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ALUMINUM OXIDE
Figure 9. Thin-layer chromatographic separation of 5H-benzo(b)carbazole from commercial chrysene Aluminum oxide with pentane-chloroform (3:2) Spots applied contained: A. 5H-Benzo(b)carbazole 6. Commercial pure chrysene C. Commercial pure chrysene, m.p. 252-54’ C., different company D. Purified chrysene
presence of 515-benzc@)carbazole in chrysene, two samples of commercial pure chryaene (m.p. 252-54’ C.) were placed un an aluminum oxide plate with 5H-benzo(b)carbazole and a sample of purified chrysene (m.p. 250-52’ C.). All samples were disr)olvcd in N , X dimethylformamide. The plate was developed in pentane chloroform (3 to 2), System 33. Figure 9 shows the separation; the presenEe of 5H-benzo(b)carbazole in commer:ially pure chrysene is demonstrated.
The absorption spectra of a commercially pure chrysene sample were obtained in both neutral and alkaline N,N-dimethylformamide. I n addition to the spectrum of chrysene, maxima were obtained a t 394 and 375 mp in neutral solvent. In alkaline solvent the spectrum of chrysene remained unchanged; however, the two maxima due to 5H-benzo(b)carbazole shifted to 481 and 456 mp. The concentration of 5H-benzo(b)carbazole in this particular sample of chrysene was 12% by absorption spectrophotometry. Fluorescence analysis in alkaline solvent was done by exciting at 302 mp to obtain the 521-mp emission wavelength band. The excitation spectrum obtained at emission wavelength 521 mp N-as that of pure 5R-benzo(b) carbazole. The estimated concentration of 5N-benzo(b)carbazole in cominercially pure chrysene by fluorescent spectrophotometry was 10%. In future studies these methods will be applied to the separation, characterization, and determination of carbazoles and polynuclear carbazoles in airborne particulate matter. LiTERATURE CITED
(1) Brinkmann Instruments, Inc., Great
Neck, Tu’. Y., “Apparatus for TLC,” Operating Rfanual 103-A. (2) Corbellini, A., Debenedetti, E., Gazz. Chzm. Ital. 59, 391 (1929). (3) Corbellini, A,, Marconi, L., Ibid., 62, 39 (1932’). \----,-
(4)Falk, H., in Samicki, E., Camel, K., eds., “Symposium on the Analysis of Carcinogenic Air Pollutants,” Xational
Cancer Institute Monograph 9, 219 (August 1962). (5) Hartwell, .J. L., “Survey of Compounds Which Have Been Tested for Carcinogenic Activitv.” 2nd ed.. Pub. Health gervice PubL”149 (1951),’ (6) Helm, R. V., Latham, D. R., Berrin, C. R., Ball, J. S., ANAL.C m Y . 32, 1765 (1960). (7) Japp, E”. R., Maitland, W., J . Chem. SOC.83, 267 (1903). (8) Kikkawa, S., J. Chem. Soc. Japan,, lad. Chem. Sect. 54, 631 (1961); CA 47, 7195 (1952). (9) Kruber, O., Raeithel, A., Grigoleit, G., Erdol Kohle 8, 637 (1955). (10) Mabille, P., Buu-Hoi, N. P., J. Org. Chem. 25, 1937 (1960). (11) Sawicki, E., Fox, F. T., Elbert,, W. C., Hauser, T. R., Meeker, J. E. Am. Ind. Hug. Assoc. J . 23, 482 (1962). (12) Sawicki, E., Hauser, T. R Stanley, T. W.. Intern. J . Air Pollutlbn 2. 253 (1960): 3 ) Sawicki, E., Hauser, T. R., Stanley, T. W., Elbert, W. C., Fox, F. T., ANAL. CHEM.33, 1574 (1961). 4) Sawicki, E., Stanley, T. W., Elbert, W. C Pfaff, J. D., Division of Water and i”lste Chemistry, Meeting, .4CS, Sew York, September 1963. 5 ) Sawicki, E., Stanley, T. W., Hauser, T. R., Elbert, W. C., Noe, J. L., AKAL.CHEM.33, 722 (1961). 6) Schneider,. P.,, Anqew. Chem. 67, 61 (1955). (17) Shubik, P., Hartwell, J. L., “Survey of Compounds m’hich Have Been Tested for Carcinogenic Activity. Supplement 1,” Pub. Health Service Publ. 149, (1957). (18) Snyder, L. R., Buell, B. E., Division of Petroleum Chemistry, 145th Meeting, ACS, New York, September 1963. (19) Van Duuren, B. L., Bilbao, J. A., Joseph, C. A,, J . Natl. Cancer Inst. 25, 53 (1960). RECEIVED for review November 19, 1963. Accepted January 29, 1964.
