ANALYTICAL CHEMISTRY
764
separation This demonstrates that for crude urine evtracts it probably gives a measure of those steroids remaining after the Girard separation, thereby making the latter unnecessary M hen the total 17-ketosteroid content of the urine is desired. Most of the spectra from urine evtracts exhibited a minimum of 90% transmittance (Figure 3 ) and closely approvimated in shape those in Figure 2 A determination of the 17-ketosteroids of several neutral urine extracts was made The data are summarized in Table I1 together with the higher values found by the absolute alcohol technique (urines E and F are evceptions). The quantities of 17-ketosteroids obtained b\- the e\traction method before and after the Girard separations (urine samples A and B) agreed ne11 with those found b> the Callow procedure following the Girard separations. This again indicates that the extraction method measures the ketonic steroid. which are separated by the Girard reagent.
Table 11. Application of Extraction Rlethod to Neutral Urine Extracts
Urine Sample Pooled male 1 After Girard separation Pooled male 1 After Girardseparation Pooled male 2 Pooled male 2
A A B B C D
Hydrolysis and Extraction Procedure, Refeience (5)
(2)U (6)
( 2 ,h
Mg. 17-KS. Colorimetric Methods Present Callow et al. (4) 4.67 6.70
4.48
4.43
4.67
6.19 4 80 6.24 6 33
4 54 5 78
6.20
Per 24 Hours N a l e arthritic (0') 12 5 11 5 Female virilisni during cortisone therapy (8) 12.3 12.1 G Female virilism (8) 15 7 17.4 48-hour ether extraction. 6 72-hour ether extraction. Urines E , F, and G were made available through the courtesy of D. Bergenstal, Department of Medicine. L-nirersity of Chicago.
E F
(1
Table 111. Recovery of Crystalline Dehydroepiandrosterone Acetate Added to Neutral Urine Extracts Urine Sample C E
Urine Extract, y 17-KS 22 5 45 0 26 7 53 4
F
22 3
c
23 7
44 5
23 7 47 4
DEAA Added, y 20 20 20 20 20 20 20 40 20
DE.A.1 Reco\ercd 21 0 18 8 18 8 17 6 19 3 16 1 20 3 40 8 20 9
Y
Recovery, % 105 94
94 88
97 81 102 102 105 AT
96
ACKNOW LEDGMEbT
The authors are inlebted to the Schering Corp. for crystalline dehydroepiandrosterone acetate and to F. A Travers of the Ciba Pharmaceutical Products, Inc. for the crystalline epiandrosterone. EdlTard Davis, Department of Obstetrics Thanks are due to ?*I. and Gynecology, Cniversity of Chicago, for the crystalline estrone. urinary pregnanolone, and etiocholan-3-or-ol-l7-one David Fukushima kindly supplied crystalline androsterone and etiocholan-3-ol-ol-l7-one. Discussions with George V. LeRoy have been very helpful. LITERATURE CITED
(1) Beher. IT,T.. and Gaebler. 0. H.. AN-\L. CHEM..23. 118 (1951). . , (2j Buehler, H. J., Kataman, P. . i , , ' a n d Doisy, E. a:,Proc. SOC. Esptl. Biol. 'Wed., 78, 3 (1961). (3) Cahen, R. L.. and Salter, W. T., J . Biol. Chem.. 152, 1 8 9 (1944). (4) Callow, X . H., Callow, R. K., and Emmens, C . IT.,Biochem. J . , 32, 1312 (1938).
(6)
Jlasuda, 31., and Thuline, H. C., J . Clin. E~tclooinol.and Meteb-
oli'rrn, 13, 581 (1953). (6) Robbie, IT7. d.,and Gibson, R. B., J . Clin. Endocrinol., 3, 200
The average recovery of dehydroepiandrosterone acetate which as added to diffeient amounts of urine evtract was 96% (Table 111) The recovery from the larger evtract tended to be lot\ er than that from the snialler aliquot. This was also noted by Masuda and Thuline ( 5 ) .although no quantitative data were presented
(1943).
