1763
V O L U M E 24, N O . 1 1 , N O V E M B E R 1 9 5 2 (2s) JIcKee. R . H.. ‘,,, and enters automatically operated reducing valve E . The air emerging at a constant pressure of about 4 pounds per square inch passes through solenoid-operated stop valve F to a scrubbing train consisting of bottle G containing concentrated sulfuric acid, drying tube H containing Xscarite, bottle I containing concentrated sodium hydroxide solution, and drying tube J containing cotton. The purified air passes a eei,ies of mercury manostats, in each of which the head of liquid is adjusted to a particular level equivalent to a desired pressure. Eycess air passing through a manostat escapes to the atmosphere. The first manostat, K , is connected permanently t o the air main. The other manostats ( P , Q , R, S ) are connected t o the pressure system through solenoid-operated valves, thus permitting automatic electrical selection of any one of
ANALYTICAL CHEMISTRY
1764
the five preset air pressures. The solenoid-operztedvent valve, T, functions synchronously with stop valve P, thus relieving the pressure when F is closed. The adjusted air pressure is applied through a series of tubes to eight solvent reservoirs (only two shown in the figure) represented by U. Solenoid-operated values L, M , A', and 0 , respectively, are the same as SL9 to SL12 shown on Figure 11. The sequence of solvents, the total volume of solvent; and the pressure (rate of flow) of each solvent are predetermined by settines on the control panel (Figure 3) and me controlled auto-
and the cell assembly is inserted into the slot in the cell holder, E, which holds the cell in a fixed position by means of internal sorine strips. A neoprene gasket, F , is placed a t each end of the cei? holder and the assembled cell .is inserted into the cylindrical channel in the Dural cell block. G. A elitss tube. H . is a,liennrl ~~~-, ~~, with the opening in thelower&,gk& P , t o serve as an outlet tube. A cover plate, I , containing the waxed-in glass inlet tube, J , is D
~~
~
~~
The liquid flaws from the absbsorution cell to a measurine dumn ~
~~
~~
~ ~~
~~
~~
~
~~~~
~
~
connecting &lenoid-&er&ed valves, B~(sameas SL1 t o SL8 in Figure 11). All eight valves (only three shown) are connected
b, are operated hy a"sihngle
~~~~~
~
~~~. . ~
between the electrodes. The solenoid coil (& shown) is energized and an iron slug sealed in the glass valve stem is drawn
solenoid which simultaneouslG open; the cup from being wetted. 'The delivery bolumes are more reproducible from coated, than from uncoated, tubes.
tively, and aremaintained a t constanttempe;ature by the liguid which is pumped from bath J .
A spectrophotometer (Beckman Model D U R ) is indicated in the upper left-hand corner on Figure 8. The resistors of the output voltage divider are lettered to correspond to those in the Beckman diagram. Switch S8 is the "record-adjust" switch shown in the "record" position. The output of the photocell is amplified and applied to a Leeds and Northrup Speedomax reoorder which prints dots, correspondingt o the per cent transmittance of the sample stream, on a 10-inch tape at 20-second intervals. A four-point rotary switch is synchronized with the recorder mechanism to operate a t
1
chadged and the spectrophotometer stmdmdieed.
WL
Figure 1.
Simplified Outline Diagram of Apparatus
A monochromatic beam of light. K . emanating from the mouo-
CUD.
0. where {he volume'snd rate o f h u
df the licluid are meas:
sto&xk N t o the fraction collector, S, w t h h consists essentially of a. pivoted delivery tube shifted to different positions hy means of four solenoids T (only one shown) over funnels U 8 s desired. Each funnel leads t o a receiver. Ti. Dissolved air in the salvent'is removed before it reaches the chromatographic column by means of a deaerator (Figure 6).
the noazle, 6.into tKe separating chamber. H . fromnhich air h&~
~~
~
Figure 2.
Front View of Apparatus
The parts of the recorder are shown a t the bottom of the diagram. M1 is the chrt-drive motor and M2 is the slide-wire
~~~
ing jacket L 2 M ana esckping a t N. The cool~d,~&-freesolvent is discharged a t 0. The distilled water in J is replaced as needed
photometer. -Two stainless'steel, gold-gated spacers, 'A, are fitted and waxed between two plates of optically polished fused silica, B and C , to form a cell of 3-mm. light path and approximately 0.25-ml. volume. This cell is backed by a spacer slug, D,
the recorder vower switch and S2 is th;? chart-drive motor
nkw
contact every 20 seconds. A potential of 24 volts, direct current, is applied to one of the relays (RY3,RY4, RY5, and R Y 6 ) causing i t to apply the output voltage of the spectrophotometer amplifier across one of the potentiometerz ( P 3 , P4, P5,and P6).
