Determination of Micromolar Quantities of Primary Amides by Flame Emission Spectrometry Raphael P. D’Alonzo and Sidney Siggia’ Department of Chemistry, University of Massachusetts, Amherst, Mass. 0 1002
The analysls of prlmary aromatic and alkyl amides has been accomplished by means of flame emlsslon spectrometry. Barium hypobromite, generated by the addltlon of liquid bromine to a solution of barlum hydroxide, reacts with prlmary amides to form the amlne of one less carbon, and Insoluble barium carbonate. Excess barium from the reagent is removed by flltering, and the separated barium carbonate Is dissolved in nitric acid and determined by flame emission spectrometry. Selectlvlty is a key feature of this procedure allowing the determination of prlmary amides In the presence of esters and anhydrides. The precision of the method is f1.5-6.5”tt in the range of 1.0-4.0 pmol/ml of primary amide. The procedure is rapld allowing the complete determination of SIXsamples in less than 45 minutes.
Mitchell and Ashby (6) developed a volumetric method involving the reaction between 3,5-dinitrobenzoyl chloride and amides, followed by titration with standard sodium methoxide solution. RCONHz
+ (N02)~C&COC1 RCN + (N0z)~C&COOH + HCl -+
This method requires the determination of free acid and water present in the original sample before analysis. The quantity of primary amide present is equal to the increase of acidity of the sample over the blank. Amides have been determined colorimetrically at trace levels by the hydroxamic acid-ferric complex (7). RCONHz
Carboxylic acid amides have been determined by several methods. The most common of these methods is saponificaton with strong alkali (1). The liberated ammonia or amine is dissolved in excess standard acid and the excess acid titrated or, in the case of primary amides, the ammonia can be easily boiled off and the excess alkali used for hydrolysis can be titrated with standard acid. RCONHz
+ NaOH
-
RCOONa
+ NHR
(1)
Long reaction times are generally required and many amides are resistant to quantitative saponification including a fair number of primary amides. The desirable sample size for saponification is about 10 mmol of amide. Methods for determining amides as bases by direct titration in nonaqueous media have been developed. Wimer (2) employed the use of acetic anhydride as a solvent and perchloric acid as a titrant. The titration has been regarded as a reaction between a Lewis acid “acetyl perchlorate”, and the amide to form a salt. RCONHz
-
+ CH3CO+C104-
RCONH2CH3COfC104-
(2)
In addition to the large sample size required, (6-9 mmol), the acetic anhydride is a reactive solvent and tends to react with certain amides and hydrazides. Glacial acetic acid instead of acetic anhydride has been employed for direct titration of amides by Higuchi et al. ( 3 ) . In this method, simple potentiometric titration cannot be used. Photometric titration is required since amide basicity is weak in acetic acid. Other nonaqueous solvents such as nitromethane and acetonitrile have been employed ( 4 ) . Lithium aluminum hydride has been employed ( 5 )to reduce the amide to the corresponding amine which is then steam-distilled from the reaction mixture into excess standard acid. The excess acid is then titrated with alkali. 2RCONHz
+ LiAlH4
-
2RCHzNHz
+ LiAlOz
(3)
In addition to its lack of sensitivity, as in most volumetric analyses, nitriles, imides, and aliphatic nitro compounds are also reduced to amines which may lead to high results. 262
ANALYTICAL CHEMISTRY, VOL. 49, NO. 2, FEBRUARY 1977
(4)
l / n Fe3+
+ NHZOH
+ RCONHOH
OH-
-
----t
RCONHOH R-C-N-H
II o\
+ NH3 + H+ I
(5) (6)
o,
Feln Conditions for maximum colorimetric value are different for each compound, and other functional groups such as esters, acid chlorides, anhydrides, and nitriles interfere (8-10). Direct determination of amides by gas chromatography has been accomplished, although difficulties with the more polar carboxamides have been reported (11). P205 in dioxane has been used to convert the amide to the corresponding nitrile which can be easily gas chromatographed (12). pZo5
RCONHz + R C = N + H z O Dioxane
(7)
Carboxylic acid amides have also been derivatized for gas chromatographic quantitation using T M S (13). These gas chromatographic methods all require a separate calibration curve for each amide assayed. Amides resistant to solution saponification have been assayed by reaction gas chromatography (14).In this procedure, solid potassium hydroxide is fused with the amide to liberate ammonia or the amine which is then determined by gas chromatography. Although caustic fusion reaction time is much faster than solution saponification, only one sample a t a time can be determined; thus, multiple determinations become time consuming. Furthermore, this procedure will not lend itself to the quantitation of amides in solution. A procedure by Leader (15) using nuclear magnetic resonance has been employed in amide analysis. However, this method requires l9F NMR capabilities which are not always readily available. The present work makes use of the Hofmann reaction for the synthesis of amines of one less carbon than the starting amide. The reagent used is barium hypobromite generated by the addition of liquid bromine to an aqueous solution of barium hydroxide. Barium hypobromite reacts quantitatively with primary amides to produce the amine plus insoluble barium carbonate.
