absorbance of 0.422, 0.3%. It is difficult t o make a quantitative comparison between the two machines, since the Beckman instrument in the authors' hands does not give as good results as those obtained by Lowry and Bessey (8). The results obtained with the present machine appear to be more reproducible, by a factor of 2 to 5, than those routinely obtained on the Beckman instrument under the conditions of operation. The larger part of the improved performance is ascribed to the accurate positioning of the cuvettes in the present instrument. However, the absence of dark current and the stability of the signal also contribute to the improved performance, particularly a t low absorbance. The machine has been in use for about a year, and has performed reliably over this period. The instrument has been used for the microanalysis of inulin by the Lowry
(1) modification of the method of Roe, Epstein, and Goldstein (8). I n one experiment, seven 0.37-pl. samples of inulin mere taken from a solution containing inulin in a concentration of 4 y per pl. The results of the colorimetric analysis had a standard deviation of o.9yO. A similar replicate analysis (seven 0.37-p1. samples) from a solution containing 1 y of inulin per microliter gave a standard deviation of 2.2%. I n addition to the excellent reproducibility, the new microcolorirneter has the advantages of freedom from dark current, the accommodation of a larger number of samples, the provision of a written record, and great rapidity of measurement (five samples and a blank can be measured and recorded in less than 200 seconds). Furthermore, the results obtained do not depend on the skill of the operator.
ACKNOWLEDGMENT
Lloyd Cail of Laboratory Associates, Inc., has provided welcome and necessary assistance in the design of the mechanical part of the apparatus and is entirely responsible for its construction. Joan Lord and Bradford D. Pearson have helped by providing the cobaltous chloride measurements. LITERATURE CITED
( 1 ) Lowry, 0. H., Washington University,
S t . Louis, Mo., personal eommunication.
(1949):
RECEIVEDfor review July 31, 1957. Accepted September 21, 1957. Work supported in part by the Atomic Energy Commission.
Microdetermination of Vo ati e Aldehydes IRVING R. HUNTER and EARL
F. POTTER
Western Regional Research laboratory, Albany, Calif.
b In the microdetermination of volatile aldehydes, particularly those generated by and distilled from ninhydrin oxidations of amino acids, the aldehydes are absorbed in and made to react with an excess of sodium bisulfite to form stable complexes. Excess bisulfite is removed b y oxidation with iodine. A measured amount of iodine in excess of the aldehydebisulfite equivalent and an alkaline buffer are added to the residual aldehyde-bisulfite complex. The iodine quantitatively oxidizes the bisulfite and aldehydes. Excess iodine is then determined b y back-titration with standard thiosulfate. A blank is run a t the same time and the difference in titration values is used to calculate aldehyde equivalent.
C
of rice that may be related to its behavior under various processing treatments are under investigation a t this laboratory. One of the groups of compounds being studied is the free amino acids, 18 of which have been identified by paper chromatography ( 3 ) . I n developing a method for determining some of the free amino acids of parboiled and other forms of rice by oxidizing them with ninhydrin and separating the volatile aldehydes by gas-liquid chromatography (i?),it became necessary to know whether the system of generation and collection ONSTITUENTS
used yielded aldehydes quantitatively prior to the actual partition phase of the analysis. Although numerous methods are available for determining aldehydes, one involving an initial reaction between aldehydes and sodium bisulfite was considered the most suitable for this purpose. This reagent reacts rapidly with aldehydes to form stable complexes. Other reagents-e.g., hydroxylamine and phenylhydrazine derivatives -react completely with aldehydes only after prolonged contact and would be difficult to use for absorbing very volatile aldehydes-e.g., acetaldehydefrom a gas stream. The method adopted proved more satisfactory from the standpoint of accuracy and precision than other procedures (1, 4, 5 ) . It is currently in use a t this laboratory as an aid in developing a method for determining amino acids. Aldehydes generated from amino acids are collected in an excess of 1% bisulfite solution. The solution is treated with iodine t o oxidize excess bisulfite. A measured amount of standard iodine and an alkaline buffer, added t o the solution, quantitatively oxidize both the bisulfite and aldehyde moieties of the complex. Excess iodine is determined by titration with standard thiosulfate. A blank is run a t the same time and the difference in titration values is used t o determine aldehyde equivalents.
