V O L U M E 26, NO. 6, J U N E 1 9 5 4 and 5 or 6 drops of the sodium hydroxide are added to give a p H of about 13. About 75 mg. of sodium perborate tetrahydrate are added immediately, and the sample is shaken well to mix, brought again to boiling, and another 25 mg. of sodium perborate are added. The sample is then boiled for about 30 seconds and allowed to cool. The sample is transferred to a 100-ml. centrifuge tube, and the Kjeldahl flask is rinsed with an 0.01% Aerosol OT solution and then v, ith water. The inside of the centrifuge tube is rinsed down with a small amount of the Xerosol O T solution, and the sample is centrifuged for 15 minutes. The supernatant liquid is removed by suction, using a glass filter stick (Pyrex S o . 3953510F), the inside of the tube washed down with about 10 ml. of Aerosol OT solution to wash out any remaining soluble salts, the sample again centrifuged for 15 minutes, and the supernatant liquid removed using the same filter stick. In handling the centrifuged samples, great care must be taken to prevent jarring the tube, as the precipitate is very easily stirred up. The filter stick is left in the centrifuge tube, which is washed down with 2 ml. of concentrated nitric acid to dissolve the precipitate, a few drops being run into the inside of the filter stick. The centrifuge tube is then placed in boiling water and allowed to stand until cool, during which time 1 ml. of 6X hydrochloric acid is added to the sample. When cool, the acid is blown out of the filter stick into the centrifuge tube with compressed air, and the filter stick filled ~ i t hwater and blown out again, and finally rinsed off and removed. The inside of the tube is washed with about 10 ml. of Aerosol OT solution, and the sample placed in an oven a t 95 to 100 C. to dry (too high a drying temperature produces an insoluble precipitate). When thoroughly dry, about 5 ml. of water are added to aid in the removal of any residual acid, and the sample is again taken to dryness, after which it can be stored indefinitely before proceeding with the analysis. Plating. A very light copper flash (1 ampere for 30 seconds) is applied to the inside of the stainless steel cup, using a solution of cupric fluoborate. The cup is rinsed with distilled water and dried with a clean cloth. The surface must be smooth and the plate unbroken. (In putting the Lucite plug into the bottom of the tube, care must be taken to see that all touching surfaces are clean and that the metal cap is screwed on tightly, to prevent some of the sample leaking into the metal ca .) The centrifuge tube containing the sampfe t o be analyzed is rinsed with 5 ml. of saturated potassium fluoborate buffer, of
1035 pH 3.1. The tube is placed in boiling water for a few minutes, and then in cold water. The lip of the tube is lightly greased, the solution is poured into the plating cup, and then the centrifuge tube is rinsed with another 5 ml. of potassium fluoborate buffer, using 3-, 1-, and 1-ml. portions. The tube is centrifuged briefly to collect all drops of solution hanging on the sides of the tube, and this solution is added to the plating cup. The plating is carried out a t 3.5 volts, which in this assembly gives a current of 40 to 80 ma. (current density 2.1 to 4.2 ma. per square cm.). After 1.5 hours of plating, 1 ml. of 1.5N sodium hydroxide is added, and the plating is continued for another 1.5 hours. The cup is then removed from the plating setup, the solution poured out quickly, and the cup rinsed with 95% alcohol and drained dry. I t is then ready to be counted. The total elapsed time from start to finish of the assay is 3 days. By careful programming, 50 samples can be handled in a working week. LITERATURE CITED (1) Ballentine, R., and Stephens, D. G., J . Cellular and Comp. Physiol., 37, 369 (1951). (2) Bernstein, W., and Ballentine, R., Rev. Sci. Znstr., 20, 347 (1949). (3) Bernstein, W., Brewer, H. G., and Rubinson, W., Sucleonics, 6, KO.2, 39 (1950). (4) Comar, C. L., and Davis, G. K., Arch. Biochem., 12, 257 (1947). (5) Comar. C. L., Davis, G. K., and Taylor, R. F., Ibid., 9, 149 (1946). (6) Copp, D. H., and Greenberg, D. AI., Proc. S a t l . Acad. Sci. U . S . , 27, 153 (1941). (7) Elmore, W. C., and Sands, M., “Electronics, Experimental Techniques,” p. 254, Kew York. JIcGraw-Hill Book Co., 1949. (8) Higinbotham, W. A., Reu. Sci. Instr., 22, 429 (1951). (9) Hoekstra, H. R., U. S. Atomic Energy Commission, AEC Rept. AECD 2144 (1949). (10) Sheline, G. E., Chaikoff, I. L., and Montgomery, 11.L., Am. J . Physiol., 145, 285 (1946). RECEIVED for review ripril 6, 1953. Accepted March 6, 1954. Contribution No. 42 froin McCollum-Pratt Institute. Work supported i n p a r t by a contract, AT(30-1)-933, between t h e Atomic Energy Commission a n d The Johns Hopkins Cniversity.
