V O L U M E 23, NO. 8, A U G U S T 1 9 5 1 filled cable is manufactured, the dryness of the nitrogen is monitored by a de\y point reccrder. Other uses for the equipment have been found in manufacturing operations. ACKNOWLEDGMENT
Included in the group active in the development and design of the automatic dew point equipment are Herbert Robinson, R. C. Leever, and S. S. Stack of the General Engineering Laboratory, General Electric Co. The suggestions of E. R. TT7eaver of the National Bureau of Standards have been helpful in preparing the h a 1 manuscript of this paper.
1089 BIBLIOGRAPHY
(1) Awberry and Griffiths, Proc. Phys. SOC.London, 47, 684 (1935). (2) Ewell, A. D., Refinery Eng., 27, 131 (1934). (3) International Critical Tables, Vol. 111, p. 211, Xew York, 11rGraw-Hill Book Co. (vapor pressures over water at 1 atmos-
phere).
(4) Thornthwaite and Owen, Mo. Weather Rev.,68, 315-19 (1940); Z;.S.Patents2,240,082 (April 29, 1941),2,268,785(Jan.6, 1942).
( 5 ) Zimmerman, 0. T., and Lavine, Irvin, “Psychrometric Tables
and Charts,” Dover, K. H., Industrial Research Service, 1945. RECEIVED September 5 , 1950. Presented before the Division of Analytical Chemistry a t the 118th Meeting of the AMERICANCHEMICALS O C I E T Y . Chicago, Ill.
Automatic Measurement of Optical Rotation GABOR B. LEVY Schenley Laboratories, Inc., Lawrenceburg, Znd. A technique involving an automatic recording polarimeter has been established for studying the kinetics of reactions in whichachangeinoptical activity occurs. It lends itself to various analytical chemical applications. In some instances the presence of a catalyst or enzyme can be detected by the reaction
T
HE measurement of optical rotation affords a valuable analytical method because it is nondestructive. It is of particular potential advantage in kinetic studies, in which analyses should be performed without interference with the reaction under investigation. Although these advantages have been generally recognized, the technique is not employed frequently in research work, because of the tedium of reading values of optical rotation and time simultaneously. Furthermore, only isolated data are obtained and the precision of the readings of continually changing rotation falls significantly short of optimum. To eliminate these objections a continuous recording polarimeter was constructed ( 6 ) , which registers optical rotation within a range of 5’ mith a precision of about f0.005 O. The advantage of automatic operation with respect to precision and economy is apparent. In additioii, it permits quantitative evaluation of reaction rates even when they change n the course of the reaction, and determination of the exact location of these changes. In nianual operation this would be possible only if an infinite number of points were determined. Therefore this automatic operation, for which the author suggests the name ‘Lrotography,J’permits new applications to analytical problems. Some of these applications are discussed below, When the course of a reaction is follow~edby a recording polarimeter, a permanent record, “a rotogram,” is obtained, which shows optical rotation on the abscissa and time on the ordinate (Figure I). (The arrangement of scale expansion causes a return to zero after a travel of every 0 . 5 O . Figure I represents the inactivation of a solution pf sodium benzylpenicillin in 0.2 JI phosphate buffer of p H 7.0. Concentration is 3.6 mg. per nil.; 15,000 units of penicillinase were added.) The zero or starting point on the ordinate is conveniently made by starting the recorder when the reactants are mixed, but the first pertinent values recorded on the abscissa are obtained only after the mixture has been placed in the instrument and ha1nnc.e is reached. By conducting the reaction over a long period of t h e , this initial region of uncertain values of rotation may be reduvd to an insignificant portion, Thus, essentially the entire course of the reaction is mapped. The evaluation of the reaction rate from the curvature of the
order. The activity or concentration of this agent can be determined by the slope of a ‘(rotogram.” The concentration of a reactant or reaction product may be determined simultaneously. De terminations show a high degree of accuracy and precision and surpass many analytical methods in simplicity.
