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 be 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 b y 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 a n y 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), t h a t are individually pumped into a small mixing vessel. The mixture then flows through the column b y 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. T o determine the height of the potentiometer arm required for the elution of individual hormones, i t 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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76 \5
/ .2c
T U B E NUMBER
Figure 3. Programmed separation of compounds A, S, and B Different gradient from Figure 2
Figure 4. Programmed separation of compounds A,. S.. and B Different gradient from Figure 2
TUBE NUMBER
got
Figure 1. Separation of compounds A, S, and 6 using a linear gradient Gradient change expressed as D C M in PE per tube
/---
%
TUQE NUMBER
Figure 2. Programmed separation of compounds A, S, and B Upper. lower.
Elution gradient, % D C M per tube Absorbance per tube
I
b Figure 5. Programmed separation of seven physiologically active adrenocortical hormones Upper. Lower.
O
a
I-
m
Gradient change, % D C M in PE per tube Absorbance per tube
TUBE NUMBER
2020
0
ANALYTICAL CHEMISTRY
t
.,IJ
‘3 .2t
W U
5
BEEF ADRENAL EXTRACT LINEAR GRADIENT
a
F
B E
I
0
I
I
1
E
I
D
S
.I
TUBE NUMBER
Figure 6.
Separation of adrenal steroids in 6 ml. of beef adrenal extract Linear gradient, started a t 100% PE
ml. of solvent, having the composition shown under “Elute.” This is equal to the holdup volume of the column. Thereafter, the polarity of the eluent is decreased below the composition shown under “Hold.” The interval between the peaks is governed by the length of time that the i3olvent composition is kept below the “Hold” concentration.
I
BEEF ADRENAL EXTRACT
I
I
I
I
I
AtS+B
Table 1. Per Cent DCM in PE Required to Elute or Irlold Individual Hormones
Compound
Elute
Hold
2
33 64 70 75 80 86 91
20 55 60 65 70
S
13
E Aldosterone F
75
80
Figure 1 illustrates the separation achieved for a mixture of A, S, and 13, using a linear gradieni,, concentration change of 0.66% per tube. Under these conditions, the three compounds are eluted in succession without intervals. The distance between peaks is readily controlled by selecting an appropriate elution program. I n Figure 2, the interval between S and B has been increased by a drop in polarity of the eluent. I n Figure 3, this is shown for the interval between A and S, and Figure 4 illustrates an extremely wide spacing between the three peak$, produced by a special elution program. The programmed separation of all seven reference compou ids is presented in Figure 5. The distance between peaks and the sharpne:,s of each peak c:m be coiitr olltd by belthctiiig 311 alq1ropriate gradient curve. This analysis was accomplibhed in l i hours, using a
AtStB
TUBE NUMBER
Figure 7. Programmed separation of adrenal steroids in 6 ml. of beef adrenal extract Upper. lower.
Elution gradient, % DCM in Absorbance per tube
IO-minute collection cycle on the analyzer. Subsequently, the time was reduced to 8.5 hours by adjusting the collection time to 5-minute cycles. One of the prime advantages of (:olurnn ctirom:itograhy over other t y ~ i e s of chromatography is its greater load capacity. This permits the use of
PE
per tube
samples large enough for the determination of minor components in the presence of excessive amounts of other components or impurities. Even so, analysis is often hampered by overlapping of closely related compounds, especially if such an overlap involves a minor component. Figure 6, representing the VOL. 35, NO. 13, DECEMBER 1963
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analysis of 6 ml. of adrenal extract, shows that the column is overloaded under the conditions used (linear gradient, concentration change of 0.333% per tube). Yet, this amount of sample is required for the determination of small peaks, such as those occurring betmeen B and E and between E and F. The problem was solved by programming the gradient elution as shown in Figure 7 (top). T o save time, compounds A, S, and B were deliberately eluted in a single peak, while increasing the resolution of subsequent compounds, particularly the BT-reducing compound between E and F. This compound was shown by thin-layer chromatography to be Reichstein's Compound D (allopregliane - 38,17c~,21- triol - 11,20 - dione). Tlius, any portion of the elution pattem can be selectively compressed or cs-
panded by programmed gradient elution. The steroid analyzer ( 1 ) is a prototype instrument, representing a considerable investment of time and cost. The degree of automation achieved probably exceeds the requirements of a routine laboratory. However, the principle of gradient elution and particularly polarity reversal will undoubtedly be found generally useful for difficult resolution problems and may be applied either manually or by a simple combination of tape programming and dual pumping. Details of this simplified system will be described elsewhere.