Colorimetric Determination of Propionaldehyde LAWRENCE R. JONES and JOHN R. RlDDlCK Commercial Solvents Corp., Terre Haute, lnd.
Aldehydes are a principal or minor product of many chemical and biochemical processes. Often, it is desirable not only to determine the amount of aldehyde(s) produced, but the specific aldehyde(s). Propionaldehyde reacts with ninhydrin in concentrated sulfuric to produce a deep red-blue color suitable for spectrophotometric analysis at a wave length of 595 mg. The method is sensitive to 1 to 3 y of propionaldehyde with an accuracy within about and a precision within about *I%. The method is specific for propionaldehyde in the presence of several other aldehydes and it may be used for the determination of propionaldehyde in solutions and in air.
A
SPECIFIC method was desired for propionaldehyde in the presence of other aliphatic aldehydes, Numerous techniques have been proposed for the determination of micro and macro amounts of aldehydes, but no specific method for propionaldehyde was reported in the literature. The determination of one aldehyde in the presence of another is difficult because of their closely related chemical properties; but some specificity in reaction of certain aldehydes was obtained
with colorimetric methods. Because of this, various reactions were investigated that might form a specific color with propionaldehyde. Propionaldehyde reacts n i t h 1,2,3-triketohydrindene (ninhydrin) in the presence of concentrated sulfuric acid to yield an intense red-blue color suitable for quantitative measurement a t a wave length of 595 mfi. This reaction Itas found to be almost specific for propionaldehyde. The chemical reactions between propionaldehyde and ninhydrin that develop color are not known but this test is similar to the reaction of ninhydrin n hen used as a reagent for amino acid determinations (1, 3,5 ) . BASIC PROCEDURE
Apparatus. Spectrophotometer, Beckman Model DU. Tubes, Lei&-Benedict, graduated a t 12.5 and 25.0 ml., Corning No. 7860. Flowmeter, Fisher Laboratory Model No. 11-163. Cooling bath, 25’ C . , any type. Pipet, 0.5 ml., Ostn-ald type. Aeration apparatus, Figure 1. Reagents. Propionaldehyde Standard, obtained from Eastman Kodak and purified by method of Smith and Bonner (4). Analyzed 99.997, by the bisulfite method. Sulfuric ilcid, specific gravity 1.84, Mallinckrodt, low nitrogen.