A Continuous Flow Analyzer for Recording of Light Absorption and Radioactivity in the Eluates from Chrolmatographic Columns C. I W A RSJOBERGam3 GUNNAR AGREN Institute o f Medical Chomistry, University of Uppsala, Sweden
b The description 0 : an automatic recording device for location and quantitative measurement of ultraviolet absorption of nucleotides in the fluid emerging from ion exchange columns is given. The absorption signal i s fed into a strip chart multipoint recorder which cilso accepts the output signals from a dual rate meter with two separate OLItputs for linear and logarithmic readings of radioactivity,
B
ECAUSE OF THE TEDIOUS and
timeconsuming work o f collecting and analyzing a large number of nucleotide fractions, automatic recording of chro-
matographic Feparations has been carried out here for many years. Commercially available analyzers (Gilson, LKB), as well as those constructed and built by the institute, have been used (7). A photometric analyzer which might be of general interest will be described in detail. It is useful especially in the ultraviolet region for the location and quantitative measurement of nucleotides in the eluates from chromatographic columns. Since this type of analysis is a relatively slow process, an analyzer for such a purpose need not be standardized more than a few times an hour, without sacrificing accuracy. This also was considered
in designing the analyzer, to include simplicity in construction as well as troublefree operation. A picture of:the analyzer is shown in Figure 1. Instrumentation. The block diagram in Figure 2 outlines the principle and operation of the analyzer. Light from the hydrogen lamp source passes through a Beckman DU Quartz Monochromator. The radiation from the monochromator enters the absorption cell compartment, where, b y means of a movable pair of mirrors, it is alternately directed through the sample cell and the comparison cell onto a photomultiplier tube. During the standardizing period, the servo system adjusts the sensitivity of VOL 36, NO. 6, MAY 1964
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1017
Figure 1. Front view of continuous flow photometric analyzer the photometer arrangement to the proper value for 100% transmission of light. The strip chart recorder in the first version of construction, was a onechannel pen recorder (Speedomax G). The recording and standardizing periods were timed by a cyoling mechanism, specially made lor the purpose. The timing intervals were set to 2 minutes 50 seconds for the recording period followed by a 10-second standardizing period. Simultaneous recording of ultraviolet ahsornt,iou and radioact,ivit,v wm reX72, ElectroniK &point, range 0 to 2.5 mv.). The ultraviolet analyzer was connected to channel No. 1. Two rate meters with the inputs in parallel were connected to channels Nos. 2 and 3. One of the rate meters was set on a high sensitivity range and the other on a low sensitivity range to cover a wide range of counting. Recently a dual rate meter with two separate outputs for linear and logarithmic readings (Baird-Atomic Precision Dual Count Rate Meter, Model 412, with 10 linear ranges covering 0 to 30 c.p.m. to 0 to lo6 c.p.m., and a
I~garithmic %cycle log s ~ x l e 10 to 10’ i,.p.m.), l i n d reyisced the rate nirtrrs mentioned. To synchronize the cycling periods of the analyzer with those of the strip chart recorder, the functions of the cvcline mechanism mentioned above were &en over by two microswitches actuated by cams, mounted on the shaft 01 the point selector switch in the machinery of the recorder. The printing intervals for the striu chart record6 now in use are 18 seconds and the selector switch shaft makes one revolution in 4 X 3 X 18 seconds-i.e., 4 channels X 3 printings lor each channel for every shaft revolution X 18 seconds. The cams of the point selector switch shaft are arranged in such a way that the switches are engaged only once in three printings of channel No. 1. This means that there is a standarduing period in every 216 seconds. The standardizing period is set to about 30 seconds, and it starts and ends when channels Nos. 2 and 3 are in action. The print-carriage speed is 4% seconds for fnll deflection. When the analyzer is used only for qualitative identifications, the chart speed for convenience is usually chosen as low as l/s inch per hour (corresponding to a chart length of 1 foot lor every 24hour run). However, when quantitative measurements are carried out, a somewhat higher speed is to be recommended-for example, 1 inch per hour.
ELECTRICAL CIRCUITRY OF ANALYMost of the electronic parts
ZER.
