyridine at approximately 100’ C.-have been used to determine primary and secondary alcohols, “unhindered” phenols, and primary and secondary amines. Other acylation reagents may be advantageous if tertiary alcohols or pyrroles must be determined or selectivity of reaction is desired. Applicability of different acylation methods for determining functional groups has been tabulated. The methods of deterniining reactive hydrogen have thus been divided into two major groups-determinations of active hydrogrn and determinations of acylatable hydrogen.

DETERIIIINATION OF ACTIVE HYDROGEX In 1902, Tschugaeff (168) reported that Grignard reagents reacted quantitatively with free hydroxyl groups of acids, alcohols, phenols, and oximes, and when methylmagnesium iodide was used, the amount of hydroxyl could be calculated from the volume of methane gas formed. Shortly thereafter, Hibbert, and Sudhorough (63) and Zerewitinoff (185) published similar methods for the determination of hydroxyl, amine, imine, and amide groups. This analytical technique has been called the Zerewitinoff determination, probably as a result of his continued investigat,ion of this method and the caonipoiind.;; which react with Grignard reagents (186-189). Recently, a new reagent-lithium aluminum h y d r i d e w h i c h is very similar to Grignard reagents in its chemical behavior has heen applied in the determination of active hydrogen. Other reagents have also been suggested. G R I G h i R U R E \GENTS

Reaction. Xethylniagnesium halides react with compounds containing active hydrogen according to the following equation:

RH

+ CH3MgX --+ RMgX + CH, t

Alcoholfi, phenols, mercaptans (thiols), inorganic aa well as carboxylic and sulfonic acids, secondary amines including pyrrole (41), and monosubstituted amides liberate 1 mole of methane;

1426 water and primary amines and amides liberate 2 moles of methane for each functional group. RIonosubstitutedacetylenesreactmole for mole with Grignard reagents (1889, but no analytical values were found reported in the literature. In addition to the compounds which react to form methane, a further group of compounds react with Grignard reagents by addition or coupling. These substances remove reagent from the reaction mixture, but do not liberate gas. Thus, if the total amount of Grignard reagent added in a given determination is known, (1) the amount of active hydrogen can be determined from the methane formed and (2) the amount of other structures reactive to Grignard reagent can be calculated by subtracting the amount of unreacted reagent and the amount used to react with the active hydrogen present from the total amount of reagent added. The amount of unreacted reagent is determined by adding water or aniline to the mixture after the reaction is completed; the amount of methane evolved is a measure of the reagent which remained after the reaction. The types of compounds which react with Grignard reagents without the formation of methane are aldehydes, ketones, alkyl halides, nitriles, and isonitriles, consuming 1 mole of reagent per mole, and esters and acid halides, consuming 2 moles of reagent per mole (150). Organic acids fall into this group also-i.e., besides liberating methane, an additional 2 moles of reagent are consumed. The differentiation b e k e e n these two groups of compounds is not always clear, owing to keto-enol tautomerism of certain carbonyl and hydroxy compounds. For example, the P-diketone, acetylacetone, may show as much as one active hydrogen when treated with methylmagnesium iodide (186). In the other direction, resorcinol, which is conventionally written with two hydroxyl groups, reacts as if it contained one active hydrogen and one ketone group when the reaction is carried out in pyridine ( 7 9 ) , although in anisole as solvent the expected two active hydrogens are found (87). Results vary m-ith the solvent and temperature (see below). Zerewitinoff (188) reported that nitroalkanes indicated 0.65 to 0.97 atom of active hydrogen per mole. The interference of nitro, nitroso, and diazo groups has been investigated by Gilman and Fothergill(S9), who found that compounds such as nitrobenzene show one to two “active hydrogen” atoms when treated with various Grignard reagents, As conclusive evidence that the reaction is an inherent property of the nitrogen-containing group, a later paper (40)reported gas evolution from compounds such as tetranitromethane and pentabromonitrobenzene, which contain no hydrogen atoms at all. Reagents. ?rIethylmagnesium iodide has been most widely used. However, both the biomide and the chloride have given satisfactory results (65, 127, 164). Methylmagnesium bromide was found stable in diisoamyl ether solution for several months. When the bromide was used, the reaction with primary amines was not complete, as the intermediate product was less soluble than with the iodide. Methylmagnesium chloride was less active and not as stable in solution. Butylmagnesium bromide has been suggested to increase the precision in one procedure vhere the liberated alkane gas was burned in oxygen and the resulting carbon dioxide and water were determined gravimetrically (29). I n general, the work described in the following sections was done with methylmagnesium iodide a t concentrations from 0.5 to 1.5 M . Solvents. The Grignard reagent is usually prepared and used in di-n-amyl or diisoamyl ether, although other ethers are sometimes used. The sample can be dissolved in a large variety of Bolvents, depending on its solubility properties. h-o solvent is necessary if the sample is soluble in the reagent solution (74,911. Diethyl, di-n-amyl, diisoamyl, and dibutyl ethers, anisole, anethole, dioxane, pyridine, benzene, xylene, mesitylene, chloroform, dichloroethane, and mixtures of diphenyl ether-xylene, pyridine-xylene, and di-n-amyl ether-pyridine have been S U C C ~ S B -

ANALYTICAL CHEMISTRY fully used. However, the results obtained may vary with the solvent, especially if keto-enol tautomerism is possible. .4 blank determination should be carried out on each solvent used. Several studies of the effect of solvent on the results obtained by Grignard analysis have been made. Lieff, Kright, and Hibbert (86) investigated xylene, diisoamyl ether, pyridine, and dioxane as solvents for a variety of substances. They showed that the results of active hydrogen determination and of other groups reactive to the Grignard reagent are not consistently the same for any two of these solvents. For example, acetophenone showed essentially no active hydrogen when determined in xylene, 12% of one active hydrogen in diisoamyl ether, and 23% in either pyridine or dioxane, while a-diphenylacetophenone showed 2% in diisoamyl ether, 11% in pyridine, and 48% in dioxane. Apparently, pyridine and dioxane both promote enolization. In general, the total amount of reaction (active hydrogen plus other reactive groups) was greater, and closer to theoretical, n ith both pyridine and xylene than with dioxane. Schmitz-Duniont and Hamann (146) used three different solvents-xylene, diphenyl ether-xylene mixture, and pyridinexylene mixture-in analyzing some polymeric indoles and their acyl derivatives, and also obtained results varying with the solvent. However, for many compounds, essentially identical values may be obtained in various solvents. Terent‘ev and Shcherbakova (163), using an apparatus and method (described below) that required a carbon dioside atmosphere, obtained consistently good active hydrogen values (&2% of theory) on acids, amines, and phenols in benzene, xylene, chloroform, and 1,2dichloroethane. Chloroform and 1,2-dichloroethane might be considered unsatisfactory solvents because of their reaction with the reagent. However, these reactions are very s l o ~ and , sincc no gaseous products are formed, they can be used in the determination of active hydrogen but not of other groups which react with Grignard reagents. Some of the differences attributed to enolizing properties of solvents may be concentration effects. Hollyday and Cottle (61) observed increasing active hydrogen from acetophenone as its concentration in the reaction mixture was decreased. Even the lower aliphatic alcohols showed similar effects of concentration, but only when the alcohols !\-ere so concentrated that precipitates formed when the reagent \\-as added. The use of pyridine as a solvent has been a controversial subject. Zerewitinoff (186) observed that dry pyridine formed a precipitate with methyl magnesium iodide in amyl ether and that a considerable amount of gas was formed a t once, and more upon standing or heating. He suggested that pyridine reacted with the excess methyl iodide in the Grignard reagent, producing S-methylpyridinum iodide vhich, in turn, acted n-ith methylmagnesium iodide t o give magnesium iodide and ethane. Eliminating the methyl iodide, he obtained satisfactory results by working quickly and a t room temperature. Rut Tanberg (159) was unable to obtain consistent results with pyridine as solvent. Flaschentrager (32) reported reasonably accurate results from a micromethod using an amyl ether-pyridine mixture, but some blanks were as high as half of the gas evolved from the sample. Later, Marrian and Marrian (91) advised against the use of pyridine for micro work because of the large and variable blanks even when methyl iodide was excluded from the reagent. SchmitzDumont and Hamann (145) operated a t room temperature and plotted the volume of gas against time. The portion of the resulting curve beyond 10 or 15 minutes’ time was straight and represented the continuing slow reaction of pyridine after reaction with the sample was complete, By extrapolating the straight line back to the ordinate asis, the volume of gas due to the sample only was determined. Lehman and Basch (79) reviewed the use of pyridine as a solvent for active hydrogen determination and developed a method for using pyridine a t 90” C. The pyridine and Grignard reagent were first allowed to react together until the initial gas evolution subsided; then a blank determination

V O L U M E 2 4 , NO, 9, S E P T E M B E R 1 9 5 2 was made a t 90Dto determine the amount of continuing gas formation, the determination w a ~run in the same mixture, and finally a second blank was determined. The average of the two blanks was subtracted from the samplevalue. Low results were obtained a t first using a nitrogen atmosphere, apparently due to solubility of the methane in pyridine. When purified methane was used as the inert atmosphere, good results were obtained -e.g., 1.00 active hydrogen for picric acid, 2.01 for hydroquinone, 2.80 for pyrogallol, and 0.98 for phthalimide. Resorcinol and phloroglucinol showed only one active hydrogen each and one and two ketone groups, respectively. The solubility of methane in xylene used for a solvent was observed by Villars (172), who applied a correction factor, calculated from the absorption coefficient, to the observed volumes of methane produced. Time and Temperature. Reaction times used in most cases have ranged froin 5 minutes t o 0.5 hour. An exception is the work of Terent'ev, Shcherbakova, and Kremenskaya ( 1 6 4 ) , in which 2 hours were required when 0.72 N methylmagnesium chloride was used as the reagent, although 0.5 hour was sufficient when 2 h'methylmagnesium bromide was used. The temperature a t which the determination is performed appears to influence markedly the extent of reaction of methylmagnesium iodide with a large number of substances. l l a n y compounds containing active hydrogen give the calculated amount of methane when alloued to react at room temperature. A fev of t h e s e f o r example, some acids (29, 153) and polymeric indoles (146)--gave high results when heated to 50' or 100" C. Roth (133),on the other hand, found that the results wereusually more quantitative a t 95'. Primary amines and amides generally exhibited only one active hydrogen at room temperature, the second one reacting when the mixture ~3a$ heated ( 18 6 ) . Trichloroacetamide is exceptionally reactive and liberated 2 moles of methane a t room temperature. Carbamide and thiocarbamide each liberated 2 moles of methane at room temperature and one more upon heating, but phenylcarbamide, phenylthiocarbamide, and menthonesemicarbazone (HeKCOSHS:R liberated only 2 moles of methane regardless of temperature. Terent'ev and Shor (165) reported two active hydrogens from primary amines even in the cold using their carbon dioxide atmosphere method. Keto-enol tautomerism is also affected by temperature ( 1 8 6 ) . Acetylacetone, bt,nzoylacetone, ethyl acetoacetate, and ethyl malonate each showed one reactive hydrogen a t 100". but a t 20" leqs methane was formed, indicating less of the enol form. Apparently no one temperature is satisfactory for all substances. Some investigators have allowed the reaction to proceed to completion at room temperature, noted the volume of methane, and then raised the temperature to 95" to 100" C. for 5 to 15 minutes (111).