rotogram is an easy task and changes in rate can often be detected by simple inspection. Of particular interest to analytical applications are the zero-order reactions which appear as a straight line on the rotogram. Deviation from the straight lint. can easily be detected by inspection. Because catalytic or enzymatic reactions frequently exhibit zero-order reaction rates in some ranges of concentration, the presence of such catalysts can be detected. Furthermore, in these ranges, the reaction rate, and consequently the slope of thr rotogram, are proportional to the concentration of the catalysts or enzymes. This offers a convenient method for their quantitative determination. An abrupt change in reaction order indicates a change in the reaction mechanism. In a closed system, this is usually caused by the disappearance of a reactant. The location of such a point can be determined with great precision by rotography. If the characteristics of the reaction are sufficiently known and if the reaction is specific to a reactant, the concentration of this component can be determined accurately by the change in rotation between the initial value, corresponding to the control, and the value a t the “break” in the rotogram. Optically active impurities, inhibitors, subsequent rearrangements, etc., do not interfere, as this second type of rotographic analysis is based on the determination of absolute differences in optical rotation within a certain phase of a complex reaction. To illustrate the various rotographic techniques, results obtained with the penicillin-penicillinase system are presented. INACTIVATION OF PENICILLIN
Abraham and Chain have found (1) that penicillin is inactivated rapidly a t room temperature by the action of penicillinase. The enzymatic degradation is assumed to be due to the hydrolysis of penicillin to penicilloic acid ( 2 ) . A typical curve representing the reaction is shown in Figure 1. I t is immediately apparent that the reaction is of zero order, essentially, throughout its entire course-Le., the reaction rate is independent of the penicillin concentration. According to the theory of Nichaelis and Menten ( 7 , 1 1 ) , thiP is due to the fact that thc rate-determining process is thr hydrolysis proper rather than the formation of the enzyme-
1090
ANALYTICAL CHEMISTRY
substrate complex. The existence of zero order rates for this Table I. Dependence of Reaction Rate on Penicillin reaction has been reported ( S ) , but by rotography it was possible Concentration t o ascertain this condition over a lvider range of penicillin conSlope, centration (up to 40 mg. per ml.). Relative Enzyme Reaction R a t e , Degrees /Min./ Concentration Degrees ”in. Relative Concentration It is generally accepted that the alkaline hydrolysis of peni0,033 0.132 0.25 ’ cillin and the enzymatic inactivation are identical reactions, in 0.132 0,066 0.5 that they yield penicilloic acid or its salts. However, it was 0.126 0,095 0.75 0 127 0 127 1 0 found (Figure 2, representing the inactivation of a sodium benzyl1 5 0 204 0 176 0 143 2.0 0 268 penicillin solution of 1.8 mg. per ml. a t pH 11.0) that the alkaline inactivation is a first-order reaction, 1%hile the enzymatic hyTable 11. Dependence of Change in Optical Rotation on drolysis is essentially a zero-order reaction. This illustrates the Penicillin Concentration possibility of detecting the presence of an enzyme as compared Difference in Rotation to a “chemical” agent, both destructive to penicillin. sodium Per sample. Corrected A peculiarity of the enzymatic inactivation of penicillin is that for enzyme Slope, scale -,/inI. Xg./ml. divisions 17 divisions) o/mg./nil. initially the optical rotation drops rapidly a t a steady rate, then 0 786 0 96 171 154 1603 there is an abrupt change, and the rotation continues to drop a t 0 763 1 92 316 231 3206 0 767 468 451 2 88 4809 a much reduced and diminishing rate (Figure 1). The former is 3 85 0 769 62 1 624 6412 0 i67 assumed to be due to the destruction of penicillin and formation 4 81 770 8315 T33 of penicilloic acid, n hile the latter is considered to be due to the s e c o n d a r y reactions. Three independent facts support this assumption. F i r s t , t h e v a l u e s of optical rotation indicate that penicilloic acid is the intermediate reaction product (8, I O ) . Second, there is no residual penicillin found a t the end of the first phase of the reaction. [This n-as proved by separating the enzyme and p e n i c i l l i n immediately after the break in the r o t o gr a m o c c u r r e d . This n a s done by ext r a c t i o n , substantially as described ( 5 ) . The final aqueous e x t r a c t consistently showed no microbiological activity. I Third, reactions characteristic of penicilloic acid (8, I O ) , were obtained. (This was done by adding an equivalent a m o u n t of m e r c u r i c chloride to the mixture after completion of the i n i t i a l reaction. This caufed a suhstantial acceleration of the second phase of the reaction, with the optical rotstion approaching z e r o v a l u e . - 1 d d i t i o n of aqueous iodine solution was found t o have the same effect.) T h e s e findings indicate that 1.1 1 .o the “initial” zero-order 0.6 0.5 0.9 0.8 0.1 1 .o reaction represents the 0.1 0.4 0.3 0.2 0.5 hydrolysis of penicillin OPTICAL ROTATION, DEGREES t o penicilloic acid or Figure 1. Inactivation of Penicillin its salt and that the A . Starting point B . Balance reached change in optical acB-C. Main reaction C. Inflection point tivity is proportional to
