V O L U M E 26, NO. 1 2 , D E C E M B E R 1 9 5 4 similzr t o the maximum absorption of the hydrazine p-dimethylaminobenzaldehyde condensation product obviates the use of the method for the determination of one in the presence of the other. Pesez and Petit ( 1 0 ) report that a slight coloration is obtained with urea and adrenalone, while no interference occur\ with mono-, di-, and trimethylamiries, di- and triethylamiries, ethanolamine, glycocoll, choline, urethane, aminoguanidine, or hydroxylamine. Furthermore, semicarbazide and urea (again) were shown t o interfere b y W a t t a n d Chrisp ( 1 3 ) . These same authors state t h a t ammonium chloride or nitrate in a 5000 to I mole ratio to hydrazine does not change the per cent transmittancy or shift the minimum. -Accordingly, the method for methylhydrazine determination would be subject to similar interference from those substance. PI hich interfere with hydrazine determinations. T h e observed effectiveness of p-dimethylaminobenzaldehydefor colorimetric determination of methylhydrazine assumes especial interest, since Pesez and Petit ( 1 0 ) report that phenylhydrazine does not interfere with the determination of hydrazine.
1963 drying for 50 hours a t 105’ C., or upon drying at room temperature over potassium hydroxide in vacuo. Methylhydrazine sulfate crystals appear to suffer oxidative changes similar t o those observed for solutions of the salt. Yellow impurities which accumulate on the exterior of c r y ~ t a l swere removed by recrystallization of methylhydrazine sulfate from aqueous alcohol. ACKKOWLEDGMEST
T h e authors wish to thank Elizabeth J. Adair for her very able technical aisistance during these studies. LITERATURE CITED
Idanis, It., and Coleman, G . H., Ore. Syntheses, 1, 214 (1941). .ludrieth. L. F., and Ogg, R . d.,“Chemistry of Hydrazine,’’ S e w T o r k , John Kiley B- Sons, 1951. Clark, C. C., “Hydrazine,” Baltimore, Mathieson Chemical C O r D . . 1953. Fox, H. H., Science, 116, 129 (1952). Ibid., 118, 497 (1953). Kelly, J. A I . , and Poet, R. B.. A m . Rer. Tuhcrc., 6 5 , 454 (1952). Knoufer, G., iWoitatsh. Chem., 30, 29 (1909). Kolthoff, I. II.,J . Am. Chem. Soc., 46, 2009 (1924). JIcBride, W.R., Henry, R. d.,and Hkolnik. S.. ASIL. CHEY., 25, 1042 (1953). , 132. Peaes, AI., and Petit, A , , BUZZ.soc. c h i n . F ~ a n c e 1947, Prescott, B., Kauffman, G., and James, IT. D., Proc. SOC. Ezptl. B i d . Med., 84, 704 (1953). Troyan, J. E., Ind. Eng. Chem.. 45, 2608 (1953). Watt, G. W., and Chrisp. J. D., ANAL.CHEM.,24, 2006 (1952). Weatherby, J. H., and Witkin, L. B., private communication. White, D. G., Agr. Chem., 7 , KO.1, 40 (1952). K o o d , P. R., ASAL.CHEM.,25, 1879 (1953).
M ETHYLHY DR.4 ZINE STANDARDS
For most purposes it was found expedient to use commercially available methylhydrazine sulfate directly as received from the supplier. The high purity of the reagent is shown by iodate titration (Table I ) and neutralization equivalents. Figure 3 shows the plot of the potentiometric titration of a sample of methylhydrazine sulfate neutral equivalent; cslculated, 141.15; found, 144.28. Better samples of commercial material had apparent methylhydrazine content of 100 f 0.57, on the basis of titration data. Samples of this materisl lost no neight upon
R E C E I V Efor D review llpril 5 , 1954.
Accepted July
12, 1954.
An Isothermal Distillation Method for Determining Molecular Weights C. E. CHILDS Parke, Davis & Co., Detroit, M i c h .
The isothermal distillation method for determining molecular weights has been modified by including a reservoir of the pure solvent in the system, so that results within 5% of theory may be obtained in 3 days.