APPARATUS A N D REAGENTS
Oxidation apparatus shown in Figure 1 is custom-made with standard borosilicate laboratory glassware. All chemicals are reagent grade unless otherwise specified. Ninhydrin, Eastman Kodak Go. white label. Sodium bisulfite, 1%, freshly prepared. Iodine, 0.1N and 0.02N in water (standardized). Soluble starch indicator, 0.5y0 protected from deterioration with elemental mercury. Sodium thiosulfate, 0.02N, freshly preDared from 0.1N reagent bv dilution. * Sodium carbonate;2N. " Sulfuric acid, 1N. Xitrogen, compressed, water-pumped. Potassium dihydrogen phosphate. Sodium chloride. Amino acids, commercial samples. All showed single ninhydrin reacting spots on a two-dimensional paper chromatogram, when the method and solvents described by Hunter, Ferrel, and Houston were used ( 3 ) . Aqueous solutions were prepared and protected with 0.01% sodium ethyl mercurithiosalicylate. Silicone oils, Dow Corning 703 and 550. PROCEDURE
Amino acids were oxidized to aldehydes by ninhydrin essentially by the method of Virtanen and Rautanen (6). Ten milliliters of an amino acid solution containing approximately 3 mg. of amino nitrogen were pipetted into the flask and 1 gram of potassium dihydrogen phosphate and 2.1 grams of sodium chloride were added, either as solids or as saturated solution. VOL. 30, NO. 2, FEBRUARY 1958
293
Two milliliters of silicone oil D.C. 703 and 114.3 mg. of solid ninhydrin (1.5 times the stoichiometric amount calculated by multiplying milligrams of nitrogen in sample by 38.1) were added. The flask was then connected t o the water-cooled condenser (cleaned with detergent), whose outlet was joined through a capillary stopcock t o the base of a long column (Figure 1) containing 25 ml. of sodium bisulfite solution. A fast stream of water in the condenser kept the upper two thirds cool. The solution was heated by a silicone D.C. 550 bath maintained at 100" t o 105" C. During reaction nitrogen was passed through the side arm of the flask a t about 80 to 90 ml. per minute, to transfer evolved aldehydes into the sodium bisulfite. Heating was continued for 45 minutes in most instances. Phenylalanine, however, required 2.5 hours and methionine reauired 4 hours of heating. A t the end of the distillation the absorber was disconnected and the aldehyde-bisulfite solution was quantitatively transferred t o a 50-ml. volumetric flask and made u p t o the mark. Ten milliliters of the bisulfite solution were pipetted into a 126-m1. Erlenmeyer flask, a few drops of starch indicator were added, and excess bisulfite was destroyed by titrating with 0.1N iodine solution just short of the end point. The end point was reached by careful titration with 0.02.47 iodine from a 10-ml. buret. Ten milliliters of 0.02N iodine were then added, followed by 5 ml. of 2N sodium carbonate. The solution was placed in the dark for 20 t o 30 minutes (Table I). Ten milliliters of 1.V sulfuric acid were then added, and the flask was lightly stoppered and allowed to stand for 10 minutes in the dark. A blank sample of bisulfite was treated in the same manner as the sample. All procedures were run in duplicate. At the end of the 10-minute period, unreacted iodine was titrated with 0.02N thiosulfate.