Application and Accuracy. In general, the Grignard method is useful for determining active hydrogen in samples containing OH, S H , and SI1 groups. And n i t h little extra effort. other groups TT hich react with the reagent, but do not produce methane. can be determined in the same analysis. When only one or tn o types of functional groups are present, other more selective methods are usually preferable. Xhen unknown samples are analyzed, results obtained using different solvents and temperatures may provide valuable information. If the results are independent, of solvent and temperature, the active hydrogen-containing structures are limited to relatively simple alcohols, phenols, mercaptan., acids, sccondary amines and amides, and water. TVidely disagreeing values may indicate primary amines or amides, keto-enol tautomerism, or structures that react slowly. Qualitative knonledge of the sample \$ill then aid in interpretation. Although several investigators have reported results within 1 to 2% of the theoretical values, in general reproducibility and accuracy arr probably =k3 t o 5%. Hon-ever, tertiary alcohols

1421 give high results due to partial dehydration and reaction with the liberated water. Apparatus and Methods. The apparatus used for the determination of active hydrogen have been largely of a general type, but there are seveiai variations worthy of note. EARLY.The original work of Tschugaeff ( 1 6 8 ) was carried out in diethyl ether in an air atmosphere, and the methane was collected over mercury in a Xnop's nitrometer. Correction was made for the vapor pressure of the ether. For about 20 years, succeeding investigators used the same type of system but with other less volatile solvents and usually a nitrogen atmosphere. A41thoughmore complex setups have been used generally since then, Uraude and Stern ( 1 5 ) in 1946 returned to the use of a simple type similar to Tschugaeff's which gave results within 2% of theoretical for compounds that are relatively nonvolatile and react completely at room temperature. COSVENTIOSAL.In 1927 Kohler and coworkers ( 7 3 , 7 4 ) described an assembly containing a one-piece glass dispensing system which could be charged with Grignard reagent sufficient for 100 or more determinations. The reaction flask was attached by a ground joint and the gas buret by rubber tubing. This apparatus and its modifications have become the conventional apparatus for the determination of active hydrogen with the Grignard reagent. Soltys (163) described a similar apparatus for microdetermination in which one charge of reagent served for 50 analyses. The method and apparatus of Soltys are described by Niederl and Kiederl(111). The Naginnity and Cloke (88) version of the Kohler apparatus was made of several pieces of standard laboratory apparatus connected by standard-taper joints and has the advantages of convenience in cleaning and replaceability. -411 these variations in apparatus n-ere designed for the determination of other structures which react with Grignard reagent in addition to the determination of active hydrogen. VARIATIOKS.Three new ideas in apparatus were published in 1940. The apparatus of Fuchs, Ishler, and Sandhoff (36) differed from the conventional type principally by the use of a steel cup in which the sample was weighed and which xas hung on a capillary tubing over the Grignard reagent. The sample was added to the reagent by the use of an electromagnet. The second modification was the gravimetric method of Evans, Davenport, and Revukas ( 2 9 ) in which the evolved methane (or butane, when butylmagnesium iodide was used for increased precision) was burned and the resulting water and carbon dioxide were absorbed in Dehgdrite and Xscarite weighing tubes and weighed as in the carbon and hydrogen determination. Ailso described in 1940 xas the method of Terent'ev and Shcherbakova (161) in which the reaction \vas carried out in an atmosphere of carbon dioxide. -4fter reaction, the system was swept 17-ith carbon dioxide and the methane collected in an azotometer over potassium hydroxide solution, then transferred to an eudiometer \$-hereit was measured as it is often done in a Dumas nitrogen determination. Since carbon diovide reacts with Grignard reagents, this method cannot be used to determine other groups which react in addition to active hydrogen. Further work of Terent'ev and coworkers was described in later papers (160, 162-165), including a modification of their earlier technique (165). A recent (1948) apparatus of Horner and Ehrich (62J incorporating features of both the original and the conventional types, was designed for series determinations with methylmagnesium bromide. Results varied considerably. The latest designs in apparatus are greatly simplified by the use of a hypodermic syringe to introduce reagents into the reaction flask. The micro apparatus of Siggia (150) eliminated the complev manipulations for dispensing the reagent to the reaction flask and consisted of only a gas buret and the reaction flask into which the sample was veighed and solvent added. The system \vas swept with nitrogen and the reagent added from a hypodermic syringe inserted through a rubber serum Cdp and the bore of a stopcock. Aniline could be added later in t h e same

1428

ANALYTICAL CHEMISTRY

manner if the determination of other groups which react with the reagent was desired. Two other somewhat more complex assemblies have been reported. A buret for aniline introduction and a pressure-compensating chamber were the primary differences in the semimicro setup of Zaugg and Lauer (184). A gas sampling section for use with dehydrogenation and other gas-forming processes waa included by Orchin and Wender (121)in a versatile macroapparatus. All three modifications can be easily adapted for quantitative hydrogenation analyses. LlTHIUM ALUMINUM H Y D R I D E

Reaction. This new reagent has been used for the determination of active hydrogen and reducible groups in organic compounds. Its reactions closely resemble those of the Grignard reagents, but are generally more vigorous. A general equation for the reaction of lithium aluminum hydride with active hydrogen compounds can be written:

4RH

+ LiAIH,

----f

LiAIRa

+ 4H2

Thus 1 equivalent of active hydrogen compound produces 1 mole of gas, but only 1 mole of the reagent is required to do the job done by 4 moles of Grignard reagent-Le., 1 active hydrogen requires only 0.25 mole of lithium aluminum hydride. In 1947, Finholt, Bond, and Schlesinger (SO) reported the first preparation of the compound, now commercially available, and its reaction with ammonia, amines, and water. They observed hydrogen evolution equivalent to 1 active hydrogen per mole of water rather than 2 as with the Grignard reagent'. On the contrary, Hochstein (58)obtained values indicating 1.7 to 1.9 active hydrogens in water, while Baker and MacNevin (8) reported a molar ratio of 1.5 to 1.6 for hydrogen from water in the presence of excess lithium aluminum hydride. In general, lithium aluminum hydride has been used to determine active hydrogen in primary, secondary, and tertiary alcohols, phenols, acids, enolizable ketones, amines, and amides. Nitro compounds also produce gaa (77). Lithium aluminum hydride, like Grignard reagents, also reacts with compounds other than those containing active hydrogen, It will reduce aldehydes, ketones, carboxylic acids, esters, acid chlorides and acid anhydrides (115, 116), alkyl halides (69, 117), quinones, nitriles, nitro compounds, azoxy compounds, aldinlines, epoxides, and amides (117'),amino acid esters ( 7 1 ) ,chlorosubstituted acids and acid derivatives (155), cyclic quaternary ammonium salts (144), and sulfones (11). The stoichiometry of these reductions requires 0.25 mole of lithium aluminurn hydride per mole of aldehyde or ketone, 0.50 for esters and acid chlorides, 1.00 for acid anhydrides, 0.50 (in addition to 0.25 rnole for active hydrogen) for carboxyl groups (11.5, 116), 0.50 for nitriles (1831, and 0.25 for alkyl halides (69). The literature indicates varying degrees of completeness for these reactions by refluxing in various ethers. While lithium aluminum hydride does not reduce carbon to carbon double bonds in general, other functional groups in the molecule (68, 115-117) and/or the use of elevated temperatures (58, 140) can cause significant hydrogenation to occur. The presence of functional groups apparently influences the reduction of carbonyl groups also; thus carbonyl in amides may be reduced to methylene rather than to secondary alcohol (117, 134). Carhon dioxide was reduced to methanol in the presence of excess lithium aluminum hydride, but with excess carbon dioxiefe formaldehyde derivatives were obtained (118). Solvents. The lithium aluminum hydride may be dissolved in any one of several ether solvents. Finholt, Bond, and Schlesinger (SO) found the solubility of lithium aluminum hydride to be 25 to 30, 13,2, and 0.1 gram per 100 grams of solvent in diethyl ether, tetrahydrofuran, di-n-butyl ether, and dioxane, respectively. Di-n-propyl ether was used by Lieb and Schoniger (84). Diisoamyl ether is not suitable as a solvent because of the low .solubility of the hydride (183). For reactions above room tem-

perature, tetrahydrofuran (boiling point 65 -66" C.) and di-n-butyl ether (boiling point 140.9' C.) ale suitable, although external temperature control is necessary with the latter solvent to prevent decomposition of the reagent above 100" C. (69). The sample may be left dry or an ether solvent added. Hochstein (58) found N-ethylmorpholine more satisfactory than dibutyl ether; dioxane, anethole, and K-methylmorpholine were less satisfactory. Application and Accuracy. In general, the action of lithium aluminum hydride is equivalent to that of methylmagnesium iodide, determining active hydrogen in OH-, KH-, and SHcontaining compounds, and also reducible groups. However, it reacts more vigorously than the Grignard reagent, and competitive side reactions and steric effects are therefore reduced (58). The values vary with solvent in the case of enolizable compounds, but less enol form is found with the hydride than with the Grignard reagent, probably because the reaction is more rapid vith the hydride and there is less time for enolization to occur. It thus appears that the use of both reagents on some samples may give helpful information, lover active hydrogen values with the hydride than R-ith the Grignard reagent indicating enolizable cornpounds and higher values with the hydride showing slowly reacting groups such as those with steric hindrance. Reduction also occurs more readily nrith the hydride, but even so, elevated temperatures are often necessary for complete reduction. Reproducibility and accuracy in determination of active hydrogen are probably f3 to 5%, the same as with Grignard determination. Determination of reducible groups is not so accurate. Actually, more work is necessary to present a clear quantitative picture of the reactions of both methylmagnesium iodide and lithium aluminum hydride, especially in cases where there are steric hindrance, keto-enol tautomerism, activated methylene groups, or other functional groups in the molecule. Methods and Results. Apparatus and methods have generally been adopted from those used with the Grignard reagent, although two new forms of apparatus have been described. A nitrogen atmosphere is more desirable than air (54, 68). The reagent can be standardized by measuring the hydrogen evolved on addition of water (SO, 76), aniline, or n-amyl alcohol, the alcohol being preferred (185). The Gilman and Schulze ( 4 2 ) color test can be used to estimate the approximate concentration of the reagent (117). Zaugg and Horrom (183),using a semimicro Grignard apparatus (284), compared the actions of lithium aluminum hydride and methylmagnesium iodide on a number of relatively inert or enobzable compounds which react abnormally with the latter reagent. I n general, lithium aluminum hydride proved superior with these types of compounds. Although active hydrogen values were usually consistent whether determined a t 25' or 98" C., reducible groups reacted more completely a t the higher temperature. However, in some cases lithium aluminum hydride produced abnormally high results on heating and the Grignard reagent gave more accurate results. The authors stated that the probable limit of experimental error of the procedure was &3%. Hochstein (58) compared his results from the hydride method in the microapparatus of Soltys (153) and in his own semimicro modification with some literature values of Grignard active hydrogen on enolizable compounds and observed consistentljlover results with the hydride, although solvent dependence was apparent with the hydride as well as with the Grignard reagent-e.g., acetophenone with lithium aluminum hydride showed 0.03 to 0.05 active hydrogen in no solvent, 0.02 to 0.03 in dibutyl ether, and 0.38 to 0.42 in dioxane. Ordinary active hydrogen compounds-alcohols, glycols, phenols, and aminesgave satisfactory results. Compounds which undergo reduction as well as replacement of active hydrogen showed fair agreement with theory in most cases except where carbon-to-carbon double bonds reacted.

