Potentiometric Acid- Base Titrations in Tetramethy Iurea Siegmond L. Culp and Joseph A. Carusol Department of Chemistry, University of Cincinnati, Cincinnati, Ohio 45221 SOLVENTS SUCH AS dimethylsulfoxide (DMSO), dimethylformamide (DMF), dimethylacetamide (DMAC), and methyl isobutyl ketone (MIBK) have achieved importance in recent years as evidenced by the considerable number of acid-base equilibrium studies in these media (1-7). Included in these are the studies by Ritchie and coworkers in dimethylsulfoxide ( I , 2) and of Bruss and Wyld in methyl isobutyl ketone (3). They have demonstrated that these solvents are very useful media for studying acid-base titrations due to the much broader range of acids and bases which can exist in them as compared to solvents like ethylenediamine or glacial acetic acid. Their weakly basic character as well as the absence of the usual type of autoprotolysis contribute to the above phenomena. Tetramethylurea (TMU) is one of the few urea derivatives which is liquid at room temperature, yet it has been used only sporadically as a solvent despite its functional group similarity to the above. This is particularly true when its use in analytical chemistry is considered. Several years ago Liittringhaus and Dirksen published an authoritative review article in which they discussed TMU as a solvent and reagent (8). With regard to analytical applications Wawzonek and Duty conducted polarographic studies of polyhalogenerated compounds in TMU (9). Bull and Stonestreet studied the polarographic reduction of various metal ions in TMU (10). In view of tetramethylurea’s functional group similarity to DMSO, DMF, and MIBK, its commercial availability at a moderate cost, and its ease of purification, a study of acidbase titration behavior was undertaken. EXPERIMENTAL
Apparatus. Potentiometric measurements were taken with a Beckman Expandomatic pH/millivolt meter. For readings on the expanded scale the instrument was calibrated by means of a Heath calibration source, Model EUA-20-12. Titrations were performed in a heavy-walled 125-ml beaker with tapered base similar to that recommended by Huber ( I ] ) . The top of the beaker was sealed with a rubber stopper which could accept two half-inch electrodes, an argon-gas inlet line, and a burette tip. The titrant was dispensed from a 10-ml Machlett burette graduated in 0.05-ml divisions with a storage vessel capacity of one liter. The To whom all communications should be addressed. (1) C. D. Ritchie and R. D. Uschold, J . Amer. Chem. SOC.,90, 2821 (1968). (2) C. D. Ritchie and R. D. Uschold, ibid., 89, 1721 (1967). (3) D. B. Bruss and G. E. A. Wyld, ANAL.CHEM., 29,232 (1957). (4) K. K. Barnes and C. K. Mann, ibid., 36,2502 (1964). (5) R. H. Cundiff and P. C. Markunas, ibid., 30, 1447 (1958). (6) T. W. Sturney and R. Christian, Talanta, 13, 144 (1966). CHEM., 24, 306(1952). (7) J. S. Fritz, ANAL. (8) A. Luttringhaus and H. W. Dirkson, Angew. Chem. Znternat. Edit., 3, 260 (1964). (9) S . Wawzonek and R. C . Duty, Rec. Polarog. (Kyoto), 11, 1 (1963). (10) W. E. Bull and R. H. Stonestreet, J. Electroanal. Chem., 12, 166 (1966). (1 1 ) W. Huber, “Titrations in Nonaqueous Solvents,” Academic Press, New York, N. Y.,1967.
