0
ANALYTICAL CHEMISTRY
274
thiocyanate will also yield cyanogen chloride. The reaction between thiocyanate and chloramine T, however, is slow and a maximum color developed from the cyanogen chloride formed is not reached until the chlorinating agent and the thiocyanate have been in contact for 30 minutes. Addition of 20 micrograms of the catalyst, ferric chloride, to the thiocyanate solution prior to or simult'aneous with t'he addition of 0.2 ml. of 1% chloramine T decreased t'he time for maximum reaction between thiocyanate ion and chloramine T a t 25" C. to 3 minutes. Amounts of ferric chloride up to 50 micrograms did not further decrease the reaction time.
Table V. No. of Determinations 10 8 10
12 13 8
.
Added Microorams 0.2 0.4 0.6 0.8 1.0 1.2
0.7
Average Recovery
Standard Deviation
5%
%
99 99 100 100 98 99
4 1.3
2.5 1.9
2.8 2.0
DISCUSSION
0.6
Ninety-eight to 1 0 0 ~ oof cyanide in quantities ranging from 0.2 to 1.2 microgram may be recovered by this method with a standard deviation of no greater than 470,as shown in Table V. This method has not been tried for determining cyanide in distillates from animal organs.
0.5 0.L
9
3
Recovery of Cyanide
0.3 0.2 0.1
0
ACKNOWLEDGMENT 0.2
0.4
0.6
0.8
1.0
1.2
1.4
YI~CGRA!S X'AhTDDE ION
Figu e 1.
Calibration Curve
The author wishes to express his gratitude to S. D. Silver, chief, Gassing and Analytical Section, Medical Division, and B. Gehauf, Technical Command, for valuable suggestions. Grateful acknowlednment is also due Mrs. Marv " Rumert Van Hollen and Mrs. Aurora Bransford for technical assistance. I
The procedure for thiocyanates is the following:
.
To 1 ml. of a solution containing up to 2.5 micrograms of thiocyanate ion are added 0.2 ml. of 0.1% ferric chloride solution and 0.2 ml. of 1% chloramine T solution. The tube is stoppered and shaken. After 3 minutes' contact time, 6 ml. of the pyridinepyrazolone mixture are added. The tube is stoppered again and the reagents are mixed. After 20 minutes, readings are taken in a spectrophotometer set a t wave length 630 mp with the optical density of the reagent blank set zero. The color is stable for a t least 30 minutes a t room temperature. RESULTS
Figure 1 gives a typical calibration curve. Table 1V shows the recoveries of known amounts of cyanide.
..
LITERATURE CITED
(1) Aldridge, W. N., Analyst, 69, 262 (1944). (2) Gehauf, B., Witten, B., and Falkof, M.,personal communication, June 1944. (3) Knorr, L., Be?., 17, 2044 (1884). (4) Kolthoff, I. M., 2. anal. Chem., 57, 11 (1918). ( 5 ) Kolthoff, I. M., and Sandell, E. B., "Textbook of Quantitative
Inorganic Analysis", revised edition, New York, Macmillan
Go., 1943. (6) Nicholson, R. I., Analyst, 66, 189 (1941) (7) Viehoever, A., and Johns, C. O., J . Am. Chem. SOC.,37, 601 (1916). (8) Waller, A. D., Ibid., 35,406 (1910).
Volumetric Semimicrodetermination of Sulfur in Organic Compounds By Use of the Oxygen Bomb E. C. WAGNER
T
AND
SARAH H. MILES, Department of Chemistry and Chemical Engineering, University of Pennsylcania, Philadelphia, Pa.
