Since various compositions can be compensated by simply changing attenuations, the method is applicable to a wide range of concentrations. Also, no reagents or special techniques are necessary. The apparatus could easily be adapted, by means of solenoid valves and timers, so that cnly one master switch would be needed for the entire determination. The procedure would then consist only of loading the sample, activating the master switch, and reading the result. Use of calibration curves would result in an analysis requiring only about 5 minutes if a technician’s time per run. The time per analysis could be reduced by shortening the time required t o elute the sulfur dioxide from the
column. An oven capable of being programmed at a faster rate could conceivably reduce the total analysis time to 15 miniitcs or less.
(4) Cain, J. R., Max Eng. Chem. 1- --(5) Charpenet, No. 153, 39 (1961). (6) Hale, C. I T . , Jr., Muehlberg, W. F., IND. ENG. CHEM.,ANAL. ED. 8, 317 (19.?fi\
ACKNOWLEDGMENT
The authors gratefully acknowledge assistance from Thomas Dunphy, presently of Colorado State University, in preparing the computer program. LITERATURE CITED
(1) Bennet, E. L., Harley, J. H., Fowler, R. M., ANAL.CHEM.22, 445 (1950). (2) Bouton, P., Hoste, J., Anal. Chim. Acta 27,315 (1962). ( 3 ) Burke, K. E., David, C. &I., ANAL. CHEM.34, 1747 (1962).
(7)
3132
(
(8)€10
23, 1696 (9) Kuo, C J. M., Ibid., (10) Pepkowitz Ibid., 26, 102 (11) Walker, J. 2017 (1963). RECEIVEDfor review July 11, 1963. Accepted October 9, 1963. Abstracted from a thesis by W. K. Stuckey, submitted
.
in partial fulfi1.lment for the degree of Master of Science at Kansas State College of Pittsburg, Pittsbiirg, Xan.
Carbon Determination in Ferrous Metals by Gas Chromatography J. M. WALKER and C:.
W. KUO’
Department of Chemisfry, Kansas State College of Pitfsburg, Pittsburg, Kan.
b An extremely sensitive and highly precise method for the determination of carbon content in ferrous metals b y gas chromatogrlgphy was developed. Samples were combusted in a high frequency induction furnace, their gaseous products passed through a &foot 5-A. molecular sieve column, and detected b y a thermoconductivity detector. The c a r b m dioxide was trapped in the coluinn a t 100’ C. while oxygen was swept out b y the helium carrier gas after complete combustion. The carbon dioxide peak came off the column a t about 275” C. b y means of temperature programming while its area was measured b y a disk chart integrator. Eight different NBS steel and iron saniples with carbon contents varying from 0.01 1 to 3.28% were run. This metk80d permits detection of 0.0005% carbon a t the maximum detector sensitivity. The time required for a single run i s approximately 20 minutes. Some advantages of this technique are its simplicity of operation, broad detection range (0.0005 to 20% carbon), and high sensitivity.
T
HE PROCEDURE clf carbon determination in ferrous metals has been well established. Many methods and their modifications have appeared in the literature (1-8, 6, 11, 18). They can be classified mainly as the following categories: the wet chemical method, the direct combustion method, the vacuum
fusion (low-pressure combustion) method, the electroconductometric method, and the spectrographic and mass spectroscopic methods. The trend of development in this field is to seek a rapid and highly sensitive method which would enable one to detect not only very low carbon content, but also very high carbon content as well. The gas chromatographic technique appears to be a step in the right direction. Although there has not been any published paper concerning the carbon determination in ferrous metals by gas chromatographic technique, papers were given a t the 1962 and 1963 meetings of the Pittsburgh Conference of Analytical Chemistry and Applied Spectroscopy (6, 8). I n addition, several workers did utilize this technique in the determination of carbon and hydrogen in organic compounds. Duswalt and Brandt (4) combusted their sample in an oxygen stream. Sundberg and Maresh ( l a )using copper oxide as internal oxidizing agent, burned their sample in a helium atmosphere. I n both cases, the combusted gases were led through a liquid nitrogen trap and the carbon dioxide was trapped. Since silica gel columns were used in both cases, the liquid nitrogen trap is a necessity so as to get rid of the excess oxygen and to concentrate the gases for quick injection into the sample column. Recently, Nightingale and Walker (9) developed a simultaneous C-H-N determination using a gas chromatographic technique. They introduced the coupling of the high fre-
quency induction furnace and the gas chromatograph. These workers made use of a 5 A. molecular sieve column and eliminated the troublesome liquid nitrogen trap. Still, an internal oxidizing agent, silver permanganate, was used instead of burning the sample in an oxygen atmosphere. Parsons, Pennington, and Walker (10) also utilized the highfrequency furnace-gas chromatograph combination in the determination of nitrogen. The work described here is based on the retention of carbon dioxide by the 5 A. molecular sieve. The carbon dioxide in the combusted mixture was trapped and “stored” in the molecular sieve column under isothermal conditions while the excess oxygen passed on through. Helium, the carrier gas, was used to purge the oxygen from the column hence the need for an internal oxidizing agent and the liquid nitrogen trap was eliminated. The carbon dioxide peak was obtained through temperature programming. EXPERIMENTAL
Apparatus and Materials. A schematic diagram of the entire system is shown in Figure 1. The apparatus consisted of a Leco high frequency induction furnace Model 523, a F & 11 Scientific Model 500 linear-programmed temperature gas chromatograph, with a 4-foot 5-A. molecular sieve column, and two Hevy D u t y Present address, The Dow Chemical
Co. ,Williamsburg, Va.
