Table II. Evaluation of W a v e Length Shifters with Simple Liquid Scintillator Systems in Presence of p-Nitrotoluene
.
Vnlta
System"
p-Kitrotoluene (0.008 mmole None per ml.) in 2,5-diphenyloxa- lJ6-Diphenylhexatriene (10 mg./ zole-toluene-C14 (3 grams per liter) 2-(l-Naphthyl)-5-phenyloxazole liter) (50 g./liter) Tetraphenylbutadiene (100 mg./ liter) 1,4-bis [2-(5-phenyloxazolyl)]benzene (100 mg./liter) pNitrotoluene (0.008 mmole per None ml.) in terphenyl-toluene-C14 1,6-Diphenylhexatriene (10 mg./ (saturated solution a t 20" F.) liter) '
a
1100
0.49
1100
0.63
1100
0.61
1100
0.45
1040 1200
0.81 0.12
1140
0.46
2-(l-Naphthyl)-5-phenyloxazole 1100 (.50 g./liter)
Tetraphenylbutadiene (100 mg./ liter)
0.56
1220
0.18
1,4-bis[2-(5-phenyloxazolyl)] benzene (100 mg./liter)
1050
0.79
-
Total sample, 30 ml.
* Efficiency relative to pure 2,5-diphenyloxazole-toluene (3 grams per liter). applications, the effect of absorption can be neglected by counting the same concentration of labeled material for all experiments. Where different sample Feights of the same absorbing compound have to be radioassayed, a suit-
ACKNOWLEDGMENT
_^"I
a t Peak Relative Counting EffiRate ciency"
Wave Length Shifter
light attenuation primarily by ultraviolet absorption.
able correction can be made by first performing a study of 1/10 us. concentration, as illustrated in Figure 2. These conclusions should be applicable to the liquid scintillation counting of all colorless materials that exhibit
The authors wish to thank James R. Arnold, Princeton University, for his helpful discussions. Acknowledgment is given to T. C. Castorina and F. S. Holahan for preparation of the toluene0 4 . The authors also are indebted to the Ordnance Corps for permission to publish this manuscript. LITERATURE CITED
(1) Haves, F. N., U. S. Atomic Energy Commission, Rept. No. LA-1639,
May 1954. (2) Hayes, F. N., Gould, R. G., Science 117, 480 (1953). (3) Hayes, F. N., Hiebert, R. D., Schuch, R. L., Zbid., 116, 140 (1952). (4) Hayes, F. N., Ott, D. G., Kerr, V. N., Nucleonics 14, No. 1, 42 (1956). ( 5 ) Hayes, F. N., Ott, D. G., Kerr, V. N., Rogers, B. S., Ibid., 13,No.13, 38 11955). (6) Hayes, 8. N., Rogers, B. S., Sanders, P. C., Ibid., 13, No. 1, 46 (1955). ( 7 ) Kallman, H., Furst, M., Zbid., 8,KO. 3, 32 (1951). ( 8 ) Kallman, H., Furst, M., Phys. Rev. 79. 857 (1950). (9) Ibid.; 81, 853 (1951). (10) Tracerlog, No. 67, Tracerlab, Inc., Boston, Mass., February, 1955. RECEIVEDfor review June 8, 1956. Accepted September 29, 1956.
Photometric Determination of Aliphatic Amines HERBERT M. HERSHENSON' and DAVID N. HUME Department o f Chemistry and laboratory for Nuclear Science, Massachuseffs Institute of Technology, Cambridge
b Two modifications have been developed of a procedure for the determination of aliphatic amines, based on the characteristic absorption band from 750 to 950 mp of the complex which forms when the amine solution is added to an alcoholic solution containing excess cupric chloride. The first is a direct procedure for the determination of aliphatic amines in alcoholic solution or in aqueous solution of sufficiently high amine concentration to permit dilution with alcohol to a point below the limit where water interferes. The second is an extraction procedure in which chloroform is used to effect a separation from other interfering basic substances and from water.
of absorption (Figure 1) in the near infrared, about 750 to 950 mp ( 1 ) . The formation of the absorbing complex is independent of the nature of the aliphatic amine, and the very near infrared absorption occurs only when the cupric chloride is maintained in excess. The structure of the reaction product is not known, but it is definite that the reactants combine in the proportions of two cupric ions. four chloride ions, and one basic molecule or ion. This reaction has been made the basis of a method for the determination of aliphatic amines. All aliphatic amines produce the same product, and a single calibration curve serves for all of them in direct determinations.
