Then when the experimental values of - d[AZ]/dt/[AZ][H+] were plotted as a function of [HP04-*], a linear correlation was obtained that indicated that the kinetics were consistent with the suggested mechanism (Figure 5; each point is the average value of three to four tests lasting 100 minutes with samples withdrawn from the solution every 5 minutes). Then, the evaluation of the constants in Equation 7 (from Figure 5) resulted in the following relationship :
k-i k? = slope - = slope (&) ki from Figure 5,
slope = 8.25 X lo4 l./mol min
(9)
This value of kz for HP04-' is in the range of other nucleophiles that attack the aziridinium ion (13, 14). The differences of the rates of disappearance of aziridine in our work and in that reported by other workers ( 5 ) can now be explained. At the higher p H of the saturated phosphate buffer system used by these investigators, the high concentration of the HP04-2 ion (acting as a stronger nucleophile than water) will attack the protonated aziridine at a faster rate, even though the concentration of protonated aziridine (aziridinium ion) is low. At the more acidic pH, the concentration of the HP04-* ion is low and probably makes the hydrolysis of aziridine the predominant mode of ring opening.
(10)
ACKNOWLEDGMENT
Therefore, when the value of K, for the aziridinium ion (12) is taken at 22 "C (assuming that this value holds at 27 "C and at high ionic strength), Equation 9 becomes: k2
=
8.25 X lo4]./mol min)
x
I
60 sec min-1
x
1 ~
0.955
x 10-8 (11)
which can be reduced to :
k2 = 14.3 X (12) G. J. Buist and H. J. Lucas, (1957).
l./mol sec
(12)
J. Amer. Chem. Soc., 79,
6157
The authors acknowledge the technical assistance of Carol Guilbert, Parnell Freeman, Barbara Weir, and also thank T. S. A d a m for his statistical analysis. RECEIVEDfor review February 2, 1970. Resubmitted and accepted June 24,1971. Mention of a proprietary product or company name in this paper does not constitute a n endorsement by the USDA. (13) B. Cohen, E. R. Van Arstdalen, and J. Harris, ibid., 74, 1878 (1952). (14) J. E. Early, C. E. ORourke, L. B. Capp, J. 0. Edwards, and B. C. Lewis, ibid., 80, 3458 (1958).
Tape-Controlled Gradient Elution Chromatography System for Steroid Analysis David F. Johnson,' Nancy S. Lamontagne,' Grant C. Riggle,2 and Frank 0. Anderson2 National Institutes of Health, U. S . Department of' Health, Education, and Welfare, Bethesda, Md.
AN AUTOMATED PUNCHED-TAPE system for gradient elution chromatography ( I ) , developed in our laboratories, has been used successfully for the analysis of adrenocortical steroids in biological mixtures (2-6). Application of the method to analysis of the residues from incubation of tissue with radioactive steroids and precursors revealed a need for further refinement in the apparatus. Radioactive compounds from such incubations were, in many cases, so similar in polarity that even the use of polarity reversal was not effective in resolving certain 1 Steroid Section, Laboratory of Chemistry, National Institute of Arthritis and Metabolic Diseases. * Instrument Engineering and Development Branch, Division of
closely associated peaks. The refinement described in this paper includes an improved pumping system for the accurate delivery (0.2%) of preselected volumes of eluting solvents, and a new solvent mixing chamber design which minimizes volume carry-over when solvent ratios are changed. These improvements have resulted in: a greater resolution of individual peaks in our chromatographic method for separating adrenocortical steroids on water-impregnated silicic acid columns ( I ) using petroleum ether (PE) and dichloromethane (DCM); and the development of a new combined method for analysis of adrenocortical and ketosteroids (7). EXPERIMENTAL
Research Services. ( I ) D. F. Johnson, D. Francois, G. C. Riggle, and C. I. Ramsden, Ann. N . Y . Acad. Sci., 130,792 (1965). (2) D. Francois, D. F. Johnson, and H. Y . C. Wong, Steroids, 7, 297 (1966). (3) D. Francois, H. Y . C. Wong, and D. F. Johnson, ibid., 8, 289 ( 1966). (4) Ibid., 9, 1 5 (1967). ( 5 ) Ibid., 10, 115 (1967). (6) D. Francois, R. W. Bates, and D. F. Johnson, Endocrinology, 81, 246 (1967).
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Apparatus. The complete apparatus is shown in Figure 1 . The main units, some of which are described in detail under procedure, consist of: water-jacketed solvent reservoirs, 1 and 2; chromatographic column, 4; tape punch and readout, 10 and 1 1 ; tape control electronic system, 12; and syringe pumps, 13. I n addition to the reservoirs and column, the outflow lines to the mixing chamber, 3, are also water-jacketed with 19.5 "C water from the cooling unit, 5. (7) N. S. Lamontagne and D. F. Johnson, Steroids, 17, 365 (1971)
ANALYTICAL CHEMISTRY, VOL. 43, NO. 12, OCTOBER 1971
11
5
10
7
Figure 1. Tape-controlled chromatographic apparatus 1. Sealed reservair, DCM 2. Sealed reservoir, PE
3. 4. 5. 6. 7.