V O L U M E 23, NO. 8, A U G U S T 1 9 5 1
1091 0.0000089 per minute corresponds to one unit of penicillinase (taking into account the dilution of 50 ml. 1 ml. and 40-em. cell). Thus, the values obtained can be incorporated in the calibration curve shown in Figure 4. A penicillinase preparation is analyzed as follows:
+
A stock solution of a pure alkali salt of benzylpenicillin is prepared in aqueous 0.2 JI phosphate buffer of pH 7.0. The range of concentration of this stock solution is preferably between l and 5 mg. per nil., but its strength need not be known. To 50 nil. of this solution in a beaker, 1 ml. of the unknosn penicillinase solution is added. The solutions are thoroughly mixed, arid a 40-cm. polarimeter tube is filled mith the mixture. -4 rotogrnm is prepared at 25' C. and its slope is measured. For this purpose it suffices to run the rotograph for a few minutes and count the 0.5 0.9 0.8 0.7 0.6 number of divisions for 0.5 0.4 0.3 0.P 0.1 s e v e r a l corresponding OPTICAL ROTATION, DEGREES values along the abscissa a n d o r d i n a t e . Figure 2. Alkaline Inactivation of Penicillin The only p r e c a u t i o n Partial rotogram necessary is to t a k e the readings of the slope tlie penicillin concentration. c o n s e q u c n t ] v , only this on a straight-line (zero reaction order) section of the rotogram. The penicillinase activity, corresponding t o the slope, is read off vhase of the reaction x-as investigated in detail. the calibration curve. The effect of pH, temperature, and the riatureof buffer \vas studied. I t was found that, the effect of buffer concentration on the reaction rate is small-viz., about 1% deviation between 0.1 and 47, phosphate 45 concentration-and that two lots of mixed penicillins (G Z570, K 35%, F 40yc) n-ere inactivated at a rate significantly lower than pure benzj-lpenicillin. Details of these studies are omittrd; however, they 5P 30 were essential in establishing the analytical methods described below and they form the basis for the nen- rational unit of W ' I ' penicillinase activity (4). F
\
I
ANALYTICAL APPLICATIOKS
Determination of Penicillinase. I n the zero-order reaction range, the rate of hydrolysis (the slope of the rotogram) is expected to be directly proportional t o the penicillinase concentration. Experimental proof of this is shown in tracings of a set of rotograms (Figure 3). The corresponding values are shown in Table I. I n accordance with the neiv definition of the penicillinase unit (4),a slope of
15
3.0
Figure 3.
2.5 2.0 OPTICAL ROTATION, DEGREES
1.5
1.o
Unfolded Tracings for Enzymatic Inactivation of Penicillin
Vertical lines indicate plaees where chart sections were joined. Relative enzyme concentration from top curve to bottom curve: 1, 1.5,2, 3, and 4. Penicillin concentration 4 mg. per ml. i n pH 7.0 phosphate buffer
1092
ANALYTICAL CHEMISTRY
Determination of Penicillin. The “initial” reaction represents the hydrolysis proper of penicillin. At the break or inflection point some penicillin is still present, but this quantity is less than 2 units per ml., as found by assay. It is assumed that the values of rotation at the point of “inflection” do not correspond to the equivalent amount of penicilloates but are somewhat reduced by further degradation. However, the intersection of the two straight lines (corresponding reaction rates) represents a geometrical “end point.” The difference in values of rotation between the control-i.e., penicillin solution prior to inactivation-and this end point is a measurable, and as found, a reprodurible quantity. The only additional datum necessary to carry out the analysis is the rotation of the penicillinase solution. This cannot be incorporated into the control and therefore its rotation is determined separately.
trial importance. Thus, with volume as the variable, it should be possible to record the quantity of an optically active compound in a varying product stream. When a varying mixture of optically active compounds is involved, the concentration of one could be determined by the use of two instruments linked iii such a fashion that the difference of optical rotation is recorded before and after completion of a reaction that is specific to only one component.
