Steroid Analyzer FRANK 0. ANDERSON,’ LAURENCE R. CRISP,’ GRANT C. RIGGLE,’ GERALD G. VUREK,’ ERICH HEFTMANNI2 DAVID F. JOHNSON,2 DANIEL FRANCOIS,2 and THEODORE D. PERRlNE* National /nsfifufes o f Health Public Health Service, U. S. Department of Health, Education, and Welfare, Bethesda, Md.
b Apparatus for the automatic quantitative determination of individual adrenocortical hormones in mixtures i s described. It is based on the separation of the mixture on a partition column b y gradient elution. Fractions are collected and divided in two equal aliquots. One i s analyzed b y ultraviolet absorption, the other b y the reduction of blue tetrazolium. As many as 300 analyzed samples may be stored and the results recorded on a strip chart. The design of the apparatus emphasizes flexibility and safety of operation.
silicic acid column. Gradient elution with petroleum ether, containing increasing amounts of dichloromethane, separates the steroids. The eluate fractions are analyzed by both ultraviolet spectrometry and a colorimetric procedure, based on the reduction of blue tetrazolium. The two methods of analysis are referred to in the text as the “CV” and the “BT” or “color” method. The samples analyzed by UV can be further used for the sulfuric acid test (4),paper chromatography (6),or radiochemical assay ( 7 ) . PRINCIPLE
W
one compares the relative merits of the two main forms of liquid-liquid partition chromatography as applied to corticosteroids, i t is difficult t o avoid the conclusion that columns are more suitable for quantitative and preparative work than filter paper (3, 8 ) . If column chromatography has not enjoyed the same popularity as paper chromatography, it is largely because of the labor involved in analyzing individual fractions. Severtheless, experience in the amino acid field has shown that i t is easier to automate column chromatography than paper chromatography (12). Over the last three years we have, therefore, concentrated our efforts on developing a n automatic steroid analyzer, based on column partition ( I O ) . An automatic device for analyzing adrenocortical hormones has been described earlier by Haines ( 2 ) . His apparatus continuously monitors the ultraviolet absorption of the effluent from a partition column. The signal from the recorder activates a valve system, permitting a series of eluent mixtures to pass through the column. I n 1954, vie described a method for the quantitative determination of individual corticosteroids (4),which is applicable to the analysis of urine (6) and tissue (7’) extracts, and, with minor modifications, to 17-ketosteroids as well ( 5 ) . The stationary phase in our partition system is water, supported by a HEN
* Instrument Engineering and Develop ment Branch, Division of Research Services. 2 Steroid Section, ,Laboratory of Chemistry, National Institute of Arthritis and Metabolic Diseases. 1606
ANALYTICAL CHEMISTRY
The steroid analyzer, (Figure l), consists of the apparatus proper (A) occupying a floor space of 5 x 3’/2 feet and two associated control relay racks (B and C). The solvent and reagent containers are filled and the test tubes placed in containers. After the packed column is placed in position, the master programmer is energized and cycling begins. Petroleum ether, 1, and dichloromethane, 2, are pumped into a mixing chamber, 5 , according to any desired gradient elution program. A linear change of dichloroniethane concentration in petroleum ether is used in the illustration, but convex, concave, or sigmoid gradients ran be produced by substituting the correaponding gradient cam for the linear cam, 19, shonn. The mixture of steroids is separated on the column, 6, and the effluent is collected in test tubes. Each effluent fraction is divided into t n o equal portions-one for UV and one for BT. The solvents are evaporated, and the residue is taken up in ethyl alcohol. h blank tube, containing the same amount of ethyl alcohol, is prepared for comparison with the BT color. -4fter suitable agitation, the color reagents, blue tetrazolium and tetramethylammonium hydroxide, are added to the color and blank tubes, and their contents are again mixed. Twenty minutes later, the absorbances of the two tubes are compared a t 520 mk, and the ultraviolet absorption a t 240 mp in the untreated aliquot is determined. The contents of t h e L V and B T tubes are stored, while the blank is discarded. The signals for the two spectrometric determinations are recorded on a strip chart.
