Polarographic Determination of Formaldehyde in Biological Material Application to the Determination of Serine M. JOHN BOYD AND KARL BAMBACH, College of Medicine, University of Cincinnati, Cincinnati, Ohio
F
the half-wave of the formaldehyde step occurs at about - 1.63 volts (normal calomel electrode). Accurate temperature control is important; a constant-temperature water bath thermostatically controlled to 0.1 C. was used. The authors’ results confirm Barnes and Speicher’s findings (1) that the wave height changes about 6.5 per cent for each 1’ C. change in the temperature of the solution. The height of the step is measured in the usual way and compared with a calibration curve made with known quantities of formaldehyde at the same temperature and drop rate, and with the same capillary.
ORMALDEHYDE has been determined quantitatively by the polarograph according to Jahoda (S),Winkel and Proske (b), and Barnes and Speicher (1). Recently Boyd and Logan (2) have developed a colorimetric method for the determination of formaldehyde as a step in the procedure for estimating serine. Their method consists essentially of quantitatively separating by distillation the formaldehyde resulting from the action of periodate on protein hydrolyzates containing serine, and condensing this formaldehyde with chromotropic acid (Eegrewe’s reagent) to form a colored compound.
Discussion
TABLEI. DETERMINATION OF FORMALDEHYDE BY P O L a R O GRAPHIC AND COLORIhfETRIC METHODS
Sample 1 mg. of serine 1 mg. of serine 5 mg. of serine 5 mg. of serine 5 mg. of serine 50 mg. of serine 50 mg. of serine
++ 100 mg. of threonine 100 ma. of threonine
Gelatin hvdrolvzate Gelatin hidrolyzate Salniin hydrolyzate Salniin hydrolyzate Salmin hydrolyzate Casein hydrolyzate Casein hydrolyzate Horse hemoglobin (crystallized) drolvsate Horse hemoglobin (crystallized) drolyzate D o g hemoglobin (crystallized) h> sate
Polarogra hic Methog Colorimetric I n 3 ml. of Method distillate Per ml. Per ml. iMicrograms 8 2.7 2.7 2.9 2.8 8.7 14.1 41 13.7 14 14.1 42 14 14.2 42 382 127 128.8 131.6 385 128 155 52 49.5 52 155 48.7 20.5 6.8 6.5 21 6.7 22 j.3 6.5 21 7.0 7.0 22 7.0 7.3 19
6.3
19.5
6.5
6.4
19
6.3
6.1
6.1
I n order to ascertain whether the polarograph can be used to determine the formaldehyde found in the distillate in the above procedure, the authors compared polarographic analyses against those run colorimetrically. The polarographic procedure proved suitable for this type of determination, since no substances which interfered with the production of smooth current-voltage curves came over in the distillation.
The results obtained by the polarographic method agree with those obtained by the colorimetric method of Boyd and Logan (2) as shown in Table I. Quantities of formaldehyde as low as 3 micrograms per ml. of distillate can be determined by the polarographic procedure. According to Barnes and Speicher, when potassium hydroxide is the only supporting electrolyte, the full height of the formaldehyde step is not given in concentrations of potassium hydroxide below 0.5 per cent, and so those workers used 1.25 per cent solutions in their determinations. I n the authors’ experience, satisfactory curves with the full height of the formaldehyde wave are obtained with 0.05 N potassium hydroxide in 0.1 N potassium chloride, so that it is not necessary to employ stronger alkali, thereby decreasing the possibility of condensation of the formaldehyde. Acetaldehyde (which arises from the action of periodate on threonine, 4) does not interfere in the analysis unless it is present in extremely large amounts, since it is not reduced until a potential of -1.8 volts is reached (Figure 1). This method cannot be used for the determination of small amounts of acetaldehyde unless the potassium ion in the supporting electrolyte is replaced by lithium, because the potassium wave interferes with that of acetaldehyde. However, good results can be obtained with acetaldehyde in lithium hydroxide. Other aldehydes, higher in the aliphatic series, are reduced a t still higher voltages.
Procedure The formaldehyde is separated from the protein hydrolyzate and the periodate reaction mixture by distillation, as described by Boyd and Logan (3). Three milliliters of the distillate are mixed with 1 ml. of a solution of electrolytes (0.4 N potassium chloride, 0.2 N potassium hydroxide), and nitrogen is bubbled through the mixture for 3 minutes. This does not result in loss of formaldehyde, since repeated runs on the same solution showed that gassing for as long as 15 minutes did not change the height of the ntep. The olarogram of the solution is then taken Prom -1.4 to -1.8 volts with the Leeds & Northrup Electro-chemograph;
FIGURE1. POLAROGRAM OBTAINED WITH LEEDS& NORTHRUP ELECTROCHEMOGRAPH
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May 15, 1943
ANALYTICAL EDITION
This method need not be limited t o the determination of formaldehyde liberated by the action of periodate On serine* It can probably be applied t o the determination of formaldehyde in other types of samples; as long as the compound can be isolated by distillation, reasonably free from interfering substances, i t can be estimated polarographically.
