stream of gas can he made> either to bypass or to siveep the C-tube by a simple turn of the stopcock. After calibrating the volume precisely a t a selected temperature-e.g., by mercurp-each cell in turn is filled with pure nitrogen under the st:tndard conditions, and then swept with the carrier gas stream at a selected and measured standard rate, which rate, Hithin lOy0 of its value, is then used in all determinations. These calibrations are repeated with three or more carrier gas mixtures representing the range of relative pressures over which determinations are to be conducted. In use, all values of relative pressure must be within this range. The volumes are plotted against the product of the peak areas obtained, the
appropriate attenuation factors previously determined, and the rate of flow measured during the generation of the calibration peak. The volumes of sor-
coniniercinl instrument and colleirted some of tlic original data.
tions can be carried out satisfactorily if necessary. Figure 5 shows such a family of curves for carrier gas of different compositions. These curves were constructed from 12 different volumes of nitrogen injected into each of the carrier streams.
Applied Spectroscopy, Pittsburgh, Pa., March 1. 1960. (3) Joy, A. S., Vacuum 3, No. 3, pp. 254-78, (1953). (4)Lee, C. F., Stross, F. H., Division of Analytical Chemistry, 135th Meeting ACS, Boston, April 1959. (5) Nelsen, F. M., Eggertsen, F. T., ANAL.CHEM.30. 1387 (1958). (6) Porter, P. E.,' Deal, ' C. H., Stross, F. H., J . Am. Chem. SOC. 78, 2999 (1956). ( 7 ) Roth, J. F., Ellwood, R. J., ASAL. CHEM.31, 1739 (1959).
ACKNOWLEDGMENT
w e acknowledge the work of c. F. Lee, who built the first prototypes of the
LITERATURE CITED
A Controller for Maintaining a Constant Rate of Vaporization in Fractional Distillation EDWIN C. KUEHNER and ROBERT T. LESLIE National Bureau of Standards, Washington, D. C.
b A controller for maintaining a constant rate of vaporization from the pot of a fractionating column i s described. To show the performance of the controller, a comparison is made of the constancy of efficiency of a still during controlled and uncontrolled test runs.
T
HE CONSTANCY of the efficiency of a still and the extent of separation of a mixture during a fractional distillation are greatly dependent on maintaining optimum operating conditions. At total reflux, the efficiency varies chiefly with changes in the boil-up rate (amount of material returning t o the still pot per unit time), changes in the through-put (amount of material rising to the head of the still per unit time), or changes in the rate of vaporization in the still pot. Other factors affecting still efficiency are not considered in this work. The constancy of the velocity of the vapors passing from the vaporizer through an orifice in the path leading to the column is closely related t o the constancy of the A thermistorother two variables. actuated control device is useful for controlling the rate of vaporization. EXPERIMENTAL
Apparatus. The apparatus developed for controlling the rate of vaporization is shown in Figure 1. T h e thermistor J is located in the stream of vapors in the vaporizer. Thermistors used i n this work were Western Electric Type 14A having a resist-
ance of 4000 to 6000 ohms a t 100" C. The variation of the resistance of the thermistor due t o dissipation of heat is used t o sense changes i n the velocity of the vapor. This thermistor is surrounded by a heater coil L to supply a small, constant amount of heat so that the thermistor will be a t a higher temperature than the vapor. The larger the difference in temperature between the thermistor and the vapor flowing past it, the greater will be the cooling effect of a given mass of vapor and the more sensitive the thermistor will be to changes in flow rate. Sensitivity is also increased by decreasing the size of the orifice with a Teflon sleeve M surrounding the heater. The restricted orifice increases the amount of vapor
Figure 1 .
brought into close contact with the sensing unit in unit time. If the pressure in the still and the composition of the vapor are constant, thermistor J indicates the rate of vapor flow. However, if the pressure in the still varies, the temperature of the vapor will change. Such a temperature change will then have the same effect on ther-
Vaporizer for still
A. Teflon-gasketed taper joint (wirer sealed through) al. Tension spring B. To still column C. Tefion-gasketed spherical joint (wirer sealed in) CI. Tension coil spring c2. Tension screw CJ. Teflon gasket cc Metal follower D. Thermistor well E,F. Compensating thermistors G,H. Liquid level thermistors I,K. Sensing thermistors L. Heater coil M. Teflon sleeve N. Maqnetically operated valve 0. Internal heater P. Sampling port VOL 34, NO. 9, AUGUST 1962
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ential refractometer, which was calibrated with a series of samples prepared from weighed amounts of each of the two constituents of the test mixture. The differential refractometer readings of these samples were plotted, and the best equation to fit this curve was derived by applying the method of least squares ( 3 ) . The relation of refractive index to composition was A = 29.45 X 10-4X (2.96 X 10-4)X2, where A is the difference in refractive index when compared with 2,2,4-trimethylpentane and X is the mole fraction of n-heptane. For the differential refractometer used in this work, the refractive indices of n-heptane and 2,2,4trimethylpentane differed by 4600 scale divisions and readings were reproducible to about three divisions. The number of equivalent theoretical plates was calculated from the composition of the samples of distillate and material returning to the vaporizer by use of the Fenske equation ( I ) , and the value of 1.0240 (4) for the relative volatility of the components of the test mixture.
