V O L U M E 19. NO. 1 2
1006 Upjohn Company, using a n adaptation of the hollow-cup agarplate method (IO). ACKNOWLEDGMENTS
The author is indebted t o Fred Hanson and Mrs. Gayle Hinckley for the bioassays; t o Schenley Laboratories, Inc., for the penicillinase; and to the following people for the samples of pure crystalline penicillin sodium salts: Roberta Harris for penicillins F and G, D. W. RlacCorquodale of Abbott Laboratories for penicillin K, and A. F. Langlykke of the Xorthern Regional Research Laboratories for penicillin X. LITERATURE CITED
(1) Alicino, IND.ENO.CHEM., ANAL.ED.,18, 619 (1946). (2) Allinson, Proc. SOC. Exptl. Biol. Med., 60, 293 (1945).
Committee on Medical Research, O.S.R.D., Sn'ence, 102, 627 (1945). Foster, Zbid., 101, 205 (1945). Herriott, J . Biol. C h a . , 164, 725 (1946). Karrer, "Organic Chemistry," 2nd English ed., p. 212, New York, Elsevier Publishing Co., 1946. Lipmann and Tuttle, J. Biol. Chem., 159, 21 (1945). Mundell, Fischbach, and Eble, J . Am. Pharm. Assoc., 35, 37'3 (1946). Murtaugh and Levy, J. Am. Chem.SOC.,67, 1042 (1945). Savage and VanderBrook. J . Bad., 52,386 (1946). Scudi, J . Biol. Chem., 164, 183 (1946). Shriner and Fuson, "Identification of Organic Compounds," 2nd ed., p. 68, New York, John Wiley & Sons, 1940. Staab, Ragan, and Binkley, Abstracts of 109th Meeting, AM CHEM.SOC., p. 3B (April 1946). RECEIVEDRIay 15, 1947
Determination of the Alcoholic Hydroxyl Group in Organic Compounds Phthalic Anhydride Method PHILIP J. ELVING' A N D BENJ. WARSHOWSKYz,Publicker Industries, Znc., Philadelphia, Pa. Phthalization with phthalic anhydride'in hot pyridine solution can be readily applied to the determination of alcohols or of the alcoholic hydroxyl content of complex mixtures of the type obtained in vapor-phase catalytic reactions involving alcohols. Aqueous alcoholic solutions can be analyzed. Aldehydes and other compounds found in such condensates do not interfere. Phenolic hydroxyl groups do not react. Amines react quantitatively or to excess. Polyhydroxy compounds with primary and secondary hydroxyl groups give satisfactory results; tertiary hydroxyl groups do not give satisfactory results. Volatile alcohols can be analyzed without difficulty.
I
N THE course of a comprehensive investigation of methods for the quantitative determination of the hydroxyl group in organic compounds, the reaction of phthalic anhydride with various hydroxyl-containing compounds was studied, since i t seemed to offer certain advantages over the acetic anhydride and other methods usually used for the estimation of this functional group. A simple method for the determination of the alcoholic hydroxyl group content of organic compounds, based on estcrification with phthalic anhydride in pyridine solution, was developcd and tested. It waa found that the method can be used for the determination of the alcohol content of dilute aqueous solutions, and that such substances as ketones, saturated and unsaturated aldehydea, acids, esters, and phenols do not intcrfere. This method has been successfully applied t o monohydric aliphatic alcohols from C, to Ca. various polyhydric compounds, and complex liquid condensates obtained from organic reactions. The principal advantages of the method described in this paper are its specificity for alcoholic hydroxyl groups and its applicability to aqueous solutions. Its principal limitations are its inapplicability to tertiary hydroxyl compounds due to the dchydration of the latter, and the apparent excessive reaction of certain amines as discussed below. The extensive literature up to 1037 on the dctcrmination of the hydroxyl group is reviewed by Meyer (10). The method most frequently used for the determination of hydroxyl groups, which was first proposed in 1901 by Verley and Rolsing (ZS), is esterification by acetic anhydride in pvridine Jolution. More reccnt modifications by this method are dcscribed by Oyg, Porter, and Rillits ( I d ) and Christeusen arid his coPresent address. Purdue University, Lafayette, Ind. tpresent addreas, Camp Detrick, Frederick, RId.