V O L U M E 24, NO. 1 1 , N O V E M B E R 1 9 5 2
1765
By operation of switch S7 it is possible t o employ only one wave length. Under these conditions, slide-wire P6 is connected continuously and only dutch C4 is used during standardization. Power for the whole assembly is supplied by the control panel. The wave length of the spectrophotometer is changed every 20 seconds hy means of a servomechanism coupled t,o the wave-length control shaft of the monochromator and a set of four rheostats alternately connected into the circuit bv the rotarv sn-itchof the recorder, to g& e' a sequence of four desired wave~lengths(Figure 9). The spectrophotometer amulifier. recorder bridee. recorder ' amplifier, mot%; MI and M2, and relay RY2 are the same as shown in Figure 8. Switch 8 6 B fFieore 9) is the second section o r t h e motor-driven rotary switch and operates synchronously with the first section S6 A , shown in Fieure 8. Figure 3. Close-up Vicw of Control Panel The w a v h w t h changer iS designed t o &Perate f0; 5 Chromatographic column is shown at right with by.*& valve assembly at its base. The monochromator with mounted wave-length changer is shown at the bottom and the recorder at the left. seconds at the beginning of each 20-second period. Each time the recorder prints and switch S6 moves to a new Dosition, microswitch 820 is merated The four relavs. four uotentiometers. and the four rotam switch momentarily by the reeofd'der motor, initiating the $second Dositions cor&band tb four wave IeLaths. wave-length change period which is timed by the time delay relav RY9. Durine this 5-second neriod relav RY7 onerat,er. .~~~ ~-~ t,o ~sw&h the recorder amplifier input'from t h e spectrophotometer amplifier t o the wavelength changer bridge. Relay RYE also tacts of the recorder stkdardi&g relay, R Y I , to'the input of the recorder amplifier. This input voltage is bucked hy an equal
~.~~~~
~
recbrder slid&wlre, P2. The resultant of the recorder bridge output and the speetro-
A
the net input t o the amplifier is zero. The recorder print wheel, coupled to the slide-wire, indicates and reoords the speetrophatometer output voltage, which is proportional to the position of the slide-wire after it has been balanced. The recorder standardizes itself every 48 minutes by operating relay RY1, which applies standard cell E2 in place of the signal. At the mme time, mechanical connection is made t o slide-wire P1 and it is driven until the output of the dry cell is balanced. Standardization of the spectrophotometer is initiated and controlled hv relay RY15 which causes relay RY2 t o change the i n m t ~~~
~
motor M 3 . ?he operationbf &lay RY15 also causes the magnetic clutch coils (C1, C2, C3, and C4)to he operated in turn undercontrol of switch S6 A . During the 20 seconds each clutch is operated, a mechanical connection is made between its associated potentiometer and motor M3. In this mmner, motor M3 drives eaoh potentiometer in turn until it is adjusted t o an output of precisely 50 mv., corresponding t o 100% transmittance. Switches S9 to SI5 are also operated by motor M3, depending upon the position of potentiometers P3 t o P6. Switches S9 to S12 are arranged so that they close when potentiometers P3 to P6 me new the mid-points of their permissible travel. This causes pilot light P L to light and facilitates initial adjustment of the compensator without requiring visual inspection of the potentiometer. Switches SI3 to S16 operate when the potentiometers are near the end point of their permissible travel, giving warning by means of buzzer B that manual adjustment is required. The pilot light and buzzer are operated by bell transformer T2. The mechanical detail? of the standardization compensator are shown in Figure 10.
Figure 4
length control shaft 01 MIL. a p e c u r u p n u u u n ~ r ;I I~I U U U C I ~ I U E I ~ ~ W C . Relay RY8 is connected in such a manner that it disconnects wave-length change motor M4 a fraction of a second before the input is changed by relay RY7 thus protecting the wave-length setting during switching operahns. Switch 18 is the wave-length changer "on-off" switch. Switch
ANALYTICAL CHEMISTRY
1766 19, shown in the automatic position, holds the wave-length changer in operation while the settings are adjusted. The wavelength changer is operated by what is essentially a Wheatstone bridge powered by a %volt battery, E3, in series with 1000-ohm rheostat P18; 1000-ohm rheostats P16 and P17 constitute the normally fixed legs of the bridge. The 1000-ohm slide u ire, P i , is balanced by the 100-ohm rheostats, P8 to P11, each in turn as determined tiyswitch S6B. The 2-ohm vernier adjustment rheostats, P12 to Plj, are connected in series with each 100-ohm rheostat. The four desired wave lengths are set by means of rheostats P8 to Plj. P l y is a 300-ohm rheostat used to match the impedance of the bridge with that of the recorder amplifier.