HE procedure described depends upon combustion of the sample in the Parr oxygen bomb and determination of the resulting sulfuric acid by precipitation as benzidine sulfate and titration with standard alkali. Similar macro- and microprocedures are already available (3,6, 14); in the development of the method presented, the conditions appropriate to the analysis of samples of semimicro size were determined. The semimicro scale of operation appears to be more favorable to satisfactory execution of this volumetric procedure than either the macro or micro scale. In the macroprocedure the size of the precipitate, combined with its tendency to become matted and compacted upon the filter, results in clogging, difficulty in filtration, and often high results caused by occluded benzidine hydro-
chloride not satisfactorily removable by washing. In the microprocedure accuracy is decreased because the end point of the titration is only moderately sharp and the titration is small. Neither disadvantage is apparent in the semimicroprocedure; filtration is rapid, washing is easy and apparently effective, and the titration yields results which are not affected by any noticeable irregularity. A preliminary study of the volumetric beyidine method on a semimicro scale, with respect to several points on which the literature records disagreements, led to conclusions which were applied in elaboration of the procedure, and which for brevity are stated without supporting data;
V O L U M E 19, NO. 4, A P R I L 1 9 4 7
275
A procedure for the volumetric semimicrodetermination of sulfur depends upon combustion of the sample i n the Parr oxygen bomb and determination of the resulting sulfuric acid by precipitation as benzidine sulfate and titration with standard alkali. Similar macro- and microprocedures are available.
1. The presence of acetone in the liquid during precipitation, claimed (3, 5) to accelerate settling of the precipitate and to facilitate washing, was judged to be without such advantages or to have the reverse effects, accompanied by a tendency toward high results.' 2. The optimum acidity (pH = 2.75) (13) is approximated in the procedure described. 3. To obtain a precipitate that settles rapidly and is easy to filter and wash, the benzidine hydrochloride solution should be added by drops during several minutes and with agitation, a recommendation made twice previously (6, 14) but often disregarded; the advantage of this procedure is marked. 4. The amount of precipitant'added should be such that the excess benzidine hydrochloride is 0.002 to 0.005 M in the mixture. Results become higher as the concentration of benzidine hydrochloride is much increased, t ecause of increased adsorption on the precipitate (9, 12). The adsorbed salt can be removed (12) by numerous R ashings (ten to twenty) with saturated benzidine sulfate solution, but this inconvenience is to be avoided. 5. After addition of the precipitant as recommended the mixture can be filtered without standing, but as the suspended precipitate may retard filtration rand washing it is better to allow the precipitate to settle sufficiently so that most of the liquid can be decanted. S o advantage is gained by allon-ing precipitation to continue overnight. 6 . The precipitate obtained from a sample of semimicro size is too large to be collected in a Fiske filter tube (3, 5 ) or on a filter stick, either of which is quickly clogged by the compacted solid. A 7-cm. paper filter used as described was found to be satisfactory. 7 . The precipitate is best transferred to the filter and xashed by uFe of saturated benzidine wlfate solution (cf. 5 ) . Final washing of the precipitation flask and the precipitate with acetone, though dispensable, was found to decrease the magnitude and inciease the uniformity of the blanks. 8. Both phenolphthalein and phenol red ( 5 ) proved to be usable indicators, the former yielding somewhat the better end points and affording the further advantage that it can also be used in standardization of the alkali.
The accuracy and range of the semimicroprocedure for precipitation and titration of sulfuric acid are indicakd by t h e results in Table I, representing analyses of known amounts of pure potassium sulfate. It is concluded that the method yields acceptable results for amounts of sulfur from 2.5 to 15 mg., the accuracy being highest (99.6 to 100.2%) with 5 to 15 mg. The method is not useful for less than 1 mg. of sulfur (8), the titrations being too small for accuracy. Amounts larger than 15 mg. were judged to be outside the range of semimicroanalysis.
Table I.