'

V O L U M E 2 4 , N O . 9, S E P T E M B E R 1 9 5 2 Lieb and Sch6niger (84), using the apparatus of Soltys (153) f o r 15 minutes a t room temperature, reported results from 100 to 107% of theoretical with benzoic, o-bromobenzoic, salicylic, and 1-naphthoic acids, tribromophenol, and 1- and 2-naphthols; the average was 102% of theoretical. Krynitsky, Johnson, and Carhart (76,77)used a macroapparatus in which the gas was evolved from diethyl ether solution a t vonstant temperature and the amount calculated from the invrease in pressure. Consecutive samples were run in the same reagent solution. A large number of organic compounds including primary, secondary: and tertiary alcohols, diols, phenols, :ccds, amines, and amides were analyzed with resulting values largely within 2 to 573 of theory. Nitrocompounds showed ii,om 2 t o 3 active hydrogen atoms per mole. Again, enolizable compounds generally Fhoned less active hydrogen than by Gri~ n a r danalysis. Higuchi, Lintner, and dchleif (65) have described an elcctrometric titration met’hod for the determination of functional g~oups. The samples were allo~vedto react for 15 to 30 minutes with a known excess of lithium aluminum hydride in tetrahydrofuran solution. The excess hydride was measured by potentiometric titration with either 20% ethanol or 5% propanol in lienzene. -4marked drop in potential occurred a t the end point. Preliminary work suggested p-aminobenzene as a possible colorinietric end-point indicator for further investigation. The authors :analyzed only alcohols and phenols, but the method should be applicable t o the analyeis of all compounds that contain active hydrogen or readily reducible groups. However, the method makes no distinction between the two types of reaction with lithium aluminum hydride. The reported results were within 2% of theoretical for eleven compounds; however, cholesterol was 6.5% high, tert-butyl alcohol 12% high, thymol 3.8% high, and mmthol 5.1 and 2.7% high. OTHER REAGENTS

Triphenylmethylsodium and phenyldiisopropylmethylpotassium nete used by Ziegler and Dersch (190) in 1929 for the determination of active hydrogen in alcohols and amines. The sample for analysis, without a solvent, was titrated with a solution of the intensely colored alkali-organic substance to the point where the color was permanent. The results for triphenylcarbinol, borneol, and L,3-dipheny1-2-methy1-3-indanol \?-ere 101 t o 102% theoretical; diphenylamine, 97 and 103%; but aniline ran consistently high (l180j0,. In 1942, Corwin and Ellingson ( 2 4 ) titrated pyrroles with triphenylmethylsodium, Although some of the results were 94 to 117%, the majorit>- were between 97 and 104% of the calculated values. A compound, such as 2,4,5-trimethyl-3-carbethoxypyrrole, which is inert to sodium, reacted n-ith triphenylmethylsodiuni, showing 1 acstive hydrogen. Sodamide will react ~ i t some h active hydrogen compounds to liberate ammonia which can be titrated. Although this method was used successfully for phenols ( 1 4 7 ) , later work (162, 123) showed the results on alcohols to be unsatisfactory as excess amounts of ammonia were liberated in most cases. Deuterium oxide was suggested by Williams (179) in 1936 for water-soluble compound,- The sample was dissolved in deuterium oxide, then evaporated to dryness, and the increase in weight due to replacement of hydrogen by deuterium \%-asused as a measure of the active hydrogen. Organometallic compounds, other than Grignard reagents and alkali organic compounda can be used for determination, detection, and differentiation of active hydrogen groups (50, 109). The order of relative reactivities of organometallic compounds i s K > N a > L i > C a > M g > ? Z n > A l > Cd > B > Pb > Hg > Bi > Sn > Si for -OH, -XH, and -C=CH. The order varies slightly for -SH and -SeH, being Bi > Pb > Hg. Reagents that are less active than the Grignard reagents generally

1429

give only 60 to 80% reaction and are not used for quantitative work. Various groups, especially -NO, --NOz, and --N=N-, interfere with some of these reagents. Terent’ev and Shor (166) compared methylzinc iodide with methylmagnesium iodide for the quantitative determination of active hydrogen and found the zinc compound less reactive with primary amines. DETERMINATION OF ACY LATABLE HYDROGEN In contrast to total reactive hydrogen, which can be determined by the methods described above, acylatable hydrogen methods determine, in general, only primary and secondary alcohols, phenols, mercaptans, and primary and secondary amines. Not all the methods determine quantitatively all these groups, and only a few methods determine structures such as pyrroles, imino nitrogen, and tertiary alcohols. Slso, the radicals attached to the functional groups may greatly influence the rate of acylation and thus the accuracies of the methods. The large majority of acylation methods depend on the acetylation of the reactive group-with acetic anhydride, acetyl chloride, or acetic acid, alone or with various catalysts. These acetylation methods are discussed individually below, and the fen other acylation methods are also described. Most of the methods of determining acylatable hydrogen can be divided into four general types according to the experimental means of measuring the amount of acylatable hydrogen. These means and their basic principles are described here, so that later discussions need mention only the type of method and specific reagents and conditions. CLASSIFICATION OF ACYLATIOS PROCEDURES ON BASIS OF h l E 4 R S OF ME4SUREMENT

The older methods, and in particular those developed by oil, fat, and soap analysts for the determination of hydroxyl, involved the isolation of the acylated product. This was followed by determination of acyl content or saponification number of the product, and of the sample, too, in certain cases. Generally, these procedures are not desirable for routine analysis, as they are time-consuming and often inaccurate. Errors can be introduced by incomplete acylation; incomplete removal of reagent by too little washing; hydrolysis of the esters by too much washing; the presence of fatty acids, which react with the reagents to form mixed anhydrides which are relatively stable in boiling water during isolation but are hydrolyzed during the saponification and measured as acylated groups: and the procedures for the determination of acyl content. More recent general methods allon the measurement of acylation to be performed in the reaction mixture or on a product distilled from the reaction mixture as the reaction proceeds. These methods measure excess acylating reagent, volatile acid formed, water formed by the reaction, or exceqs water added to hydrolyze the unused acylating reagent. Isolation of Acylated Product. Most of the procedures which require isolation of a product have been developed for acetylation methods and are discussed here as such. However, some have been adapted to formylation and benzoylation procedures. In general, the water-insoluble acetylation products are recovered from the reaction mixtures after acetylation by addition of water, separation, and further iTashings with Jvater. Procedures for the determination of acetyl content of the isolated product can be divided into three types: (1) determination of saponification equivalent-Le., alkaline hydrolysis and titration of the unused alkali; (2) acid or alkaline hydrolysis folloived by separation of the acetic acid by distillation or “filtration” (separation of the aqueous from the oily layer) and alkalimetric or iodometric titration of the acetic acid; and (3) transesterificatiori to ethyl or methyl acetates which are distilled and saponified, and acetic acid determined. The determination of the saponification equivalent of an acet-

ANALYTICAL CHEMISTRY

1430 ylated product was used by Benedikt and Ulzer (10) to analyze fats as early as 1887, but TYas shown to be unsatisfactory for samples containing fatty acids (82, 83). Stable esters can be analyzed if the saponification equivalent is determined on the original sample as well as on its acetylated product. The And& Cook formulas (6, 22) were developed for the rapid calculation of acetyl index or alcohol content of ester-containing samples. Nomographs for the solution of the Andr6-Cook formula were published by Patton (124) in 1947. The acetyl index or value is based on a weight of acetylated product; Andrews and Reed (6) suggested its replacement by the hydroxyl value, based on the original sample and calculated from the same saponification equivalents. To avoid the errors of the Benedikt and Ulzer method, Lewkowitsch (81-83) developed an early modification using the second type of acetyl determination. His isolation technique required several successive half-hour boilings with water to decompose mixed anhydrides. The acetylated fats were then analyzed by either of two procedures: In one, the saponified mixture was acidified Kith sulfuric acid and the acetic acid distilled and titrated; in the other, the saponified mixture was treated with sulfuric acid in an amount equivalent to the alkali used for the saponification and the aqueous acetic acid solution was separated from the fatty acid oily layer and titrated. While this showed some improvement over the Benedikt and Ulzer method primarily because of the improved isolation procedure, mixed anhydrides were not always completely decomposed and esters could be partially hydrolyzed during isolation (176). It appears that great care is necessary with the isolation procedure in order to obtain consistent results especially when fatty acids are present. However, for those cases where it is desirable to isolate an acetylated product, total acetyl, including acetylated oxygen and nitrogen compounds, can be determined according to the general methods given above or in analytical texts such as Niederl and Niederl (110),which includes a discussion of the literature up to 1942, or Clark (20), or in the more recent literature ( 7 , 14, 21, 38, 64, 66, 92, 158, 174, 177). Weisenberger (174) reported an average error of =kO.l% in 47 determinations. The selective determination of 0-acetyl in the presence of N-acetyl has been accomplished by saponification with dilute alkali in aqueous acetone a t 0’ C. or atroomtemperature (3, 78, 181). The use of methoxyacetic anhydride for the acetylation followed by analysis of the product for methoxyl instead of acetyl was suggested by Hill ( 5 6 ) . Excess Acylating Reagent. These methods involve the use of either an acid anhydride or acid chloride as the acylation reagent. After the acylation reaction is complete, the excess reagent is hydrolyzed with water and the solution is titrated with standard base. A correction must be made for any acid originally present in the sample. The difference between the corrected titration value and that from an identical treatment omitting the sample represents the acyl group introduced into the sample. A few other variations are described under acetylation methods. Volatile Acid Formed. These methods apply specifically to the use of acid chlorides of higher acids. RCOCl