Figure 2 shoiis a portion of the iecording. Each steroid produces a peak a t a gi\en fraction number. The total amount of each is determined by adding the amounts in all tubes belonging to a single band from either the UV or B T plot. The adrenocortical hormones, containing both an a$-unsaturated 3-keto group and an a-keto1 side chain, respond to both tests, while some of their metabolites show no ultraviolet absorption. Other steroid hormones, e.g., progpsterone, do not reduce the blue tetrazolium reagent. The presence of both responses confirms the identity of the adrenocortical hormones and illustrates the advantage of separation by gradient elution. Details on the sensitivity and reproducibility of the method and other performancc data will be published separately. APPARATUS
Gradient Elution System. The gradient elution system: n-hicli is essentially the same as previously described (9), operates independently of the cyclic operation of the remainder of the apparatus. Figure 3 diagrammatically shows the method of translating a change in gradient cam position into an electrical signal, n-hich is further converted to control pumping speeds. The gradient cam is a metal replica of a plot of the solvent ratio as the ordinate i:s. time as the abscissa. It m o w s laterallj. a prescribed distanve a t t,he bcginriing of each cycle. The a,rm of tlie rectilinear potentiometer tlic vhmgc iii ordinate position of t,he CIII’T~C and converts this change into an electrical voltage dircctly proportional to the position of the arm. The signal is fed into a servoamplifier whose motor is coupled to a dual variable autotransformer. The alternating current voltage outputs of the transformers are oppositely connected, i.e., onc is a niaximum when the other is a minimum. The resultant alternating current voltages are rectified and applied to direct current shunt motors, driving hypodermic syringes, n-hich act as pumps for the two eluent’s. The pump delivery rates are in direct proportion to the direct current potential applied to the motor armatures. The servoloop is balanced by feeding a portion of the derived direct current voltage to the amplifier input through a
Figure 1. A.
Apparatus
1, 2. 3, 4. 5. 1.
12. 13. 14. 15. 16. 17. 18.
Solvent storage bottler Solvent pumps Mixing chamber ,-^1.."."
8.
I .
21. 22.
Master c~ntrol Master timer 23. Droplet counter opd fraction divider controlr 24. Column cut-off d y e control 25. Vibration control C. Relay rock 26. Strip chart recorder 77 I I V nnci rnlnr omplifler balancing controls and power rupply power wpply
Ethyl alcohol pump Reagent slotion Reagent pumps Readout and tronrfer rtotion Spectrophotometers Test tube removal station Choin drive
Relay rock 19. 20.
potentiometer, mecnanicany connectea t o the servomotor. To prevent formation of vapor locks due to pressure changes, it was found preferable to displace the solvents in the storage bottles with water instead of pumping them directly. The solvents are conireyed to the mixing chamber through Kel-F and glass tubing. The delivery rate of solvents to the mixing chamber is designed to equal twice the outflow to provide a smooth gradient transition and a 50% duty cycle on the pumps. Pump syringe sizes and stroke lengths can be varied to meet this requirement. The pumps are activated by a photoelectric detector when the meniscus in the side arm of the mixing chamber reaches the lower level position (Figure 3). They ston when the unner level nhotoelectric system senses the meniscus. An-
Side view of steroid analyzer
Gradient cam Gradient cam follower
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. . . .
,
- 1 -
. . . . .
. . . -.
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.
Figure 2.
Portion of strip chart recording
Ordinate. absorbance Abscissa, fraction number Seporotion of two-positionisomers, 4-pregnene-17or.21 -diol-3,20-dionelleft peak) from 4-pregnene-1 10, 21-diol-3.20-dione (right peokl.
Each frac-
tion give, a pair of recordings, UV (left1 and BT (right1 VOL. 33,
NO.
11, OCTOBER 1961
* 1607
,I
I .- I
5 cc 3 cc
METERING PUMP MOTOR
'7
PUMP MOTOR
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SERVO
AMPLIFIER
L -
Figure 3.
Gradient elution system
CV TRANS 15/6 V
Figure 4.
C.3LLMh
LOW
HIGH
CLTOFG
LEVEL
LEVEL
Wiring diagram of meniscus detectors
A, B, C, leads to time delay relays which control the gradient pump operation
,1 /---
I
*I
REP.GENi INTRODUCTION ISTliiOU
Figure 5.