Volumetric Benzidine Sulfate Semimicromethod Applied to Potassium Sulfate
Sulfur Taken 0,525
0.03KQ Sodium Hydroxide
1 ,000 1.0304 2.498 2.498 2.498 5.002 5.002 5.002 10.004 10.004
APPARATUS
The Burgess-Parr oxygen bomb (single valve type) is used in a room reserved for pressure apparatus, on a table fitted with the bench seat used to assist locking and unlocking the bomb and installed in a corner, with a steel guard plate separating this space from the room proper. Ignition of the charge in the bomb is effected by remote control (around an angle in the wall), using a circuit which includes a rheostat and which, at the control position, is divided into two paths, each controlled by a switch. One path includes a 25-watt light bulb; when the current is passed through this circuit the light glows, the fuse wire in the bomb being able to carry this load. On shunting in the other circuit the fuse wire burns out and the light is extinguished. The rheostat is so adjusted that the current passed renders the fuse wire incandescent for a second or two and then melts it.
Sulfur Found
% of S Taken
0 4993 0.89 (A) 95 0.47f 0.87 (B) 1 0099.9 1.83 (A) 1 04100.5 1.90 (B) 2 48f 99.4 4.55 (A) 2 5l+ 100.6 4.61 ( B ) 2 4999.5 4.56 (B) 4 9899.6 9.13 (-4) 5 oo+ 100.1 9.18 ( B ) 4 9999.8 9.15 ( B ) 10 03100.2 18.37 (A) 10 00 99.9(6) 18.34 (B) 15.006 14 99 99.9 27.46 (A) 15,006 100.0 15.01 27.53 (B) * Sormalities: (h)0.03405 (B) 0.034015. b Buret readings corrected fdr temperature and calibration, and blank of 0.19 ml. deducted. 0,500
For the decomposition in the oxygen bomb Kujol was found to be a satisfactory combustible liquid vehicle for the charge of sample and ammonium nitrate, replacing the paraffin oil (4)or decalin ( 3 ) often used. Interference by iron present in the liquid from the bomb is more advantageously eliminated by reduction with hydroxylamine (4,9) than by precipitation with ammonia water (3, I%?),which latter involves filtration, washing, and subsequent neutralization. The presence of some undecomposed hydroxylamine has no observable effect upon the results. The oxidation of sulfur to sulfuric acid in the bomb is not invariably complete, some sulfur dioxide apparently persisting even when the bomb is allowed to cool slowly ( 2 , S ) , as indicated by occasional low results. Error from this cause can be avoided either by adding bromine water ( 1 ) to the water in the bomb immediately after opening, or by including some hydrogen peroxide with the water put into the bomb. Excess bromine or hydrogen peroxide is destroyed by the hydroxylamine added later to reduce iron. In tests of the completeness of the removal of sulfate from the bomb by the working procedure adopted, a solution containing 5.002 mg. of sulfur (as potassium sulfate) was run over the inner surfaces of the bomb and was then xashed out and the sulfate present was determined; the result vias 5.015 mg. of sulfur, or 100.25C; of that taken. In similar trials a charge of Kujol and ammonium nitratewas burned in the bomb in presence of the same amount of potassium sulfate, and the sulfate n a s washed out and determined. Three trials indicated the presence of 5.016, 4.973, and 5.000 mg. of sulfur-i.e., 100.29, 99.42, and 99.97% of that taken. Consideration of these results and those in Table I indicates that the accuracy of the procedure, exclusive of the oxidation of sulfur during the decomposition of a sample, ranges from 99.4 to 100.270when the amount of sulfur is from 5 to 15 mg.