+ R’H +RCOR’ + HC1

The hydrogen chloride is easily distilled from a hot reaction mixture and is carried by an air or nitrogen stream into either water or standard alkali. The resulting solutions are titrated with standard alkali or standard acid, respectively. The acylatable hydrogen is equivalent to the hydrogen chloride measured, Water. These are the methods in which the Karl Fischer reagent (31) is used to titrate the amount of water in the system. Mitchell and Smith (98) have recently described various analytical procedures in which water is titrated. Two of these procedures are concerned with the determination of acetylatable

hydrogen-the determination of primary, secondary, and tertiary alcohols by acetylation with acetic acid using boron trifluoride as catalyst (100) and the determination of primary and secondary amines with acetic anhydride and pyridine (102). The Karl Fischer reagent is a solution of iodine, pyridine, sulfur dioxide, and methanol. The end point can be observed visually as the first appearance of unused iodine (direct titration with the reagent) or by electrometric methods-potentiometric direct and back-titration methods and “dead-stop” direct and backtitration methods (99). Seaman, McComas, and Allen (148) have recently proposed a modified titration procedure based on the work of Johansson (67) and using the reagent as two solutions rather than one. Empirical standardization corrects for water content of the reagent, but is not necessary daily as with the usual Fischer reagent, which deteriorates rapidly a t first. Ricciuti and Willits (131) have further investigated the Fischer reagent and its standardization. ACETYLATION WITH ACETIC ACID ASD BOROSTRIFLUORIDE CATALYST.The simple esterification of an alcohol with an acid produces a mole of water for each equivalent of alcohol. ROH

+ CH3COOH s CHaCOOR + HzO

By employing a large excess of acetic acid, the equilibrium is shifted almost completely in favor of the ester and water; boron trifluoride increases the reaction rate so that equilibrium is reached in a relatively short time. After the reaction is complete, pyridine is added to destroy the catalytic power of the boron trifluoride so that the methanol of the reagent will not be esterified. The water produced by the reaction is then titrated A ith the Fischer reagent. ACETYLATIOX U-ITH ACETICAKHYDRIDEIX PYRIDIKE. This method actually amounts to another means of determining t h e excess acetylating reagent. After reaction, the solution is treated with a measured quantity of standardized hydrolysis reagent and the remaining water is titrated with Karl Fischer reagent. The amount of acetic anhydride used in acetylating the sample, equivalent to the acetylatable hydrogen, is calculated. ACETYLATION METHODS

Acetic Anhydride and Sodium Acetate. This combination of reagents has been used for determining primary, secondary, and tertiary alcohols and phenols in procedures where the acetylated product was isolated and the acetyl content measured. Early methods are discussed by hieyer (94). There is little in the recent literature; however, in 1932 de Mingo (96) referred to acetic anhydride and sodium acetate as the usual reagent for the estimation of alcoholic and phenolic constitutents by acetylation. Liebermann and Horniann (86) in 1878 described the acetylation of rhamnetin by heating with acetic anhydride and sodium acetate. The amount of acetyl in the acetylated product was calculated from carbon and hydrogen analysis. Acetic anhydride and fused sodium acetate &-ere adapted to the determination of terpene tertiary alcohols by Boule2 (12) in 1907, using oil of turpentine as solvent and 3 hours’ refluxing. The acetyl contents of the isolated products were calculated from the saponification equivalents. The usual value of about 477, of the theoretical hydroxyl in linalol, obtained with no solvent, was increased to 98 to 100%. rn-Xylene was later substituted for the oil of turpentine ( l S ) , again yielding theoretical values for tertiary alcohols-Iinalol 99.5% and terpineol 100%. Dupont and Lebaune (27) in 1927 confirmed the catalytic effect of sodium acetate and determined the minimum necessary for consistent results. Allan and coworkers (4)in 1928 published a method 11-ith precise directions, including a four-step washing procedure, in an attempt to avoid the serious difference in results previously reported by different workers. Han (49) in 1940 reported good results on several essential oils using Allan’s procedure. Acetic Anhydride and Strong Acid Catalysts. Sulfuric, phoc-

V O L U M E 2 4 , N O . 9, S E P T E M B E R 1 9 5 2 phoric, fluoboric, perchloric, and 2,4-dinitrobenzenesulfonicacids have been used as catalysts in analytical acetylation procedures for alcohols and phenols. Tertiary alcohols gave low results with phosphoric acid (137). Aliphatic -SH, pyrrole -NH-, and imino =NH were determined by the special procedure described in this section, using acetic anhydride and perchloric acid in glacial acetic acid (167). SULFURIC ASD PHOSPHORIC ACIDShave been used in procedures where the product is isolated and acetyl is determined. These methods are claimed to be as reliable, simpler, and more rapid than the acetic anhydride-sodium acetate acetylations and, in addition, they can be run a t lower temperatures and yield a relatively pure product (96, 137, 173). However, Naves (105) warned that this type of “acce1erated”acetylation gives inaccurate results with some essential oils due to side reactions. One of the earliest t o use an acid catalyst was Franchimont (33)in 1879, who added concentrated sulfuric acid dropwise to a solution of the product in acetic anhydride. The mixture was chilled quickly, the product isolated, and the acetate estimated by saponification with alcoholic potassium hydroxide. de illingo (96) reported the determination of thymol, menthol santalol, and borneol in oils using this method. Sabetay (137) in 1934 preferred phosphoric to sulfuric acid and treated the reaction mixture for 15 minutes below 50” C. A comparison of his results on primary and secondary alcohols in essential oils with those from the classical acetic anhydridesodium acetate procedure showed fair agreement-7 of 10 values checking within 2%. Tertiary alcohols gave low results due to dehydration (25). FLCOBORIC ACID. Valentin (169) has recently reported the use of fluoboric acid catalyst with acetic anhydride in dioxane for determining hydroxyl and/or water in organic compounds. In either case, the reaction mixture was heated 5 minutes on the water bath. To determine hydroxyl, the solution was titrated with standard sodium hydroxide, and hydroxyl calculated from the difference betrveen this and a blank titration. To determine water, the excess anhydride after reaction was destroyed with an alcoholic solution of triethanolamine and the acid titrated with standard sodium hydroxide. Good results n-ere reported for ethanol in dioxane, hydroxyl in castor oil, and water in dioxane. PERCHLORIC A N D 2,4-DIN1TROBENZENESULFONIC ACIDS. Toennies and coworkers (166, 167) have developed two methods using perchloric or 2,4-dinitrobenzenesulfonicacid catalysts and determination of the excess reagent in organic rather than aqueous media. The first method was published by Toennies and Elliot (166) in 1935 for the separate determination of water and alcohols present in inert solvents. The determinations are based on the facts: (1) that the reactions of water and alcohols with acetic anhydride, which are negligibly slow in organic media, are completed a t room temperature in the presence of a strong acid such as perchloric or 2,4-dinitrobenzenesulfonic acid in less than 48 hours, the time depending on the nature of the solvent and on the reagent and catalyst concentrations; (2) that 1 mole of water reacts R-ith 1 mole of acetic anhydride to form 2 moles of acetic acid n hile 1 mole of alcohol reacts with 1 mole of acetic anhydride to form only 1 mole of acetic acid; and (3) that 1 mole of sodium methoxide reacts with one mole of either acetic anhydride or acetic acid.

+

-

+

(CH3CO)20 N a + OCH3CH3COOCH~ CHaCOO- N a + CH,COOH S a + OCHs- + CH,OH CH,COO- N a +

+

+

The amount of alcohol in the sample was determined by reaction with acetic anhydride, hydrolysis with water in acetonitrile, and titration of the reaction mixture as in other methods in which the excess reagent is determined. The difference lies in the titration in an organic medium with sodium methoxidein methanol. The amount of water was determined by allowing the sample to

1431

react with acetic anhydride and measuring the increase in acid titer toward sodium methoxide. The increased titer comes from the formation of 2 moles of acetic acid for each mole of acetic anhydride which has reacted with water. The acetic anhydride and the 2,4-dinitrobenzenesulfonicacid were made up as separate solutions in dry acetonitrile and the solutions mixed immediately before an experiment to give initial concentrations of 0.04 to 0.2 M and 0.0002 to 0.0004 M , respectively. Equal amounts of mixture were used for tests and blanks. Results reported for the determinations of water and alcohols (methanol and ethanol) in acetonitrile, ether, benzene, and chloroform in low and moderate concentrations were good. The error in concentrations of 1% wm less than 1%, while the lower limit of response was less than 0.001%. The second method is that of Toennies and Kolb (167), based on the work of Sakami and Toennies (139) and was developed for the determination of hydroxyl and analogous groups-for example, the indole -KHof tryptophan, the imino =NH of arginine, and the mercaptan -SH of cysteine-in amino acids: Acetylation was accomplished with acetic anhydride and perchloric acid catalyst in glacial acetic acid. Under these conditions, amino groups are protected by salt formation with perchloric acid (76). However, these free basic groups must be titrated separately, as they are a part of the acid-base system in glacial acetic acid. Reaction was allowed to proceed for 2 hours a t room temperature, and the mixture was then made basic with a known amount of o-aminobenzoic acid-a base in the acetic acid solvent system. After standing for 3 more hours to allow the base to react with all of the remaining perchloric acid and acetic anhydride, the excess amino groups were titrated with a standardized solution of perchloric acid in glacial acetic acid, using crystal violet as indicator. The difference between this titration, corrected for amino groups of the sample, and that of a blank run represented the nonbasic acetylatable hydrogen. The actual method and calculations are complex, owing to an unusual aliquot removal and the resulting calculations. The method is unique in determining acetylatable hydroxyl, etc., in the presence of amines. It may also be valuable for the determination of pyrrole and imino nitrogen compounds which are not determined by the usual acetylation procedures. Results for eighteen amino acids were fair to good. The average precision of the hydroxyl determinations was about & l % ,and the authors claimed this to represent the average accuracy also. Hon-ever, poor agreement between acetylated groups and basic groups was observed in a number of cases. Acetic Anhydride Alone. Aketic anhydride has been used without any catalyst by a number of workers for the determination of hydroxyl groups in natural products. The accuracies of most of these methods are difficult t o evaluate, because the results reported were for natural products whose exact compositions may have varied with isolation and purification treatments The early methods of Benedikt and Ulzer (10) and Lewkowitsch (81-83) prescribed acetylation by refluxing with acetic anhydride and the respective isolation and acetyl determinations described above. Normann (113) in 1912 isolated the products simply by distilling off the acetic acid and excess acetic anhydride after the acetylation and carried out the saponification in the same flask. In 1930, Somiya (154) described a method for determining the extent of acetylation of oils from natural products as the difference between the amount of 3 N acetic anhydride added, and the amount titrated after reacting at 140” for 2 hours with an equal amount of dried oil. The unique feature of his method was determination of excess reagent by “thermometric” titration of an aliquot of the reaction mixture with 3.1 N aniline, instead of hydrolysis with water and titration with an alkali hydroxide as in the usual procedures for the determination of excess acylating reagent. Two years later Roberts and Schuette (132)published a method for determining the hydroxyl numbers of oils, fats, and waxes by acetylation with acetic anhydride in a sealed tube for 70