1036
ANALYTICAL CHEMISTRY
Kinhydrin Reagent, 3% solution Eastman Kodak Co. KO. 249.5, 1,2,3-triketohydrindene crystals in a 57, aqueous sodium bisulfite solution which is stable a t room temperature. Sodium Bisulfite, 5% aqueous solution. Glycine Solution. Dissolve 5 grams of U.S.P. glycine (aminoacetic acid) in 250 ml. of Baturated sodium bicarbonate solution. Sodium Bicarbonate Solution, saturated aqueous. Preparation of Calibration Curve. Prepare a solution of propionaldehyde standard in 5% sodium, bisulfite solution to contain 1 mg. per ml. Transfer 0.0, 0.5, 1.0, 2.0, and 3.0 ml. of the standard solution to five 50-ml. volumetric flasks and dilute each to volume with water or bisulfite solution. Transfer 0.5 ml. of each dilute standard to Lewis-Benedict tubes, add 0.1 ml. of distilled water, cautiously add 4.0 ml. of concentrated sulfuric acid, and mix. Stopper tubes as a prot,ection against dilution due to water vapor and cool to 25" C. in a water bath. Add 0.2 ml. of ninhydrin reagent and mix Stopper tubes and allow them to stand for 1 hour a t 25' C. to develop full color intensity. Dilute contents to 12.5 ml. with concentrated sulfuric acid and mix. Transfer to a 1-cm. Corex cell and read the absorbance a t 595 mjc with the spectrophotometer, using the zero prepared standard as the blank for the zero absorbance setting of the instrument. Plot concentration against absorbance on linear graph paper. The above standards equal 0, 5, 10, 20, and 30 y of propionaldehyde, respectively. Determination. The det,erminatiori was made according to the described method, but the sample was prepared eo that a 0.5-ml. aliquot of bisulfite solution does not contain more than 30 y of propionaldehyde. Applications. Formaldehyde does not react with ninhydrin to give a color complex, but inhibits the formation of color with propionaldehl-de and ninhj-drin (Table I). Therefor?, formaldehyde must be removed prior to determination of propionaldehyde. The presence of formaldehyde can he conveniently proved IIJ- the method of Eegriwe ( 2 ) .
Table 11. Determination of Propionaldehyde in Formaldehyde Mixtures Composition of Mixture, Mg. FormalPropiondehyde aldehyde 3.0 0.5 5.0 0.5 3.0 1.0 5.0 1.0 3.0 2.0 3.0 3.0
Acetaldehyde
Composition of RIixtures, TVt. n-ButyrIsobutyraldehyde aldehyde 19.32 0.0 20.60 0.0 5.32
29.28 71.63 49,50 35 00 15.06
22.20 0.0 10.10 21.15 30.10
Propion: aldehyde
Propionaldehyde Found, Kt.
29.20 28.37 19.80 43.85 48.93
30,40 28.35 19 85 43,84 49.00
I N A RIIXTUREOF ALDEHYDES IN THE ABSENCE OF FORYALDEWeigh a sample of an aldehyde mixture into a volumetric flask containing 594 bisulfite solution and dilute to volume with bisulfite solution. From this solution, prepare a dilute sample such that a 0.5-ml. aliquot will contain 30 y or less of propionaldehyde and analyze. HYDE.
Data for the determination of propionaldehyde in mixtures of aldehydes other than formaldehyde are presented in Table 111. E
E
E S
Effect of Formaldehyde on Determination
Conon. of Propionaldehyde, y 20 20 20 20 20 20 20
0.48 0.47 0.99 1.00 2.05 3.02
Table 111. Determination of Propionaldehyde in Aldehyde Mixtures
E
Table I.
Propionaldehyde Found, M g .
