Stabilization of Biotin Solutions by Acid DOROTHY L. GALLANT AVD GERRIT TOEKXIES Lankenau Hospital Research Institute and Institute for Cancer Research, Philadelphia 30, Pa.
laboratory and elsewhere (personal communications) INit THIS has been observed that dilute aqueous solutions of biotin
kcpt in the refrigerator, as used for bacterimetric work, may rapidly lose their biological potency. In view of the reported stability of biotin toward aeration! n-ell as its relative resistance to heating, in acid or alkaline solutions ( 1 ) . the authors have made some observations on the effect of acidity on the longterm stability of aqueous biotin solutions.
Using the ampoules of the General Biochemicals Company, which contain 25 micrograms per nd. of water, nonsterile solutions containing 1 microgram per nil. were prepared in ordinary distilled water, and in S, lo-? .V, 10-l .Y,and 1 -1-hydrochloric acid. The glass-stoppered 25-ml. flasks containing these solutions werq stored a t approximately 4' C. and opened only a t the time of testing. For the potency determinations appropriate dilutions were compared with solutions made from freshly opened vials, for their power to cause growth of Streptococczis faecalis 9790 in the buffered medium of Toennies and Gallant (W),using the described bacterimetric technique. The following results were obtained. In the first series of tests the standards contained 2 micrograms of biotin per 10 ml. of
finished medium-i.e., more than the minimal requirement. I n the other determinat'ions several levels of standard, up to 0.5 microgram, and test solutions were used. Time, M o n t h s 6.5 9.5 18 Biological activity. Z ' of fresh standard 25 0 0 -100 112 102 -100 -100 108 103 -100 -100 98 89 -100 -100 -100 73 44
7 -
3
Medium H?O
~O-~SHCI 10-*SHCI 10-1.VHCl S HCI
-
The results indicate the advisability of making stock dilutions of biotin in 0.001 S or 0.01 -V hydrochloric acid rather than in water. The data also attest to the stability and relative uniformity of the commercial aqueous solutions sealed in ampoules. LITERATURE CITED
Brown and du Vigneaud, J . Biol. Chem., 141,85 (1941). ( 2 ) Toennies and Gallant, Ihid., 174, 461 (1948).
(1)
RECEIVED January 28,1949.
Convenient Melting Point Apparatus ALEXANDER MAY Sorcthwestern Louisiana Institute, Lafayette, La.
4 1 7 E R T simple, yet accurate melting point apparatus was developed to meet two basic requiienients: to cover a wide range of temperatures from that of the room to about 500" C. and to be rapid and clean in use, avoidlng hot liquid baths or calibrated metallic blocks. These t n o needs weie met admirablj- in an apparatus which consists of only tn o concentric test tubes, a thermometer, and cork stoppers. Although this 14 similar to arrangements previously described ( 2 , 4,7 ) , the exclusion of all capillary tubes while retaining a double air bath resulted in a remarkably accurate a n d efficient apparatus. 1
T
Figure 1. Apparatus