1432

ANALYTICAL CHEMISTRY

a vigorous and practically irreversible reaction, yet does not inminutes at 120'; for solids, 120 minutes a t 120' or 60 minutes terfere with titration of the arids with standard alkali" (89). a t 130" were used. The excess reagent was hydrolyzed and acid Pyridine is also a negative catalyst for the acetylation of tertiary titrated as described above. Results of determinations on some alcohols (25, 107); it is a some\vhat better negative catalyst than oils and m x e s indicated an accuracy of 2%. A modification by quinoline, although dimethylaniline give8 similar results (107). Helrich and Rieman ( b 2 ) using a reaction period of 1 hour at 180" gave similar results. The presence of more than 0.5% water in the reagent retarded the reaction sufficiently to make acet,ylation incomplete i n some Even aslate as 1934, Furth, Kaunitz, and Stein ( 3 7 ) described a cases (180). On the other hand, resin formation was observed method for fatty acids using the isolation technique. The sample upon prolonged boiling when the reagent contained less than 0.3% was acetylated in pure acetic anyhdride under a nitrogen atmosKater (51, 180). However, t'he resin apparently is not formed phere, and the product extracted with petroleum ether. Acetyl in t,he presence of an acetylatable sample, even upon prolonged was determined by the method of Friedrich and Rapoport (361, boiling of a very dry reagent, and an accurate blank without in which the acetic acid distilled from the acidified saponification resin formation can be obtained if heating is omitted (26). mixture was determined by iodbmetric titi ation. Purified ii relatively fresh reagent is desirable. Ogg, Porter, and Killits samples of ricinoleic acid gave nine out of tnelve values which (119) reported low results with a 4-day-old reagent mixture. were n-ithin 2% of theoretical. Brignall (16)obtained very good results (99 to 101% of theoDelYalt and Glenn ( 8 6 ) obtained quantitative results with reagents as much as 60 days old, but recommend a mixture not older retical) on known mixtures of menthol by acetylation in 1 to 4 than 30 days. acetic anhydride-N-butyl ether at reflux for an hour, followed by Conditions. The reaction is usually run a t or near 100' C., for a half-hour hot hydrolysis and titration with potassium hydroxide. Acetic Anhydride and Pyridine. GEKERALIKFORIUATION. approximately an hour, and is then applicable to the determination of all hydroxy and amino compounds stable in the reagent a t The use of pyridine with acetic anhydride to get a rapid quantita100" and n-hose acetates are not easily hydrolyzed (157). Procetive acetylation was introduced in 1901 by 1-erley and Bolsing dures designed especially for sugars (18, 226), cellulose dei,iva(171). The reaction required only 15 minutes on a water bath, tives (89), and unstable compounds ( 9 0 ) have specified lower and essentially quantitative results were obtained mith a number temperatures and longer reaction times-1 to 5 days. of primary and secondai y alcohols and phenols. Jones and Fang (70) have studied the effects of time, excess of This is probably the most aidely used reagent for deterinining reagent,, and reagent concentration on t'he reaction of essential arylatable hydrogen a t the present time, and certainly is the oils a t room temperature, and have confirmed the catalytic one about which most has been written in the past 20 years. effect of pyridine. Hawke (51) reported studies on the influence There are many variations, but in general the procedures involve of reagent purity, concentration, and m-ater content, and the time hydrolysis of the excess anhydride after the reaction and titraand temperature of hydrolysis on determination of hydroxyl tion of the liberated acid with standard alkali, as described above. values of fats. DeWalt and Glenn (26) have reviewed the literaOne exception is the method of Mitchell, Hawkins, and Smith ture and/or experimentally studied the eflects of reagent purity, (97)using the Karl Fischer reagent to titrate the excess watw water content, concentration, and age, sample size, reaction after hydrolysis. time and temperature, hydrolysis temperature, and titration Applzcation. The reagent has been successfully applied to the of colored solutions, particularly as applied to phenols, a feu- alcodetermination of primary and secondary alcohols, phenols, and hols, and coal hydrogenat'ion products. primary and some secondary amines. Pyrroles and diary1 GENERALMACROMETHODS. Most of the variations of the secondary amines did not react at room temperature and on]? Verley-Bolsing method have been designed for special types of slightly a t 60' C. (10.3). Honever, Hoi and Royer (60) acetycompounds or involve specifically different apparatus or techlated some 2,3-disubstituted indoles by refluxing 24 hours with niques. However, several invest'igators have described procedures acetic anhydride without pyridine. Acetic anhydride with varying little from the original Verley and Bolsing procedure pyridine has been used for the determination of polj hydric (111 ) which used a 1 to 7 by weight acetic anhydride to pyridine alcohols and phenols including sugars (18, 34, 125, 186, 128), reagent, and required 15 minutes' heating on a water bath in an cellulose derivatives (89),flavanones and flavanols (128), glycopen flask, addition of water, and titration with alkali. erol and glycol (149,, for the hydroxjl groups of essential oils In 1932, Delaby and Breugnob (24) reported using a 1 to 2 by (25, 108, 171), fats and oils (112), fatty acids (67, 119, 176), and volume reagent and a heating period of 30 t,o60 minutes. Delalhy lignin ( 2 ) ,and for amines (26, 26, 97, 102, 167). Although data and Sabetay (26) used this method and obtained values of 99% are lacking, it is probable that mercaptans can be quantitatively of theoret,ical on primary alcohols, 03 to 97% on secondary alcodetermined. hols, and the excellent value of 99.6% on the primary amine, Tertiary alcohols and aldehj des react partially, thus interfering methyl anthranilate. Tertiary alcohols gave 0 to 2.3% of theoR ith accurate determinations. Tertiary alcohols reacted as much as lO%at 1OOOfor 1hour withaveryconcentratedreagent(26,107~. retical and aldehydes reacted from 0 to 7.5%. Rabat6 (1281, using the same procedure, reported results from 70 to 101% of Values up to 9% have been reported for some aldehydes (25,107). theoretical for 42 compounds including substituted and dihyAccording to the work of Wiley (178), or-amino acids n.ould be dric phenols, flavanones, and glucides; 26 of these were deterexpected to give erroneous results, since acylamino acids react mined between 96 and 101% of theoretical. Low results were further w ith acetic anhydridein pyridineto giveacylamido ketones obtained when compounds were insoluble in the reagent and in Accuracy. Generally, an accuracy of 3% is obtainable, but all some cases m-hen carbonyl v a s present in the molecule. the procedures produced varying results with different types of The use of a funnel to cover the react,ion flask during the heatcompounds and complexities of structure. Freed and Rynne ing period of 1 hour was proposed by Normann and Schildknecht ( 3 4 ) claimed excellent results, zt0,3% for primary and secondary (114) in 1933 (1 to 2 reagent). l f t e r cooling, the funnel was alcohols and phenols. However, their values for most sugar rinsed x i t h water, and the reaction mixture was diluted with alcohols were about 2.5% loa. The method of Mitchell, Hawneutral alcohol and titrated with alcoholic potassium hydroxide. kins, and Smith (97,102) determined most primary and secondary Duplicate results reported for octadecanediol were both 101% amines with a precision and accuracy of 1 0 . 2 % . of theoretical. Reagent. The concentration of the reagent has ranged from GENERALMICROMETHODS. Freed and Wynne (1936) ( 3 4 ) 5% acetic anhydride by volume (89) to 100 to 3 acetic anhydrideused either 12 (1 to 7 ) or 20% ( 1 to 4, preferred for sugars) acetic pyridine by weight (107). However, most procedures reconianhydride in pyridine. The sample with 2 ml. of reagent in a mended reagents of 1 to 2 or 1 to 3 anhydride-pyridine by volume. test tube was heated to boiling, allowed to boil 1 minute, :tnd The pyridine acts as a solvent as well as the catalyst, "produces

V O L U M E 24, NO. 9, S E P T E M B E R 1 9 5 2 cooled to room temperature. The reaction mixture was diluted with water, transferred to a small flask with water and ethanol washings, and titrated with sodium hydroxide. If fatty acids were present, another 1 minute boiling was recommended after dilution with water in order to hydrolyze mixed anhydrides. Extra ethanol was added when lipoidal (fatlike) substances were being analyzed. Results reported on alcohols, phenols, and some sugars showed an accuracy of +0.3%. Most sugar alcohols analyzed about 2.5% low, DeU-alt and Glenn ( 8 6 ) have modified the Freed anti \VynnrL procedure by the use of a 3 M (approximately 1 to 2 ) reagent, a condenser, a boiling acetic acid bath, extended reaction time. and addition of carbon tetrachloride before titration 01' cololed solutions. They recommended two procedures, identical except for 5-minute and 1-hour reaction times, for the analysis of coal hydrogenation oils and other samples containing "hiiidered" phenols. hlost phenols react completely in 5 minute$; t h w , i f the reaction is greater in 1 hour than in 5 minutes, the difference is a qualitative measure of hindered phenols or other acetylatable compounds which react slom-ly-e.g., cyclohesanols. Mannitol and sucrose reacted completely in 15 minutes. The reported results (except hindered phenols and c~yc~lohesanols) were almost' all within f1% of theoretical, including 3-aniinophenol in which the amino group also reacted quantit,ntively. The niicroadaptation of Stodola (193i) ( 1 5 7 ) required a 1 to 4 by volume reagent. The sample and then the reagent %-ere weighed into a tiny reaction flask which was placed in a glycerol bath a t 95" to 100" for 1 hour. The reaction mixture was rinsed into a titration f l a k with pyridine, diluted with n-ater and neutral alcohol, and titrated Kith alcoholic sodium hydroxide. The reported results were good-resorcinol, 98 and 99.5% theoretical; tetraniethylhydroquinone,09y0;methy1-9,10-dihydrosystearate, 97, 101, and !IS%; and paeudocuniidine (2,4,5-trimethylaniline), 100%. SEALED-TUBE METHODS.A micromethod ivas described in 1943 by Petersen, Hedberg, and Christensen (185). The sample and a t least twice the necessary quantity of acetic anhydride were weighed into a glass tube and pxridine about equal in volume to the acet'ic anhydride was added. The tube was immediately sealed and allowed to stand 24 hours, then broken under water, and the solution n-as titrated. A large number of compounds including primary and secondary alcohols, polyhydric alcohols, chlorine-substituted alcohols, benzoin, cholesterol, phenols, substituted phenols, and sugars gave generally satisfactory values-within 4% of theoretical or better. Isoeugenol, geraniol, and citronellol were 6, 10, and 13y0 low, respectively. Jones and Fang ( 7 0 ) adapted this method to the macro scale but closed the test tube with a xaxed cork and used 48-hour reaction time. .1 fen minutes heating before the titration end point x a s reached assured complete hydrolysis of anhydride. Results on various peppermint oils indicated an accuracy of 98 to 100% of theoretical. A very concentrated reagent, 100 parts of acetic anhydride and 3 parts of pyridine by weight, was used by Naves (1947) (107) in a sealed-tube procedure for determining alcohols in essential oils. Approximately 0.03 mole of alcohol and 0.6 ml. of reagent were weighed into an ampoule of 2.5- to 3-nil. capacity. The sealed ampoules were heated for an hour in boiling water, then broken under 50 nil. of cold water in the titration flask and titrated with alcoholic alkali after 3 minutes. Results were '37 to 100% theoretical for primary alcohols and 95 to 9Sy0 for secondary alcohols, including those from essential oils. Cyclohexanol gave results about 15yo loa. Tertiary alcohols of essential oils gave 5 to 10% reaction. Replacing pyridine with dimethylaniline gave similar values. hldehydes reacted 0 to 9% using pyridine, but only 0 to 2.6y0 using dimethylaniline. Xaves (108) later reported a reaction time of 15 minutes a t 100" for primary alcohols, but, either 3 hours a t 100" or 15 minutes at 140' when secondary alcohols were present.