VOL. 35, NO. 13, DECEMBER 1963
2017
Electric Co. Miiltiple Unit tube furnaces. Two 3/4-inch copper tubes, filled with Mallinckrodt wire-form analytical reagent grade copper(ic) oxide, mere heated by the tube furnaces which were set at the 3-3 position giving a temperature of 500' to 600' C. Linde hospitalgrade oxygen and helium were run through the preheaters which oxidized and carbonaceous matter present in the gases. But the oxygen and helium were passed through two copper tubes, 24 inches long and "4 inch in diameter, 17-hich mere packed with G. F. Smith Chemical Co. reagent-grade magnesium perchlorate, Arthur W. Thomas Co. Ascarite (8-20 mesh) and Leco 501-60 specially prepared manganese dioxide. Any possible moisture, carbon dioxide, and sulfur dioxide in the flow lines was thereby removed. The purified helium was then passed into the chromatographic unit as the carrier gas. The purified oxygen was introduced into the induction furnace. To maintain proper flow rate of oxygen through the molecular sieve column, a pressurized system was desired. Since the original Leco designed silicone rubber connection could hold only moderate pressures, a self-designed brass double "0" ring seal was incorporated into the oxidation chamber. This double "0" ring arrangement was first reported in a paper by Kuo, Bender, and Walker ( 7 ) . Instead of the conventional Leco quartz combustion tube, a quartz tube with a length of inches, and an outside diameter of inches was used. All of the flow lines and connections were made of '/&ch copper tubing and fittings. Needle valves were purchased from the Matheson Co. (No. 107). Leco 501-76 carbonfree tin accelerator (20-40 mesh), 528-35 crucibles, and 528-40 covers were used in this study. Procedure. The following conditions were maintained during each analysis: Helium flow rate at sample side: 80 ml. per minute at 100" C. column temperature. Helium flow rate at reference side: 50 ml. per minute at room temperature. Oxygen flow rate at sample side: 80 ml. per minute at 100' C. column temperature. Program rate: 42' C. per minute setting. Block temperature: 200" C. Injection port temperature: off, room temperature. Bridge setting: 130 ma. Temperature limit setting: 400" C. Oxygen pressure: 11 p.8.i. Helium pressure: 11p.s.i. Attenuator setting: 2-128, depending on carbon content of samples. Table 1.
NBS
sample 55e 170a
133a 1 0g I Oe
10d
82a
122d 2018
3
Figure
1.
Schematic diagram of apparatus
7. Oxygen cylinder 2. Helium cylinder 3. Pressure regulators 4. Copper oxide tubes and preheaters 5. Magnesium perchlorate scrubber 6. 7.
Ascarite scrubber Manganese dloxide scrubber High frequency indudion furnace Molecular sieve column, 4 feet 5 A.