(7) Ruppert, d.,Z . g e s . e.rpt2. M e d . , 119, 229 (1952). ( 8 ) Zimmermann, W,, Anton, H. V.,and Pontius, D., H o p p e - S e y l e r ' s Z. phU8iol. Chem., 289, 91 (1952). RECEIVED for reviem August 17, 1953.
Accepted .Janiiary 26. 1954.
Improved Fractional Melting Apparatus S. V. R. MASTRANGELO' and J. G. ASTON Pennsylvania State University, State College, Pa.
I
S VIEW of the utility of fractional melting as a means of purification (1), an improved fractional melting apparatus
with compressible conduction vanes has been developed which is sturdier than the others described previously (1). This apparatus is referred to as fractional melting apparatus 5. The reader is referred to the earlier publication for background and experimental detail. A test of performance of the apparatus with and without using the vanes to press out the melted substance is described. APPARATUS
A scale drawing of the apparatus is shown in Figure 1. The apparatus consists of four sections: the melting chamber, 18, and vane system, 27, the shield supplied with a heater, 17, the 1 Present address, Barrett Division, Allied Chemical and Dye Corp., Glenolden, Pa.
shield or temperature equalizer, 16 (which is not supplied with heater), and the external cryostat envelope, 15. The melting chamber is provided with a compressible spiral vane system 27, which is the basis of the high efficiency of the apparatus. This type of vane system proved to be mechanically superior t o and simpler in design than the one used in the fractional melting apparatus 3 ( 1 ) . The vanes are made by cutting disks from S o . 36 gage sheet steel, perforating them a t random, and drilling a central and a radial hole for the vane guide tube, 28, and the transfer tube, 30, respectively. The disks are cut along one radius (opposite the radial hole) and twisted approvimately 15'. The twisted disks are then hardqoldered end t o end t o form a continuous spiral. The pitch of the spiral is about 0.25 inch when the spiral is expanded the full length of the melting chamber. The lower end of the spiral vane is soft-soldered t o the bottom of the melting chamber, and the top is hard-soldered t o the main activator vane, 20. The main activator vane, 20, consists of a copper disk l / 1 6 inch thick. This vane is hard-soldered t o the plunger tube, 19, which has a perforated cap, 12. The plunger tube, 19, is partially slotted along one side and the slot is made t o ride on a key mounted in the cryostat pumping tube, 14. This device preZI
V O L U M E 26, NO. 4, A P R I L 1 9 5 4
765 vents thr spiral vane system from turning. The perforated cap, 12, is hard-soldered to the stainless steel activator rod, 11. The activator rod, 11, passes out of the apparatus through the stuffing box, 4. The stuffing box, 4, is packed with a Teflon washer, 3, compressed by a screw cap acting through a brass gland, 1, and a stainless steel washer, 2. The Teflon stuffing box proved to be vacuum-tight, even when the vanes were being activated. The needle valve, 38, on the transfer tube, 30, is also provided with Teflon packing. The heater element, 29, is wound in four sections on the outside of the melting chamber. The absolute thermocouple used for measuring the temperature of the melting chamber is soldered to the side wall of the melting chamber. The e l e c t r i c a l supply wires to this thermocouple are wrapped (10 turns) around the wall of the melting chamber and fastened there with Bakelite lacquer. This last measure prevents false readings of the thermocouple by reducing heat leak. The transfer tube, 30, and filling line, 5, for the melting chamber are wound with heaters, 13 and 14, respectively. These heaters are not necessary for the operation of the apparatus; they serve merely as a convenience when time is short and for inexperienced operators who have not yet learned to control the liquid air level in the refrigerant bath Dewar flask. They are to be used when the plunger tube, 19, sticks owing to solidification of sample in that region. Also, if the liquid air level is not controlled properly, the transfer t u b e , 30, m a y b e c o m e plugged in the region wrapped with the auxiliary heater, 13. Figure 1 shows the arrangement of the receiving flask, 42, for receiving the impure liquid fractions. The other details of construction and operation are similar to those of. the fractional melting apparatus 3 (1). It is the purpose of this paper not only t o present the improved design but to show how essential are the compressible vanes.