As shown by Figure 1, the apparatus consists of a double air bath of two test tubes, A and B, held concentric by means of cork stoppers and the thermometer, C. The entire unit is held in a horizontal position by means of a buret clamp. The sample, whose melting point is to be determined, is placed directly on the thermometer bulb, as Stahl ( 7 ) suggested, and heat is applied evenly along the outer edge of the larger tube by means of a Bunsen burner. The thermometer is cleaned after each use by wiping the melted sample off with a cloth. If any traces remain, they may be removed Kith acetone. The corks are grooved to prevent internal pressure. Both total and partial immersion thermometers have been used with equal satisfaction. For routine work, the student grade thermometers with ranges up to 110", 250", or 310" C. are most useful; for higher values or greater accuracy, a thermometer extending to 530" C. or high quality A.S.T.M. instruments were employed. The size of test tubes A and B is not critical. Equally good results have been obtained with a 6-inch (1.8-cm. dismeter, 15-cm. length) or an %inch (2.5-cm. diameter, 20-rm. length)
outer tube, d, and a 4-inch (1.2-cm. diameter, 9.5-cni. length) or a 5-inch (1.5-cni. diameter, 12.5-em. length) inner tube, B. When taking a melting point, it has been found convenient to place the buret clamp on the laiger stopper. This allows the test tubes to be put in position or removed quickly, eliminating the time between successive melting points, which otherwise irould be needed to allow the tubes to become cool enough for reuse. The only precaution needed is to heat the larger test tube evenly and be sure that the tubes are held concentric. The actual point of melting is very easily seen through the clear tubes and the thermometer stem is completely unobstructed. PERFORMANCE O F THE APPAR4TUS
In checking this instrument, two methods were employed: an absolute accuracy determination, and a comparison with the usual capillary method. For the first, benzoic acid was employed as a standard, inasmuch as this substance is now accepted as aofi.\;edpoint in thermometry, with a melting point of 122.362 A 0.002' C. (6). Coleman and Bell C.P. acid was used, and was purified by recrystallizing from water, subliming, and finally fractional freezing by the procedure of R. S. Jessup ( 5 ) . By this procedure, the acid \vas obtained as a 99.99a/, pure product, which was then used to calibrate an A.S.T.M. high softening point total immersion thermometer. The thermometer range was from 30" to 200" C. graduated every 0.5'' C. For the calibration, cooling curves were determined, while the transition point was noted with a 1OX magnifying lens, equipped with a cross hair, and so held in a stand as to prevent any error of parallax. The uncorrxted melting point recorded on the thermometer was 121.5' C. with a stem temperature of 44" C. The corrected value was 122.4" C., in excellent agreement with the accepted value. The identical thermometer and magnifying system was then used with the air bath apparatus, and the melting point of the same purified bcnzoic acid was determined. An average of six determinations gave an uncorrected melting point of 121.7" C. with a stem temperature of 37" C. which, when corrected, yielded the melting point of 122.6" C. The discrepancy between the cooling curve value and that of the present 1427
1428
ANALYTICAL CHEMISTRY Table I.
Melting Point Comparisons Melting Points Capillary Present tube Method method 49.3-49.5 50.5-51.0 92.5-95.2 92.8-95.6 109.5-110.0 109.0-110.5 130.5-132.0 132.8-133.9 132.8-133.0 132.5-133.2 138.8-139.4 141.6-143.5
Substance Benzalaniline Benzil Acetyl o-toludine Acetyl %naphthylamine Cinnamic acid m-Nitrobenzoic acid p-Aminobensenesulfonyl amide 162.8-163.5 Silver nitrate 211.5-212.0 Potassium chlorate 362.0-361.0 Potassium dichromate 398.0-399.0 Cupric chloride 496.0-500.0
162.8-163.8 212.0-213.0
......