Table 1. Effect of Reaction Time on lsobutyraldehyde Prepared from Valine by Ninhydrin Oxidation
Reaction Time, Min. 4
Aldehyde Found,
%
96.1 98.5 99.3 99.5 99.6 99.6
10
15 20 25 30
Aldehydes were calculated by applying the equation: Millimoles of aldehyde
=
5/4N (V
ANALYTICAL CHEMISTRY
Aldehyde Obtained from Amino Acids b y Oxidation with Ninhydrin
Amino Acid Alanine
Aldehyde, Millimole' Calcd. Found 0.0224 0.0224 0.0223 0.0220 0.0220 0.0148 0.0148 0,0151 0.0150 0.0153 0.0154 0.0150 0.0305 0.0304
Aldehyde Formed Acetaldehyde
Valine
Isobutyraldehyde
Leucine
3-Methylbutanal
Isoleucine
2-Methylbutanal
0.0162
Korleucine
Valeraldehyde
0.0155
a-Aminobutyric acid
Propanal
0.0272
Methionine
Methional
0.0101
Phenylalanine
Phenylacetaldehyde
0.0121
Yield, yo 100.2
99.5 99.2 99.2 100.0 102.0 101.3 100.6 98.0 99.6 99.6 101.2 100.0 100.0 98.1 98.7 97.4 97.4 99.5 100.5 99.0 100.0 100.0 98.3 98.3
0.0304
0.0240
0.0164 0.0162 0.0165 0.0152 0.0153 0.0265 0.0265 0.0100 0.0101 0,0099 0.0121 0.0121 0.0236 0.0236
Valine Isobutyraldehyde 0.0108 100.9 Leucine 3-Methylbutanal 0.0107 0.0109 101.9 Phenylalanine Phenylacetaldehyde Alanine Acetaldehyde Valine Isobutyraldehyde 0.0141 100.8 Leucine 3-Methylbutanal 0,0140 0.0140 100.0 Phenylalanine Phenylacetaldehyde h'orleucine Valeraldehyde Prooanal a-Aminobutyric acid 0.0140 100.0 Methionine Methional 0.0140 0.0137 97.9 Isoleucine 2-Methylbutanal a Based on 100% purity of amino acid and 100% aldehyde generation and recovery.
11 I
'0.
RESULTS AND DISCUSSION
The method described was used in conjunction with ninhydrin oxidation to determine eight amino acids individually and in groups. The amounts of aldehyde titrated varied from 0.01 to 0.03 mmole. Substantially complete recovery of aldehydes (100 & 2%) was obtained from all of the original amino acids. Representative results are shown in Table 11. I n the iodine oxidation of aldehydebisulfite, reaction velocity rises with increasing pH, which must be about 10 before reaction will proceed quantitatively in a reasonable time (15 to 30 minutes). This p H is maintained by the sodium carbonate buffer. Table I illustrates the effect of reaction time on determination of isobutyraldehyde by this method.
8 m m x 71 cm
LJ hi;jtted
Dlsk
(yarse)
I& m m
ACKNOWLEDGMENT
- VI)
where N is the normality of thiosulfate, V is volume of sodium thiosulfate used t o titrate t h e blank, and T." is volume of sodium thiosulfate used t o titrate unreacted iodine in the unknown.
294
Table II.
The authors wish to thank R. E. Ferrel for helpful advice in the experimental work, N. Floy Bracelin Figure 1. Ninhydrin oxidation apparatus
y l
for preparing the illustration, and K. T. Williams, E. B. Kester, and Yoshio Tomimatsu for technical advice. LITERATURE CITED
(1) Feinberg, B. G., Am. Chem. J . 49, 87 (1913).
(2) Hunter, I. R., Dimick, K. P., Corse, J. W.,Chem. & Znd. (London) 16, 294 (1956). ( 3 ) Hunter, I. R., Ferrel, R. E., Houston, D. F., J . A q r . Food Chem. 4, 874 (1956). (4) Parkinson, A. E., Wagner, E. C., ISD. EXG. CHEM., .4sa~. ED. 6 , 433 (1934).
(5) Tomada, J., J . SOC. Chem. Znd., Japan, 30, 747 (1927); J. SOC. Chem. Znd. 48, 79 (1929). (6) S’irtanen, A. I., Rautanen, N., Soumen Kenaistilehtz 19B, 56 (1946).
RECEIVEDfor review June 10, 1957. Accepted October 4, 1957.
Semimicro Hydrogenation with Electrically Generated Hydrogen JOHN W. MILLER’ and DONALD D. DeFORD Department of Chernisfry, Northwestern University, Evanston, 111.
b As hydrogenation apparatus previously described does not allow simple pressure regulation or easy measurement of reaction rates, a simple apparatus was designed. The conventional gas buret and leveling bulb were replaced b y a U-shaped electrolysis cell, which served as both a source of hydrogen and a pressureregulating device. The electrical current used to produce the hydrogen consumed in the hydrogenation was measured by an electronic coulometer, which also operated a stepping recorder. Curves were automatically plotted. A variety of organic compounds were hydrogenated with an accuracy to f 3 % and a standard deviation of 2%. If room temperature or atmospheric pressure does not change during hydrogenation, a hydrogen uptake of less than 2 ml. can be accurately measured.