RCONH2
+ 2Ba(OH)z + Brz
-
RNH2 + BaC034
+ BaBr2 + 2 H2O
(8)
Similarly, barium hypobromite reacts with imides to produce BaC03 and the barium salt of the resulting amino acid. The carbonate is separated from the reaction mixture by filtration. The filtered barium carbonate is then redissolved in nitric acid and t h e resulting solution is analyzed for barium content by flame emission spectrometry. The method allows for t h e determination of primary amides both aliphatic and aromatic in the presence of secondary and tertiary amides. T h e sensitivity of t h e procedure described is greater than or equal t o most existing methods. Selectivity is a key feature of this reaction since few functional groups result in the production of an insoluble barium salt. As many as six determinations may be accomplished in less than 45 min.
EXPERIMENTAL Reagents. Barium hydroxide (certified ACS) was purchased from Fisher ScientificCompany. Solutions of this reagent to be mixed with bromine to generate hypobromite were prepared by adding 200 ml of distilled deionized water to 0.03 mol of barium hydroxide (Ba(OH)Y9HzO).After stirring for 1h, the solution was filtered using a fine frit (4-5.5 pm) Pyrex glass funnel. Barium hypobromite was then prepared by mixing 0.01 mol of reagent grade liquid bromine (Fisher) with the freshly prepared alkali solution. The final concentration of barium hypobromite generated should range between 0.025 and 0.075 M. Higher concentrations result in serious side reactions with aromatic amides. The hypobromite solution is not stable. It should be used immediately after its preparation and discarded after 24 h if not used. Nitric acid reagent was prepared by mixing one volume of concentrated nitric acid (Baker Analyzed Reagent) and one volume of distilled deionized water. A stock solution of barium was prepared by weighing out 0.10-0.15 g of dry barium carbonate (Fisher), transferring to a 1000-ml volumetric flask, dissolving the carbonate with the least amount of 1:4 concentrated nitric acid-water and diluting to volume with distilled deionized water. A second stock solution of potassium chloride was prepared by dissolving 38 g of potassium chloride (Fisher-Certified ACS-0.001% Ba) with sufficient distilled deionized water to make 11. of solution. Standard barium solutions for calibration were then prepared by taking appropriate aliquots of barium stock solution plus 10 ml of stock potassium chloride and diluting to volume with deionized water in 100-ml volumetric flasks. Acetamide, 2-chloroacetamide, valeramide, isovaleramide, adipamide, salicylamide, o-toluamide, p toluamide, p-nitrobenzamide, succinimide, and phthalimide were all purchased from Eastman Kodak Company. Propionamide, butyramide, isobutyramide, acrylamide, methacrylamide, benzamide, nicotinamide, and pyrazinamide were all received from Aldrich. All amides purchased were the best grade available. Benzamide (Aldrich Gold Label-99.9+%) and acrylamide (Aldrich Gold Label-99+% were used as received without further purification. All other amides were purified twice by vacuum sublimation. Standard amide solutions were prepared by weighing out 10-50 mg of freshly sublimed amide on a microbalance transferring to a 100-ml volumetric flask and diluting to volume with distilled deionized water. Apparatus. A Perkin-Elmer 403 Atomic Absorption Spectrometer operated in the flame emissibn mode was used to measure intensities in the visible region at a wavelength of 276.8 nm. A nitrous oxideacetylene flame was used and operating conditions were as outlined in reference (16). A vacuum filtering apparatus consisting of six 15-ml capacity fine frit (4-5.5 bm) Pyrex glass funnels was used for filtering. Eppendorf pipets (Brinkmann Instruments) were used t o pipet all aqueous reagents and samples. A Mettler microbalance was used for weighing all amide samples. Procedure. A 2-ml sample of standard amide solution is pipetted into a 15 X 150 mm test tube. The concentration of amide should range between 1.00-4.00 kmol/ml. An equal volume of barium hypobromite reagent is added and the top of the test tube sealed with parafilm. The contents of the test tube are then mixed and a small pin hole is made in the parafilm. The test tubes are then placed in a water bath maintained between 70-75 "C for 10-15 min. After heating the test tubes are removed from the bath and placed in an ice-water bath for 5 min. After cooling, the test tubes are removed from the ice water and allowed to come t o room temperature before filtering. Filtering cold solutions results in longer filtering time and therefore higher blank values since the reagent is in contact with atmospheric carbon