I
workers (IS). While this method has been successfully applied t o the determination of most primary alcohols and many secondary alcohols, i t is not readily applied to volatile compounds; aldehydes, especially those of relatively low molecular weight, interfere by reacting with the acetic anhydride; phenols react partiall? or completcly, interfering with the direct determination of the alcoholic hydroxyl content of the sample; and the presence of more than a small amount of water in the sample renders the determination inexact or impossible (26). The use of an acetyl chloride-pyridine complex suspended in toluene aa the esterifying agent is described by Smith and Bryant ( 2 1 ) ,who extensively investigated the applicability of the method to various types of hydroxyl-containing compounds. This method seems to offer some advantage over the use of acetic anhvdride-eg., the interference of aldchyde is lens, although the presence of aldehydes has an unfavorable effect on the cnd point. Smith and Bryant give the average precision of the accbtyl chloride method as '0.2%; the absolute accuracy of the two methods is said to be comparable and, based on the data given, about 1% relative. Christensen, Pennington, and Dimick ( 2 ) obtained satisfactory results using acetyl chloride without a solvent. Bryant, Rlitchell, and Smith (I) have described a procedure based on the determination by the Karl Fischer method of the water produced on csterification of the hydroxyl group with acetic acid i n the presence of boron trifluoride as catalyst. While the method can be applied to dilute aqueous solutions, phenols react incomplctely, and carbonyl and somc other types of compounds interfere by rearting with the reagent or the catalyst,. Other recently suggested methods for the determination of hydroxyl groups involve reaction with hydrogen iodide ( 1 1 ) , with acid chlorides of the higher fatty acids (15), or with triphenylmethyl chloride ( 1 7 ) .
DECEMBER 1947 The nature of the reaction of phthalic anhydride with various types of alcohols in benzene solution was investigated in 1899 hy Stephan (62) and it.s use for determining primary alcoholic hydroxyl groups was suggested. Based on this work, Schimmel and Co. ($0)developed and used a method for the approximate determination of primary alcohois such as geraniol and citronellol in essential oils. D e Jong ( 7 , 8 ) examined critically this procedure for geraniol and found that the reaction was not quantitative but that compensating reactions may occur. The use of phthalic anhydride in benzene solutions has also been critically examined by others (4,6, 14, 18), who indicated that this procedure had no particular advantages to warrant its use and in many cases possessed definite disadvantages. In 1926 Radcliffe and Chadderton (14) suggested the use of phthalization overnight a t room temperature in pyridine solution for geraniol and other alcohols found in essential oils. These authors found that under the experimental conditions carboxyl compounds, esters, ethers, and phenols did not react a t rooin temperature: primary amines apparently reacted quant,itatively, Yecondary amines varied in the extent of their reaction, while tertiary amiries and amides did not react. Analytical data for pure alcohols of the type found in essential oils were satisfactory; the results obtained for a number of industrial grade oils were, in many cases, different from those obtained by the acetylation method. Glichitch and Naves (4,6) found thp method satisfactory for the estimation of essential oils. While this technique Jeemed more effective than phthalization in benzene, the required reaction time of 18 hours or longer as well as other disadvantages made improvement desirable. Sabetay and Naves (16, 19) described phthalization in hot pyridine solution, which gave B more rapid and manageable method. These authors examined A comprehensive group of compounds, although in most cases only tt single determination on each compound was reported. They found the method to be applicable to primary alcohols (93% to 96% of the calculated hydroxyl content being found) and to certain secondary alcohols; glycerol and 1,2-propylene glycol, among others, gave poor results. Glycerol, calculated as ponsessing three hydroxyl groups, gave 63Yc of the calcillated hydroxyl content, which is equivalent to 96% of the expected hydroxyl content if only the two primary hydroxyl groups are assumed to react. Unfortunately, the phthalization procedure involving reaction in hot pyridine solution, which seemed to the present authors the most suitable method for their purposes, has been applied almost entirely to t h r determination of the.hydroxvl-containing compounds which occur in essential oils. I t has not been sufficiently investigated as regards its applicability to the determination with an accuracy of one part in a hundred of cthanol and other lower alcohols as well as glycols and other compounds containing sccondary hydroxyl groups. No study has been made of the interference which may be expected from water and organic compounds wch as aldehydes which might be present in typical liquid products resulting from the reaction of ethanol or other alcohols-e.g., no results are given by previous workers for the anolgsis of mixtures of compounds. The purpose of this paper was to develop a technique which would answer the points mentioned and which would, it was hoped, call to general attention the applicability of phthalization in pyridine solution for analyzing complex organic mixtures. REAGENTS AND APPARATUS
The reagents used in the studies described were I\fallinckrodt's analytical reagent grade phthalic anhvdt ide, B w e t t ' s refined grade 2h pyridine, 0.35 N standard sodium hydroxide solution, and a 1% alcoholic solution of phenolphthalein indicator. The hydroxyl-containing compounds analyzed n w e distillrd in most cases and a narrow-boiling fraction was used; the probable purity is indicated in Table I. Barrett's grade 2-4 pyridine a s obtained commercially contains a significant amount of water and other substances which intcrfere with the accuracy of the determination. I n order to elimi-
lo07 nate these substances, the pyridine should be distilled from over barium oxide and only the portion distilling a t 115' C. used The phthalization mixture is prepared by dissolving 20 grams of phthalic anhydride i n 200 ml. of purified pyridine; this solution is prepared fresh daily. The apparatus required depends partly on the procedure selected. I t was found satisfactory t o carry out the reaction either under reflux or by the use of pressure bottles. Inasmuch as the pressure'bottle technique is somewhat simpler and more rapid, this is described in detail below. For this technique, citrate of magnesia bottles of 1-pint capacity are used. An air bath set a t 100" =t 2" C. is required; a n ordinary laboratory drying oven will suffice. It is preferable that the pyridine used be anhydrous and the reaction equipment, whether pressure bottles or flasks and condensers, be thoroughly dried. Unless these precautions arc observed low values m.ty be obtained; water hydrolyzes the phthalic anhydride and may thereby reduce the concentration below the excess required. Table I.