UMEROUS methods for determining molecular Tveight have been developed, but the isothermal distillation method of Signer ( 2 ) is outstanding for its accuracy and simplicity. Clark ( 1 ) improved the method, and White and Morris ( 3 ) made i t more practical, but the time required to complete a determination was generally one week or longer. The method described, though perhaps not so accurate, will give results within 5% of theory in 3 days.
Table I.
Molecular Weights in Acetone
Sample Acetanilide p-Chlorobenzoic acid Azobenzene Benzidine Phenylindanedione 3-Xitroiodotoluene Phenolphthalein Curcumin
Mol. Weight of Sample 135 156 182 184 222
Mol. Weight Found 137
138 156 152
181 182 180 182 .__ 225 ~
~~
217
263 315
368
When two solutions of the same solvent are enclosed in an evacuated system with only their vapors in contact, the solvent will distill from the weaker to the more concentrated solution, depending upon their respective molarities, and if completed under isothermal conditions the two solutions \Till eventually become equimolar. The principle involved in the present method is the same, except that the distillation is enhanced by means of a reservoir of the pure solvent which is included in the system. Because the vapor pressure of the pure solvent, is higher than that of either of the solutions, the solvent n-ill distill into both solutions in proportion to their respective molarcoricentrations. After a period of time the volumes can be measured, and b\. comparing with the known. the molecular weight of the unknonn may be calculated. Table I gives the experiniental data obtained \\it11 acet,oiie as the solvent. but ethyl ether, ethyl bromide, and ethyl formate have also been used successfully. Either azobenzcne or benzoic acid may be used as the reference compound, but i n gcnerrtl a higher degwe of accurary was observed n-ith benzoic :witl.
256 250
310
313 366
382
EQUIP1\IENT
The apparatus as pictured (Figure 1) consists of three bulbs connected by a bridge in the form of a Y . Bulbs A and B, which contain the reference and unknown solutions, are corinccteti to the solvent bulb, C, by a 19/38 ground-glass joint on the vapor bridge, D. -4stopcock, E, 1-mm. bore, through which the vacuum is applied, is connected to the top of bulb C. The two 1-ml, sealed-tip serological pipets, F-F, which are used to measure the Iand volumes of the solutionsr,are connected to the tops of bulbs :
B.
ANALYTICAL CHEMISTRY
1964 The apparatus must be airtight, and the ground-glass joint presented a problem, but it was found that a thick paste of graphite and Dow Corning high vacuum grease worked very well. For best results the solvents must be pure, and the samples completely dry. PROCEDURE
\Veighed samples, 5 t o 15 mg. of the reference and unknown compounds, are placed in bulbs A and R, respectively, with 0.10 t o 0.30 ml. of solvent t o put each into solution. The amount of solvent required will depend upon the solubility of the compound, but it should be kept :it a minimum. The weights of the samples should be somewhat proportional t o their molecular weights. S e s t , 1 ml. of the pure solvent is placed in bulb C, which is then attached to D by means of the ground-glass joint and a fresh supply of the graphite rease. A heavy Figure 1. Apparatus rubber band is pyaced around the hulbs t o hold the joint firmly in place, and a water pump vacuum is gradually applied through stopcock B until there is a slight evaporation of the solvent. Ordinary stopcock grease is suitable for the stopcock, but it should be held in place with a clamp of some type. The stopcock is turned off, and the ahole apparatus is immersed in a constant temperature bath with the pipets up. A temperature of 40’ C., used for the
determinations listed, has several advantages; it prevents condensation within the apparatus, speeds up the procedure, and helps t o keep difficultly soluble compounds in solution. \Then all of the solvent from bulb C has been absorbed, and this usually occurs within 48 hours, the first reading is made. The apparatus is removed from the water bath, dried, and turned slowly t o a horizontal position, allowing the solutions t o run into their respective pipets. The pipets are pointed down and jarred slightly t o remove any bubbles, then allowed t o drain for 6 to 10 minutes before the solution volumes are read. Bt the end of this period the isopiestic point usually will have been reached, but the apparatus should be replaced in the bath in its original condition for 24 hours as a check. When the second reading is taken, the volumes should have remained constant, indicating that the end point has been reached. If the sample tends t o come out of solution upon cooling, or the bath temperature is high, it may be necessary t o replace the apparatus in the bath before the volumes are read. The molecular weight, Mu,of the unknown is calculated from the formula Mu = Wu X V X .%!/Vu X W , where Wu is the weight of the unknown, Vu the volume of the unknown, V t h e volume of the reference solution, W the weight of the reference solution, and M its molecular m i g h t . ACKNOWLEDGMENT
The author is indebted to Claire Johnston and Ed. Meyers for their technical assistance, and to Hurshel Hill for making the apparatus. LITERATURE CITED
(1) Clark, E. P., A N ~ LCHEN., . 13, 820 (1941). (2) Signer, R.,Ann., 478, 246 (1930). . ~ A L CHEM., . 24, 1063 (1952). (3) White, I,. If..and Morris, R. T., RECEIVED for review April 1, 1954.