1433 SPECIAL PROCEDURES FOR SEGARS, CELLULOSE DERIVATIVES, TEMPERATURE-UXSTABLE SUBSTANCES, ASD LIGNIN. Peterson and \Vest (1927) (126) analyzed a number of sugars and sugar derivatives and a few other compounds, employing a 1 to 2 reagent by volume and heating the reaction mixture in a test tube for 24 to 48 hours at 60' to 80" C. Their experimental values for sugars were 97 to 102% of theoretical. Other results were hydroquinone 100.3%, benzoin 96%, resorcinol 98q70, and 2naphthol 101.7%. In a modification of this procedure, Marks and PIIorrell ( 9 0 ) used a 1 to 3 reagent and varied the time and temperature with the substance being analyzed. Good results were obtained with castor oil and 2-naphthol a t 100' for I 5 minutes and at room temperalure for 24 and 48 hours, respectively, with guaiacol and vanillin at 100" for 15 nlinutes or room temperature for 120 hours. With the methyl ester of oxidized p-elaeoatearic acid and its hydrogenation product,, 37' for 120 hours producedresultsof 100 and 91.5%, but lower values Fere obtained at room temperature. ;idkins, Frank, and Bloom (1941) ( 8 ) developed a procedure for the determination of hydroxyl in hydrogenated lignin. The sample with a 1 to 7 reagent Tvas heated in glass-stoppered tubes at 90" to 100" C. for 4 hours or overnight,. They claimed less than 0.1 % error for alcohols such as 4-~n-propylcyclohexanol. Poor results lvere obtained when the hydroxyl content was very IOW.

For the determination of hydroxyl in cellulose derivatives, Malm, Genung, and Williams (1942) ( 8 9 ) emp!oyed a very weak reagent-1 to 19 acetic anhydride to pyridine by volume. This reagent and a reaction time of 24 hours at 75" to 80' C. were determined optimum conditions for cellulose derivatives. The values reported for cellulose acetate propionates and cellulose acetate butyrates agreed well with those calculated by difference from the determinations of acetyl, propionyl, and butyryl contents. Both shorter and longer reaction times result'ed in gradually lower values. Christensen and Clark (1945) ( 1 8 ) modified the sealed-tube microprocedure of Petersen, Hedberg, and Christensen ( 126) (described above) to include sugars and glycosides by using a reagent with a 1 to 2 ratio of anhydride to pyridine and doubling the reaction time t o 48 hours. The results were generally within 3% of theoretical but the value for fructose as 15% low, SPECIAL PROCEDURES FOR HYDROXY ACIDS. West, Hoagland, and Curtis (176) developed a satisfactory macroprocedure for the determination of hydroxyl in lipides and especially hydroxy fatty acids. Previous methods were improved by including a 1.5- to 2-minute heating after addition of n-ater to assure complete hydrolj-sis of mixed anhydrides and addition of sufficient butanol to yield a homogeneous solution before titration. Other det'aile were similar to previous methods, a 1 to 7 reagent and 45 minutee on the steam bath in glass-stoppered Erlenmeyers being specified. Titration was with alcoholic sodium hydroside. An alternative procedure required 24 hours at room temperature instead of heating on the steam bath. Ilesult? for known compounds were cholesterol 101% theoretical. ricinoleic acid 96.5% (hot) and 9iy0 (cold), lithium lacntate ((i5 minutes) Oi.5%, and palmitic a d no reaction. The modification of Ogy, Porter, arid \\.illits (1945) (119) consisted in the use of iodine flasks having two side arms with standard-taper joints through which electrodes could be inserted for potentiometric titration of colored solutions. Their macroprocedure used a 1 to 3 reagent, since low results were obtained with the 1 to 7 reagent of \Vest, Hoagland, and Curtis, and a 45-minute reaction period on the steam bath; the heating after a.ddition of water was extended to 30 minutes if free fatty acids xere present. Sfter cooling, butanol was added, the electrodes bvere inserted, and the solution was titrated to pH 9.8, or to a color end point using an indicator of mixed cresol red and thymol blue. slightly different procedure was described for a similar seminiicro method. Results of both indicator and potentiomet-

1434 ric titrations and macro and semimicro procedures agreed well. Hydroxystearic acids and oleyl alcohol showed results within 1% of theoretical; the values for cyclohexanoland benzyl alcohol were 1.5 to 2.5% low. Another modification decribed by Hawke (51)in 1948 required a 1 to 3 reagent, 2 hours on a boiling water bath, and a 15-minute hot hydrolysis. He also studied methods for determining the acidity of the sample and recommended titration of the sample in pyridine and neutral butanol with alcoholic potassium hydroxide. Results were reported for seven natural products. In 1936, Hinsberg (57) reported a different approach to the problem of determining hydroxyl in hydroxy fatty acids: The acetylation was first carried out in a glass-stoppered volumetric flask by heating the sample with 5 N acetic anhydride in pyridine (-1 to 3) for 5 to 7 hours on a water bath. After cooling, the contents of the flask were diluted to volume with pyridine and an aliquot was transferred to a s ecial distillation apparatus. This consisted of tlyo small vessels, l a n d B, connected by an inverted V-shaped tube. Vessel B contained a measured quantity of 2.5 N potassium hydroxide and was kept a t 15" C. The system was evacuated to 20 mm. of mercury and the aliquot of reaction mixture was added to vessel A through a buret, which was the? rinsed tvith pyridine. Vessel A was immersed in a bath a t 100 for about 30 to 40 minutes, while acetic acid and acetic anhydride were distilled over into the alkali solution. The excess potassium hydroxide was determined by titration with hydrochloric acid. Dihydroxystearic acid gave values of 102.8, 97, and 101.1% theoretical. Stearic acid indicated 0% and linoleic 1% of a hydroxyl group. SPECIALPROCEDURE FOR PRIMARY AND SECONDARY AMINES. The macroprocedure of Pllitchell, Hawkins, and Smith (97, 102) differs from all the others using acetic anhydride and pyridine in its method of determining excess acetylating reagent after the reaction by titration of the unused water after hydrolysis as described above. At least 200% excess of a 1.5 M (approximately 1 to 6) reagent was added to the sample and reaction allowed to proceed for 30 minutes a t room temperature, followed by 30 minutes' hydrolysis a t 60" C. and titration of water with Karl Fischer reagent. The hydrolysis reagent contains water and sodium iodide in pyridine, the sodium iodide being an effective catalyst in the hydrolysis of acid anhydrides. Water in the sample must be determined separately and subtracted. Results for both primary and secondary amines were very good; precision and accuracy were generally within +0.2%. However, diarylamines and pyrroles did not react; diphenylamine, carbazole, and phenothiazine did not react at all in 1 hour a t 60°, and pyrrole showed only 0.6 and 2% acetylation at 60' after 30 minutes and 2 hours, respectively, and no reaction a t room temperature. Values of 4.9 =k 0.1% were determined for dicyciohexylamine. Unfortunately, alcohols were neither quantitatively acetylated nor completely inert under the specified conditions. By adding a 30-minute heating a t 60" C., quantitative results were obtained with primary alcohols and phenols, but secondary alcohols were only 70 to 80% acetylated and tertiary alcohols only slightly. Heating for an hour a t 60" increased the reaction of secondary hydroxyl, but quantitative results still were not obtained. The reaction could be carried out for an hour a t 60" without affecting the results on amines. Judging from the results of other investigators R-ho have used acetic anhydride and pyridine for determining primary and secondary hydroxyl groups, some further variation of conditions might accomplish essentially complete acetylation of secondary alcohols as %.ell as primary alcohols and phenols, without disturbing the stoichiometric reaction of primary and secondary amines. SPECIAL REAGENT COKTAINING 5% ACETYLCHLORIDE.Verley (1928) (170)prepared a reagent from 2 parts of pyridine mixed with 1 part of acetic anhydride which contained 570 of acetyl chloride. Reaction time of 1 to 2 hours on a water bath was specified for primary alcohols and 2 to 4 hours for secondary alcohols. The interference of tertiary alcohols was increased by the presence of the acetyl chloride.