700
600 500
0.3 c Y 3
I
400
f5
300
9.00
L w P v)
4 9
0.2
w
100
P w
; 5 w‘
1
0.1
Figure 5.
6F U
P 3 4 5 SODIUM BENZYLPENICILLIN, M G . / M L .
Calibration Curve for Determination of Pctiicillin
4
z
10,000 P0,OOO 30,000 UNITS OF PENICILLINASE PER S A M P L E
Figure 4. Calibration Curve for Determination of Penicillinase
Rotography is dehnc d to include dl applications based on the continuous recording of optical rotation with relation to a variable-usually elapsed time. Rotographic analysis is defined a~ a special case, in Lvhich the record or rotogram is used to determine the concentration of a catalyst or enzyme (slope) or concentration of a reactant or rcwtion product (width).
A penicillin sample is analyzed as follows:
h solution containing the penicillin sample, 0.5 to 5 mg. per ml. with 0.2 M phosphate buffer a t pH 7.0, is prepared. A
portion of this solution is diluted with buffer and is used to balance the instrument. Another portion is diluted in the same pro rtion by a penicillinase solution (whose strength need not be E o w n ) and a complete rotogram of the ensuing reaction is prepared as described above. The rotation of the aliquot of penicillinase solution used in the analysis is determined separately. The change of rotation between the initial value and the value a t inflection is determined by counting the divisions along the abscissa in the rotogram. The value found for the penicillinase solution is subtracted. The penicillin concentration corresponding to this value is read off the calibration curve shown in Figure 5 (based on the experimental points shown in Table 11). DISCUSSION
The proposed method for the determination of penicillinase is romparable in precision to other available methods ( 4 ) , and surpasses them in simplicity and ease of operation. The method for the determination of penicillin shows a precision of the order of 1% and is comparable in specificity (accuracy) and precision to the earlier chemical method (9) and is complementary to it, in that it permits the analysis of buffered solutions. These analytical methods point to general applicability whenever optically active compounds are involved. Polarimetry and rotography are distinct methods and they are related, to use an analogy, as p H measurement is to potentiometric titration. By modifying the apparatus and technique, it should be possible to record optical rotation with relation to variables other than time. Some of these modifications are of potential indus-
ACKYOW LEDGMEIVT
Suggestions of several members of these laboratories are gratefully acknowledged, particularly those of Philip Srhwed and David Fergus, as well as the latter’s valuable assistance in the experimental work. The author also wishes to thank Kurt Ladenburg and Bruno Puetzer for their interest and encouragement and Schenley Laboratorie?, ILK, for permission to publish this paper. LITEKA’I‘URE CITED
( 1 ) Abraham, E. P., and Chain, E., Brit. J . Exptl. Path., 23, 103 f 1942). --,\ -
(2) Abraham, E. P., Chain, E., et al., Medical Research Counci2 Rept. 21 (1944). (3) Henry, R. J., and Housewright, R. D., J. Biol. Chem., 166, 465
(1946). (4) Levy, G. B., Nature, 166, 740 ’(1950). (5) Levy, G. B., Fergus, D., and Caldas, J. M., ANAL.CHEM.,21,
664 (1949). (6) Levy, G. B., Schwed, P., and Fergus, D., Rev. Sci. Instruments, 21, 693 (1950). (7) Michaelis, L., and Menten, M. L., Biochem. Z., 19, 333 (1913).
(8) hloaingo, R., and Folkers, K., “Chemistry of Penicillin,” p. 537, Princeton, N. J., Princeton University Press, 1949. (9) blurtaugh, J. J., and Levy, G. B., J. Am. Chem. Soc., 67, 1042
(1945). (10) Peck, R. L., private communication.
(11) Van Slyke, D. D., Advances in Enzymol., 2, 33 (1942). RECEIVED September 28, 1950. Presented before the Division of Biological Chemistry a t the 118th hfeeting of the AJIERICAN CHEMICAL SOCIETY, Cliicago, Ill. Submitted t o the Institutum Divi Thomae in partial fulfil1,uant of the requirements for the degree of doctor of philosophy.