1608
Collection and preparation of fractions
ANALYTICAL CHEMISTRY
$1
other detector maintains the liquid level on the chromatographic column by energizing the column cut-off solenoid in the event the meniscus falls below the detector position. The schematic wiring diagram of the system is shown in Figure 4. Fraction Collector. T h e fraction collector (Figure 5) consists of three parallel rows of 18 X 125 mni. test tubes, moved a t 10-minute intervals through 12 stations by a n intermittent motion chain drive system. The test tubes are supplied t o t h e conveyor system from three reservoir hoppers. T h e effluent from t h e column passes through a droplet counter ( I I ) , which also provides a pulse to close the column cut-off valve when the preset number of drops has been collected. The function of the fraction divider is to produce two equal aliquots of each fraction. It consists of two funnels, which alternately deflect drops into the UV and color tubes at Station 1. The divider is actuated by solenoids, energized alternately from the droplet counter. The number of drops delivered to each test tube between divider movements can be varied from 1 to 10 drops. Master Timer. The apparatus operates on a 10-minute cycle. This time interval F a s selected because it permits optimum color development of the B T reaction (2 x 10 minutes) and collection of u p to 400 drops per fraction. Three hundred fractions are collected and analyzed in a 50-hour period. Figure 6 shows the timed sequence of operations performed by the apparatus. The master timer controls several auxiliary timers Pr-hich perform a variety of functions in the preparation and analysis of the fractions. Preparation of Samples. Figure 5 is a schematic representation of all steps performed in preparing the samples for analysis. T o conserve space in t h e figure, not all stations are s h o m . A t Stations 3 and 4, the solvents are evaporated b y passing heated nitrogen into the test tubes. At Station 6, 5-ml. portions of 95% ethyl alcohol are introduced sequentially into the UV, color, and blank tubes (cf. Figure 6). The delivery tubes for nitrogen and ethyl alcohol are lowered together into the test tubes before their valves open. 'To dissolve the residue, the tubes pass over a vibrating table between Stations 4 and 10. Blue tetrazolium and tetramethylammonium hydroxide reagents, in 0.5ml. portions, are introduced into both color and blank tubes a t Station 9, and their contents are mixed by rapid rotation. A special device for the automatic operation of glass Teflon valves was developed for this application ( 1 ) .
3 OR
REAGENTS INTRODUCED
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BLANI
I 1
Analyzing System. .\tltlitional mixing o(ac*urs a t 3tatioii 10 (Figurc' 5 ) whrw nitrogen is l)\ibl)l(,(l through t h e solutions prior to roudout a t Station 11. The fluid t,ransfer system, which is structurally coupled with the nitrogen mixer, lowers the dip tubes into the three test tubes and withdraws their contents into the cuvettes. The meclianism for creating tlie suct,ion and transfer motion is n i o u u t d hencatli the table top. The B'T deterrniIixtioii is niadc in a simple comparative d u r i m r t e r using interferrnce filters (Fishw Scientific Co., S o . 220 or equivalent) to obtain a responstx a t 520 m p , Tlie difference output of the inatchetl ihotovoltaic cells (General Electric Co., Type 81'T'10FAA4, mat'ched or ecjuildent) is fed into a direct current amplifier (Houston Instrument' Corp.. Model M10-:1 or equivalent) (Figure 8). The nnipiifitsr out,putsignal is a,ttennated to mxtch the desired sensitivity range. The absorbancae of the (T' sample is read in a quartz cuvrtte. The ultraviolet light sourw is a n ozone lamp (Kwtinghouse Type 794-1-1 or eyuivaltrit) n-ith a 240-111p filter (IhirdAtomic,, iiiterfererwe filter, 230 mp, band width 10 ~ i i p . or equivalent). The :Ittenuated output of t'he 935 phototube is coiiipared with a merrury bnttcry source Icference ~-olt:tge and the difference is amplifid and atteimtted as in tlie colorimeter. To obtain the sequential plotting of the UT nnd BT signals ou a single channel recorder, the amplifier outputs are alternately connected to t'he recorder through a timer-prograninied relay system (Figure 8 ) . .1 simple method
of obtaining absorbance without the use of integrating amplifiers will be described elsewhere. The recorder span has been adjusted to read directly in units of absorbance. The amplifier signals may be attenuated to produce as much as 4 X amplification. After spectrometric readings are made, the UV and the B T samples are transferred laterally to a storage rack, shown in Figure 7. Three hundred samples (600 tubes) may be stored The dip tubes are then moved to a washdry station where the cuvettes and dip tubes are rinsed with ethyl alcohol and then dried by nitrogen. This
4
,
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operation is not required in the present application and is therefore not shown. After drying, the dip tubes are returned to their initial position. At Station 12, the three empty tubes drop into water-filled polyethylene containers beneath the table. Calibration of Analyzing and Recording System. T h e UV amplifier i n p u t signal from the 935 phototube is balanced with a constant voltage reference source when a blank of 9517' ethyl alcohol is in t h e cuvette. After balancing with a second blank, a sample of known concentration is introduced, and the amplifier output signal is attenuated to produce a graphic record equal to the absorbance of the solution. A second standard sample of different concentration provides a reference check. B T amplifier calibration follows the same procedure, except that all blanks and standard samples are recorded 20 minutes after the introduction of the reagents. Safety Features. T h e apparatus has been designed t o operate completely unattended after initial setup. I n the event of malfunction of a n y of the rritical operations, the apparatus \rill automatically stop and display warning lights. Fail-safe circuitry is included in those portions of the apparatus which may be damaged by malfunction. The chromatographic column is protected by the cut-off valve, and the gradient pump system stops in the event that any of the following fail: armature or field on the pump motors, servoamplifier power, pump mot'or time limits, or main h e power. The evaporator and the fluid transfer tubes are protected from damage by a circuit which senses that test tubes
-INCANDESCENT
1
LAMP
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I
1
' I
I CLUTCHES
-+-
RE4DOUT MOTOR
Figure 7.