dioxide for a longer period of time. The contents of the test tube are transferred to a glass funnel along with two 5-ml washing portions of water from the test tube. Each washing portion is allowed to pass completely through the filter before the next wash portion is added; otherwise, high blank values will result due to incomplete washing of the excess barium hydroxide. Once filtering is complete, a clean suction flask is transferred to the vacuum filtering apparatus and 10 ml of 1:1concentrated nitric acid-water is added to the filter funnel from the test tube to dissolve the barium carbonate. After 1-2 min, an additional 5 ml of distilled deionized water is added to the funnel and suction applied. Two 15-ml portions of water from the test tube are then used to wash the filter of the final traces of barium and nitric acid. The contents of the suction flask are then transferred to a 100-mlvolumetric flask containing 10 ml of potassium chloride solution and diluted to volume with rinsings from the suction flask. A blank must be run for each glass filter funnel used in the determinations. The reasons for this will be discussed in a later section of this paper. Blanks are prepared in the same manner as the samples, using distilled deionized water in the place of amide solution.The water used in the blank must be the same used for preparing the sample amide solution in order not to produce error due to differences in dissolved CO? content. The solutions from both the sample and blank are then analyzed for their barium content using flame emission spectrometry.
RESULTS AND DISCUSSION Optimum Concentrationof Reagents. T h e concentration of bromine in t h e reagent is critical in this procedure. A high concentration of bromine will result in high recoveries for aromatic amides due t o t h e serious side reaction of bromination which yields a n insoluble organic precipitate. T h e resulting mixed precipitate of a brominated aromatic and barium carbonate becomes difficult to wash free of excess barium hydroxide in the reagent. A final concentration of 0.025 M barium hypobromite after t h e addition of reagent t o sample was found to give satisfactory results for selected amides and imides as indicated in Table I. The recommended low concentration of bromine and high alkaline concentration was found to eliminate the problem of bromination on all aromatic amides assayed with the exception of salicylamide. The phenolic moiety interferes because of its ability t o readily form an insoluble brominated phenol which includes barium from the reagent resulting in high recoveries. If the sample does not contain any aromatics, then the concentration of bromine may be as high as 0.15 M without any serious effects. Calibration Curve. The optimum working range used for Ba was 2-10 ppm which corresponds t o approximately 1.5-7.0 pmol of amide. Since barium is partially ionized in the nitrous oxide-acetylene flame, potassium chloride solution is added t o all standards and samples t o suppress ionization. T h e optimum concentration of potassium was determined experimentally t o be 2000 mg/l. for a 2-ppm solution of barium. Solubility of Barium Carbonate. The solubility of BaC03 in pure water at 20 "C is reported to be 0.002 g/100 cm3 ( I 7). This corresponds t o 0.10 pmol BaCOa/ml. However, because of the high concentration of barium in the reagent, the actual solubility of barium carbonate is much less than that reported for pure water. Water used t o wash t h e precipitate was maintained at 0 "C t o minimize losses due t o solubility. Source of the Blank. Two main factors were found t o contribute t o the unexpected high blank: barium carbonate production due t o contact of the reagent with atmospheric C02 and adsorption of barium from solution on the glass frit filter. Adsorption of barium on the glass frit was found to be directly related t o the flow rate of t h e filter used (Figure 1). The large difference between t h e slowest and fastest filters indicated that adsorption was the chief contributor to the blank. A second study was done t o determine the amount of blank responsible due t o adsorption. The extrapolated value of the y-axis of a blank vs. time curve is the amount of barium in the blank when the reagent is exposed t o atmospheric COz a t zero time or simply the blank due t o adsorption. T h e data ANALYTICAL CHEMISTRY, VOL. 49, NO. 2, FEBRUARY 1977
263
Table I. Determination of Primary Amides by Flame Emission Spectrometry Concentration, bmol/ml
Compound
3.41 5.58 2.85 5.37 2.19 3.50 2.08 5.64 7.67 5.09 4.70 5.50 4.31 4.10 4.53 3.61 3.23 2.76 1.61
1.705 2.79 1.425 2.685 1.095 1.75 1.04 2.82 3.835 2.545 2.35 2.75 2.155 2.05 2.265 1.805 1.615 1.38 0.805
Acetamide Propionamide Butyramide Isobutyramide Valeramide Isovaleramide Adipamide 2-Chloroacetamide Acrylamide Methacrylamide Benzamide p-Nitrobenzamide p-Toluamide o-Toluamide Salicylamide Nicotinamide Pyrazinamide Succinimide Phthalimide a
Micromoles Taken Found 3.41 5.31 2.80 5.17 2.19 3.44 2.07 3.19 8.05 5.03 4.64 5.61 4.44 4.14 6.80 3.58 3.29 2.76 1.63
Recovery, %a 100 f 5.8 (5) 95.2 f 5.1 (9) 98.2 f 4.8 (4) 98.3 f 5.5 (5) 100 f 8.6 (6) 98.3 f 6.5 (5) 99.5 f 5.5 (5) 56.6 f 13 (6) 105 f 1.8 (5) 98.8 f 6.0 (4) 98.7 f 3.2 (5) 102 f 3.0 (6) 103 f 5.1 (5) 101 i 4.1 (6) 150 f 2.6 (6) 99.2 f 6.5 (6) 102 f 3.6 (6) 100 f 6.5 (5) 101 f 2.7 (5)
Figures in parentheses indicate number of determinations.