SOLUTIONS
Benzidine Hydrochloride, 2 % Solution. Prepare a filtered solution containing per liter 20 grams of benzidine hydrochloride (Merck's Reagent grade was used) and 5 ml. of 6 N hydrochloric acid and store it away from light. This solution is not saturated, though it contains more benzidine hydrochloride than is stated by hleldrum and Xewlin (11) to represent the solubility of the salt. Benzidine Sulfate, Saturated Solution. Precipitate a quantity of benzidine sulfate, wash the precipitate repeatedly by decantation following thorough mixing with water, and transfer to a large bottle. Fill with water, invert several times a t intervals during a day, and store away from light. -4s needed filter the supernatant liquid into a wash bottle, replacing by pure water the solution withdrawn. Sodium Hydroxide, 0.03 N. In a large flask boil the water to be used, and allow to cool with a soda-lime tube attached. Trans-
ANALYTICAL CHEMISTRY
276
fer about 25 ml. of a warm saturated solution of sodium hydroxide Filtration and Washing. A suction filtration apparatus of the to a narrow cylinder, stopper tightly, and allow to cool. Thrust Witt or Filtrator type, provided with a relief valve, is recoma pipet through the surface crust and withdraw some of the clear mended. The filter consists of a long-stemmed "quantitative" carbonate-free alkali. For each liter of 0.03 N alkali to be prefunnel, a filter cone, and a 7-cm. paper of thin and fairly open rams of this saturated solution into a pared weigh 2.3 '0.1 texture (Munktell No. 2 was used). If the paper is folded so as to small tared Erlenmeyer fask containing some carbon dioxide-free yield a snug fit against the funnel wall, no suction need be applied during the filtration and washing except to draw the liquid from water. Dilute at once with the carbon dioxide-free water, and store the solution in a Pyrex bottle or flask provided with a the peak of the filter each time-Le., the liquid below the rim of siphon draw-off with a capped tip, and with a soda-lime tube the filter cone or on that portion of the paper which is covered by protecting the air inlet. A solution so stored showed little alterathe precipitate. tion in alkalinity (from 0.03405 to 0.03400 N ) during 106 days. Decant the supernatant liquid upon the filter as rapidly as it Another solution, stored in a lime-glass bottle, underwent a runs through. When about 5 ml. of liquid remain, rotate the steady increase in alkalinity-viz., from 0.03024 to 0.03083 N in flask to suspend the precipitate, and transfer the suspension to 45 days at approximately 20'. the filter. Rinse the inside wall and mouth of the flask five sepaStandardization of 0.03 N Sodium Hydroxide. Weigh 0.1 rate times with saturated benzidine sulfate solution applied as a gram ( '0.02 mg.) of potassium hydrogen phthalate of primary fine stream from a wash bottle, and set the flask aside, Wash the standard quality, transfer it to a small beaker, dissolve in about 5 precipitate and paper with saturated benzidine sulfate solution ml. of water, and transfer the solution to a 150-ml. Erlenmeyer five times, each time applying the fine jet first to the precipitate flask. Add 2 drops of 0.5% solution of phenolphthalein in 50% so as to loosen and disintegrate it and to cause it to collect in the ethanol, and introduce the alkali from a 25-ml. or a 50-ml. buret. apex of the filter. When the precipitate has been loosened apply Near the end point boil the liquid vigorously, and while still boilthe wash liquid to the upper edge of the paper. Now complete ing titrate to the first pink color. Boil for about 10 seconds, and the washing of the Erlenmeyer flask by use of two 1-ml.portions of then cool under the tap to room temperature or below, and acetone, each applied (conveniently from a 1-ml. pipet) so as to titrate the cold and now colorless liquid to a faint pink end point. rinse the entire inner surface and then transfer to the filter. The volume of the liquid a t the end should be about 25 ml. Run Finally wash the filter and precipitate with three 1-ml. portions of a blank titration in the same way and to the same end color. The acetone, and maintain suction for several minutes until the aceend point is reached too soon if the liquid is hot, and the normality tone has evaporated and the paper is dry or nearly so. Transfer values may then be about 8 parts per thousand too high, and less the paper and precipitate to the Erlenmeyer flask. In the trials precise. Any irregularity introduced when the cold-standardized alkali is used in the analysis, the end point of which is reached with the liquid hot, is compensated by the blank Table 11. Semimicrodetermination of Sulfur in Some Organic analysis described below as part of the analytical Compounds procedure. ANALYTICAL PROCEDURE