T
Photometric measurements mere made with a Beckman Model B spectrophotometer, using 1.000-em. Corex cells. Appropriate cell corrections were applied to the observed readings whenever required. The absorbance measurements were made against P reference solution containing all the components except one (usually the basic substance).
APPARATUS AND REAGENTS
of aliphatic amines and certain other basic substances to an alcoholic solution containing a n excess of cupric chloride produces a visible color change from green to yellow, and the appearance of a characteristic zone HE ADDITIOK
Present address, Prntt and Whitney Aircraft Co., East Hartford, Conn. 16
ANALYTICAL CHEMISTRY
39, Mass.
This meant that the reference solution, as well as the sample, was normally highly colored. All inorganic chemicals were the best grade obtainable, usually reagent grade. Commercial absolute ethyl alcohol was used as the solvent without further purification. The amines used were obtained from a variety of sources and mere of varying purity. Solutions were made by weighing out quantities of the amine and diluting to the appropriate volume with solvent in a volumetric flask. These solutions were standardized by titration with hydrochloric acid, using phenol red indicator. Alcoholic amine solutions were standardized after dilution with an equal volume of water. The cupric chloride reagent solution was prepared to contain 50 mmoles (8.525 grams) of cupric chloride dihydrate per liter of absolute ethyl alcohol. METHOD
Direct Procedure. Dilute the sample with ethyl alcohol so t h a t a 1- t o 5ml. aliquot contains between 10 and 80 pmoles of amine. Place a 5-ml. portion of stock cupric chloride in a 25-ml. volumetric flask and add 15 ml. of absolute
.
ethyl alcohol. Add a suitable aliquot of sample (1 to 5 ml.), make up to 25.00 mi. with alcohol, and allow the wellmixed solution to stand for 20 minutes a t room temperature. Measure the absorbance at 860 mp using as a blank a cupric chloride solution containing 10.0 pmoles per ml. of absolute ethyl alcohol. When the sample contains inorganic bases and/or large amounts of mater, it is necessary to effect a separation of the amine prior to performing the determination. As shown below, moderate amounts of a number of solvents do not interfere in the determination; hence, it is possible to extract many aminese.g., with chloroform- and perform a slightly modified determination on the extracted sample. Extraction Procedure. If the sample is a n aqueous solution, take a 5-ml. aliquot or dilute a suitable aliquot t o 5 ml. with water. If the sample is alcoholic, dilute with water to reduce the alcohol content t o 20% or less and take a 5-ml. aliquot. T o the aliquot in a small separatory funnel, add 1 gram of potassium carbonate and dissolve with shaking. Extract with 5 ml. of chloroform previously saturated with water from a 20% potassium carbonate solution. Shaking for 1 minute is sufficient. Add a 1-ml. aliquot of the chloroform extract to 5 ml. of the stock cupric chloride which has been diluted with 15 ml. of absolute ethyl alcohol in a 25-ml. volumetric flask. Make up to volume, mix well, and let stand for 20 minutes a t room temperature. Measure the absorbance a t 860 mp, using as a blank a reference solution containing 10.0 pmoles per ml. of cupric chloride and 1.00 ml. of chloroform per 25 ml. in absolute alcohol. The calibration curve must be prepared by carrying known amounts of amine through the entire procedure.
EFFECT
OF
more than a few degrees from the temperature a t the time of standardization. AMINE COXCENTRATION. The colorforming reaction is complete even with no excess of cupric chloride, and excess cupric chloride has no effect upon the color. Preliminary experiments indicated that 4 pmoles per ml. of amine in the final solution produced an absorbance slightly greater than unity. il concentration of 10 pmoles per ml. of cupric chloride was, therefore, taken as a standard amount which would ensure that an excess was present for any amount of amine which would still give a readable absorbance value. The optimum range of amine concentration in the final solution, as indicated by the essentially linear portion of a Ringbom plot (d), was 0.5 to 3.0 pmoles per ml. using 1.00-cm. cells. The use of 10cm. cells makes it possible to extend the range downward by a factor of 10 without difficulty. By use of a 20-ml. sample in alcohol, as little as 1 pmole of amine may be determined. Beer's law is obeyed.