Table 11. Product Recovery Comparison for the Hofmann Reaction
Compound
9
I3
/7
Acetamide Propionamide n-Butyramide Isovaleramide Benzamide Salicylamide Acrylamide Methacrylamide 2-Chloroacetamide
2l
N o w Rote f m / / m i n I Figure 1. Effect of filter flow rate on concentration of barium in the
blank
for the curve were obtained using the same filter and were found to be reproducible. The blank value due to adsorption divided by the corresponding value for the same filter in Figure 1 is the percent of the total blank due to adsorption. This value was found to be 83%or 17%of the blank resulted from atmospheric carbon dioxide interference. Thus 5% of the total barium carbonate produced from a 5-wmol sample of amide is due to carbonate production from atmospheric C02. Since the blank due to adsorption is reproducible and the blank due to atmospheric COZ may vary, the actual “working blank” is due only to atmospheric CO2. This value, as was stated, is on the order of an acceptable 5%which is reflected in the good accuracy of the results. Efforts to reduce this figure by working in CO2-free atmosphere were not taken because of the undesirable increase in total analysis time which would result. Utility of the Hofmann Reaction. The Hofmann reaction in the past has never been regarded as a useful analytical reaction for two reasons: 1)it has always been considered as a synthetic method and 2) the resulting amine is not always produced quantitatively. Table I1 compares literature values (18) for isolated amine produced to values for BaC03 pr9duced for the same amides. I t can be seen that for many aliphatic and aromatic amides, both the amine and the carbonate are produced in excellent yield. However, in many cases, 264
Recovery, % BaC09, Amine, flame emissions literatureb 100 95 98 98 99 150 105 99 57
70-80 85 90 90 Good 70 Poor Poorc Poor
Values reported are from Table I. Values reported are for isolated amine from reference 18. Yield obtained in aqueous media.
particularly where other functionalities are present in the molecule, the production of carbonate is quantitative while the production of the amine is poor. This is particularly true when the other functionality is subject to the reactive bromine in t h e reagent such as halohydrin formation or even oxidation of double bonds by hypobromite (19). 2-Chloroacetamide was the only compound assayed which gave a low recovery of barium carbonate. This is not surprising since a-haloamides have been reported to give low yields of the expected amine with many side reactions producing aldehydes, ketones, and gem-dihalides as the common products (20). Other Interferences. No interferences were observed from secondary and tertiary amides and nitriles as long as the reaction temperature did not exceed 75 “C. At higher temperatures (95-100 OC) these compounds have been observed to result in high barium carbonate recoveries. Isocyanates will interfere by hydrolysis to produce BaC03 but these are seldom found in combination with primary amides. Amide Mixtures. Successful determinations for total amide content on three amide mixtures have been accom-
ANALYTICAL CHEMISTRY, VOL. 49, NO. 2, FEBRUARY 1977
Table 111. Determination of Total P r i m a r y Amide in Mixtures
Mixture Acrylamide 3.83 ymol
Concentration, Micromoles pmol/ml Taken Found Recovery, O/oa 6.38
6.38
6.34
99.4 f 2.4 (6)
+
absorption spectrometry (21-28), only one calibration curve is needed. The sensitivity of the method is limited by the blank and the solubility of the precipitate. However, it may be possible to greatly increase sensitivity by working in a COzfree atmosphere and changing the solvent to alcohol to decrease barium carbonate solubility.