.4nalysis of Hydroxyl Compounds by the Ph thalization Method Puritv a8 DeterGined by Physical Constants
Compound
Methanol Ethanol
99.3 100.0
1-Propanol 2-Propanol 1-Butanol 2-Methyl-I-propanol alcohol) Cyclohexanol 2-Ethylhexan-1-01 2-Octanol Ethylene glycol Propylene glycol Glycerol
98.4 99.6 100.0
(isobutyl
Purity b y Phthalization Method 100.0, 100.7, 100.3, 98.1, 97.6, 100.6,
100.2 100.7 100.4 97.9 97.6 100.6
101.8, 1 0 1 . 2 05.1, 9 5 . 1 99.1, 99.1 96 8.0 7 , 9 86 . 73
si&
99 97-98 99 100 95.5
100 Benzyl alcohol 0 T w o hours at 100' C. allowed for reaction.
99.4. 87.9, 94.9", 99.4,
99.6 87.9 94.3" 99.7
Recoverj by Phthalization Method 100.8 100.5 99.5 98.0 100.6 9Gioo 100 ' 98-99 99.5 99.5 92.0 99.1 99.6
ANALYTICAL PROCEDURE
For samples containing a high percentage of ethanol, a sample weighing 1.0 t o 1.5 grams is carefully pipetted into a 50-ml. volumetric flask containing 30 t o 40 ml. of the purified anhydrous pyridine which hss been weighed, care being taken to avoid wetting the neck of the flask: for monohydric alcohols of higher molecular weight and dilute solutions of ethanol. samples of correspondingly lzrger weight should be taken. After reweighing, the sdution is made up t o volume with pyridine and mixed thoroughlv by shaking. In certain cases where the sample is extremely volatile-i.e , contains corisidcrable material boiling below 40" C.-the sample is wrighed direct lv in thin-walled glass ampoules. The ampoule is then transferred to the volumetric flask containing pyridine and is crushed under the surface of the pyridine by a glass rod The rod is rinsed with pyridine on withdrawal, the solution is made up to volume with pyridine, and the contents of the flask are mixed. Into a clean, dry, pressure bottle are pipetted 25 ml. of the phthalic anhydride solution by means of a n automatic Llachlett pipet or buret or a Lowy pipet. To this are added 10 ml. of the s3lution containing the ssmple. The sealed bottle containing the mivture is placed in an air oven set a t 100' C. and is heated a t that temperature for one hour. At the end of this time, the pressure is carefully released and 50 ml. of distilled water are added. After mixing, the sdution is cooled under the cold water tap and titrated immediately with standard 0.35 N sodium hydroxide. phcnolphthalein being used as a n indicator. A blank determinatjon is made in the same manner on the reagents employed. The difference i n amount of alkali consumed between the sample and the blank reprrsents the amount of esterifiable hydroxyi present in the sdution: V X N X 1.70 yo hydroxyl =
W
where TV = weight in grams of the sample in the aliquot taken V = volume in milliliters of standard sodium hydroxide solution used by sample, which equals thc difference in volumes required by the blank and sample titrations; and N = normality of the standard sodium hydroxide solution.
1008
V O L U M E 19, NO. 1 2 DISCUSSION OF PROCEDURE
Size of Sample.