aqueous solution which is suitable for direct determination is about 1 mmole per ml. or a 1M solution. If the amine Concentration is lower than this, the water will interfere and the extraction procedure must be used. Inorganic bases such as sodium and potassium hydroxides react in the same manner as the aliphatic amines. Therefore, they constitute an interference which requires the use of the extraction procedure. Although aromatic amines give a reaction product with essentially the same spectrum as that obtained with aliphatic amines, they do not yield the same color intensity per mole from amine to amine. Aniline and p-toluidine gave Beer's law plots slightly steeper than that of the aliphatic amine-alkali hydroxideammonia curve, while those of methyl- and dimethylanidine were much less steep. Aromatic amines are, therefore, an interference in this determination. Samples of a single aromatic amine may be determined by use of a special calibra-
VARIABLES
Direct Procedure. TIME AKD TEMPERATURE. Color which developed in mixtures of cupric chloride and small amounts of amines in alcohol was unstable for the first 10 t o 15 minutes, and the absorbance tended t o drop several per cent below the initial value. After 15 minutes, however, the color was stable, and no change was observed over periods as long as 6 days. The effect of temperature was determined by letting made-up samples stand in water a t various temperatures during the 20-minute waiting period specified. I n the range from 0' to 50' C., differences were noted only when the samples were not brought to the same temperature before measurement. This is due to the high (compared to water) coefficient of expansion of organic solvents-approximat.ely 0.2% per O C. Calculated values agreed well with observed values over the temperature range studied. It is evident t h a t corrections must be applied whenever laboratory temperature differs by
10 Wove
Length, m p
Figure 1. Absorption spectrum of aliphatic aminecupric chloride-ethyl alcohol complex 1.64 pmoles per ml. of 1 -butylamine, 10 pmoles per ml. of cupric chloride; reference, same concentration of alcoholic cupric chloride
INTERFERENCES. As much as 13 mg. per ml. of water could be present in the final diluted alcohol solution before any serious interference resulted; larger amounts destroyed the color. Thus, it is possible to determine concentrated aqueous amine solutions by dilution with sufficient ethyl alcohol to reduce the final water concentration to less than 13 mg. per ml., which is about 2% by volume. For example, an aqueous solution of 70% ethylamine may be determined directly in this manner. The lower limit of amine concentration in
tion curve for that amine. Polyamines, amino alcohols, and amino acids form different complexes with cupric chloride and may not be determined this way. The interference of acids is prevented by neutralization with potassium carbonate in the extraction procedure. SOLVENTEFFECTS.The effect of a number of common organic solvents w a s tested by carrying out the direct reaction in solutions containing 10 and 50% by volume of the solvents in alcohol. The results in Table I are based on 200 tests a t three levels (0.28, 0.84, and VOL. 29, NO. 1, JANUARY 1957
17
~~
Table 1.
Effect of Solvents on Color intensity Developed with sec-Butylamine Extent of Absorbance Changea
-
Solvent Methanol 1-Propanol 2-Propanol 1-Butanol 2-Butanol 2-Rlethylpropanol 2-Methyl-2-propanol 1-Pentanol Cyclohexanol Chloroform Carbon tetrachloride Acetone Methyl ethyl ketone Cyclohexanone Diethyl ether Nitromethane 0 to 3% change, no Q
~
Against Ethyl Alcohol Blank Against Mixed Solvent Blank 10% by V O ~ . 50% by V O ~ . 10% by V O ~ . 50% by V O ~ . Slight Great Great Slight Kone None None None Great Moderate h-one None Great Great Slight None Slight Great Great Great Yone Slight Xone Slight Great Great Great Moderate Great Great Great Great Great Great Great Great Sone Slight Xone None hTone None None None None Sone Slight Yone Slight Moderate Slight Sone Great Great Great Great None Slight Slight Slight Great Great Great Great effect; 3 to 6%, slight; 6 to 10%. moderate; >lo%, great.