Analyzing system VOL. 33, NO. 11, OCTOBER 1961
1609
are in proper position a t Station 1, evaporator tubes clear the test tubes, and that fluid transfer tubes and syringes are in correct position. If any of these conditions are not met, the fraction collector will not advance and successive operations will stop. DISCUSSION
The apparatus is a prototype, designed for maximum flexibility and is adaptable to a wide variety of applications. Thus, the present gradient system is designed to control the mixing ratios of t\vo solvents, but modification of the present system will permit the programming of additional solvents. Only 8 of the 12 stations are used in the present application, and only Stations I, 11, and 12 are fixed. This arrangement permits changes in the time allowed for each operation and also the addition of new operations. Additional determinations (e.g., of radioactivity) can be accommodated by the present system with slight modifications. If desired, a complete spectral scan of each sample can be accomplished with conimercially available attachments. It is planned to make multiple sensitivity plots of each sample so that recordings in the optimum range will be obtained regardless of concentration. The area under the peaks will be integrated and displayed digitally on a printed tape. ACKNOWLEDGMENT
The authors thank George L. Laurence, Vincent T. Almasy, John K.
Figure 8.
Wiring diagram for UV and BT amplifiers
A, Chart drive motor B, Recorder timer motor M, 100-0-100 pa. d. c. microammeter
Cullen, Jr., and David A. Rector for fabrication suggestions. LITERATURE CITED
( 1 ) Anderson, F. O., Crisp, L. R., Chemisl Analyst 50, 22 (1961).
12) Haines, W. J., Recent Progr. in Hormone Research 7, 225 (1952). ( 3 ) Heftmann. E.. Chem. Reus. 55. 679
(1955)
f l ) Heftmann. E.. Johnson.' D. F.. ' CHEM.2 6 , gl9 (1954).
ANAL. ~
(5) Johnson, D. F., Heftmann, E., Francois, D., J . Chromatog. 4, 446 (1960).
(6) Johnson, D. F., Heftmann, E., Hayden, A . L., Acta Endocrinol. 23, 341 (1956) ; Johnson D. F., Francois, D.,
Heftmann, E., Itnd., 32, 8 (1959). ( 7 ) Johnson, D. F., Snell, K. C., Francois, D., Hpftmann, E., Zbid., 37, 329 (1961). (8) Neher, R., J . Chromatog. 5 , 184 (1961). 19) Riaale. G. C.. Winter Genl. Meet. A.I.EyE.', Feb. 7, 1058, Paper 58-396, (10) Riggle, G. C., Anderson, F. O., Crisp, L. R., Heftmann, E., Johnson, D. F., 13th Annual Conf. on Electr. Techniques in Med. & Biol., IRE, A.I.E.E., ISA, Oct. 31, 1060, Waqhington, D. C., p. 88. (11) Riggle, G. C., Crisp, L. R., ANAL. CHEM.28, 1799 (1956). (12) Spackman, D. H., Stein, K. €I., Moore, S., Ibid., 30, 1190 (1958). RECEIVEDfor review June 16, 1961. Accepted July 11, 1061.
Identification of Aldohexuronic Acids, Free and in Polymers J. R. HELBERT Veterans Administration Hospital, Downey, Ill.
K.
D. BROWN
Veterans Administration Hospital, Downey, Ill., and Northwesfern University, Medical School, Chicago, 111.
b The reaction of aldohexuronic acids with anthrone is affected in a characteristic manner b y variations in temperature and heating time. Mixtures of aldohexuronic acids obey Beer's law and are additive in all proportions. Simple polymers of aldohexuronic acids and mixed polymers of aldohexuronic acid and hexosamine exhibit the same time-temperature pattern in the reaction as uronic acid alone. This report describes a chemi1610
ANALYTICAL CHEMISTRY
cal method for distinguishing one aldohexuronic acid from another, both free and in polymers without prior hydrolysis.
E
work with uronic acids and anthrone (9-4) suggested that each uronic acid tends to react in a characteristic manner to variations in experimental conditions. Because of the manifest analytical utility of such ARLIER
behavior, D-mannuronic, n-glucuroiiic, D-galacturonic, and L-iduronic acids have been examined systematically over a wide range of esperimental conditions. Since the results confirmed our initial inference, the investigation was extended to includc mistures and polyniers of t,hr above uronic acids. We know of no other chemical method for identifying aldohesuronic acids either frre or in polymers. The method described here permits such identi-