8. 9. 10. Block 7 7 , 72, 73. Valves 74. Reference detector side 75. Sample defector side
With valves 11 and 13 opened and valve 12 closed, oxygen was released to the atmosphere and helium passed through the column, the sample detector, and into the atmosphere. The helium flow of the reference side was maintained constant at all times. The pen on the recorder was then set a t its electrical zero by adjusting the control node. At this point, the disk chart integrator would show no area. The sample, 0.5 f 0.001 gram, was weighed on a Mettler Gram-Atic Balance and mixed with 1.0 gram of Leco tin accelerator in the prefired crucible. The sample crucible was now loaded into the induction furnace with valve 11 open so the carbon dioxide from the air would have no chance of getting into the column. After making sure that all the carbon dioxide in the combustion chamber was purged out, valve 11 was then closed. -4t the same time, valve 13 was closed and valve 12 opened. Oxygen from the induction furnace was now passed through the column. The pen of the recorder would go out of scale due to the passage of oxygen through the sample detector. The induction furnace was now turned on and sample combusted. The combusted gases were
Results of Carbon Determination in Ferrous Metals b y Gas Chromatography
YBS
Carbon, % Found
0.011 0.052 0.120 0.240 0.406
1.01 2.24 3.28
ANALYTICAL CHEMISTRY
0.0102
0.0524 0.1178 0.2345 0.3968 1 ,0269 2.3239 3.5666
Mean error
- 0.0008 +0.0004 - 0.0022 -0.0055 - 0.0092 +0 ,0169 +0.0839
+0 ,2866
Standard deviation *0 .000104 *0, 00015 zk0.00056 fO.00110 f0.00309 f 0.00200 +0. 00750 f O ,00877
passed through a manganese dioxide tube which retained all the sulfur dioxide in the gas mixture, and then into the 4-foot 5-A. molecular sieve column where carbon dioxide was trapped. The furnace was turned off after the oxidation was complete. The combustion time for each sample was found depending on the amount and the kind of samples in the crucible and varied from 10 to 12 minutes. Valve 12 was closed 2 minutes after the furnace was turned off so there would not be any carbon dioxide left behind in the oxidation chamber or in the copper connection. At the same time, valve 13 was opened and the helium carrier gas was sweeping the excess oxygen out of the column. In doing this, the pen of the recorder would slowly be drifting to lower scale and finally back to the original position-the electrical zero. This indicated that all of the oxygen was expelled. The temperature limit of the oven was now releastd and column temperature programming initiated. (To save time, once the time required for the pen to reach the equilibrium is observed, one can program it before the pen is completely back to zero, so long as the equilibrium will be reached just before the carbon dioxide peak shows up.) The area of the peak was read from the disk chart integrator. The over-all time required for each single run is approximately 20 minutes. RESULTS AND DiSCUSSlON
Eight kinds of EBS standard ferrous metals, with carbon contents ranging from 0.011 to 3.28%, were run, with results tabulated in Table I. For the lowest carbon sample, NBS 55e, the mean error is a -0.0008. For the highest carbon sample, NBS 122d, the
mean error is a +0.2866. I n actual unknown determination, a series of correction factors of counts x attenuation per p.p.in. carhon, m u 4 be found for individual gai chroniatogrsphic uiiits by firing fractional weights of a standard sample at different attenuation settings. The latter was done for the work reported in this paper. =It its maximum sensitivity, attenuation of I , 0.003y0 of carbon would give n full scale deflection. l\'ith thi; ietting. one can ra.ily detect O.Oi)OS% of carbon. In fact, 1 gram of Leco tin in the prefired crucible has been fired in thi> system, a n area n i t h a peak height of about 0.5 inch was seen which indicates the presence of trace carbon in the Leco tin accelerator. -1major advantage of this technique is its broad detection range. Its lower limit ib nbout 0.0005% while its upper
limit (extrapolated) is of the order of 2070 absolute carbon. Any sample with carbon content within this range can be determined simply by shifting the setting of thP attenuator to the proper sensitivity. This method also eliminates the necessity of absorption cells, reagents, solutions, etc. Its simplicity and neatness in operation should be noted, including its suitability for routine analytical work. The materials necessary for connecting the induction furnace to the gas chromatographic unit would c o d no more than a few dollars. By u*ing the quartz enclosed, carbon crucible instead of the porcelain crucible, this method can be applied to the microcarbon determinationf or organic compounds. LITERATURE CITED
(1) Bennett, E. L., Harley, J. H., Fonder, R. >I., ANAL.CHEM.22, 445 (1950).
