A Simple .Method for B, Determination H. H. BUNZELL, Bunzell Laboratories, New York, N. Y.
0
WING to the high cost of equipment available for B1 determinations, the author has developed a comparatively simple and rapid method which involves the use of the catox apparatus (2). The principle is the stimulation of yeast by B1 as described b y Schultz, Atkin, and Frey (3). The method gives reliable results with very small amounts of material; 0.01 microgram of B1 can be determined with fair accuracy.
Description of Apparatus The reaction is carried out in an apparatus similar to the one used for oxidase determinations ( f ) , and differs from the other apparatus mainly in two respects: It is used for measurements of increased pressure and the calibration of the manometer indicates directly the weight of carbon dioxide produced. The apparatus is shown in Figure 1. Compartment 1 contains 1 cc. of nutrient solution (8) and 1 cc. of the vitamin-containing extract or thiamin solution of known concentration. Compartment 3 contains 1 cc. of 0.4 per cent yeast suspension (free from added vitamins). Compartment 2 is used only when the effect of other substances on the reaction is t o be studied. 4 fulfills a function in catalase determinations and is eliminated for the purpose here described. 5, the manometer, is graduated so that every division is equal to 100 micrograms of carbon dioxide, when apparatus is charged as indicated. 6 is a ground joint provided with corresponding vent holes, 7, to allow for equalization of pressure when manometer is a t ri ht angles to the body of the apparatus. When manometer is hled with mercury, the apparatus can be closed by rotating the manometer through 90". It is then as shown in illustration. 8,8'is a rubber band which, with the aid of the glass hooks shown, ensures tightness of the apparatus during the reaction.
Method Six experiments are usually run concurrently. In some of these standard thiamin solutions and in some the unknown, with and without sulfite correction, were used (Table 11). The apparatus is clamped on a shaking machine mounted in a constant-temperature chamber maintained at 30" C. The air vents are adjusted to allow for equalization of internal and external pressure.
After temperature has been maintained at 30' C. for 25 minutes, the apparatus is closed as described, making use of a special trap door in the constant-temperature chamber. The shaking machine is set in motion and readings are made a t the end of 90, 120, 150, and 180 minutes. The readings of the unknowns are interpreted in terms of B1 concentration by interpolation from the readings obtained with the standards. The author's shaking machine has a stroke of 6 cm. and a period of two excursions per second. A check sample of flour furnished by the American Association of Cereal Chemists was reported as having a BI content of 2.9 I. U. per gram (average of three other laboratories). One vitamin-testing laboratory in New York City obtained 2.76 I. U.per gram (average of 3) for the same sample. Results obtained by method here described were 2.75 and 2.83 I. U. per gram. Table I shows results of a test of hard candy.
Constant-Temperature Chamber While the temperature should be in the vicinity of 30" C., a constant temperature chamber is not essential if all experi-
TABLE I. HARDCANDY (Laboratory No. 13,046) 1 2 3 0.5 cc. of 0.2570 0.20 cc. o i thia- 0 40 cc. of thiamin s o h . of Sammin soln. ( 0 . 1 ~ 30111. (0.1 y per ple per cc.) cc.) 1 cc. of,nutrient 0.30 cc. of HIO 0.10 cc. of Hz0 solution 1 cc. of nutrient 1 cc. oi nutrient soln. soln. Contents of com- 1 cc. of 0.4% 1 cc. of 0.47, 1 cc. of 0.470 yeast partment B y.east suspenyeast suspensuspension sion sion Temperature, C. 31.0 31.0 31.0 Reading,at end of 180 minutes 17.9 19.4 16.5 BI in 0.5 cc. of 0.25% solution (0.00126 gram of sampie) = 0.02 4X (17.9 16.5) = 0.0297 microgram 19.4 - 16.5 B1 in 1 gram of sample = 23.76 micrograms
Apparatus Contents of compartment A
o.020
-
I
FIGURE 1
FIGURE2. CONSTANT-TEUPERATURE CHAMBER 279
INDUSTRIAL AND ENGINEERING CHEMISTRY
280
TABLE11. READINGS AT THE ENDOF 3 HOURS ’ cc. of Unknown 0.005 y 0.015 y 0 . 0 2 5 y 0 . 0 3 5 y
No Thiamin Extract (0.1%) Bi Bi Bi Bi 4.7 8.5 6.4 7.3 8.4 9.2 Accordingly, the 0.1 per cent extract contains 0.026 microgram per cc., or 26 micrograms per gram of material.