Figure 1. Improved Fractional Melting Apparatus 1. 2. 3. 4. 5. ti.
7. 8.
4.
in. 11.
12.
13. 14. 15. 16. 17. 18. 19.
20. 21. 22. 33.
Brass eland Stainless steel washer Teflon packing Stuffing box for stainless steel activator rod, 11. Filling line t o melting chamber for evacuation a n d pressure Electrical supply wires pass through de Khotinsky seal Connection for transfer tube Filling line to melting chamber, continued Electrical supply wires exit tube Cryostat pumping tube for evacuation of cryostat envelope Stainless steel vane activator rod Perforated plate connected t o vane activator rod 11 a n d guide tube 19. Perforations allow melting chamber td be evacuated or filled with nitrogen gas under pressure. Transfer tube heater (12 ohms) Filling line heater (22 ohms) Cryostat envelope (Monel metal) Copper temperature equalizer Copper shield provided with heater Melting chamber Guide tube for vane activation Master vane to which is connected upper end of spiraled vane. Master vane is also connected t o the guide tube 19. Electrical supply wires t o sdield heater (not shown) Electrical supply wires from calorimeter heaters Electrical suppIy wires to difference couple and thermocouple
Thermocouple soldered t o melting chamber a n d wrapped around i t 10 times for thermal contact. Bakelite lacquer is used for making wires fast 25. Difference couple junction on shield (soldered) 26. Difference couple junction on melting chamber (insulated electrically) 27. Spiraled vane system 28. Guide tube 29. Four calorimeter heaters. T h e resistance of each heater is approximately 34 ohms. 411 are connected in series with t a p wires taken o u t between them. There are five heater leads in all 30. Transfer tube 31. No. 16 bare copper wire for conduction heater 32. Constriction 33. Glass wool 34. Drilled solder plug 35. Electrical supply wires from calorimeter heater 36. Corks for supporting shield 37. Radiation shield 38. Needle valve 39. Kovar glass seal 40. Three-way stopcock to evacuate receiving flask, 42, or to fill i t with nitrogen gas under uressure 41. Constriction 42. Receiving flask for impure liquid fraction 43. Glass tube with elass wool filter. 44 44. Glass wool filter45. Drain for receiving Bask 24.
RESULTS
TKO identical samples were prepared by mixing 475 ml. of Phillips’ iso-octane (2,2,4-trimethylpentane) (lot 126) with 25 ml. of Phillips’ nheptane (lot 159). The samples were used to test the effect of compression of the vane system on the efficiency of purification. Table I summarizes the progress of purification when the vanes are compressed after each operation to squeeze out the liquid from the crystals. Column 1 lists the amount of liquid withdrawn after observing the corresponding equilibrium temperature listed in column 2. Column 3 lists the fraction of material existing as liquid compared to the total amount of material remaining in the melting chamber during measurement of the equilibrium tempera.ture, and column 4 lists the purity,
766
ANALYTICAL CHEMISTRY chamber. Column 2 lists the corresponding equilibrium temperatures before the liquid was withdrawn, and column 3 lists the purities calculated by means of the melting point equation. Column 4 lists the actual yields and column 5 lists the percentage of the theoretical yield obtained based on Raoult’s law and eutectic composition. When the vanes were not compressed, larger quantities had to be melted before each withdrawal as the amount which could be removed without squeezing
Table I. Progress of Purification of Iso-octane with Compressing of Vanes Liquid Withdrawn,
MI.
.
0 28 28 36 23
36 30 0 0
Equilibrium Temp., OK.
Fraction of Liquid
161.05 163.36 163.76 164.15 165.48 165.71 165.71 165.71 165.71
0.10 0.10 0.10 0.115 0.101 0.095 0,050 0.141 0.347
Purity Calod. from Temp
%
94.4 99.1 99.4 99.40 99.91 100.00 100.00 100.00 100.00
Purity Calcd. from Washing,
%
...