The weight of sample taken should be such of 100yomolar excess of phthalic snhydride over the amount required. The presence of a sufficient excess of anhydride can be noted by the formation of a yellow 'color in the solution after heating for the prescribed length of time. Unless this color appears the results may not be reliable. Failure of t'he color to. appear indicates an insufficient excess of the reagent' due to too large a sample or the presence of too much water. Acidic Samples. Separate portions of samples containing tree acid or acidic groups on the hydroxyl-containing compounds should be titrated with the standard alkaline jolution a t room temperature, using phenolphthalein as indicator, and a suitable correction made in the volume of alkaline solution consumed in the IJ Kat( phthalization procedure. Contact Time. While an hour is specified for the phthalization reaction, in many cases this time can be reduced to 30 minutes or less. I n dealing with mixtures containing substances which react with phthalic anhydride on prolonged heating, it xvould be north while to determine the minimum reaction period necessary for the hydroxyl-containing compounds involved. The reaction mixture, after the addition of the water, should be cooled and Litrated in a minimum amount of time t o avoid the possibility of the phthalate esters formed hydrolyzing to any appreciable extent; ordinarily, the danger of such reaction is almost nil. There was no measurable consumption of phthalic anhydride due t o polymerization, decomposition, or other reactions in blank samples heated up to 4 hours. End Point. hlt.hough the pink phenolphthalein end point is normally easily detected, it is masked to some extent in this determination by the yellow color of the final solution. Therefore, instead of a color change from colorless to pink, there is a gradual transition from yellow to brown to orange to pink. I t is felt that a representation of the true end point is the first noticeable permanent color change of the solution-without having t,o continue the titration until the color is definitely pink. After several titrations this point can be detected without any difficulty. The use of the mixed indicator, thymol blue-cresol red, offered no appreciable advantage over the use of phenolphthalein. In the case of dark-colored solutions the end point can be determined electrometrically. I n a comparative series of runs on ethanol, the three methods of det,ermining the end point, gave the following per cent recoveries: phenolphthalein: 99.2, 99.3, 99.3, 99.3, 99.3; thymol blue-cresol red: 99.0, 98.8; electromet,ric (Beckman Model G p H meter) : 98.7, 99.3, 99.3, 98.6. Nature of the Reaction. Xlthou.gh the authors have not determined the nature of the intermediates formed during the reaction, 'it seems probable that' the reaction proceeds in an analogous fashion to that postulat'ed for acetylation in pyridine solution by Smith and Bryant ( 2 2 ) . The phthalic anhydride reacts with the pyridine to form an addition compound which rea& with the hydroxyl-containing compound as shown: as t o have present a minimum
On addition of water the excess of compound I is hydrolyzed, forming a compound (111) analogous to compound I1 where the organic residue, R, is replaced by a hydrogen ion. Titration with alkaline solution converts compound I11 as well as compound I1 to the sodium salts and pyridine: H
H
+
H
\\ '0
H
,O
H H
H
+
4 2H2O
(4)
From these equations it can be seen that each equivalent of hydroxyl group present decreases by one equivalent the amount of alkaline solution required to neutralize the phthalization solution. D4TA AYD DISCUSSION
Known Mixtures. A sample of Publicker absolute ethanol, which was found to contain 99.541, ethanol on the basis of its specific gravity and refractive index, was used throughout all the preliminary experiments. The water content of this sample was 0.447, by the Karl Fjscher procedure. Various modifications of the procedure recommended by Sabetay and Naves (16, 19) were tried with varying degrees of success. The method described, employing pressure bottles, was found the simplest and gave satisfactory results. Good accuracy and precison were also obtained by refluxing the reaction mixture for one hour in a boiling water bath. I n Table I are given the results obtained by the phthalization method on various monohydric and polyhydric organic compounds. Low results were obtained when glycerol was permitted to react for 1 hour; a 2-hour period of heating gave a recoverv of 99.1%. Phen'ol, o-cresol, and 1-naphthol showed a zero esterifiable hydroxyl content, indicating the specificity of the phthalization procedure for alcoholic hydroxyl under the conditions used; excellent results were obtained with benzyl alcohol. Acetaldehyde dibutyl acetal showed a hydroxyl content of about 9% based on that available by complete decomposition of the acetal, indicating some dissociation of the acetal during the phthalization procedure. \T7hile diethylamine showed a satisfactory reaction by the phthalization procedure, 100.1% recovery being obtained, 107 and 112% recoveries were found for small samples of aniline, and 134 and 135% for large samples; 103 and 109% recoveries were found in the attempted determination of ethylenediamine (1,Z-diaminoethane). The abnormal results obtained with some amines may be due t o phthalimide formation.
.