Decomposition. Weigh and transfer to the cup of the oxygen bomb a sample equivalent to about 5 to 10 mg. of sulfur (the determined limits for the procedure are 2.5 to 15 mg. .af sulfur). Add 0.05 *0.01 gram of finely divided ammonium nitrate (8, 6 ) and 20 drops of Nujol, and rotate the cup until the solids are well distributed in the oil. Pour 10 ml. of water into the bomb. If hydrogen peroxide is to be used as auxiliary oxidant add 5 ml. of a 3y0 solution, prepared by dilution of pure perhydrol (Super0x01 Hyperoxide). Assemble the apparatus, and introduce oxygen to a pressure of 30 to 40 atmospheres. Set the bomb in cold water in a bucket and ignite the charge by remote control. After about an hour relieve the pressure slowly, then remove and open the bomb. If decomposition was satisfactory there will be visible not more than a trace of soot. If bromine is to be used as an auxiliary oxidant add to the liquid in the bomb a saturated aqueous solution of bromine until the color persists (about 1.5 ml.). Transfer the liquid from the bomb through a funnel (no paper) into a 150-ml. Erlenmeyer flask. Rinse the inner surfaces of the bomb and its cover with hot water applied as a fine stream from a wash bottle. When the volume reaches about 50 ml., remove the flask (placing the bomb temporarily under the funnel), add hydroxylamine hydrochloride (free from sulfate), using 0.1 gram in absence of hydrogen peroxide or bromine, or 0.25 gram in presence of either, and boil the liquid for about 30 seconds. Replace the flask under the funnel and complete the washing of the bomb parts. When the volume approaches 100 ml. cool the liquid and dilute to approximately 100 ml. Precipitation of Benzidine Sulfate. Add by separate drops, preferably from a buret, 10 ml. of 2% benzidine hydrochloride solution while rotating the flask continuously. The rate of addition should be slow a t first, the precipitate being allowed to form in quantity before a large excess of precipitant has accumulated. When the precipitate appears to reach a maximum, the rate of addition may be somewhat increased. The addition of the reagent requires 2 or 3 minutes. Allow the mixture to stand for 30 minutes or longer.
Gravimetric Volumetric Analyses % 8, Analyses', '$ 9, Auxiliary Oxidant Auxiliary Oxidant Compound % Noneb Brominec HrOd None' HtOd Sulfonal 2 8 . 0 8 27.6227.7727.8727.6827.90Crystallizoed from Hz0 28.03 28.13 27.94 28.03 28.10 M.P. 127 Av. of Av. of Av. of Av. of Av. of 8:27.83 2:27.95 2:27.90 2:27.85 2:28.00 Di henylthiourea 1 4 . 0 3 13.56.... 13.86.. . . 13.89grystalliaoed from E t O H 14.09 13.73 13.98 M.p. 153 Av. of Av. of Av. of 5:13.64 4:13.97 3:13.92 p-Toluenesulfonamide 1 8 . 7 2 18.3618.37.... Crystallized from EtOH 18.59 18.59 M.p. 135O Av. of Av. of 5: 18.47 2: 1 8 . 4 8 A'-Phenylsulfanilamide 1 2 . 9 1 12.5412.78.... Crystallized from E t O H 12.81 12.68 M.p. l96O Av. of Av. of 4: 12.71 2 : 1 2 . 7 4 Sulfur Calcd.,
....
.... .
I
.
.
25.81Bencyl disulfide 2 6 . 0 2 25.7825.87 Cryatalli~ed~from EtOH 26.02 M.p. 71-72 Av. of Av. of 5:25.95 2:25.84
....
....
Saccharin Crystallized from HzO or EtOH M.p. 228O
....
....
....
....
....
....
..
....
....
....
....
....
....
....
....
1 7 . 5 0 17.3717.2517.71 17.66 Av. of Av. of 4:17.52 2:17.43
27.37All lthiourea 27.59 26.9127.12 Zrystallized from HtO 27.52 or EtOH Av. of Av. of M.p. 72-74' 3: 27.03 2: 2 7 . 4 4
....
Thioformaldehyde 2 3 . 1 9 22.9023.08 crystallized from EtOH Av. of 4:23.03 Thianthrene 2 9 . 6 4 29.33Crystallised from EtOH 29.87 M.p. 157O Av. of 7:29.60 Diphenylsulfone 1 4 . 6 9 14.45M.p. 128O 14.72 Av. of 3: 14.60 Ethyl benzenesulfonate B.p. 157.5-8.5' (14-15 mm.)