8. Fraction collector 9. Refractometer
Solvent mixing chamber Water-jacketed chromatographic column Refrigerated water bath Distikd water reservoir Fraction collector timer
Vith the solvents used, PE and DCM, this precaution is ecessary to prevent volume changes caused by thermal znsitivity which significantly affects outflow rates. The
Figure 2. Solvent mixing chamber
10. Tape punch 11. Tape reader 12. TaDe control electronics 13. ti& driven syringe pumps
solvent reservoirs, 1 and 2, are placed at different elevations relative to the discharge level at the mixing chamber, 3. This arraneement is dictated hv the differences in svecific gravity of the two solvents (PE, 0.64 and DCM,1.32): The head pressures are thus equalized on each reservoir, assuring that the outflow lines remain filled at all times. The refractometer, 9, (Bausch & Lomh, Model 33-45-56)is used to read concentrations of pumped solvent mixtures by referring to a plot of refractive index us. concentration. Figure 2 shows a specially designed glass mixing chamber, with the two photosensors mounted on the capillary standpipe. During the tape changes, the pump motors are deactivated until the reservoir level has dropped below the bottom photosensor. The chamber design minimizes gas bubble entrapment and provides a minimum volume carryover when solvent ratios are changed. DCM and PE are combined at the end of the curved glass capillary discharge tubes, and fall into the chamber as a single drop. The resultant "streaming" of the drops down the side of the chamber further ensures thorough mixing of the solvents before pooling in the reservoir. A 4-mm T-bore stopcock provides for withdrawal of solvent mixture samples for confirmation of the programmed solvent ratio by refractive index measurement. The mixing chamber reservoir volume is 3 mi and is designed for use with the 2-cm diameter column used in our studies. Reagents. Stock solutions of 100 Fg per ml of absolute ethanol were prepared from the following steroids: A'pregnene-3.20-dione (P), A4-pregnen-21-oI-3.2O-dione (Q), A'~pregnen-21-01-3,11,20-Vione(A), A'-pregnene-17a,21-diol3,20-dione (S), A'-pregnene-ll&21-diol-3,20-dione (B), A'pregnene-17a,21-dio1-3,11,2O-trione (E), A4-pregnene-11S,17~,21-triol-3,2O-dione(F),3a-hydroxyandrostan-l Fone (androsterone, Andro), 3a-hydroxyethiocholan-17-one (etiocholanolone, Etio), 3ar-hydroxyandrostane-lI,l'l-dione
ANALYTICAL CHEMISTRY, VOL. 43, NO. 12, OCTOBER 1971
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Figure 3. Block diagram of electronic system
,
P E.
sum of the digital values for DCM and PE may equal 100; or the per cent determined from the number of units of one solvent as related to the sum of the two numerical values i.e., DCM = 80, PE = 88, Sum = 168; therefore per cent DCM = 80/168 or 47.62%. This latter option allows adjustment of fine concentration settings rather than 1 incremental changes. The punched tape is inserted into the tape reading unit “B” (Friden, Model SP-2), and the “Timing Synchronizer” is switched to the “Read” (decode) portion of the electronics system. Tape reading is initiated by a pulse from the “Fraction Collector Timer” during sample collection, or it may be activated by the “Manual ”push button override. Because the coded information for the solvent values is read sequentially from the tape, memory units (Storage DCM and Storage PE) store the binary information for further processing after the tape reading cycle is completed. Upon “Read Command,” the stored values are converted to digital values through binary code to decimal (BCD) logic, which in turn determines the equivalent dc voltages to be applied to the inputs of appropriate voltage controlled oscillators (OSC. Freq. Selector). Frequency stability is maintained within 0.2zthrough use of highly regulated dc power supplies for the logic and oscillator circuits. The oscillator pulses are shaped by the “Motor Speed Control” (Superior Electric Co., Type STM-1800V translator), and drive the variable frequency pulsed stepping
(1 1-ketoandrosterone, 11-KA), 3a-1lp-dihydroxyandrostan17-one (1 1-hydroxyandrosterone, 11-HA), 3a-hydroxyetiocholane-11,17-dione (11-ketoetiocholanolone, 11-KE), 3a,1l~-dihydroxyethiocholan-l7-one (11-hydroxyethiocholanolone, 11-HE), 3P-hydroxyd5-androsten-17-one (dehydroepiandrosterone, DHA), and A4-androsten-17P-ol-3-one (testosterone, T). Preweighed 1OO-Kg ampoules from Calbiochem used for A‘-pregnen-18-al-1 lp,21-diol-3,20-dione (Aldo) solution. Procedure. The functional operation of the electronics system is shown in Figure 3. Tape punching (preparing the numerical values of pulsed motor speeds), and tape reading (converting the punched code to pulsed frequency) are controlled through the “Timing Synchronizer”. The “Timing Synchronizer” selector switch is placed in the “Punch” (encode) position. A digital-to-binary coded thumbwheel switch, “Data Input” (Engineered Electronics Co., Series 300, 10 position) encodes the selected numerical values of two sets of numbers, representing the ratio per cent of D C M and PE, into a memory storage bank. Upon command from the “Timing Synchronizer,” the binary values are entered serially into a tape punch “A” (Friden, Model SP-2). Sequential sets of ratio per cent values are entered, and automatically punched into the tape by depressing the “Print Command” push button. Ratio values may be selected in either of two ways: the
TO M I X . CHAMBER
-I
1-
VOLT.