Frank 0. Anderson, Grant C. Riggle and John K. Cullen, Jr., Instrument Engineering and Development Branch, Division of Research Services. They thank William J. Haines, Upjohn Co., for the generous gift of beef adrenal extract. LITERATURE CITED
(1) Bnderson, F. O., Crisp, L. R., Riggle,
ACKNOWLEDGMENT
G. C., Vurek, G. G., Heftmann, E., Johnson, D. F., Francois, D., Perrine, T. D., ANAL.CHEM.33, 1606 (1961). ( 2 ) Bennett, R. D., Heftmann, E., J . Chromatog. 9,348 (1962). (3) Heftmann, E., A N A L . CHEM.34, 13R (1962). (4) Heftmann, E., Johnson, D. F., Ibid., 2 6 , 519 (1954).
Tlic authors gratefully ackiioivlcdge the technical advice and assistance of
I ~ L C L ~ V for L D review Ma\ 16, 1903. .Iccepted Yepteinber 16, 19G3.
Qua ntitative Analysis of Aromatic Hydrocarbons by Capillary Gas Chromatography JOHN Q. WALKER' Barber-Colman Co., Pasadena, Texas DAN L. AHLBERG Research Division, Signal Oil and Gas Co., Houston 72, Texas
b The use of polar substrates with capillary columns has made the separation o f m- and p-xylene less difficult. m Bis(m phenoxyphen0xy)benzene (b.p. 273' C. at 1-mm. pressure) can b e utilized as a capillary column substrate for quantitative analysis of c6 through CI aromatics. If base line separation in the c6 through CS region i s desired, modification of the liquid phase with squalane i s required. Comparative elution data and spectra of several polar substrates are given Reliable quantitative analytical data in this aromatic range can b e obtained using these columns in a gas chromatograph employing a linear sample splitter and high temperature flame ionization detector. This investigation includes over 30 available compounds in the c6 through c11range. Twentyfive of the first 28 aromatics were resolved in 17 minutes using this method.
-
-
results of the authors' investigation of several liquid phases are given in Table I. The present paper deals with the utilization of m-bii(m-phenoxyphen0xy)benzenc (EPB) (b.p. 273" C. at I-mm. preziure) as a capillary substrate. Thiq material is easy to coat on capillary columnh, is very stable, and can be utilized for the analysis of CS through C,, aromatics. If better than 90% separation in the C6 through CS region is desired, modification with squalane (a relatively nonpolar substrate) is required. This, however, limits the ~
Table 1.
T
2022
ANALYTICAL CHEMISTRY
Apparatus. Barber-Colman Model 20 a n d LModel 61-C instruments equipped with linear stream splitters
Liquid Stationary Phases Investigated for Aromatic Separations
.Iromaticparafin resolution B,P'-C)xydiprupioiiitrile Carbowax 1500 (1 00 )
analysis of aromatic hydrocarbons has been studied b y a number of workers ( I - @ , and in the last three years the use of capillary columns has made the separation of nz- and p-xylene less difficult. The 1 Present address, Wilkene Instrument and Research, Inc., Houston, Texas.
EXPERIMENTAL
~~~~
Oxvbis-Zethyl benzoate with (;E96
HE
column to this particular range because of excessive elution time for higher boiling materials. A discussion of column preparation, operating parameters, etc., illustrates that reliable quantitative analytical data in the Cg through Cli aromatics range can be obtained using this column system on a gas chromatograph employing a linear sample splitter and ionization detector.
m-Bis(m-phenoxyphenoxy )benzene with squalane m-Bis(m-p henoxyphenoxy )benzene Rating system. 1. Poor 2 . Fair 3. Satisfactory 4. Excellent
Sylene
rcsolutioii
Thcrd
Y tnbili t y