1 7 . 2 1 16.7717.46 Av. of 6:17.10
....
a Procedure: decomposition in oxygen bomb, and determination of sulfur a8 Bas04 PB described in ( 1 0 ) . b N o bromine water or Hs0,; 0.1 gram of NHrOH.HC1; blank 0.19 ml. of 0.03 N sodium hydroxide: 0 Bromine water (satd.) 1.5 ml., 0.23 gram of NHIOH.HC1; blank 0.19 ml. of 0.03 A' NaOH. d H drogen peroxide, 3%, 5 ml.; 0.25 gram of NHzOH.HC1: blank 0.22 ml. of 0.03 A' NaOd 6 Blank 0.00036 gram of BaSOd. I Blank 0.00052 gram of BaSO,.
,
V O L U M E 19, NO. 4, A P R I L 1 9 4 7
277
the operations of filtering and washing required from 8 to 10 minutes, and the volumes of the washings ranged from 47 to 59 ml. Titration. If the amount of sulfur is believed to be not much in excess of 5 mg., add 5 to 10 ml. of water, close the flask tightly with a rubber stopper, and shake vigorously to disintegrate the filter paper. Remove the stopper and wash it and the neck of the flask with a stream of hot water from a wash bottle. Introduce 2 drops of 0.5y0 phenolphthalein indicator and, manipulating the flask by means of a holder, titrate with 0.03 N sodium hydroxide, with vigorous agitation and occasional heating, until the end point is obviously near. If the amount of sulfur is much greater than 5 mg. the initial addition of water should be omitted, and the disintegration of the paper deferred until about 10 ml. of the alkali have been added; this procedure avoids an unduly large volume of liquid a t the end point. Just short of the end point, heat the rmxture to active boiling, and then titrate to an unmisfakable and moderately deep pink color which survives a final boiling of several seconds. The titration may be completed by whole or half drops, but the end point is not sufficiently sharp to warrant attempted further rehement. I n the trials the volumes a t the end of the titrations ranged from 25 to 32 ml. Blank Analysis. Transfer to a 150-ml. Erlenmeyer flask 100 ml. of water. As appropriate add 5 ml. of 3% hydrogen peroxide or 1.5 ml. of bromine water, and then 0.1 or 0.25 gram of hydroxylamine hydrochloride. Introduce 10 ml. of 2y0 benzidine hydrochloride reagent, and proceed thereafter as in the analysis, titrating finally to the same end color. The color identity must be judged from memory, as the color does not persist long enough t o permit matching. The blanks obtained in the trials were uniformly close to 0.2 ml. of 0.03 N sodium hydroxide. It is advisable to conduct also an over-all blank which includes decomposition of some sucrose or benzoic acid in the bomb. I n the trials this did not increase the blank obtained as above. Test Analyses of Organic Compounds. Table I1 presents the results of analyses of a number of organic sulfur compounds by the procedure given above. There are included also results obtained when no auxiliary oxidant was used and, for several compounds, results obtained gravimetrically (barium sulfate), to confirm the accuracy of the volumetric benzidine method. It will be noted that analyses of a number of compounds in absence of an auxiliary oxidant gave results which are acceptable
and which in some cases were not improved by use of hydrogen peroxide or bromine water. The need for an auxiliary oxidant is perhaps to be presumed when sulfur is present in unoxidized condition, but it seems advisable as a general procedure always to include one of the supplementary oxidizing agents tested. The report (7, cf. 16)that sulfur compounds oxidizable to sulfonic acids may not be properly decomposed in the oxygen bomb was not confirmed in the cases that might test this claim, both benzyl disulfide and ethyl benzenesulfonate giving acceptable results. A single analysis requires about 2.5 hours. By use of two sample cups and by suitable arrangement of the work six analyses can be completed, and a seventh sample decomposed, in 8.5 hours. ACKNOWLEDGMENT