Accepted July 2, 1954.
Silica Qel Microcolumn for Chromatographic Resolution of Cortical Steroids MAX L. SWEAT’ Department o f Pharmacology, University o f Utah College of Medicine, Salt Lake City, Utdh
A silica gel microcolumn is described for the chromatographic analysis of pure mixtures of several steroids in amounts as small as 0.25 y . The column is for use in conjunction with the fluorescent and phenylhydrazine analytical techniques for the determination of steroids.
T
H E application of column chromatography to the resolution of mixtures of corticosteroids in blood and biological fluids has been delayed owing to the lack of methods suficiently sensitive to detect and analyze minute concentrations of these substances. The recent development of the fluorescent method of Sweat, ( 4 ) which analyzes corticosterone, l’i-iiydroxycortivosteronc, and 4-pregnene-11-8, 17-a, 20-p, 2l-tetrol-3-one, and the phenylhydrazine method of Porter and Silkier ( 3 ) which is relatively specific for thr 17-, 21-hydroxy-20-ketone group of +teroids, has made such a Ptudy poPaihle. The present report descrihes a silica gel microcolumn which, \\.hen used in conjunction with the fluorescent and phenylhydrazine analytical techniques, makes possible the analysis of pure mixtures of several steroids in amounts as small as 0.25 -,. The purification and preparation of blood extracts for chromatography and the application of the combined fluorescentsilica gel method for the evaluation of corticosteroids in man and laboratory animals will be described elsewhere. 1 Present address, Department of Physiology, Western Reserve University School of Medicine, Cleveland 6, Ohio.
APPARATUS
Two sizes of chromatographic columns are employed: one having a n over-all length of 230 mm. is used for chromatographic analysis of 1 to 10 y of steroids; the other, 126 mm. in length, is used for 0.25 to 1 y. Water jackets are necessary to keep the adsorbent cool. Stopcocks regulate the rate of “packing” in the column. Too rapid packing or too high temperature cause small vapor bubbles to form in the silica gel. Both columns are illustrated in Figure 1. REAGENTS
Adsorbent. A mixture of 250 ml. of 95% ethyl alcohol, 750 ml. of chloroform, and 200 grams of silica gel (Davison Chemical Corp., Baltimore, Md., silica gel No. 922-08-08-226TT200) is allowed to stand several hours. The solvent mixture IS filtered under gentle suction on a Buchner funnel. The silica gel is washed successively with 400 ml. of 3 to 1 ethyl alcohol-chloroform and 1 liter of 95% ethyl alcohol. The partially dried gel is now transferred to a 4-liter beaker containing 2 liters of distilled water. The contents are stirred in a circular motion to concentrate the black particles in the center and bottom of thc beaker. The major fraction of the silica gel is decanted while it is in motion. The process of stirring and decanting is repeated approximately eight times in order to remove most of the black particles. An additional twenty washings with distilled water are necessary to free the silica gel of a water-soluble gray-brown pigment. The silica gel is partially dried on a Buchner funnel, spread on a clean surface to complete drying, and activated in a muffle furnace at 500” C. for 3 hours. Ethyl Alcohol. Absolute ethyl alcohol is distilled from a waterfree system; the first and the last 10% of the distillate are dis-