Concn. of Formaldehyde, Y
Bbsorbance
10 20 30 60 100
0.800 0.740 0.690 0.600 0,505 0.386 0.200 AIR-
The formaldehyde may be separated from the propionaldehyde effectively by passing the aldehyde vapors through a glycine (aminoacetic acid) solution which viill combine the formaldehyde: HCHO
+ SHzCHzCOOH -,CH~SCHZCOOH+ Hz0
?L-Butyraldehyde T d l interfere if present in larger quaritities than propionaldehyde. Equal or smaller amounts of n-butyraldehyde in relation to propionaldehyde may be determined successfully. Determination of Propionaldehyde. I N A MIXTCRE OF ALDEHYDES CONTAINING FORMALDEHYDE. Prepare an aeration train consisting of four tubes (Figure 1). Add 20 ml. of saturated bicarbonate solution to Ti, 20 ml. of 57, glycine-bicarbonate solution to 7'2, 20 ml. of 5% bisulfite solution to T,, and 15 ml. of bisulfite solution to Ta. Prepare a sample of aldehyde mixture in sodium bisulfite solution such that a 1.0-ml. aliquot will contain 1 to 3 mg. of propionaldehyde. Bdd to 7'1 a 1.0-ml. aliquot of the prepared mixture, connect all tubes in aerat,ion train, and adjust air-flow, delivered either by vacuum or pressure, through the train at the rate of 0.2 liter per minute as measured by the flov-meter. Continue aeration for 3 hours. Combine, quantitatively, the contents of 7'8 and 2'4 in a 50-ml. volumetric flask and dilute to volume with n-ater. Analyze a 0.5-ml. aliquot of the solution for propionaldehyde. Results on the determination of propionaldehyde in mixt'ures of formaldehyde by aeration are given in Table 11.
TI
Figure 1. A.
I
I
T'2
T'3
T4
Aeration Apparatus
Air sparger, borosilicate filter sticks
C. Standard-taper 24/40 glass joints E . Tygon tubing F. Flowmeter, Fisher Laboratory Model No. 11-163 S . Three-way stopcock T. Traps, 25 X 200 mm., borosilicate tube
Table IV.
Determination of Propionaldehyde in Air Samples
Calculated.
Found,
Y
Y
1000 500 5000 50
990 500 4960 48
I x AIR. Pass the air containing propionaldehyde vapor through a train of three tubes containing 20 ml. of glycine-bicarbonate solution in Ti and.20 ml. of 5y0 bisulfite solution in T1 and Tj a t the rate of 0.2 liter per minute. At the end of the sampling period, combine the bisulfite solution from T 2and Ta into a volumetric flask, dilute to volume with bisulfite solution, and analyze a 0.5-ml. aliquot containing 30 -/ or less of propionaldehyde by the proposed procedure.
V O L U M E 2 6 , NO. 6, J U N E 1 9 5 4 Table V.
1037
Time of Reaction a t 25" C. 30 y propionaldehyde)
Ahsorb-
Time, Nin. 0 15 30 45 60 75 YO
ance
0 0 0 560
0.910 1.200 1.200 1 195 1 180
Propionaldehyde determinations in known concentrations of air samples are prePented in Table IF-. EXPERIR1ENT.A L
The effects of several variables were investigated for their influence on thr. color iorniation and quantitative applications of the reaction. The factors included the absorption curve, stability and intensity of color, time and temperature of reartion, stahility and concentration of ninhydrin, concentration of acid, conformity to Beer's law, and interference from other compounds. A Beckman 1Iodel IX-spectrophotometer was used to measure the color. Briefly. the following procedure was employed. An aqueous polution containing 20 y of propionaldehyde was transferred t o a Lewis-Benedict tube, 4.0 ml. of concentrated sulfuric acid added, and the polutions were mixed and cooled to 25" C. Xinhydrin reagent, was added and the reaction was allowed to stand for 1 hour for complete color development. The samples were diluted to 12.5 ml. with concentrated sulfuric w i d and read by means of the spectrophotometer. Absorption Spectrum. Spectral-absorbance curves of the redblue caoniplex, resulting from the reaction of propionaldehyde arid ninhydrin, shon- a niasimuni ahmrption at 595 niM. Transmittance and Concentration. Calibration curve? n-ere determincd for propionaldehj-de hy plotting absorbance against concentration. The compound gave a straight-line calibration originating a t zero conrentration and absorbance. The color obeys Beer's law for nmouiita of 1 to 30 p of propionaldehyde in the total volume of 12.5 nil. Temperature and Time of Reaction. A correlation esists hetn-ren the temperature a n d the length of time required for color tlevelopnicnt, Samples \yere treat,ed for rolor development a t O", 25O, 50", and 100' C. .It 0" C., color development Tvas riot completed for almost 3 hours; a t 25' C., color development was complete in 4