contained in the photometric analyzer are commercially available. No description in detail of these items is given, in that they need satisfy only certain specifications. Figure 3 shows the circuitry for the photomultiplier and its connections to the servo amplifier, the reference voltage source, and the strip chart recorder. The photomultiplier used is a RCA Type lP28 tube. The block diagram in Figure 3 shows that the strip chart recorder is connected to the anode of the photomultiplier over a 400-ohm resistor to ground. The anode of the 1P28 tube is also connected to 01le of the
input terminals of the servo amplifier. The other input terminal goes to the reference voltage source. This relerence voltage is manually adjusted to a value that corresponds to 100% transmission of light. The shaft of the servo motor, connected to the output of the servo amplifier, is fitted to the voltage regulator control on the extra high tension (EHT) power supply, feeding the photomultiplier dynode chain. When the off-on cam switches in the strip chart recorder are engaged by its cams, the self-balancing servo system is put in action for standardizing the analyzer. The cams also initiate the movement of the bridge carrying the mirrors in the absorption cell compartment to position for standardization. See Figures 2 and 4. If there is a difference in voltage between the output voltage from the photomultiplier and the reference voltage, the servo motor moves and adjusts the high voltage and thus varies the amplification of the photomultiplier to give an output which corresponds to 100% transmission of light. When this criterion is achieved, the voltage difference a t the servo amplifier input is zero and the servo motor stops. When the standardizing period is completed, the cam switches again are engaged by the cams. This inactivates the servo system and starts the movement of the mirrors in the absorption cell compartment back to measuring position. The EHT power supply used has a voltage range lrom 500 to 1200 volts. The servo amplifier is a Brown ElectroniK amplifier, Cat. No. 351921, and the balancing motor is a Brown Reversible Two-Phase Motor, Part No. 76750X, 27 r.p.m. There is no special arrangement for compensating the dark current as it is negligible. Provision has been made for manually operating the servo motor and the motor in the absorption cell compartment for positioning the mirrors. To operate the analyzer the procedure is a s follows. The analyzer is set in measuring position, causing the light beam to traverse the samnle absorntion cell. The strip chart recorder is t i m e d
SELFBALANCING SERVO AMPLIFIER SERVOMOTW
Figure 2. Block diagram showing principle and operation of photometric analyzer 1018
ANALYTICAL CHEMISTRY
Figure 3. Circuit diagram for photomultiplier and its connections to strip chart recorder, servo amplifier, and voltage reference source
b Figure 4. Cutaway view of absorption cell compartment
-
! T O WASTE
Figure 5. Flow diagram of setup arrangements for photometric analyzer
on. However, when channel KO.1 is to be recorded, the chart drive switch is turned off. This permits the recorder flected a t a right angle into the entrance 11 and 12 are connectors for the motor to be used as an indicator. Before any hole of the photomultiplier holder, 13, and the power cables. adjustments are carried out, the voltage which encloses the photomultiplier tube INSTRUVENT ARRANGEMENTS. A regulator is set in riid-position by in a magnetic screen of Permalloy. schematic diagram of the apparatus manually operating the servo motor. The sample cell, 5 (only a part of the set up in Figure 1 is shown in Figure Further on, only elution solvent should cell is visible), and the comparison cell, 5 . No. 1 is a reservoir flask for the be allowed to flow through the light 6, are placed so that they are alternately elution solutions. No. 2 is a gradient absorption cells. The sensitivity of the traversed by the light path when the mixer of the Mariotte flask type with its photomultiplier is adjusted to full light changes its direction between its magnetic stirrer. The solvent passes a scale deflection on the strip chart retwo fixed positions for measuring and filter, 3, and is pumped by a LKB 4500 corder (100% transmision of light), standardization. The absorption flow Mini Flow Precision Micropump, 4, by manually varying the high voltage cells used in the analyzer are as designed to the chromatographic columns, 5. with a second voltage control ?n the by Sjoquist, Rydberg, and Svensson The eluate from the reference column E H T power unit. The analyzer IS now (6). The mirrors are mounted on a passes through the comparison cell, 7, set in standardizing pxition and the movable bridge, 7, which is positioned and goes directly to waste. The eluate light beam will traverse the comparison a t two stops by a motor, 10, driving the from the sample column passes a cell. If the servo system is manually gear rack, 9, back and forth. Two scintillation flow counter tube detector, turned on, the servo motor will run in microswitches, 8, stop the motor when 6, before it goes to the sample cell, 8. either direction depending upon the the bridge is in proper position. Xos. After leaving the sample cell, the eluate sign of the polarity of the unbalanced voltage at the input terminals of the servo amplifer. The reference voltage is then adjusted to bring the motor back to exactly the Same position it was previously. For makin%the restoration easier, a dial has beer placed on the shaft of the servo mctor. It is also useful to have a vacuum-tube voltmeter connected to the servo amplifier input to indicate the unbalance of the