LITERATURE CITED
Methacrylamide 2.65 wmol Butyramide 2.41 wmol
5.02
5.02
4.77
95.0 f 4.8 (7)
+
Isobutyramide 2.61 wmol Propionamide 1.50 pmol
2.91
2.91
2.86
98.3 f 6.6 (7)
+
Butyramide 1.41 pmol a
As with methods for the assay of organic compounds by atomic
Figures in parentheses indicate number of determinations.
plished. T h e results are reported in Table 111. This method should be of particular value for the determination of total primary amide content a t trace levels in a mixture containing esters a n d anhydrides. Reaction Time. Completeness of reaction as a function of time for benzamide was studied. The results indicate that the reaction is complete after 12 min of heating a t 70 "C. Benzamide was chosen since alkylamides and benzamides substituted with electron donating groups (such as methyl) react more readily. A longer reaction time of 20 min was required in the determination of p-nitrobenzamide since the electron withdrawing nitro group retards the rearrangement.
CONCLUSION A successful procedure for the determination of primary amides by flame emission spectrometry has been developed.
(1) S. Siggia, "Quantitative Organic Analysis via Functional Groups", John Wiley & Sons, 3d ed., New York, p 140. (2)D.C. Wimer, Anal. Chem., 30, 77-80 (1958). (3)T. Higuchi, C. H. Barnstein, H. Ghassemi. and W. E. Perez, Anal. Chem.,
34, 400-3 (1962). (4)C. Steuli, Anal. Chern., 31, 1652-54 (1959). (5)S.Siggia and C. R. Stahl, Anal. Chem., 27, 550 (1955). (6)J. Mitchell and C. E. Ashby, J. Am. Chem. Soc., 67, 161-64 (1945). (7)F. Bergmann, Anal. Chem., 24, 1367-69 (1952). (8) R. E. Beckies and C. J. Theien, Anal. Chem., 22, 676 (1950). (9)W. M. Diggle and J. C. Gage, Analyst, 78, 473 (1953). (IO) S. Soloway and A. Lipschitz, Anal. Chem., 24, 898 (1952). (11) F. Acree, Jr., andM.Beroza, J. €con. Entomol., 55, 619(1962). (12)M. Maiayandi, J. P. Barrette, A. S. Y. Chau, and S. A. MacDonald, Abstracts of Papers, 6-65,154th Meeting, American Chemical Society, Chicago, Ill., 1967. (13)L. T. Sennello and C. J. Argoudelis, Anal. Chem., 41, 171 (1969). (14)S. P. Frankoski and S. Siggia, Anal. Chem., 44, 2078-81 (1972). (15) G. R. Leader, Anal. Chem., 42, 16 (1970). (16)C. R. Parker, "Water Analysis by Atomic Absorption Spectroscopy", Varian Techtron Pty. Ltd., Palo Alto, Calif., 1972. (17)R. C. Weast, Ed., "Handbook of Chemistry and Physics", The Chemical Rubber Company, Cleveland, Ohio, 1970-71,p 8-70,
(18)R. Adams, Ed., "Organic Reactions", Vol. 111, John Wiley & Sons, Inc. New York; 1946,p 267. (19)A. Weerman, Ann., 401, l(1913). (20) J. March, "Advanced Organic Chemistry: Reactions, Mechanisms, and Structure", McGraw-Hili Book Co., New York, 1968,p 817. (21)P. J. Oles and S. Siggia, Anal. Chem., 46, 91 1 (1974). (22)P. J. Oles and S. Siggia, Anal. Chem., 46, 2197 (1974). (23)P. J. Oles and S. Siggia, Anal. Chern., 45, 2150 (1973). (24)T. Mitsui and Y. Fuyimura, BunsekiKagaku, 23, 1309 (1974). (25)T. Mitsul and Y. Fuyimura, BunsekiKagaku, 23, 1303 (1974). (26)A. LeBihan and J. Courtot-Coupez, Analusis, 2, 695 (1974). (27)A. Maynard and M. J. Dodd, Aust. J. Med. Techno/., 5, 161 (1974). (28) J. B. Carisen. Anal. Biochem., 64,53 (1975).
RECEIVEDfor review September 17, 1976. Accepted November 8, 1976. This work was supported by Grant No. CHE76-07378 from the National Science Foundation.
ANALYTICAL CHEMISTRY, VOL. 49, NO. 2, FEBRUARY 1977
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