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Literature Cited (1) Barnes, E. C., and Speicher, H.W., J. I d . Hyo., 24, 10 (1942). (2) Boyd. M. J., and Logan, M. A., J. BWZ. chm., 146,279 (1942). (3) Jahods, F. G., CoZZection Czechosloo. Chem. Commun., 7, 415 (1935). (4) Shinn, L.A,, and Nicolet, B. H., J.Biol. Chem., 138,91 (1941). (5) Winkel, A.,and Proske, G., Ber., 69,693 (1936).
Determination of Small Amounts of Molybdenum in Tungsten and Molybdenum Ores F. S. GRIMALDI AND R. C. WELLS, Geological Survey, u.
A rapid method has been developed for the determination of small amounts of molybdenum in tungsten and molybdenum ores. After removing iron and other major constituents the molybdenum thiocyanate color is developed in water-acetone solutions, using ammonium citrate to eliminate the interference of tungsten. Comparison is made by titrating a blank with a standard molybdenum solution. Aliquots are adjusted to deal with amounts of molybdenum ranging from 0.01 to 1.30 mg.
of the calcium tungstate ores froin contact-metaM O Smorphic T deposits in the western states contain a t least a trace of molybdenum, and in many such ores as much as 0.5 per cent of molybdenum niay be present. As concentrates from such ores are subject to a penalty, the determination of molybdenum is important. I n its search for new supplies of the different strategic metals the U. S. Geological Survey has examined various samples of possible molybdenum content t h a t n-ere on the border line betm-een a little and no molybdenum. For such work a quick b u t accurate method was needed. One method developed for this purpose is based on fluorescence ( 1 ) ; the other depends on chemical analysis and colorimetric estimation of the molybdenum. The amber color developed by molybdenum, thiocyanate, and a reducing agent has long been used as a sensitive test for molybdenum, the colored complex usually being concentrated by extraction with organic solvents such as ether, butyl acetate, or cyclohexanol, before colorimetric estimation. Ether, the most common solvent employed, has several objections, as noted by Sandell (6). The authors have found that even a few milligrams of tungsten or vanadium cause high results with the ether extraction alone, unless tartrate or citrate is present, Hurd and Allen ( 3 ) studied the conditions for maximum stability of color, using various solvents, and stated that “butyl acetate, although a n excellent solvent for the thiocyanate, should be used with care. The color produced by a given concentration of molybdenum appears to depend to no slight extent upon the manner in which the solvent has been saturated with the reagents.” They reported t h a t cyclohexanol is difficult to handle, separates slowly from aqueous solution, and may develop a turbidity.
s. Department of the Interior, Washington, D. C.
The writers dispense with extractions altogether, but employ a n acetone-mater solution, as acetone appears to have a stabilizing action and prevents fluctuations of the color with time. The interference of tungsten is eliminated by the use of amnionium citrate. The essential features of the procedure are that the molybdenum thiocyanate color is developed in a water-acetone solution and the comparison is made by titrating a blank containing the necessary reagents v i t h a standard molybdenum solution.
Reagents STASSOUSCHLORIDE(10 PER CEXT). Dissolve 20 grams of stannous chloride dihydrate in 100 ml. of concentrated hydrochloric acid and 100 nil. of water. A M ~ I O S I CTHIOCYASSTE ~I (10 PERCEST). Dissolve 20 grams in 200 ml. of water. A~IIIOSITXCITRATE, reagent grade. ACETOSE, reagent grade, STAXDARD MOLYBDEXUM SOLUTIOX.1 ml. = 0.1 mg. of molybdenum trioxide. Dissolve 0.1 gram of pure molybdenum trioxide in a little sodium hydroxide, neutralize to litmus with hydrochloric acid, and make up to 1 liter in a volumetric flask. As the standard degenerates with time, it is necessary to make up a fresh solution every month.
Solution of Sample With substances like scheelite that contain up to 40 per cent of tungstic oxide and possibly as much as 1 per cent of molybdenum oxide, fuse in platinum 0.5 gram of the sample with 2.5 grams of sodium carbonate and about 0.05 gram of sodium nitrate ( 2 ) . Begin the heating a t a low temperature and gradually increase the heat until a homogeneous melt is obtained. Extract the cooled melt with water containing a drop of alcohol to reduce manganate, add a little paper pulp, filter, and wash with warm 1 per cent sodium carbonate solution. Make just acid with hydrochloric acid. If any chromate is present, reduce it with a little sulfurous acid. Evaporate to about 25 ml. on the steam bath to remove carbon dioxide. At this point silica may separate out. Without removing silica neutralize with 10 per cent sodium hydroxide and add 10 ml. in excess, add a little paper pulp, and digest on the bath about 10 minutes. Filter hot and wash with dilute sodium hydroxide. Add phenolphthalein to the filtrate, make it barely acid with hydrochloric acid, and transfer it to a 100-ml. volumetric flask. The solution is now ready for the estimation of molybdenum. With molybdenite ores, wulfenite ores, and ores high in silica, in which tungsten is lorn, an acid attack of the sample is preferable. The usual practice (6) involves treatment with nitric and sulfuric acids and eventual extraction of the molybdenum in a sulfuric acid solution. On a 0.5-gram sample. 2 ml. of sulfuric acid are sufficient.
Colorimetric Estimation of Molybdenum Take a 15-ml. aliquot of the neutral molybdenum solution in a 100-ml. beaker; if a larger or smaller aliquot is taken, adjust volume to 15 ml. Add to the solution a t room temperature 1.5