Figure 4. Block diagram of pumping system I
- 1 TO M I X . ’ CHAMBER
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ANALYTICAL CHEMISTRY, VOL. 43, NO. 12, OCTOBER 1971
motors (Superior Electric Co., Type SS 50-1008-P2) used to drive the glass syringe pumps. Pumping is interrupted during a tape reading cycle “Tape Advance Standby,” or when the “Mixing Chamber Meniscus Control” senses the appropriate liquid level in the solvent mixing chamber. Figure 4 shows a block diagram of the tape-controlled pumping system. The values of the desired solvent ratios, previously encoded on binary code tape, are processed by the “BCD Converters” and “Voltage Control Oscillators,” shaped by translators (Trans), and their signals then activate the pulse-controlled stepping motors. These in turn drive the two-chamber syringe pumps in a linear motion, Fourway motor-driven valves (Hamilton Co., No. 4MMMM4) rotate a t the end of each pump stroke, alternately connecting each syringe to filling and discharge lines. Limit switches (MS) control valve rotation and pump motor direction reversal. The solvents (DCM and PE) are kept watersaturated in sealed reservoirs and are displaced by the water pumped into them. This procedure eliminates solvent vapor pressure changes which develop when the solvents are pumped directly. TUBE NO.
RESULTS AND DISCUSSION
Separations utilizing solvent gradients in column chromatography depend on the gradual polarity change as compounds are eluted. I n effect, the small increments of increasing polarity prevent or minimize tailing between compounds, thus improving resolution. Precise control of the gradient is essential in resolution of compounds that differ only slightly in polarity. Less sophisticated apparatus for producing gradients is adequate for separating compounds with large polarity differences. Earlier studies in our laboratory showed that adrenocortical steroids had definite elution per cents that could be used in design of programmed separations (8) when using water-impregnated silicic acid and a PE-DCM gradient. Subsequent analysis of complex mixtures, following incubation of adrenal tissue with radioactive steroid precursors, presented a variety of radioactive products more complex than the standard mixture separated with the earlier equipment. The gradient system described here, with its capability of precisely controlling elution per cents t o i 0 . 2 facilitates separations not attainable previously.
z,
Figure 5. Programmed separation of adrenocortical and ketosteroids See Reagents for nomenclature of standard compounds
Figure 5 shows the linear gradient used t o separate adrenocortical and ketosteroids with this gradient system (7). Although each compound is not completely separated, the use of the linear gradient shown allows for the isolation of zones containing only a few compounds. These compounds can then be easily resolved and identified by thin-layer techniques. We previously reported on the use of different gradient programming and isolation of individual regions by using polarity reversal ( I , 8). The gradient elution system described has been used in our laboratory for separations of steroids, but it can easily be adapted to the analysis of other compound mixtures by proper choice of column support and solvent pairs. ACKNOWLEDGMENT
The authors acknowledge the assistance of H. E. Cascio and H. W. Tipton for part ofthe technical development. (8) D. Francois, D. F. Johnson, and E. Heftmann, ANAL.CHEM., 35, 2019 (1963).
RECEIVED for review April 26,1971. Accepted June 18, 1971.
CORRESPONDENCE Some Considerations on Stepwise Electroreduction in Volta m metry and Chronopotentiometry SIR: Quite often the compounds of elements characterized by several oxidation states yield voltammetric curves exhibiting several steps, due t o the gradual electroreduction (or electrooxidation) of the element in question. Furthermore the case is frequently encountered in which during electrolysis some disproportionation equilibria are established near the electrode among the starting substance and the products of its electroreduction (or electrooxidation). In this connection, let us consider the general case of a sub-
stance A1 which is electroreduced at the electrode surface in two successive steps, by the following overall electrode processes: nlAl
n2A2
+ nl’e + nz’e
-+
nl“A2
(la)
-+
n2”Aa
(1 b)
In Equations l a and l b A Zis an intermediate product and A3 the final product; nl, nl’, n l ” , nz, n2‘, and n2“ are stoichio-
ANALYTICAL CHEMISTRY, VOL. 43, NO. 12, OCTOBER 1971
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