sembly t o something under 360” of arc. Flexible leads (not shown) are brought out from the clutch coil and slide-wire. During standardization, the clutch is energized and drawn against the friction plate and the niotor drives the assembly until the slide-wire reaches a balance point. Three other friction plate clutch slide-wire assemblies (not shown) operated by (the same) shaft, D , effect standardization at the other three ~ a v e lengths. Leaf switch L indicates that thelimit of the compensator’s range is approaching by operating an alarm buzzer and leaf snitch M indicates the mid-point of the compensator’s range by operating a pilot light. During operation of the apparatus alternating current power IS supplied to the control panel (Figure 11), steam and tap water t o the deaerator, and compressed air to the main air valve. Power is applied to the spectrophotometer amplifier for several hours before use to ensure uniform response. A brief warm-up is sufficient for the recorder amplifier. h delay switch and pilot light
1
0
V
Figure 5.
Solvent Plumbing System
Standardization of the spectrophotometer is performed automatically a t 1-hour intervals except when the recorder is “on peak”-i.e , under 90% transmittance-during which standardization is performed a t 4-hour intervals. Standardization also occurs when the transmittance rises t o 90% and when the solvent is changed. The following operations are performed automatically during standardization. The by-pass valve ( F , Figure 5 ) is operated, permitting the eluting solvent ,to by-pass the column and run directly int o the absorption cell. When the sample has been flushed a servomechanism adjusts the output of the epectrophotometer t o a reference potential corresponding to 100% transmittance (Figure 10). This adjustment is performed a t each of the four wave lengths and the apparatus “memorizesJJthese values until the next standardization. These functions are initiated and controlled by settings on the control panel. Only essential parts of the etandardizationcoinpensatoraresho\vninFigure 10. The two-phase motor ‘4 (same as M3, Figure 8) with pinion B drives gear C, which is fixed t o shaft D. Clutch friction plate E ie pinned t o shaft D. -4magnetic coil is located in clutch housing F . A collar, G, connects the clutch housing to disk H , on the periphery of which is located slide-wire I. A contact arm, J , rides on the slide-nire. Also fastened to disk H is actuatorplate K which opera tes leaf switch L or Af when in the proper position. Rotating stop N and fixed stop 0 limit the rotation of the clutch slide-vrire as-
PEC
Figure 8.
Figure 6. Deaerator
Figure 7 . Spectrophotonieter Absorption Cell
BRIDGE
Spectrophotometer Amplifier, Standardization Compensator, and Recorder Circuits
V O L U M E 24, NO. 1 1 , N O V E M B E R 1 9 5 2
r-- -
I767
relay RY4 which operates for 2 seconds through the intermediacy of TR1, a normally closed, %second, time delay relay. During this 2-second period, the solenoids of the dump cup and the recorder marker pen are energized. The momentary impulse caused by filling the dump cup is transmitted t o relay I RY14, which in turn transmits a pulse to relays RY6 and RY7. The latter are arranged in a scale of two circuits in such a manner that only every second pulse is transmitted to the units counter, stepping relay SR1. Pilot light P21 flashes each time the cup is dumped and pilot light P22 flashes each second time the cup is dumped. Stepping relay SR1 advances one step for each second dumping, constituting one count. The number of counts from zero to nine is indicated by pilot lights P1 to P10. When ten counts have been made, relay SR1 resets and relay SR2 receives a pulse causing it to advance one step. In this manner, as many as 99 double dumpings of the cup may be counted. The volume of each of the eight solI ?--L ---J vents permitted to pass before changREG. BRIDGE ing to the next of the solvents is predetermined by means of 16 ten-posiFigure 9. Wave-Length Changer Circuit tion rotary switches. For example, the volume of the first solvent is defacilitate r$arm-up of the time delay relays in the control panel, termined by setting rotary switch RS1 for units and rotary switch RS9 for tens. As shown in the diagram, the arms of The Beckman equipment is operated normally except as follows. The four selected wave lengths are set by means of rheostats these rotary switches are connected by eight wire cables to the contact points of stepping relay SR3. Each time the pre(Figure 9) and the standardization compensator is set to a median position with the aid of a buzzer and pilot light (Figure 8 ) . The determined count is reached, this relay is advanced one step. solvent sequence is established as described, the sample is placed This relay has five arms which operate simultaneously on five banks of contacts. Level C is used to select the solvent solenoid on the column, and the run is started