96.0 97.6 99.6 99.8 99.93 99.998 99.998 99.998
calculated by means of the melting point equation, of the material remaining in the melting chamber (this includes the solid and liquid portions). Column 5 lists the purities calculated by the batch theory of washing, assuming all of the impurity to be dissolved in the melt. The amount of liquid remaining after withdrawing the melted liquid (as completely as possible) was estimated from the difference between the amount of liquid melted, as determined from the added energy, and that withdrawn into the receiving flask. Column 6 lists the actual yields (in percentage of the original charge) and column 7 lists the percentage of the theoretical yield obtained. The theoretical yield was estimated from a eutectic diagram calculated assuming the applicability of Raoult’s law and the absence of solid solution. A lower purity is calculated by batch theory of washing because the actual operation is effectively continuous, the descending ’ washings occupying a relatively small volume. For this reason the purity calculated by means of the melting point equation is considerably more accurate than that calculated by the theory of washing. That equilibrium is reached with respect to composition is indicated by the fact that the equilibrium temperature did not change with time. Moreover, after the equilibrium temperature indicated that all impurity had been removed, melting half the remaining material produced no rise in the equilibrium temperature (see the last three entries in Table I). The second purification was effected by keeping the vanes stationary in the expanded position. The results are listed in Table 11. No attempt was made to apply the theory of washing, since the melt no longer made contact with all of the crystals. Column 1 lists the amount of liquid withdrawn from the melting
Theoretical ‘70
100.0 94.4
88.8 81.6 77.0 69.6 64.0 64.0 64 0
.. .. .. ..
%
96 87 80
80 80
was greatly reduced. Comparison of the two tables shows that the purification efficiency is increased by a factor of about 3 a t the 99.98% level of purity when the compressible vane system is used. The time for equilibration was about 2 hours when the vanes were immobilized, as compared to 15 to 20 minutes when the vanes were activated. The last fraction in the purification without compressing the vanes had not come to equilibrium even after 3 hours.
Table 11. Purification of Iso-octane without Compressing of Vanes Liquid Withdrawn,
MI.
Equilibrium Temp.,
K.
Purity Calcd. from Temp., %
Yield,
5%
Theoretical Yield Obtained, %
ACKNOWLEDGMENT
The authors wish to thank the Phillips Petroleum Co. for the financial help which made this work possible. LITERATURE CITED
(1) Aston, J. G., and Mastrangelo, S. V. R., ANAL.CHEM.,22, 636 (1950). RECEIVED for review August 22, 1953.
Accepted January 18, 1954.
Estimation of Fluorine in Biological Material P. VENKATESWARLU and D. NARAYANA RAO Department of Biochemistry, M e d i c a l College, Trivandrum, M i a
I
N CONNECTION with the estimation of fluorine in blood, Smith and Gardner ( 8 ) drew attention to the loss of fluorine as a volatile iron fluoride when the blood is ashed preparatory to the Willard-Winter distillation (3). For this reason, they adopted a preliminary sulfuric acid distillation (before ashing) to free fluorine from the iron. To obtain reliable results in the case of other biological materials, it was also found that a preliminary distillation is necessary owing to the almost universal occurrence of iron in plant and animal tissues Silica in plant materials also interferes with fluorine recovery (1). This interference was reduced by preliminarv distillation of the unashed sample or by fusion of the lime-ashed sample with qoclium hydroxide before distillation of the fluorine from perchloric acid. . i n earlier observation (4)made in these labora-
tories, to the effect that magnesium oxide is a good adsorbent for the fluoride ions, considerably facilitated the fluoride analytical procedure which was thereby rendered more rapid. PROCEDURE
One-half gram of material is submitted to Willard-Winter distillation, employing sulfuric acid. The distillate, which is kept just alkaline to phenolphthalein, is brought to boiling, 0.2 gram of light magnesium oxide is added, and the boiling 1s continued for 5 minutes more. The magnesium oxide with the adsorbed fluoride is separated by centrifugation or filtration and eubmitted t o the Willard-Winter perchloric acid distillation in the presenrr of harium and silver perchlorates t o withhold the interfering sulfate and chloride ions. The fluoride in the distillate is determined by titration by the Smith and Gardner technique ( 2 ) .