1.40 kmoles per ml.) of sec-butylamine, which 1%-astaken as typical of the aliphatic amines. A substantial number of common solvents, particularly in concentrations below 10% by volume, have no effect on the results. This makes it possible to determine amines in these solvents directly upon dilution with alcohol. Better accuracy can usually be obtained by the use of a calibration curve made up from solutions containing the same proportion of solvent as found in the sample. Evperiments on the possibility of substituting other solvents for ethyl alcohol showed that essentially the same reaction takes place in systems using 2-propanol, 1-butanol, or 2-methyl-2propanol. Of the three substitutes tested, 1-butanol is the most satisfactory because it has about the same sensitivity as ethyl alcohol; 2-propanol gives a considerable loss of sensitivity, while the freezing point of 2-methyl2-propanol is too close to room temperature for convenience. Other solvents niight also be found satisfactory. Nethaiiol has an effect similar to that of nater and cannot be used. SEQUESCEEFFECTS. I n cases where the amine is in alcoholic solution and no interferences such as water are present, the sequence in which the amine and cupric chloride are mixed and diluted with alcohol is not important. But if the amine sample contains water, or water is added as a separate step, then the order of addition is important in those cases rhere the amount of water is close to the tolerable limit. ACCUrate results were obtained only when the concentration of water mas brought below the tolerable limit before the amine was added to the cupric chloride. For example, if the amine sample was added to a mixture of the cupric chloride and water and then the solution was diluted with alcohol to its final volume, erroneous results were obtained: on the 18 *
ANALYTICAL CHEMISTRY
other hand, if the cupric chloride and water mixture was first diluted almost to its final volume, the addition OF the amine produced the correct result. Therefore, in any case where water or some other interference may be present in the sample, it is important first to dilute the cupric chloride solution with alcohol almost to its final volume. Extraction Procedure. COMPLETET h e extent t o NESS OF EXTRBCTiON. which extraction had taken place under various experimental conditions was determined by comparing the absorbance found for t h e extracted sample with t h a t calculated for complete extraction using a calibration curve made with standard amine solutions containing the same proportion of chloroform. The extent of extraction was found to be constant with times of shaking from 0.5 t o 3 minutes. For convenience, 1 minute was chosen for the standard procedure. The effect of temperature was found to be negligible-somtions extracted at 0" and a t 30" C. gave the same results, within &2%, as samples extracted a t room temperature. The initial concentration of amine in the aqueous solution was likewise found to be unimportant in the range from 20 to 100 pmoles per nil. The recovery of amine in the extraction procedure is neyer complete, a single extraction yielding only about 75%. Evidently there is some loss by volatility when the potassium carbonate is added (an exothermic reaction), for even five successive extractions did not give complete recovery. However, the single 5-ml. chloroform extraction is rapid and convenient. Furthermore, quantitative recovery of the chloroform extract is not essential if the standards are carried through the same procedure in preparing the calibration curve. CARBONATE COXCENTRATIOI~. Po-
tassium carbonate is added to neutralize any acidic substances, provide a basic medium, and aid in salting out the free amine. The percentage of amine extracted varied considerably with the amount of carbonate added, as shown in Figure 2. The addition of 1 gram was selected as optimal for the standard procedure. CALIBRATION CURVE. Of the various amines used to test the extraction procedure, diethyl, triethyl, 1-butyl, and sec-amyl had almost identical extraction characteristics; in fact, a single (molar basis) calibration curve for sec-amylamine served for the determination of any of these with a n accuracy of better than &5%. Ethylamine gave much lower results (43% error from the amylamine calibration curve), indicating losses by volatility, inextractability, or both. The precision with ethylamine was normal, however, indicating that good results could be obtained if an ethylamine-based calibration curve were used. For the best results in general, the calibration curve should be made with the same amine as is being determined.