.. . .. .. . .. ,
,
Literature Refervalue ence 56 (48) (3) 95 (9) 112 (1) 132 (1) 133 (5) 140 163 212 368 398 498
method amounted to 0.2" C. which is about the accuracy with which the thermometer can be read. Also investigated mere the effects of the rate of heating, the uniformity of heating, and the position of the substance on the thermometer bulb. In the case of the benzoic acid, the rate of heating was found to have no effect on the result, other than to diminish the accuracy with n-hich the thermometer temperature and sample could be observed when both were changing very rapidly. However, the melting point of 121.7" C. was checked when the acid was heated a t a rate of 1O every 10 seconds and also a t a rate of less than 1' ever)- minute. When the heat was applied uniformly belon- the thermometer bulb, over an area extending 4 em. on either side of the bulb, the position of the sample on the bulb made no difference in the results. Lnder these conditions, the sample melted simultaneously over the entire bulb. On the other hand, when purposely only the extreme end of the large test tube n as heated, the sample melted first at the tip of the bulb. and then progiessively further up the bulb. In the latter case, the difference between the first and laqt temperatures of melting was as large as 6" C. The average of the lowest and highest values gave an uncorrected melting point of 122.0' C. for the benzoic acid. The
24. Dimethylglyoxime (Diacetyl Dioxime) CH3-C-C-CH3
I1
'
X-OH
1;
S-OH
single position of greatest accuracy was 7 mm. from the end of the thermometer, or approximately two thirds the length of the bulb. The second comparative method of checking the apparatus is tabulated in Table I. These results were obtained by the use of a variety of thermometers, over a range of temperatures. I n each case, however, the same thermometer was employed for the air bath and for the capillary tube method for any given compound. The values reported are the uncorrected melting points. This air bath method compares very favorably with the usual capillary tube, liquid bath method ( I ) , but a t the same time is less cumbersome, more rapid, and above all, has a greater range of temperatures covered. The melting point ranges reported are those associated with the purity of a compound. The sample is so small that, even when melted, it remains on the top of the thermometer bulb, thus ensuring equilibrium conditions between the solid and liquid phases. COXC LUSIOV s
The melting point apparatus described possesses many advantages, mainly its use at high temperatures and quickness of operation. The accuracy is at least 15-ithin 0.2" C. or as great as That of the thermometer used. LITERATURE CITED 1 C'heion~s,S . D., and Entrikin, J. B., "Semirnmo Qualitative Organic Analysis," pp. 29, 362, 402, 4 0 8 , 4 4 3 , Yen- York, Thomas
Y.Crowell Co., 1947. 1 2 ) Friedrichs, Fritz, 2. a n g e u . C'henz., 34, I , 61 (1921). 3: Lanse, X. .I.,"Handbook of Chemistrv," 6th ed., Sanduskv. Ohio, Handbook Publibhers. 1946. 1 4 Malowan. S.L.. Z. a?mew. Chem.. 32. I. 16 (1919). 51 Schwab, F. W., and IT'Ichers, E.. J Research .YatZ. Bici. Standards. 25, 747 (1940). 6 ) Ibid., 34, 333 (19451. 17) Stahl, G. 'T.,ISD. ESG.CHEM.,A s ~ L ED., . 13, 545 ( 1 9 4 1 ) . RECEIVED December 2, 1948.
-1Xial Ratio. a : b : c = 0.950:1:0.703. Interfacial Angles (Polar). 100 IO10 = 63"; 73"; 011 .i 100 = 80". Crystal Sngles. CY = 125"; 6 = 91 '; y = T9 Twinning Plane. 001. Cleavage. 001 and 100.
100 I O 0 1 =
'.
Structural Formula for Dimethylglyoxime Excellent crystals of dimethylglyoxime can be obtained from alcohol-water mixtures (50-50) by cooling hot saturated solutions with agitation. Twinned crystals are very common and cleavage parallel to b is pronounced. The fibrous nature of the cleavage fragments and the fact that the highest refractive index is approximately parallel to b indicate that the molecules are oriented within the crystal so that their longest direction is approximately parallel to b. CRYSTAL MORPHOLOGY (determined by W. C. McCrone). Crystal System. Triclinic. Form and Habit. Tablets flattened on lo0 and usually elongated parallel to b. The principal forms are the three pinacoids. The brachydome I011 } and macrodome I 101 } also appear __ occasionally.
Principal Lines d 6.63 5.92 5.40 5.19 3.76 3.66 3.51 3.42 3.33 3.02 2.93 2.86 2.58 2.51 2.45 2.34
I/Ii 0.05 0.6 0.2 0.05 0.1 0.1 0.1 0.2 1.0 0.1 0.05 0.05 0.2 0.1 0.05 0.1
d 2.20 2.14 2.10 2.08 2.04 1.89 1.84 1.82 1.78 1.75 1.70 1.67 1.47 1.44 1.38
I/II 0.02 0.02 0.02 0.02
0.02 0.01 0.02 0.1 0.01 0.02 0.02 0.1 0.02 0.02 0.01