Vol. 14, No. 3
with double glass and a black slide t o eliminate light. Where light effect is suspected, a black lamp, 7, is used. The motor is mounted in the rear and its shaft operates the fan through pulley 2. It also operates the shaking machine through a reducing gear. Shaking is started and stopped through a clutch arrangement operated by knob 5. 14 is a cooling coil making maintenance of box at 30” C. possible at higher outside temperatures. The author will be glad to assist investigators in securing the equipment required in connection with the method.
ments are carried on simultaneously under identical conditions. The constant-temperature chamber used is very convenient for this purpose and is shown in Figure 2. In general, its operation is obvious from the illustration. 12 is a trap door which, when opened, exposes sleeve 13. After arm is slipped into this sleeve, a slide also closing opening and operated by lever 11 admits the arm to turn the manometers. The front is a removable panel
Literature Cited (1) Bunzell, H. H., J. Biol. Chem., 17, No. 3, 409 (April 1914). (2) Bunaell, H. H., and Kenyon, Marjorie, “Prevention of Catalase Activity by Certain Organic Compounds”, Division of Agricultural and Food Chemistry, Am. Chem. SOC., Cleveland,
Ohio, 1934. (3) Schulta, A. J., Atkin, L., and Frey, C. N., J . Am. Chem. Soc., 59, 2547 (1937).
Micro-Kjeldahl Determination of Nitrogen A New Indicator and an Improved Rapid Method T. S. MA AND G. ZUAZAGA G. H. Jones Chemical Laboratory, University of Chicago, Chicago, Ill.
IXKLER (12) proposed in 1913 the direct titration of ammonia, absorbed in boric acid solution, by mineral acid for the Kjeldahl determination of nitrogen. A thorough study was later made by Markley and Hann (S), who recommended it as a standard method. It has three advantages over the regular back titration method: only one standard solution is needed, there is no danger of spoiling a determination because of insufficient standard acid for the absorption of the ammonia, and in case the content of the receiving flask is sucked back to the distilling flask, through an error of operation, distillation can be resumed without bad effect.
TABLE I. pH 2% Boric Acid
Mixed Indicator
M1.
1M1.
5 5 5
0.05 0.05 0.05
AXD
COLOR OF 2 PERCENTBORICACID
0.01 N
HCI M1.
...
0.02
...
0.01 .V KHIOH
Color
PH
Bluish purple Pink Bluish green
4.52 4.26 4.90
M1.
... ...
0.02
Several investigators have tried to adapt Winkler’s direct titration method to the micro scale, using as indicator a mixture of methyl red and tetrabromophenol blue (IO), methyl red and methylene blue (1, 9),or methyl red alone (4, 11). The end point of the titration is located by matching color with a standard, because the indicators show only a gradual transition of color. The amount of boric acid used and the volume of solution in titration have to be carefully controlled (4). Consequently, the operation becomes more complicated and time-consuming than Pregl’s procedure for micro-Kjeldah1 analysis (8). I n connection with some work in which a large number of micro-Kjeldahl determinations were required, the writers made an extensive study of the indicators suitable for the titration of ammonia in boric acid, as well as the minimum time required for the micro-Kjeldahl distillation. It was found that bromocresol green (tetrabromo-rn-cresolsulfon-
phthalein) compares favorably with the indicators mentioned above. This indicator changes from blue color in ammonia solution to greenish yellow a t the end point. While the two colors, blue and greenish yellow, are not easily distinguishable, the transition is rather sharp. A still better indicator has been found, however, in a mixture composed of 5 parts of bromocresol green and 1 part of methyl red, which gives a bluish-purple color in 2 per cent boric acid solution, changing to bluish green in the presence of a trace of ammonia and to pink with a trace of mineral acid. The intensity of color does not vary appreciably with the amount of indicator added. The transition a t the end point is very sharp and distinct, a color standard being entirely superfluous. Pregl (8) prescribed 6 minutes for the micro-Kjeldahl distillation. Niederl and Niederl (5) reduced the time to 4 minutes, while Hallett (2) remarked that 5 minutes are required for a complete distillation. I n a review article, Parnas (6) indicated that 1.5 to 2.5 minutes are sufficient. The writers found that distillation for 2 minutes with collection of 10 to 15 ml. of condensate recovered completely 1 mg. of nitrogen, the feasible upper limit of micro-Kjeldahl analysis. They also found that a Pyrex flask may be used as the receiver without being previously steamed out, and that a mixture of selenium, copper sulfate, and potassium sulfate may be used as catalyst for the digestion in preference to hydrogen peroxide. Taking advantage of these observations, the writers have developed a method for micro-Kjeldahl analysis in which the operation is simplified and the time required is considerably shortened. This method has been used in this laboratory and other laboratories, by analysts as well as students, for more than a year with satisfactory results.
Sensitivity of Mixed Indicator I n Table I are listed the pH values obtained by the addition
of 2 x millimole of hydrochloric acid or ammonium hydroxide to 2 per cent boric acid solution, together with the observed color of the mixed indicator for each pH value, determined by means of a glass electrode.