burette was fitted with a guard tube containing calcium chloride and Lithasorb (Fisher Scientific Co.). The indicator electrodes were Jena glass electrodes, Sargent S-30080-15C. These were presoaked in tetramethylurea at least 48 hours prior to use and stored thereafter in the same solvent. The calomel reference electrodes were also of Jena manufacture, Sargent S-30080-15A. These were selected because of their low leak rate (-25 microliters per hour), made possible by a porous ceramic junction, which minimizes the deleterious effect of potassium ion (12). The aqueous salt bridge was replaced with a methanol solution saturated with potassium chloride as given by the modification of Cundiff and Markunas (13). A Fisher-Johns melting point apparatus was used to determine the melting points of all solid samples with the exception of the acidimetric standards. A micrometric syringe burette (Model SB2) was used to dispense liquid samples. Reagents. Tetramethylurea was obtained from the Ott Chemical Co. The solvent as received contained 0.36x water as analyzed by Karl Fischer titrations (14). Several methods were evaluated to determine optimum dehydration procedurcs. The method eventually selected involved an initial vacuum distillation (5mm pressure and 50 “C) from a 47-cm Vigreaux column. The first and last fractions were discarded. The distillate then was stored over Drierite for at least 24 hours and subsequently was decanted through a glass wool plug into a one liter distillation flask from which it was again vacuum distilled. A water content of about 0.003% was found by Karl Fischer titrations. It also was observed that the OH bands at 3480 and 3545 cm-I virtually disappeared from the infrared spectrum. Following purification, the tetramethylurea was stored in a large diameter chromatographic column equipped with a stopcock made of Teflon (DuPont), and a guard tube filled with calcium chloride and Lithasorb. Tetrabutylammoniuni hydroxide was used as the titrant for the acidic substances and was obtained as a 25 % solution in methanol from E:astman. An approximately 0.10F solution was obtained tiy diluting about 25 ml of this reagent to 200 ml with purified tetramethylurea. When prepared in this manner the titrant was reasonably stable. (The concentration changed by about t T r e e percent over a three week period.) The titrant was standardized against National Bureau of Standards benzoic acid. A 0.10F solution of perchloric acid in p-dioxane (Eastman) was used to titrate the bases. The titrant solution was standardized against 1,3-diphenylguanidine. All of the substances examined were of analytical reagent grade or equivalent and were purified by standard techniques. All solids were stored in oacuo prior to use. Argon, which was used to establish the inert atmosphere, was dried by passing it through magnesium perchlorate immediately before passing it into the titration vessel. Procedure. A suitable amount of solid sample (less than 1 mequiv) was weighed and transferred to the titration vessel. Liquid samples were dispensed by means of a precalibrated syringe microburette. ‘Twenty-fivemilliliters of tetramethylurea were added and the vessel was closed with the stopper
(12) G. A. Harlow, ANAL.CHEM., 34, 148 (1962). (13) R. H. Cundiff and P. C. Markunas, ibid., 28,792 (1956). (14) I. Geynes, “Titrations in Non-aqueous Media,” Van Nostrand, New York, N. Y . , 1967, p 139. VOL. 41,NO. 10,AUGUST 1969
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Table I. Titration Results of Acidic Compounds Melting point Meq. Found Exp. Lit. (18) Taken pKa(H~0)(17) 0,6072 0.6072 0.6145 0.6124 114 114.0 7.15 0.6312 0.6279 97 96-97 8.28 0.5385 0.5452 107 109.5 9.47 (19) 0.6993 0.7020 39 40.9 9.89 0.3395 0.3394 159.5 159 3.86 pK1 (20) 13.12pKz 0.3073 0.3060 189.5 186-7 1.23 pKi (189.5) ( 1 7 ) 4.19 pKe
Perchloric acid p-Nitrophenol m-Nitrophenol 2-Hydroxy-1-nitrosonaphthalene Phenol Salicyclic acid Oxalic acid
-300t -400
....,I
E
LL
w z -600-
-
-700
I
t
I
I
I
I
,
I
I
I
I
I
I
I
I
I
I
I l l
rnl TBAH-
Figure 1. Titration curves of (1) phenol, (2) benzoic acid and phenol, (3) 2-hydroxy-l-nitrosonaphthalene, (4) p-nitrophenol, and (5) benzoic acid in TMU bearing the electrodes. The argon flow was started, magnetic stirring begun, and the potential monitored. With the electrode combination used initial stabilization generally required no more than 10-15 min, after which time the titration was begun. After each titrant addition about 30 sec were allowed for equilibration before the emf was recorded. The solvent blank was less than 0.02 ml and, therefore, was neglected. The effect of water upon the analytical accuracy and the shape of the titration curve was studied by titrating samples of in-nitrophenol in the presence of varying amounts of water added by means of a syringe. The effect of atmospheric contamination was determined by titrating a sample of benzoic acid in an open vessel without the inert gas atmosphere. RESULTS AND DISCUSSION
The stability of individual potentiometric measurements was good provided excessive amounts of water were not present. Normally only 30-60 seconds were required t o achieve a stable emf reading. Reproducibility for the titration curves of acids appears t o depend upon the previous history of the electrodes. The effects of previous titrations were often manifested by a displacement of the titration curve of as much as 100 mV below the same curve obtained with electrodes freshly removed from their storage solution. The effect was most pronounced in the region of the curve preceding the equivalence point; the overall effect was to compress the curve and somewhat minimize the potential break. Van der Heidje attributed such nonreproducibility to adsorption on electrode surfaces (15). (15) H. B. Van der Heidje, Aim/. Chim. Acta, 16, 392 (1957).