by pressing the reset valves and level D the air solenoid valves. Level E is used for rebutton (Figure 11). When the predetermined volume oi solvent has been used, the column is washed automatically with acid and setting water and is in readiness for the next run. After washing is completed, the air pressure is cut off from the system and the electric. power is shut off unless the “hold warm’’ switch is shut. In the latter case, the apparatus may be re-used immediately. As shown on the control panel wiring diagram (Figure l l ) , the grounded side of the alternating current line of the power circuit (lower right-hand corner) is connected through a neon pilot bulb, P24 (which indicates reversed polarity j , to the chassis, an external ground, and a floating ground. The power is controlled by a master switch, 83, and a &ere fuse. d series of receptacles indicated by RE1 supplies power for the spectrophotometer, amplifier, etc., independently of the rest of the circuit. The main power relay, RY1, is operated by a push-button switch, PS4, the “start” button, and is interrupted by PSX, the “stop” button Delay switch S5, set in the “start” position, supplies current through the ”hold warm” saitch, S4 (shown in the automatic position), to those circuits requiring an initial warm-up period. These circuits include those of the hydrogen lamp, the rectifier, the recoider, and the fraction collect hold warm (marked FCHlT7+) Mhich is always energized when the apparatus is in operation The change-over switch, S1 ( A t o E, five-pole, doubleFigure 10. Standardization Compensator throw switch shown in ”automatic” position: each of the five sections shown separately j, functions to change from automatic quantitative analysis t o fraction collection. The sequence in which the solvent solenoid valves SL1 to SL8 When the apparatus is operating automatically, current is also operate is determined by the patch panel. Pilot lights P28 t o applied initially through switch S1 to the thermostat, the main air solenoid, and the “hold warm” circuit, H W + , which ties to all P35 indicate which solvent valve is operating or ready t o operate. Toggle switches S7 to 514 permit solenoid valves to be operated of the other points in the diagram marked HW+. Manual manually. Air solenoid valves SL9 to SL12 are similarly operation of toggle switch S6 turns off the main air solenoid. \Vhen delay switch S5 is thrown to the “operate” position after operated automatically by the patch panel and manually by means of toggle switches S15 to 818. Pilot light P25 indicates the proper warm-up period, power is supplied t o all the point“solvent on.” The ninth jack socket on the solvent patch panel is marked FC+ and the “on” pilot light P27, and the thyratron used to stop the apparatus a t the end of run. For this purpose, the circuit, OA4G. The symbol indicates a direct connection to plug following that used for the last desired solvent is inserted in the “hot” side of the alternating current line. This thyratron the ninth socket and, when that position on relay SR3 is reached, fires and operates relay RY2, stopping all power when the leak the shut-off thyratron is fired. The flow of solvent is controlled cell electrodes are shorted, the air failure switch operates, the by means of the “solvent start” button, PS5, and the “solvent zero transmittance snitch operates, or a t the end of a run. When stop” button, PS6, which operates relay RT16. the hold warm switch, 84, is thrown to the “hold warm” position, The advance solvent circuits are actuated by reaching a predecurrent is applied to the hold warm bus. termined count as decribed, pressing the “reset” button, PS1, or The volume counting circuits are shown on the left-hand side pressing “solvent advance” button, PS2. Pressing “reset” of the diagram. When the dump cu fills, its electrodes are button PS1 causes stepping relay SR3 t o be reset and actuates the shorted momentarily, causing the O A 4 6 tube to fire and operate advance solvent circuits. When a n advance solvent impulse 1s relay RY3 momentarily. This operation energizes the roil of
>--------- - _ _ -
+,
+
ANALYTICAL CHEMISTRY
1768 received from any of the three sources, relay RYO is energized momentarily. By means of a normally closed, 2-second, time delay relay, TR2, relay RY9 is held energized for 2 seconds. During this period, steppers S R l and SR2 are reset and current is removed from time delay relay TR3, permitting it to close. Khen relay 9 is de-energized the advance solvent impulse is transmitted onwards, unless relay RYlO is operated, owing to the system being in “on peak” condition. This selection is determined by switch S2, a disk switch operated by the slide-wire motor of the recorder, and the normally closed 120-second time delay relay, TR12. In this manner, when the solvent advance impulse is applied while the system is “on peak,” the impulse is stored by relay RYlO until the system comes