1009
D E C E M B E R 1947 However, the phthalization proceduie can apparently be applied *o the quantitative determination of at least certain amines (cf. HISO
In Table I1 are given the results of analyzing one monohyiroxyl and four polyhydroxy1 compounds, which gave poor re-ults. The purity of the samples of 2-methyl-2-butanol and of the I-methyl-2,4-pentanediol exceeded 95%; the other three compounds were used without purification and were believed to be not less than 85y0pure. Results for anv given compound in Table I1 In the same horizontal line were obtained at the same time-Le., in the same batch of samples. The results for 2,3-butanediol are probably correct in view of the consistent values obtained for aamples heated for 1 hour and for 4 hours, JIhile the 1,3-butanediol apparently required 2 hours of reaction for complete esterifiLaation. The results for the 2-methyl-1,2-propanediol are probAbly meaningless, as in the presence of even dilute acids the compound dehydrates and rearranges ieadily to give isobutyraldehyde ( 2 5 ) . The unsatisfactory results for 2-methyl-2-butanol and 2-methyl-2,4-pentanediol are due to the ease with which tertiary alcohols are dehydrated in the presence of acidic catalysts (24). Investigation of the literature shoired that 2-methyl-2,4-penranediol can be dehydrated in the liquid phase in the presence of acidic catalysts to 2-methyl pentenols and 2-methyl pentadienes 3, 9). The results obtained using stoichiometric molar ratios of 1-2, 1-1, and 2-1 of phthalic anhydride and 2-methyl-2,4pentanediol in pyridine solution plus the data in Table I1 indicate rhat phthalic anhydride apparently causes the conversion of the methyl pentanedlol to the mothy1 pcntenol. The results obtained for 2-methyl-1,2-propanediol,which is also a tertiary dcohol, may be explicable in part on the basis of the same reaction; only about half of the expected hydroxyl content is -~vailablefor esterification. Furthermore, 2-methyl-2-butano1, which is very readily dehydrated (24), shows vrry little esterification. I t is hoped to investigate further the reaction of polyhvdroxyl compounds n ith phthalic anhydride. Table 11. Analysis of Hydroxyl Compounds by Phthalization Method
Compound I ,3-Butanediol
2.3-Butanediol 2-Methyl-1 ,2-propanediol (isobutylene dycol) 2-Methyl-2-butanol (tert-amyl alcohol) 2-JIethyl-2,4-pentanediol (preparation I ) &Jlethyl-2,4-pentanediol (preparation 11) l - M e t h y l - 2 , 4 - p e n t a n e d i o l (preparation 111)
Apparent Purity, Weight P e r Cent for Different Reaction Times a t 1000 c. 1 hour 2 hours 4 hours 84,84 96,89 79,78 92,91 93,92 75>76 89,89 91,QO 89,90 89,89 59*59 59,60
64,64
5, 5
10,11
43.42
52,5l
56,58
51,50
50,57
60,60
54,54 53,53 54,53 3, 3 45,46
In order t o determine whether other substances possibly present the liquid condensates obtained in catalytic organic reactionwould affect the accuracy of the hydroxyl determination, synthetic mixturns were prepared containing known amounts of w'ater and of representative members of various types of organic t'iinctional groups, including carbonyl compounds, acids, esters. m d unqaturated compounds. Ethanol was determined in the prwcnce of these substances singly and in mixtures; results of this study, shown in Table 111, indicate that such substances, Drerent in amounts likely to be encountered in reaction mixture-, do not interfere with the determination of the esterifiable hydroxi 1 group. Of particular interest is the accuracy attainable in mixrureb containing as much as 85y0of water. The data reported in Tables I and I11 indicate that the esterifinble hydroxyl content of the organir compounds analyzed with in
Tahle 111.
4,14).
Analyses of Synthetic 3Iiwturas Containing Ethanol
Constituents of Mixture I.
2, 8.
4
5.
Ethanol Water Ethanol Kater Ethanol Kater Acetaldehyde Ethanol 2,4-Hexadiene Water Acetaldehyde Ethanol Acetic acid Acetone Crotonaldehyde Ethyl acetate Phenol
Composition Wt. 92 79.4 20.6 14.6 85.4 75.2 19.6 5.2 71.7 4.7 18.7 4.9 11.3 27.2 13 5 24.0 17.6 6.3
Ethanol Found
70 7H.6,79.7,79.7 l4.5,14.7 75.3,74.8.75.0,75.O 71.3.71.1,71.4
11.4.11.3
Table IV. ilcohol Content of Condensate and Condensate Fractions Fraction Boiling Range