ANALYTICAL CHEMISTRY Acetyl Chloride. A4cetylchloride has several advantages over acetic anhydride as an acetylating reagent as well as some serious disadvantages. It is more reactive and therefore less time is required for complete reaction. Less interference is caused by aldehydes (151). On the other hand, it is very volatile when used without pyridine and therefore difficult to handle, and the formation of mixed anhydrides occurs to a greater degree (72, 151). Tertiary alcohols react to a greater extent (30 to 40%) even when pyridine is used (151) and without pyridine, n-idely varying results (3.5 to 89%) were obtained (19). A further disadvantage is the increased possibility of side reactions such as hydrogen chloride addition to unsaturation, rearrangement, chelation, and enolization, especially if acetyl chloride is used alone (19). However, with acetyl chloride and pyridine, primary and secondary alcohols and phenols have been determined satisfactorily and primary and secondary amines less accurately. Acetyl chloride alone has given good results with simple alcohols and phenols, but more complex compounds gave inaccurate results due to side reactions. Although acetyl chloride was first used by Adam ( I ) in 1899, the procedure of Smith and Bryant (151) published in 1935 was the first successful use of this reagent for analytical purposes: The method is similar in principle to the majority of those using acetic anhydride in pyridine, excess reagent being determined by titration of acids after hydrolysis. The acetyl chloride, a 1.5 iM solution in toluene, and pyridine were pipetted separately into the reaction flask to give a paste of acetyl pyridinium chloride. The weighed sample was then added, and the mixture allowed to react for 20 minutes a t 60' C. The addition of water gave a two-phase system, and vigorous shaking was necessary to be sure of complete decomposition in the toluene layer before titration. Smith and Bryant reported results 98 to 101% of theoretical on a number of purified compounds-all primary and secondary alcohols. Glycerol reacted to 95% of theoretical and benzoin 93%. Ten phenols, which were trade products not further purified, analyzed around 95%. In addition to tertiary alcohols and fatty acids, products such as formic esters which hydrolyze easily into acidic or basic materials interfered, and aldehydes had an unfavorable effect on the end point. Although no direct experimental data were included, the authors stated that the reactive hydrogen in mercaptans and primary and secondary amines could probably be determined by this method. However, Olson and Feldman (1937) (120) found the method of Smith and Bryant unsuitable for the quantitative determination of amines. Primary aromatic amines yielded widely varying results although secondary aromatic amines gave quantitative results; aliphatic amines gave very low results. Thiophenol reacted quantitatively. Although they were unable t o increase the acetyl values by using more pyridine, Olsen and Feldman developed a new acetylating mixture of acetyl chloride in di-nbutyl ether instead of toluene and increased the reaction temperature from 60" to '70" to obtain values better than 90% theoretical for all compounds tested that were soluble in the reagent. Amides reacted from 20 to 100%. A similar procedure was proposed for fats in 1937 by Kaufmann and Funke (72). The fat was dissolved in pyridine and a t least 100% excess of 0.5'iA acetyl chloride in toluene solution was added from a buret under the surface of the fat solution. Heating for 5 minutes a t 65" to 70" was followed by the addition of a large amount of water, a &minute refluxing to hydrolyze mixed anhydrides, and titration. ilverage results on several compounds were trihydroxystearic acid 101% theoretical, tetrahydroxystearic acid lOO%, dodecyl alcohol 97.5%, and the ethyl ester of dihydroxystearic acid 99%. Houget (1944) ( 6 3 ) used an acetylation procedure almost identical with that of Kaufmann and Funke, but tripled both the concentration of acetyl chloride in toluene and the reaction time to 1.5 M and 15 minutes, repectively. In order to avoid a hot hydrolysis period, he esterified the free acids with methanol

V O L U M E 24, NO. 9, S E P T E M B E R 1 9 5 2 before the acetylation procedure.

H e also added a large quantity

of ethanol before titration and titrated with alcoholic potassium hydroxide. Christensen, Pennington, and Dimick (1941) (19) proposed a method using pure acetyl chloride without pyridine or other solvent. The reagent was dispensed from a modified form of Linderstrgm-Lang and Holter pipet into a small test tube immersed in solid carbon dioxide and already containing the weighed sample of organic compound. It was placed immediately in a large test tube containing water and a third medium-sized test tube v a s inverted over the small one. The large tube was stoppered and placed in a 40” bath for 20 minutes. The tube was inverted to hydrolyze the excess reagent, and the solution titrated. A large number oi results were reported. Primary and secondary alcohols ranged from 94 to 101% theoretical, tertiary alcohols 3.5 to 89%, some glycols and glycerol 98.8 to 99.1%, phenols 98.2 to 101.1% except 1- and %naphthols, which were 7 and 10% high, possibly owing to addition of hydrogen chloride from the acetylation reaction, borneol 100.9, menthol 100.7, geraniol 140.4, and linalool 131.0%, the last two possibly highoi+ingto reaction with the liberated hydrogen chloride. Since nitroso and aldehyde groups interfered v i t h the end point, good results could not be obtained with substituted phenols containing these groups. Salicylic acid showed only 3.3% of theoretical, possibly because of chelation, picric acid only 1.670, while benzoin showed 124 1% of theoretical (the authors suggested enolization), and borne compounds such as Rochelle salt and citric acid did not react completely, owing to insolubility in the reagent. Thus, solubility, chelation, unsaturation, enolization, and rearrangement may all be important in determining the analytical results obtained in the estimation of hydroxyl content by the use of acetyl chloride. S o experiments a ere conducted with amines. Montes (104)reported further results using this method. Johnson (68) also tried the use of acetyl chloride without pyridine. The sample was refluxed 2 hours with a 1 N acetyl chloride in toluene reagent in a Kjeldahl flask whose exit tube was protected with standardized sodium hydroxide solution. The addition of water yielded a heterogeneous system. With the common alcohols tested the results averaged 8% high. Acetic Acid and Boron Trifluoride. The principle of this method (17, 100) mas discussed above. It is applicable to primary, secondary, and tertiary alcohols. Amines do not react, and if relatively small amounts of boron trifluoride are used, acetylation of phenols is negligible. In the general analytical procedure, the sample is esterified by treating in a glass-stoppered volumetric flask with a glacial acetic acid solution containing 100 grams of the boron trifluoride catalyst per liter. The mixture is kept in a water bath a t 67” for 2 hours and then removed and allowed to cool to room temperature. Pyridine is added to destroy the activity of the catalyst and the water formed is titrated with Fischer reagent. The average precision and accuracy of determination on a large number of alcohols have been reported to be about =k0.3%,. Theseincluded primary, secondary, and tertiary alcohols as well as compounds containing unsaturated linkages. Under these conditions, phenols did not react completely. An increased catalyst concentration of 360 grams of boron trifluoride per liter (BFI .2CH3COOH)gave values varying from 63 to 100% theoretical for five different phenols; only phenol itself reacted completely. Longer heating and various substituted acetic acids also did not produce quantitative results. Lower concentrations of catalyst rrere tried, and by using only 25 grams of boron trifluoride per liter of acetic acid solution, the reaction of phenols was reduced to 4 to 5y0 (2 hours a t 60’ C.). The use of dichloroacetic acid without any catalyst (2 hours a t G i ” ) essentially eliminated the esterification of phenols. In both of these modifications, however, the reaction with alcoholic h j droxyl was less quantitative than in the general procedure.

1435

sohie COhipBrBtive values for the three procedures are given in Table I. The results for alcohols were least accurate with dichloroacetic acid. However, as phenol esterification was essentially zero, “an empirical correction could be applied with more assurance” than when boron trifluoride in acetic acid reagent was used (100).

Table I.

Comparative Acetylation Values by Various Reagents

Compound Ethyl alcohol %-Propylalcohol Isobutyl alcohol tert-Butyl alcohol Ethylene glycol Glycerol Cyclohexyl alcohol Benzyl alcohol Phenol Guaiacol

100 g. BFs/1. 99 9 100.1 99.7 100.0 100 0 99 1 98 2 98 4 75 5

% of Theory 25 g. BF:/l. 96.8 98.7 96.7 95.5 90.6 78.1 55.9 97.2 3.9 4.5

CliCHCOOH 96 0 97 9 96.9 94.4 68 5 43 0 22.4 I9 2 0 5 0 2

Thioalcohoisreactedalmost completely with the general reagent containing 100 grams of boron trifluoride per liter, giving values about the same as those obtained by the iodine oxidation method (101). Thiophenols reacted only partially, while acetals and ketals reacted almost completely. Ortho esters reacted quantitatively, but normal esters, acids, ethers, amides, and anilides generally did not react. Aldehydes, cyclohexanone (but not other ketones), and nitriles interfered by formation of Rater or reaction with water. Amines interfered indirectly by “neutralizing” the catalyst, but accurate results were obtained when the concentration of catalyst was increased. Thus, Smith, Mitchell, and Hawkins (152) developed a method for determining hydroxyl in amino alcohols using a reagent of 200 grams of boron trifluoride per liter of acetic acid solution. iin average precision and accuracy of &0.3% were reported (100). OTHER 4CYLATION iMETHODS

Phthaloylation. The use of phthalic anhydride as a reagent for determining alcoholic hydroxyl dates back a t least to 1899 (141, 156), but only the most recent workers, Elving and Warshowsky (28), using pyridine and phthalic anhydride, have reported consistently good results. The advantages of phthaloylation are (1) that phenols are not acylated, and primary and secondary alcohols can therefore be determined in the presence of phenols and (2) that aldehydes do not interfere. Amines are acylated quantitatively or to ewess by the Elving and Warshowsky method. Tertiary alcohols react incompletely nith phthalic anhydride in various inert solvents, but in the presence of pyridine, reaction is reduced to 3 to 5%. Stephan (156) in 1899 pointed out that treatment with phthalic anhydride in a solvent for 1 hour on a nater bath converted primary alcohols quantitatively to their acid phthalate esters, 1% hile secondary alcohols reacted only partially and tertiary alcohols not a t all In the Semiannual Report of October 1890 (141) and again in 1912 (143), Schimmel and Co. described the determination of geraniol, a primary alcohol, in the presence of citronellal (an aldehyde which is readily cyclized to the secondary alcohol, isopulegol) in an essential oil by 2 hours’ heating on a water bath R ith phthalic anhydride in benzene, follou-ed by neutralization of the unused anhydride and the acid ester with excess alkali and back-titration a i t h sulfuric acid. The Schimme1 method has been reported to give erratic results, requiring corrections which vary a i t h the nature and amount of the alcohols and the time of alkaline hydrolysis (46). In 1926, Radcliffe and Chadderton (129) introduced the use of a pyridine solution of phthalic anhydride a t room temperature and titrated the liberated acids after hydrolysis with water. Glichitch and Naves (46,47)viith a modified procedure using 1 to 4 anhydride to pyridine for 18 hours a t room temperature, obtained