off peak. When the solvent advance impulse is passed through time delay relay TR3, relay R Y l l operates, causing the solvent stepper, SR3, to advance one step, and actuates the pen relay, RY12, which causes the recorder pen to operate for 4 minutes. After a delay of 5 seconds, the advance solvent impulse is transmitted through the time delay relay, TR4, to rela: PI-13, which starts the solvent by operating relay RY16 and initiates standardization by operating relaS7 RT18. Standardization relay RY18 is also operated by pressing “standardize” button PS7, by the time clock, M, which makes one revolution per hour, or by de-energization of relay RY17 a t the termination of the “on peak” condition. Once energized, standardization relay is held for 2 minutes by means of the time delay relay TR10. During this 2-minute period, the recorder pen marker, the dump solenoid, and relay RYIB, which applies 24 volts direct current to the standardizing circuits shown in Figure 8, are operated. Relay RT15 may also be operated manually by means of toggle switch 820. The solvent is on only for the first 90 seconds of the
I
standardization period and is timed by the time delay relay, TR11. Solenoid-operated by pass valve SL13 is also open during the 2minute period and may be operated manually by switch 819. Pilot light P36 indicates operation of the marker pen. Stepping relay SR4 enables the standardization period to be lengthened from 1 hour t o 4 hours when the system I S “on peak.” During operation as a fraction collector, most of the control circuit is inoperative. Only those points marked F C H W T , FC+, and receive power. There is no automatic solvent selection or standardization. The air solenoid,valves, SL9 to SL12, are replaced by four fraction collector solenoids. Change-over snitch S I is thrown to the “fraction collect” position. Stepping relay SR3 is used only t o change receivers. Standardization is effected during this time by operating switches 819 and 520. When collecting fractions the eluate is directed from the absorption cell to a fraction collector ( 8 ,Figure 5 ) v.-hich is actuated by a switch on the recorder. When the per cent transmittance of the sample stream drops below a selected value, the first solenoid operates and directs the eluate to the first receiver. When the transmittance rises above this value, the solenoid is de-energized, allowing the elute to flow to a waste receiver. When the per cent transmittance falls again, a second solenoid is operated, causing the delivery tube to take position over a second receiver. Frwtions are collected by repetition of this process, each fraction representing a complete optical density peak-Le., all of one coinponent.
+
IMPROVEMEVTS I S APPARATUS
The following changes in the apparatus have been made since this paper was submitted for publication:
I L
Figure 11.
Wiring Diagram of Control Panel
1769
V O L U M E 2 4 , NO. 11, NOVEMBER 1 9 5 2 Each pair of ~inglc-turnpotentiometers in the wave-length changer has been replaced by ten-turn Helipots. All of the wiring of the wave-length changer has been shielded from stray currents. The network of resistors involved in st.andardizations has been replaced, bringing the recorder print wheel into position on the chart a t 100% transmittance. The standardization compensator has been overhauled to eliminate defective standardizations which occurred occasionally. The relays which carry the low-vol tage currents have been hermetically sealed to lessen noise. All relays niid sensitive parts have been protect,ed more effectively from corrosion, the system of electrical grounds has been rearranged to eliminate ground loops, and t,he solenoid-operated solvent valves have been overhauled to eliminate occasional internal nntl external leakage. ACKNOWLEDGMEST
The authors are indebted to Edgerton Caldwell, Ross II-.Farmer, Edn-in 11.Griffiths, Frank Sehuster, John Tanaka, and Ed-
\tin L. \\'heeler of the C.C.L.A. technical staff for assistance in constructing the apparatus and the engineers of Beckman Instruments, Inc , for much valuable advice. LITERATURE CITED
(1) Cohn, W ,E., Abstracts of Papers, Division of Biological Chemist r y , 115th Meeting, A M . CHEM.Soc., p. 26C, 1949. (2) Cohn, W. E., J. Am. Chem. Soc., 71, 2275 (1949). (3) Cohn, W.E., Science, 109, 377 (1949). (4)
Deutsch, A., Zuckerman, R., and Dunn, 31. S., ANAL.CHEM.,24, 1769 (1952).
RECEIVED for review January 4, 1952. Accepted J u l y 28, 1952. Paper 88. Presented before Section 3, Biological Chemistry, X I I t h International Congress of Pure a n d Applied Chemistry, New York, N. y . , September 10 to 13, 1951. Work aided b y grants from the Kesearch Corp., Swift and Co., and the University of California. T h e material in this paper was taken from t h e thesis of Alfred Deutsch, presented in February 1952 t o the University of California in partial fulfillment of the requirements for the degree of doctor of philosophy.