(2) Cain, J. R., Maxwell, 1,. C., I d . Eng. C h e m 1 1 , 852 11919'1. (3) Charpenet, L., Fluinme Thermzque 13, 153 (1961). (4) Duswalt, A. A., Brandt, W.K., , ~ N A I . . CHEILI. 32, 273 (1960). (5) Hickan, W. M., Ibid., 24, 36%(195%). (6) Karpathy, 0. C., Pittsburgh Con-
ference on Analytical Chemistry and Applied Spectroscopy, March 1963. ( 7 ) Kuo, C. W., Bender, G. T., Walker, J. M., A S A L . CHEM.35, 1505 (1963). (8) Mooney, J. B., Carbini, L. J., Pittsburgh Conference on Analytical Chemistry and -4pplied Spectroscoliv, March
1062. ( 9 ) Sightingnle, C. F , Walker, ,J. XI., A s ~ L CHEV. . 34,1435 (1962). (10) Parsons, 11. I,:, Pennington. S. X., JValker, J. hl., Ibzd., 35, 842 (1963). (11) Pepkowitz, L. P., Moak, W. ll , Ibzd., 24, 889 (1952). (12) Sundberg, 0. E., Maresh, C., Ibid., 32, 274 (1960). (13) Wooten, L. A , , Guldner, W. G., IND. ENG.CHEkl , A N A L . ED. 14, 835 (1942).
RECEIVEDfor review April 11, 1063. Accepted August 15, 1903.
Programmed Gradient Elution Chromatography with the Steroid Analyzer DANIEL FRANCOIS, DAVID F. JOHNSON, and ERICH HEFTMANN National Institute of Arfhritis and Metabolic Diseases, National lnstitutes of Health, Public Health Service, U. S. Department of Htmlth, Educotion, and Welfare, Bethesda, Md. b Programmed separcition of adrenocortical hormones by gradient elution chromatography with the steroid analyzer is described. Controlled separations are accomplisheld by means of a gradient pumping system, which permits the polarity of the eluting solvent mixture to b e increased or decreased at will. The effect OF selected programmed gradients on the separation of seven adrenocortical hormones and beef adrenal extract i!; demonstrated.
auto natic device for analyzing adrenocortical hormones by gradient elution chromatography on columns has recently btben developed in this laboratory ( I ) . The steroid analyzer assays aliquots of eluate fractions and is capable of producing any desired elution gradient. A nL.mber of devices for programmed gradient elution have previously been described ( 3 ) . COMPLETELY
PRINCIPLI:
The steroid analyzer, described in detail earlier ( I ) , consists of two integral units. The gradient elution system operates independently of the cyclic operation of the remainder of the apparatus. A gradient cam, a metal replica of a plot of eolvent ratio as ordinate vs. time as absAssn, is followed
by the arm of a linear potentiometer. The position of this arm, as it traces the cam, governs the amounts of the two solvents, light petroleum ether (PE) and dichloromethane (DChf), that are individually pumped into a small mixing vessel. The mixture then flows through the column by gravity. il desirable feature of thi. differential pumping method of producing the elution gradient is that the concentration of one solvent in the other may be increased or decreased a t will. The remainder of the apparatus performs the automated procedures for dividing, collecting, drying, and analyzing the fractions by both ultraviolet spectrophotometry (UV) and colorimetry after reaction with Blue Tetrazolium (BT). EXPERIMENTAL
The steroid analyzer was used as previously described, with the following exceptions. To determine the height of the potentiometer arm required for the elution of individual hormones, it was positioned manually for the preliminary experiments involving reversal of solvent ratios, as described below. The column used differed slightly from that described in our original method for the quantitative determination of individual corticosteroids (4). The stationary phase was water, supported by a silicic acid column (hferck, dried at 100' for 3 hours). The ratio
of support to ftationary phase n a s 2.5 to 1 by weight. Identity and purity of fractions \vere monitored by thin-layer chromatography ( 2 ) . Stock solutions of 100 pg. per ml. of absolute ethanol were prepared from the following steroids: A4-pregnen-21-ol3,20-dione (Q), A4-pregnen-21-01-3,11,20-trione (A), A4-pregnene-ll&21-diol3,20-dione (B), Ad-pregnene-17 a,21-diol3,20-dione (S), A4-pregnene-17a,21-diol3,-11,20-trione (E), A4-pregnen-18-olllp,-21-diol-3,20-dione (dldo.). and A4pregnene- llp,17a - 21 -triol- 3,20-dione
(F).
Adrenal cortex extract (Upjohn) with a biological activity equivalent to 100 pg. of F per ml. vias dried under nitrogen and applied to the column nithout purification. Samples of 5 pg. of each reference compound were automatically determined with a n accuracy of 98 =t2%. Preliminary esperimentc with the seven adrenocortical hormones revealed that the solvent ratio. required for the elution of each are critical. If the concentration of DChI in PE is decreased by as little as 1%, the elution pattern is altered, and a 5 t o 10% decrease will retard the succeeding zones. Table I lists the concentrations of DCRI in PE which will elute or hold each steroid. Any given steroid is eluted by delivering t o the cohimn 30 I 'OL. 35, NO. 13, DECEMBER 1963
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