Grateful acknowledgment is made t o the Faculty Research Committee of the University of Pennsylvania for grants to aid this study. Preliminary studies by Doris Koch Cavalieri were useful in the development of the procedure. LITERATURE CITED
(1) Alicino, IND.ENQ.CHEM.,ANAL.ED., 13, 506 (1941);Am. 800. Teating Materials, D271-43, p. 13. (2) Bradley, Corbin, and Floyd, Zlzd. Ens. C h . , 18, 583 (1926). (3) Cnllen and Toenniea, IND. ENG. CHIIM.,ANAL.ED., 13, 469 (1941). (4) Clauder, Magyar Gyogysreresrtud Tarsmag Erteoitdje, 11, 246 (1935). (5) Fiske, J. Biol. Chem., 47,59 (1921). (6) Friedheim and Nydegger, 2. angcw. C h m . , 20, 9 (1907). (7) Griffin, IND.ENQ.CHEM.,ANAL.ED., 1, 167 (1929). (8) Haese, 2 . angew. C h m . , 40,595 (1927). (9) JBrvinen, Ann. acad. aci. Fcnnicac, 2, No. 4, 22; Chem. Zentr., 192, I, p. 526. (10) Lincoln, Carney, and Wagner, IND.ENQ.CHEM.,ANAL.ED.,13, 358 (1941). (11) Meldrum and Newlin Zbid., 1, 231 (1929). (12) Miiller and Dtirkea, Bar., 35, 1587 (1902). (13) Owen, Biochem. J.,30, 352 (1936). (14) Skau and Newell, IND.ENQ.CHEM.,ANAL.ED.,5, 180 (1933). (15) Weisselberg, Petroleum Z . , 30, No. 33, 1 (1934).
VITAMIN B, GROUP Determination Extraction Procedures for the Microbiological of Vitamin B, d
JESSE C. RABINOWITZ AND EShlOND E. SNELL Department of Biochemistry, College of Agriculture, University of Wisconsin, Madison, Wis.
S
I S C E much of the vitamin B6 present in natural materials is not available to microorganisms ( I ) , the determination of this factor by microbiological assay requires preliminary treatment of the sample t o liberate the vitamin Be. Atkin et al. (1) have suggested for this purpose either hydrolysis of a sample containing 2 to 4 micrograms of vitamin Bs with 180 cc. of 0.055 N sulfuric acid for 1 hour at 1.4 kg. per sq. cm. (20 pounds per sq. in.) pressure, or enzymatic digestion with clarase. Other investigators have hydrolyzed with 1 or 2 N sulfuric or hydrochloric acid (IO,19). Melnick et al. (6) showed, however, that the vitamin B6 content of a dried yeast sample hydrolyzed with 2 N sulfuric acid was much lower than the value obtained when 0.055 N acid was used for the hydrolysis. They ascribed this t o destruction of an acid-labile substance possessing vitamin Bo activity. This investigation waa originally undertaken to compare the effectiveness of various procedures for liberating vitamin Bs
from natural materials, preliminary to a study of the relative distribution of pyridoxal, pyridoxamine, and pyridoxine in natural materials, and to determine, if possible, whether an additional, acid-labile form of vitamin Bs, as reported by Melnick el al. (4, exists. In the meantime, a preliminary note by Rubin, and Scheiner (6) reported that, although hydrolysis with 2 N sulfuric acid yields lower values than hydrolysis with 0.055 N acid or clarase, this does not result from destruction of vitamin B, by the 2 N acid, since subsequent treatment of the 2 N acid hydrolyzates with clarase liberates as much vitamin Bs as the original clarase treatment. Some of this unusual behavior may result from the presence of pyridoxal phosphate in natural materials. The growthpromoting properties of this substance for microorganisms have not been reported previously. The action of various hydrolytic procedures on a sample of this synthetic coenzyme has been studied and compared with results of the same procedures a p