0 0
0.5
1.0 1.5 Grams K2C03 Added
2 .o
Figure 2. Extraction as function of potassium carbonate added
INTERFERENCES. Any substance present in the sample which is eutractable into the chloroform and which will react with cupric chloride may interfere with the determination of aliphatic amines. Inorganic salts may causp some difficulty, if present in large amounts, by altering the extractability of the amine. A brief study on the effect of sodium acetate and sodium benzoate showed that the presence of 0.5 gram of sodium acetate in a 5-ml. aqueous amine sample caused no change in the final absorbance of the chloroform extract, while 0.5 gram of sodium benzoate caused a n increase of about 10% in the observed absorbance. Acidic substances which would interfere in the direct procedure are neutralized by the excess potassium carbonate in the extraction procedure. The presence of much alcohol in a sample containing both amine and in-
~~
organic base interferes with the extraction of the amine. Experiments also showed that the extraction of an amine from an aqueous solution containing as much as 20 or 30% ethyl alcohol could be carried out successfully, although, as would be expected, with decreased efficiency. Therefore, it is recommended that the original sample be diluted with sufficient water to give a solution about 20y0 in alcohol before attenipting the extraction. I n such a procedure, the calibration curve must be constructed by extracting solutions of standard amine which contain the same proportion of alcohol as the sample. AMINECONCEKTRATION. I n order to obtain a final absorbance reading which falls between 0.1 and 1-2, the concentration of amine in the initial 5-ml. aqueous solution which is extracted should be in the range of from 10 to 100 pmoles per ml. Thus, the minimum aqueous amine concentration which can be measured satisfactorily by the extraction method is about 20 times greater than that determinable by the direct method. However, the lower limit for the direct determination of amine in aqueous solution is 1000 pmoles per ml., or about 100 times as great as that measurable by extraction. Therefore, the extraction procedure has a quite wide range of applicability to aqueous amine solutions of intermediate concentration. RESULTS
To estimate the repeatability of the direct procedure, a series of 11 identical samples, each containing 25 pmoles of sec-butylamine, was run a t one time. The average absorbance obtained was 0.329 with a range of 0.007 and a standard deviation of 0,0018. The coefficient of variation (per cent standard deviation) was 0.55yo. For a better picture of the reproducibility on a day-to-day basis, a series was run in which three samples of a stock secbutylamine solution were run each day for 9 days. Analysis of variance applied to the results showed that the betweendays variability was, as expected, greater than the vithin-days variability. The difference just reached the 5% level of significance. I n terms of standard deviations, the within-days variability was 0.0035, the between-days rariability, 0.0057, and the over-all
Table
II.
Amine Butyl
Diethyl Triethyl
~~~~
~~
Results with Various Amines Using Single Calibration Curve
rlbsorbance 0.519 0.521 0.252 0.255 0.405 0.406 0.280 0.276 0.303
0.304
0.303 0.279 0.285 0.452 0.453 0.217 0.221
Diisopropyl Dicyclohexyl
a
Concn., polmes/Ml. Found Present5 1.64 1.64 1.65 1.64 .~ 0.82 0.86 0.82 0.87 1:31 1.34 1.31 1.34 0.94 0.99 0.93 0.99 1.01 0.99 - . ._ 0.99 1.01 0.99 1.01 0.94 0.98 0.95 0.98 1.45 ,1.47 1.45 1.47 0.75 0.74 ~
0.76
0.74
70
Error 0 $0.6 +4.8 +6.1
-2.2 -2.2 -5.0
-6.0 $2.0 $2.0 $2.0 -4.1 -3.0 -1.4 -1.4
+1.3
$2.7
HC1 standardization. Table 111.
Determination of Amine Mixtures
hbsorbance Amine Mixture Expected Found Butylamine and dicyclohexylamine 0 480 0 490 Dicyclohexylamine and triethylamine 0 537 0 542 0 565 0.580 Butylamine and triethylamine 0 791 0.830 Butylamine, dicyclohexylamine, and triethylamine
variability, 0.0034. The latter figure, R hich corresponds to an over-all coefficient of variation of 1.9%, probably represents the best appraisal of the precision of the direct procedure in daily routine use. Table I1 shows a test of the use of a single calibration curve (that of butylamine) for the determination of a variety of different amines. All the amine stock solutions were made up in alcohol and standardized by titration with hydrochloric acid. Table I11 lists the results of an experiment designed to demonstrate that the absorbance of a mixture of two or more aliphatic amines is the sum of the individual absorbances. The absorbance of each component n-as determined separately and the expected absorbance of the mixture is the sum of the individual absorbances found for its components. I n order to determine the variability introduced in the extraction step, 14 identical samples of sec-amylamine were run over a period of 3 days. Each solution contained 19.6 pmoles per ml.
%
Diff. $2 1 +O
9
+2 7 $4 9
of sec-amylamine initially, and was extracted with 5 ml. of chloroform in the usual, manner. The average absorbance found was 0.182 (theoretical, 0.250), with a standard deviation of 0.0066, corresponding to 72.9% estraction with a standard deviation of 2.640j0, or a coefficient of variation of 3.6%. The extraction procedure, although considerably more variable than the direct procedure, g.ives a precisioii which is satisfactory in view of the complexity of the manipulations involved, ACKNOWLEDGMENT
This work was supported in part by the U. S. Atomic Energy Commission. LITERATURE CITED
(1) Hershenson, H. M., Ph.D. thesis, Massachusetts Institute of Technology, January 1952. (2) Ringbom, A., Z. anal. Chem. 1 1 5 , 332 (1939). RECEIVED for review July 18, 1956. rlccepted October 5, 1956.
VOL. 29, NO. 1, JANUARY 1957
19