1436 fair results on total primary plus secondary alcohols. Menthol, a secondary alcohol, gave 88.5%. Primary amines were acylated quantitatively. Tertiary alcohols, aldehydes, phenols, and esters did not react. In a procedure designed for primary alcohols, Sabetay and Saves (158) used pyridine as a hydrolysis catalyst only, carrying out the phthaloylation in benzene a t the boiling point of the alcohol for 2 hours. After heating for 10 minutes with water and pyridine, acids were titrated. The conditions were critical and had t o be exactly followed (195). The method of Elving and Warshowsky (28), published in 1947, used a 10% phthalic anhydride in pyridine reagent which \vas added t o the sample already dissolved in pyridine and the mixture heated at 100” for an hour; water was added, and the acids were titrated. Primary and secondary hydroxyl m-ere determined 98 to 101% theoretical; glycerol required 2 hours’ heating for 99% reaction. Primary amines gave high results, possibly due to phthalimide formation, but secondary amines reacted quantitatively. Phenols did not react, tertiary alcohols shorn-ed only 3 to 5% reaction, and aldehydes and other osygenated compounds did not interfere. A precision of 3 parts per 1000 and an accuracy of 1 relative yowere reported on complex mixtures De Graef and Pierret (48) have suggested the use of 3-nitrophthalic anhydride for characterization and quantitative studieg of primary and secondary alcohols. Quantitative determinations were carried out by boiling the alcohol or mixture of alcohols in benzene and 3-nitrophthalic anhydride for 4 hours and titrating the excess reagent. Although 15% solutions of alcohols in benzene gave 99.7 to 100% of theoretical, 1 to 5% solutions gave 99.2 to 99.9% for primary alcohols and unsatisfactory results m-ith secondary alcohols. Aqueous solutions of primary alcohols gave good results (99 to 99.8%) only up to 50% water. Mercaptans could probably be determined quantitatively, as they are easily acylated with this reagent (175). Benzoylation. Both benzoyl chloride and benzoic anhydi ide have been reported for use as acylating reagents in the determination of alcohols and phenols. Good results have been obtained with phenols using a pyridine catalyst, but alcohols have 30 far given erratic and generally low results. In 1934, Meyer (95) described a method for alcohols and phenols using either benzoyl chloride or m- or p-nitrobenzoyl chloride in Tetralin. The samples were heated with the reagent at various temperatures from 110” to 180”, and for times ranging from 0.5 to 12 hours. The hydrogen chloride formed during the reaction was swept out of the reaction vessel with hydrogen or nitrogen gas into a solution of standard alkali. The results were poor, ranging from 48 to 103% theoretical with the majority near or above 90%. Low values were generally obtained with compounds having an accumulation of hydroxyl groups. Amines interfered with quantitative results, as part of the liberated hydrogen chloride ivas held by the amine in the reaction mixture. Delaby and Sabetay (1935) ( 2 5 ) attempted to use benzoic anhydride instead of acetic anhydride in their method involving a 0.5- to 1-hour refluxing with a 1 to 2 anhydride-pyridine reagent. Although the original method gave satisfactory results with primary and secondary alcohols and amines, they were unable to get good results when benzoic anhydride was substituted. Leman (1939) (80) used a reagent of benzoic anhydride and pyridine of 1 to 1 concentration by weight for the successful determination of mono- and dihydric phenols. The samples were heated with the reagent for 1 hour a t loo”, the excess reagent hydrolyzed during another 1-hour heating period, and the sohtions titrated. Average results reported for seven mono- and four dihydric phenols were w ithin 1% of theoretical. Formylation. This method is particularly adapted to Samples containing tertiary alcohols. Primary and secondary alcohols and primary and secondary amines are also formylated; phenols are acetylated. The reagent is prepared by adding 1 part of formic acid to 2 parts of chloride-free acetic anhydride while keeping

ANALYTICAL CHEMISTRY the temperature below 15”, gradually warming to 50°, and suddenly cooling (44). The determination is based on the observations of Behal (1900) (9) that the mixed anhydride of formic and acetic acids reacts with uni- and multivalent alcohols, ammonia, amines, phenylhydrazine, and urea. He prepared the formates of a number of alcohols. including tertiary alcohols. Glycol and phenylglycol gave diformates, but glycerol was converted to the acetodifarmate. Phenols were acetylated, rather than formylated. Amines and ammonia yielded formamides. In 1923, Glichitch (49, 44)proposed a procedure for determining the alcohols of essential oils by forniylation using the acetoformic anhydride a t room temperature. iZfter standing for 72 to 96 hours, the product was isolated from the reaction mixture and the extent of formylation determined by saponification. The author reported 911.15 and 99.31% of theoretical for linalool, a tertiary alcohol, after 72 and 96 hours’ treatment, respectively, The same linalool showed only 5i.41% of the theoretical hydroxyl by the “ordinary acetylation method’’ and 88.80% using the special acetylation procedure of Schimmel (1907) ( 1 4 2 ) with acetic anhydride and sodium acetate in xylene. This low comparison value of 88.80’% is unexpected on the basis of the original Boulez (1907) method ( 1 2 ) from which the Schimmel method was taken and which gave 98.00 and 100.05% of theoretical for linalool using an oil of turpentine medium. Later (1924) Boulez ( I S ) also substituted a m-xylene medium with comparably good results for linalool-99.5~o of theoretical. Sabetay (1%) reported in 1936 that Glichitch’s method esterifies a number of terpenes, C ~ H hydrocarbons, M in addition to the desired alcoholic hydroxyl. However, Xaves (106) treated ionones, which are similar to terpenes, according to the prescribed analytical procedure and obtained no esters. A study of the formylation rates of the amino groups of some amino acids with acetic anhydride-formic acid reagent in glacial acetic acid solution was reported by Kolb and Toennies (75). Reactions were generally quantitative in less than 24 hours at room temperature if sufficient reagent was present. The use of formic acid alone as a formylation reagent was described by Glichitch and Kaves ( 4 6 ) in 1933. They obtained accurate results on rhodinol, a primary alcohol, by formylating 1 hour on the water bath with 2 volumes of 90% formic acid, but high results were obtained n-ith only 1volumc of 100% formic acid on a sand bath for 1 hour. Stearylation. The use of acyl chlorides other than acetyl chloride was suggest,ed by Raymond and Bouvetier (1SO) in 1939. The acyl chlorides of the higher fatty acids react quantitat,ively with alcoholic and phenolic hydroxyl, and the hydrogen chloride produced ($anbe collected in water and measured by titration with alkali. These authors used stearyl chloride dissolved in benzene or carbon tetrachloride. Dry air was passed through the react,ion vessel while the mixture of sample and reagent XTere boiled gently. Results reported on nine alcohols, including tertiary butyl, and five phenols were all within 2.2% of theoretical. For primary and most secondary alcohols, 1-hour boiling was sufficient; tertiary alcohols and phenols reacted more slowly, and 4 hours was required for some phenols. Organic acids reacted quantitatively and amines gave quantitative results but reacted slowly. This method was unsuitable for polyhydric alcohols, as they are generally insoluble in benzene and carbon tetrachloride.

SUMM.4RY For the determination of total reactive hydrogen or active hydrogen, either methylmagnesium iodide or lithium aluminum hydride is recommended. In some cases the Grignard reagent gives more accurate results, but in general the hydride is more reactive, less side reactions are observed, and more nearly quantitative results can be obtained. Both reagentsproduce results vary-

V O L U M E 2 4 , NO. 9, S E P T E M B E R 1 9 5 2

1437

ing with the reaction solvent and temperature for certain classes of compounds, especially those subject t o tautomerism. Active hydrogen can be determined in alcohols, phenols, mercaptans, acids, amines, amides, acetylenes, and water by these reagents. They are not suitable for the determination of any one functional group unless all other types of active hydrogen atoms have been proved absent. n-hile both niay also be used t o determine other g o u p s which react by addition or coupling, lithium aluminum hydride is again more reactive than methylmagnesium iodide, and Inore nearly quantitative results can be obtained. In some cases, A comparison of results with both reagents will give information t,oncerning types of chemical structures.

the added feature of determining structures generally not acetylated, such For further seimino =xH and pyrrole

)”.

lectivity, alcohols including tertiary alcohols and thioalcohols can be determined in the presence of amines and even phenols if acetic acid and boron trifluoride are used as the acetylating reagent (100). Phthaloylation with phthalic anhydride and pyridine can be used to determine Primary and secondary alcohols and aminrs, but phenols do not react (28). Tertiary alcohols are a problem because they generally react significantly but not completely n-ith acylating reagents. Pyridine, however. is a negative catalyst for teitiary alcohols, and its

Table 11. S u m m a r y of Acylation Methods Reagent .\cetic anhydride sodium acetate 4cetic anhydride HzSOi or HaPo4 .Acetic anhydride (N0z)sCsHsSOaH icetic anhydride fluoboric acid .Icetic anhydride tIC106 in CHaCOOH

+

+ + +

+

20 Alcohoia

S

30 hlcohols

Functional Groups Determined ThioThioalcohols Phenols phenols

.___

10

Amine..

20

AInine-

Pyrroles

Others

S L Water TVater

5 N

5

s icetic anhydride alone \retic anhydride 3 S IJyridine ?.cetyl chloride s u pyridine P u .?.cetyl chloride alone .Icetic acid boron S S 8 trifluoride Phthalic anhydride N r alone Phthalic anhydride T 3 S pyridine .J-Sitrophthalic anhydride Benzoyl chloride Benzoic anhydride u pyridine Formic-acetic anhy6 3 drideb P S StParyl chloride 3. Satisfactory results S . N o or negligible reaction C . Unsntiafactory results (partial renetion) I. Interferes H. Hieh values a Reaction is under 5 % only when boron trifluoride quantity is reduced t o 25 g./L j Formylation. C Acetylated rather t h a n formylated.

=”

+

S

+

+

+

Imino

S

P

N4

S

N

S

Orthoesters

A-

H

N

I

1

S S

SC S

P

Acids

I

The determination of acylatable hydrogen cannot be sumniarized as a single method, but represents a group of methods, each wit,h different possibilities. The application of various acylating reagents to the det>erminationof alcohols, phenols, mercaptans, and amines is summarized in Table 11. The determinations are indicated as satisfactory if quantitative or near quantitative result,s have been obtained. Where no data were found, the space is left blank. The presence of sufficient strong acid essent idly prevents acylation of amino groups, while strong alkali inhibits acylation of hydroxyl groups (139). The acylation reagent of most general usefulness is undoubtedly acetic anhydride in pyridine, which under the proper conditions can give excellent result’s in the determination of primary and secondary alcohols, phenols, primary and secondary amines, and probably mercaptans, with little interference even from aldehydes and tertiary alcohols. The modification using the Karl Fischer reagent for the determination of primary and secondary amines (109) has no apparent advantages over the simpler methods which titrate acetic acid after hydrolysis of excess reagent, and has the disadvantage of incomplete reaction of alcohol^. Certain other acylation reagents have the advantage of selectivity for a specific functional group or groups. For example. other asetylatable hydrogen groups can be determined in the presence of amines when sufficient perchloric acid is present with the acetic anhydride in glacial acetic acid (139, 167). This method has

presence effectively reduces reaction to negligible proportions in most cases. Contrariwise, sodium acetate with acetic anhydride, acetic acid and boron trifluoride, the mixed formic-acetic anhydride, and stearyl chloride can all be used to determine tertiary alcohols quantitatively by acylation. In general, acylation methods which require titration of the acids after hydrolysis of excess reagent or of the volatile hydrogen chloride formed are to be preferred to isolation procedures for speed and accurac?. Other specialized techniques such as the titration of Tvater with Karl Fischer reagent and titration with perchloric acid in glacial acetic acid are justified if groups are present such that the same resultP cannot be obtained by othrr simpler methods 4CKNOW LEDGWENT

The author wishes to express her gratitude to her associates i n the Coal Research Laboratory and to Grant IVerniniont and his colleagues of the Eastman Kodak Co. for their kind cmsideration of this paper and their helpful suggestions on the final preparation of the manuscript. LITERATURE CITED

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63,554 (1941).

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