Nucleic Acid Derivatives Separation and Quantitative Analysis by Ultraviolet Spectrophotometry 4LFRED DEUTSCHI, RICHARD ZUCKER>IhN, AYD MAX S. DUSN Chemical Laboratory, University of California, Los Angeles, Calif. The ribose nucleic acid nucleotides, adenylic acid, cytidylic acid, uridylic acid, and guanylic acid, were separated in pure form and determined in yeast nucleic acid by the authors' chromatographic and autonlatic continuous flow-recording ultraviolet spectrophotometric methods. The purity of the nucleotides was judged by the criteria of chromatography, spectrophotometry, and elementary analysis. The nucleotides were determined w-ith reasonable accuracy, as indicated by the recovery in the individual nucleotides of 10270 of the nitrogen in the nucleic acid sample. These results indicate that the authors' procedures may be employed satisfactorily for the separation, isolation, and quantitative determination of nucleotides in nucleic acids.
T"'-
apparatus designed by the authors (4)has been applied to the quantitative determination of the nucleotide compo-ition of yeast nucleic acid. The prerequisites for this analysis included the purification of a sample of nucleic acid and its hycli olysis to nucleotides, the development of a quantitative separation of the nucleotides from each other, and the purification of samples of the individual nucleotides for use as spectrophotometric standards. PURlFlCATION OF RIBOSE NUCLEOTIDES
Solvents and conditions for the complete separation of the four nucleotides were determined in preliminary experiments (data iiot shown) and applied to purification of commercial samples of the nucleotides using the described apparatus. Each product was separated into fractions collected automatically under control of thr optical density of the eluate. Each fraction was initiated when the optical density of the eluate approached infinity and was cut when it dropped. The material was elut,ed as slowly as practicable t o minimize the possibility that each fraction would cont:tin inore than one component. The chromatographic column was packed with Dowex-2 anion cwhange resin which had been washed t,oremove the fine particles aud admixed with 25y0 Celite to improve the flow characteristics. The packed resin bed was 15 mm. in diameter and 130 mm. high. Purification of adenylic acid, cytidylic acid, and uridylic acid was readily effected utilizing optical densities a t the single 1 Present address, California Foundation lor Biochemical Research, Loa hngeles, Calif.
.
ave length of 270 mp, but optical densities a t four wave lengths (260, 265, 270, and 280 mr) were employed in the purification. of guanrlic acid because of the many components in the starting material. M
bj
The per cent transmittance records of the nucleotides prepared the procedures described below are shown in Figures 1 to 4.
Adenylic Acid. -4 solution, formed by suspending 0.591 gram of commercial (Schwartz) adenylic acid in 8 ml. of distilled water and adding 6 drops of concentrated ammonium hydroxide, was placed on the resin column and eluted n i t h 0.005 S hydrochloric acid at the rate of about 1.1 ml. per minute. The main fraction (Figure 1 ) was distilled rapidly under reduced pressure belox 35' C. to a few drops of yellow sirup. The latter was taken up in 5 nil. of water, the mixture was warmed, and acetone v a s added to slight turbidity. The flask was cooled for 1 hour, acetone was added to bring the volume to 45 ml., and the flask was placed i n the refrigerator overnight. The suspension Mas filtered, and the R hite precipitate was washed n i t h acetone and ether and dried overnight a t 90" C. in a vacuum oven over phosphorus pentoxide. The yield was 0.12 gram. h n additional 30 mg. was recovered from the mother liquor and Tvashings. Cytidylic Acid. A solution, formed by dissolving 1.00 gram of commercial (Xutritional Biochemicals) cytidylic acid in 10 ml. of 1 ammonium hydroxide, was placed on the resin column arid eluted u ith 0.007 S hydrochloric acid a t the rate of about 6 nil. per minute. The main fraction (Figure 2) was distilled as described before to about 20 ml. of light yellow liquid which crystallized. -in equal volume of ethyl alcohol was added and the miature was distilled under reduced pressure. The suspension was filtered. the rn hite precipitate was suspended in boiling absolute ethyl alcohol, 10% of water was added, and the mixture was cooled overnight in the refrigerator, The product was filtered and the precipitate was washed with ethyl alcohol and ether and dried ovei night as described. The yield was 0.57 gram.