+
LIQUID L E V E L THERMISTORS
LlOUlD L E V E L INDICATOR
Figure 2. Diagram of a continuous vaporization controller and monitor
mistor J as a change in the flow of gas passing over it, unless some compensation is made. Thermistor E in the opposite arm of a bridge makes such a compensation because it is located in a well D which is washed with condensed vapors and is essentially sensitive only to changes in boiling temperature. Oil was necessary in well D to stabilize the temperature. No compensation is made for the change in the thermal properties of the vapors. Generally this change is small. Figure 2 shows a schematic diagram of the controller. Thermistors E and J are the two arms of an a.c. bridge. A decade of precision resistors and a fixed precision resistor form the other two arms. The unbalanced current from the a x . bridge is amplified and operates a two-phase motor. These components are available commercially from manufacturers of chart recorders. The motor drives a variable transformer through a reduction train of about 1000 to 1 and varies the voltage to the internal heater 0 of the vaporizer in Figure 1. Only a part of the heat is supplied by this internal heater; the remainder is supplied by an external electrical heating mantle. To obtain a record of changes in the velocity of the vapors, a thermistor K wm placed beside thermistor J in the stream of vapors and a thermistor F was placed in the thermistor well D of Figure 1. These two thermistors are two arms of a d.c. bridge (as shown in Figure 2), and the unbalanced current from this bridge is recorded on a recorder having a chart speed of 2 inches per hour. The leads of the sensing thermistor and heaters are sealed through a glass cap C made from the ball section of a spherical joint. The cap is clamped to the vaporizer with tension springs CI, screws c2, and a metal follower c4, and is made vapor tight with a Teflon gasket c3 (Figure 1). The boil-up rate was determined by measuring the time required for the liquid level to rise from thermistor H to thermistor G, representing a predetermined volume, while the magnetically operated valve N of Figure 1 was closed. The leads to these thermistors are sealed through a Teflon-gasketed taper joint A (held together with a tension spring al) and connected to a 1 156
ANALYTICAL CHEMISTRY
battery and meter circuit for indicating the liquid levels. Procedure. The performance of the controller in maintaining a constant rate of vaporization can be shown by determining how constant i t can keep the efficiency of a still a t total reflux for a period of several days. A 300-cm. still column, random-packed with chrome1 spirals, was used for this purpose. The test mixture consisted of n-heptane and 2,2,4-trimethylpentane ( 2 ) which were carefully fractionated in a highly efficient 300-cm. still until the successive fractions differed in refractive index by not more than 0.000003 as determined with a differential refractometer. The 300-cm. random-packed still was charged with 1800 ml. of the test mixture containing about 10 mole % of n-heptane. The still column was preflooded and, while operating a t total reflux, samples of 2 ml. each were taken of the distillate a t the top of the still and of the returning material from the sampling port P (Figure 1) a t 24hour intervals for a period of several days. The compositions of the samples were determined by means of a differ-
300 -
TP
RESULTS
Figure 3 shows typical curves resulting from efficiency test runs with controlled and uncontrolled vaporization. The curve labeled “TP (controlled)” represents a run in which the controller for maintaining constant vaporization was used, while the curve labeled “TP (uncontrolled)” is a similar run except that the controller was not used. The efficiency was appreciably more constant, after equilibrium was reached, during the test run in which the vaporization was controlled than during a similar test run without the controller. The curve labeled “BR (uncontrolled)” represents measured boil-up rates for the uncontrolled efficiency test run. A
Iconlrolled)
a.
t v)
2004 -I
a
U d
0 I-.
w U
:tooI I-
0
24
48
72
96 I20 144 HOURS AFTER PREFLOODING
168
192
216
240
Figure 3. Effect of controlled and uncontrolled vaporization on efficiency of a 300-cm. packed still column
definite correlation bctucwi the \ ariation in the efficiency and the variation of the boil-up rate of tht. uncontrolled test run is indicated. Measured boilup rates were not taken during an efficiency test run with the controller for maintaining vaporization because there would be a possibility of temporarily changing the balance of the controller; furthermore any variations would show on the monitor recorder chart. The variation on the recorder chart, when calibrated in terms of boil-up rate, indicated an over-all variation of ap-
proximately 0.6 ml. per minute or 3% of the operating boil-up rate. This includes the short-time variations caused by sudden changes in vaporization M-hich the controller does not smooth out because of the lag in the heating system. An estimated long-range average of these variations suggested a much greater constancy in the boil-up rate, as was previously confirmed by the constancy of the curves resulting from efficiency test runs while the controller was used for maintaining a constant rate of vaporization.