H
has been used for the analysis of unsaturated materials for over 50 years. The “simplified type” of apparatus was first introduced in 1908 by Fokin (6) and Paal and Gerum (8). Many modifications of the original apparatus have been described (2, 7 , 9, l l , 12, Id), all based on volumetric measurement of hydrogen uptake. The lower limit of accurate measurements is dependent on the size of the gas buret. Most methods have a lower limit of 3 to 5 ml. of hydrogen consumption. Therefore, a n investigation was undertaken t o design and construct a hydrogenation apparatus which would not require volumetric gas measurements and would allow simple, automatic pressure regulation. With the restriction of measuring gas volumes eliminated, the method could be extended YDROGENATION
Present address, Research Division, Phillips Petroleum Co., Bartlesville, Okla.
to smaller volumes of hydrogen. The apparatus and method mere based on the use of electrically generated hydrogen as a source of gas and elimination of gas burets. Manegold and Peters (6) were the first t o use electrically generated hydrogen in place of the conventional gas buret. I t s use for the determination of relative rates of hydrogenation has been described by Farrington and Sawyer (4). The new apparatus is a modification of that of Manegold and Peters (6), which embodied a cell for electrically generating hydrogen, a gas coulometer t o measure the amount of electricity consumed, and a reaction vessel. This apparatus measured both the rate of reaction as indicated by the current intensity and the total amount of gas consumed as shown by the coulometer, but the current had t o be adjusted manually during a run, so that the rate of generation equaled the rate of disappearance of hydrogen. The apparatus was constructed t o measure large volumes of gas (liters) and was too complex for easy reproduction and maintenance. These disadvantages \yere overcome by automatic electrolysis, which regulated the pressure and varied the electrolysis current so that rates of consumption and generation of hydrogen were equal.
A schematic diagram of the apparatus is shown in Figure 1. The sample, solvent, and catalyst are contained in the reaction flask, F . The flask is connected to the U-shaped electrolysis cell, Tz. by the gas manifold and drying tube, T I . The entire system is closed t o the atmosphere by stopcocks and by placing generator electrolyte in T z . The catalyst is prereduced with hydrogen; when reduction is complete, the increase in the hydrogen presfiire in the system causes the liquid to be pushed away from the electrode. The contact b e h e e n the electrode and electrolyte is broken, and the elec-
trolysis current is automatically shut off. The pressure in the system exceeds t h e atmospheric pressure by an amount which corresponds to the difference in liquid levels in the U-tube arms. When the sample is introduced into the solvent, reaction takes place and the hydrogen pressure in the system decreases, causing the liquid to make contact with the electrode. The rate of hydrogen generation is adjusted to equal the initial rate of hydrogen consumption. After this setting is made, the hydrogenation is automatic. Some degree of current control is achieved by employing a tapered electrode, so that its depth of immersion in the electrolyte governs the current flowing. As hydrogenation proceeds, the rate of hydrogen uptake decreases, and the liquid level falls, slowing down the rate of hydrogen generation. If the rate of generation exceeds the rate of consumption because of a slow hydrogenation reaction, the instrument will cycle on and off. During the off-cycle the pressure in the system slowly drops until the liquid makes contact with the electrode again. K h e n no hydrogen is generated for a given length of time, the reaction is assumed to be complete. A stepping recorder was placed in the coulometer circuit, so that the number of milliequivalents of hydrogen consumed was plotted against time. After the initial adjustments, the hydrogenation proceeds to completion automatically with no operator attention. The results are then calculated from the coulometer reading as registered on the recorder. APPARATUS
The reaction vessel, F , u-as constructed from a 25-ml. Erlenmeyer flask to which was added a standardtaper 14/20 joint, J,. A short length of 6-mm. tubing served as a side arm for introduction of sample. A female standard-taper 14/20 joint, sealed to a 3inch length of 1-mm. capillary tubing, connected the reaction vessel to the rest of the system. The ball and socket VOL. 30, NO. 2, FEBRUARY 1958
295