1330
ANALYTICAL CHEMISTRY
Recovery,
z
loo.00 99.66 99.48 98.77 100.39 99.97 99.58
He noted differences between corresponding points of as much as 150 mV in solvents of dielectric constant less than 40. The titration curves for the weak monoprotic acids are presented in Figure 1. All of the curves shown have been displaced horizontally. The general titration behavior for these species shows phenol to be the weakest acid and benzoic acid to be the strongest of the series as based on relative emf values at the half-neutralization points. As in other aprotic solvents p-nitrophenol appears to be only slightly less acidic than benzoic acid in tetramethylurea. Benzoic acid and p-nitrophenol have aqueous pK, values of 4.20 and 7.15, respectively; in D M F these values are 10.2 and 10.9. Thus the relative acidities of carboxylic acids and phenols have been found to change significantly upon transfer from protic t o aprotic solvent (16); such changes are attributed to solvent effects upon acid-base equilibrium brought about by differences in anion solvation. The results of monoprotic acid titrations are given in Table I. The low recovery (98.77%) obtained for 2-hydroxy-lnitrosonaphthalene might possibly be attributed to the presence of impurities, as evidenced by the low melting point (2.5 degrees lower than the literature value). The recrystallization procedure yielded a fine powdery material rather than well-formed crystals. Attempts to resolve a mixture of benzoic acid and phenol were unsuccessful. The first inflection was quite poor and the individual recoveries were 101.37% and 98.51 %, respectively. The total recovery of 100.17% was satisfactory. The titrations of diprotic acids are represented in Figure 2 with the results of these titrations also appearing in Table I. Of interest is the extended emf span of the titration, although this is probably expected since these acids are considerably stronger aqueous acids than benozic acid. The recoveries for these species also were good. The two bases titrated were 1,3-diphenylguanidine (the acidimetric standard) and tri-n-butylamine (Figure 3). Both are of moderate basic strength [pKB (H20) of 4.20 and 3.68, respectively]. The recovery obtained for tri-n-butylamine was 99.85%. The potential spans for the titrations of these substances were similar in magnitude to those of salicylic and
(16) B. W. Clare, D. Cook, E. C. F. KO, Y . C. Mac, and A. J. Parker,J. Amer. Chem. Soc., 88,1911 (1966). (17) Values from “Handbook of Chemistry and Physics,” 46th Ed, 1965-1966, Chemical Rubber Publishing Co., unless otherwise specified. (18) “Handbook of Chemistry,” N. A. Lange, Ed., revised 10th ed, 1967. (19) C. M. Callahan, W. C. Fernelius, and B. P. Bloch, Aiio/. Chiin. Acta, 16, 101 (1957). (20) A . Agren, Acta Client. Scaird., 9, 39 (1955).
ml HClO,+
Figure 3. Titration curves of bases in TMU
ml TBAH+
Figure 2. Titration curves of diprotic acids in TMU oxalic acids. Sulfamic acid could not be titrated as a base in TMU, neither could very weak bases such as p-nitroaniline as no inflection points were observed for these species. Figure 4 illustrates the effect of water additions upon the titration of m-nitrophenol in TMU. The most obvious feature of this titration curve is the extension of the potential range covered. At the 1 water level the potential break is enhanced; however, this is countered somewhat by the increased rounding of the shoulders. Recoveries are not greatly affected by water additions at the lower levels. At the 1 and 2x water levels the recoveries obtained were 100.29% and 99.04x, respectively. When the water level was raised to five percent an unsatisfactory recovery of 101.97 % was obtained. With water contents of 2 Z or more emf readings became erratic as soon as the equivalence point
x
~
-3001
9
was reached and tended to drift toward more positive values. This tendency subsided as more titrant was added. Of particular interest are the additional inflections obtained at half neutralization in the m-nitrophenol titration curves. Bruss and Harlow showed that m-nitrophenol has exhibited this additional inflection point in inert solvents (21). These inflections are generally attributed to acid-anion association and commonly occur in solvents of low dielectric constant or solvents whose base strength is less than that of the acid anion. They suggested that m-nitrophenol molecules may associate as follows:
1 This type of association, which is not very likely with the ortho or para isomers (see Figure l), reflects itself in the acid-anion complexes formed in the titration of the meta isomer. With a dielectric constant of 23.1 and a very weak aqueous basicity [pKB = 121 (reference S), tetramethylurea might be expected to promote such association of solute species. The titration curve of perchloric acid (not reported) shows an additional inflection at about three-fourths neutralization. Weak acids such as phenol and benzoic acids also have produced additional inflections during titration, however, these have been accompanied by slight reversals of potential and have been ascribed to adsorption phenomena (15). In view of the highly polar character of tetramethylurea molecules and
Figure 4. Effect of water upon the titration curves of mnitrophenol in TMU
(21) D. B. Bruss and G . A. Harlow,ANAL.CHEM., 30,1833 (1958). VOL. 41, NO. 10,AUGUST 1969
0
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torted the recovery obtained (101.97 %) was unsatisfactorily high. This can be attributed to the reaction of carbon dioxide with the strongly basic titrant. It may be concluded that TMU is a useful solvent for the titration of very weak to strong acids and for bases of moderate to high basicities. The ability to differentiate this broad range of substances, availability at moderate cost, and ease of handling should lead to further uses of tetramethylurea as a nonaqueous titration medium.