LITERATURE CITED
(1) Fenske, M. R., Ind. Eng. Chem. 24,
482 (1932).
(2) Miller, C. H., Woodle, R. A., Division of Petroleum Chemistry, 134th Meet-
ing, ACS, Atlantic City, N. J., September 1958. (3) Scarborough, J. B., “Numerical Mathematical Analysis,” p. 363, The Johns Hopkins Press, Baltimore, Md., 1930. (4)Smith, E. R., Matheson, H., J. Res. Natl. Bur. Std. 20, 641 (1938). RECEIVED for review February 6, 1962. Accepted May 23, 1962.
Preliminary Isolation of 17-Ketosteroids from Urine for Analysis by Gas Chromatography SIR: A number of investigators have described gas chromatographic separation of 17-ketosteroids present in synthetic mixtures, but to our knowledge, only two brief reports have appeared regarding application of these techniques to 17-ketosteroids in urine. One (6) gives essentially no details regarding methodology preceding the gas chromatographic steps. The other (2) utilizes a Florosil column for preliminary isolation of ketosteroids. I n both cases, the gas chromatograms contain many peaks, only two of which are identified as corresponding to known urinary 17-ketosteroids, In the present procedure, preliminary isolation of a ketonic fraction is made with Girard’s reagent. Figure 1,A, showing a gas chromatogram obtained with normal urine, is much superior to results obtained by the previous investigators in that peaks corresponding to six 17-ketosteroids appear with proper retention times, and their relative areas are compatible with amounts of the individual 17-ketosteroids thought to occur in normal urine. ailso, the chromatogram contains few peaks that cannot reasonably be ascribed to 17ketosteroids.
hours. The pH is adjusted to 1.0 with sulfuric acid and continuously extracted with 250-ml. portions of ether for 48 hours. The aqueous solution remaining is then adjusted to p H 0.0 and continuously extracted for an additional 72
A
S
15
c i.
PROCEDURE
The isolation procedure is a combination of well known steps. Details are as follows: The 24-hour urine specimen is adjusted to pH 4.8 with acetic acid, and volume of M sodium acetate buffer (pH 4.8) plus volume of water-washed chloroform are added. After addition of 300 units of beef liver &glucuronidase per ml. of urine, the solution is incubated a t 47’ C. for 72
140
’
IM’
100’
ea
60
40
i
20
0
MI NU1E S
Figure 1. Gas chromatographic separation of urinary 17-ketosteroids ( 1 ) Etiocholanolone; (2)androsterone and dehydroepiandrosterone; ( 3 ) l l -ketoetiocholanolone; ( 4 ) 1 1 -hydroxyetiocholanolone; (5) 1 1 hydroxyandrosterone A. Urine extract from a normal male subject B. Simulated urine containing 17-ketosteroids C. Simulated urine blank
-
hours. Ether is changed every 24 hours. Bradosol (Ciba Pharmaceutical Co., Summit, N. J.) (0.2 gram) is added to the aqueous phase prior to extraction to inhibit emulsion formation (9). The combined ether extracts, after concentration to about 250 ml., are serially washed with volume portions of N NaOH until the aqueous phase measures p H 7 to 8 (pH paper). This is followed by two additional washes. The pooled NaOH is backextracted with volume of ether and then discarded. The combined ether extract is washed with volume of 0.01 N HCl and passed through about 35 grams of anhydrous sodium sulfate. Ether is then removed using a Rinco Evaporator. The residue is separated into ketonic and nonketonic fractions in the manner described by Girard (4): To the residue are added 2.0 ml. of absolute ethanol, 0.2 ml. of glacial acetic acid, and 200 mg. of Girard T reagent. The mixture is heated under reflux for 60 minutes. After cooling in an ice bath, 3.0 ml. of N NaOH plus sufficient ice water to make the total volume 20 ml. is added. This solution is washed twice with one volume of ether (spectro grade, Distillation Products Industries), and the pooled washings are backwashed with 5.0 ml. of ice water. The combined aqueous solution is made to p H 1.0 with N HC1 and incubated a t 47’ C. for 16 hours to hydrolyze steroid hydrazones. The aqueous solution is then extracted continuously for 24 hours with ether (spectro grade). The solution (0.3 to 0.5 ml.) remaining after evaporation of ether is taken up in 1.0 ml. of 50y0 aqueous ethanol and extracted three times with 5.0-ml. portions of benzene. The combined benzene extracts are evaporated under nitrogen, and the VOL 34, NO. 9, AUGUST I962
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