their tendency to form adducts with solute species, as evidenced by the steeply sloping plateaus of the titration curves obtained, it may be that such associations, perhaps in the form of a 1 :3 adduct, are responsible for the additional inflection observed. It also may be that the water present in the original perchloric acid sample contributes to the formation of different acidic species. The possibility that the added methanol from the titrant could act as a source of potential protons was discounted in view of the titration behavior exhibited by the other substances. Also due to the very weak basicity of TMU, it would not be expected that the methanol acidity would be greatly enhanced with respect to its aqueous acidity. However, it may be possible that methanol does act as a complicating component in the perchloric acid system. Further studies will be made in an attempt to explain this behavior. The effect of atmospheric contaminants was studied by titrating benzoic acid without a protective atmosphere. Although the shape of the resulting titration curve was not dis-
ACKNOWLEDGMENT
The authors thank the General Electric Company of Evendale, Ohio, for providing the facilities for this research and the Ott Chemical Company for providing an initial sample of tetramethylurea. RECEIVED for review March 17, 1969. Accepted May 12, 1969. __
Gas-Liquid Chromatographic Determination of Neomycins B and C Kiyoshi Tsuji and John H. Robertson Control Analytical Research and Development, The Upjohn Company, Kalamazoo, Mich. 49001 THETERM neomycin usually refers to mixtures of two stereoisomers, neomycins B and C, and their degradation product neamine or neomycin A. The determination of neomycin is made difficult by the close structural similarity between neomycin B and neomycin C, Figure 1. Biological responses of the neomycins are not constant and frequently are influenced by chemical and physical factors ( I ) . Several chemical methods have been reported to separate and quantitatively determine neomycins B and C. Kaiser ( 2 ) prepared * 4C-labeled hexa-N-acetyl derivatives of neomycins B and C and separated them by paper chromatography. The derivatives were eluted and quantitated with a liquid scintillation counter. Brodasky (3) reported the use of thin layer (carbon) chromatography for the separation of neomycins B and C. The use of column chromatography for the separation of neomycins B and C has also been reported (4-7). These methods are time consuming and are not suitable for a laboratory in which a large number of neomycin products are quantitated routinely. To our knowledge, this paper is the first to describe the separation and quantitation of neomycins B and C by gas chromatography. EXPERIMENTAL
Apparatus. F & M Model 400 with flame ionization detector was used with gas flow rates of hydrogen 20 ml/min, air 550 ml/min, and carrier gas (helium) 40 ml/min, chart (1) W. T. Sokolski, C. G. Chidester, and D. G. Kaiser, J. Phnrm. Sci., 53, 726 (1964). (2) D. G. Kaiser, ANAL.CHEM., 35,552 (1963). (3) T. F. Brodasky, ibid., 35, 343 (1963). (4) G. Nomine and L. Penasse, U. S. Patent 3,062,807 (1962). ( 5 ) H. Maeher and C. P. Schaffner, ANAL.CHEM., 36, 104 (1964). (6) S. Inoue and H. Ogawa, J . Chroniatogr., 13, 536 (1964). (7) A. E. Kaptionak, E. Biernacka, and H. J. Pazdera, Technicon Symposia, Mediad Press, New York, N. Y . 1965, p 27. 1332
ANALYTICAL CHEMISTRY
O0OH”
-HO
“2
OH
-0
“2
0
CH,OH
2,6-diamineglucose
deoayslreptamine
D-ribose
neamine
neosamine
B
neobiosomine B
4;
NEOMYCIN B
H OO
o
G
H
*
0
“2
OH
0
“2
CH,OH
2,6-diomineglucose deoxystreptomine
Y
D-ribose
neosamine
c
j -
neamine
neobiosomine C
NEOMYCIN C Figure 1. Structure of neomycins B and C
speed of 0.25 inch/min, and an oven temperature of 290 “C. Column. A glass column, 3 mm X 1830 mm (6 ft.) and packed with 0.75% OV-1 on GAS-CHROM Q, 100-120 mesh (Applied Science Laboratory, Inc., State College, Pa.) was used. The column was non-flow conditioned at 330 “C followed by the injection of Silyl-8 (Pierce Chemical Co., Rockford, Ill.) and silylated neomycin. The column thus prepared had 300 theoretical plates per foot for silylated neomycin B. Internal Standard Solution. Prepare a pyridine solution containing approximately 3 mg/ml of trilaurin (Supelco, Inc., St. Bellefonte, Pa.). TRI-SIL Z (Pierce Chemical) may be substituted for pyridine for better stability of silylated neomycin.