amplifier. By observing the meter one can make a coarse adjustml3nt of the reference voltage before the servo motor is put in action. Following this, the reference voltage is adjusted and the analyzer is srritched ov?r for automatic running. ABSORPTION CELL COMPARTUENT. A diagram of the con:;truction of the absorption cell compartment is shown in Figure 4. The cont,tiner is made of alummum, and of dimewions such that it may be attached to the monochromator casing to replace the ordinary cell compartment. Two opposite malls of the cell compartment are made on slides, so they may easily be removed for 600 convenience in exchanging cells or Figure 6. Acid-soluble nucleotides extracted from diploid tumor ascites cells inspecting the compaptment. Figure 30 minutes after incubation with radioactive inorganic phosphate 4, No. 1, shows the lamp house from which the light enters the monochromaNucleotides were separated by gradient elution with reservoir content changed ot tubes numbered as tor, 2. The light passing the slit and follows: 80, 1N formic acid; 168,4N formic acid; 260, 0.2M ammonium formate 4N formic acid; the exit hole, 3, is first reflected at a 444, 0.4M ammonium formate - 4N formic acid; 600 1M ammonium formate f 4N formic acid. right angle by one of thct pair of mirrors, Continuous line represents radioactivity values; broken line represents ultraviolet ( E z ~ o a ) bsorbonce 4, to the other, and is then again revalues. Column was 42 cm. long 3 cm. in diameter
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100 TUBENUMBER 125 150 175 Figure 7. Acid-soluble nucleotides extracted from diploid tumor ascites cells 30 minutes after incubation with radioactive inorganic phosphate Nucleotides were separated by gradient elution increasing from 0 corresponding to the position of fraction No. 1 to 3.8N formic acid in the tubes corresponding to the position of fraction No. 18 on the elution curve
is collected by a fraction collector, 10. In the scintillation detector, built by the institute, the flow counter tube is a N E 801 plastic scintillator spiral flow cell for beta and alpha emitters in liquids. The NE 801 flow counter tube contains approximately 2 feet of 1.5mm. 0.d. and 0.7-mm. i.d. capillary tubing, wound in a spiral with a maximum diameter of 13/4 inches and a volume of approximately 0.3 cc. The flow counter cell is fabricated by Nuclear Enterprises (G.B.) Ltd. I n the figure, 9 is the Beckman monochromator with light source and cell compartment and 11 is a Philips Ghl 4410 rate meter (as mentioned above, two rate meters were commonly in use), now replaced by a dual rate meter. No. 12 is the multipoint strip chart recorder and 13, the servo amplifier unit and the reference source. S o . 14 is the EHT power supply for the photomultiplier tube and contains the controls for nianual operation of the analyzer. No. 15 is the main power panel, and 16 is a pulse amplifier, Philips PW 4042, which also contains an EHT power supply for the scintillation detector. Recently a slight modification of the organization has been made. To save solvent fluid, the reference column is excluded and the solvent goes directly from pump 4 to the comparison cell, 7, and then to the sample column. The eluate from the sample column goes the same way as mentioned before.
Table 1.
RESULTS
Analysis of Trichloroacetic Acid Extract of Tumor Cells. The apparatus has mostly been used t o record the elution pattern of trichloroaceticacid-soluble, radioactive-labeled nucleotides from material such as rabbit liver (2) and ascites tumor cells ( 1 ) . The latter material especially contains a large number of ultraviolet absorbing peaks which are eluted in such proximity to each other that they would not have been detected with the previously used technique where light absorption and
7 1 lF
radioactive determinations were carried out on samples from each tube of the fraction collector (3, 4). Figure 6 presents the chromatogram of the ultraviolet and radioactive material present in the acid-soluble extract of Ehrlich tumor ascites cells separated by counter-streaming centrifugation (6). The extract had been filtered into a Dowex 1 (2Yc DVB) formate column and eluted mainly as previously described (3, 4). Other experimental details and results will appear in further publications. The large peak marked X in the first appears in part Of the the eluate when the front of the gradient system leaves the column. It is followed by a series of closely positioned peaks in the region of adenylic acid (AMP) and between ANP and inorganic radioactive phosphate (P), The details of the curves are more clearly observed in Figure 7. Many details seen in this figure could not have been obtained wit)h the older conventional technique where several of the peaks would have been collected in the same tube of the fraction collector. As previously mentioned, the analyzer has generally been used for identification purposes and for following preparative chromatographic separations. However, tests have proved that the ac-
IS
IS
I. 9
Absorbance
Planimetric values
Analyzer
Beckman from DU curve I
(CMP)G 0.40 0.23 0.26 0.39 0.40 (DPN) 0.50 (AMP) 0.66 0.58 0.57 Experimental and calculated light absorbance (260 mp) values for cytid lic acid (CMP),diphospho-pyridine nuccotide (DPN) and adenylic acid (AMP). The nucleotides were separated by gradient elution 0 1M formic acid. The column was 20 cm. in length and 0.9 cm. in diameter. It was loaded with 0.25 ~ i gof. nucleotide. 0
1020
ANALYTICAL CHEMISTRY
5 TLI3E NUMBER
IO
15
20
25
Figure 8. Curve I. Chromatogram obtained with recording unit when 0.25 mg. of three nucleotides were run through a small Dowex 1 formate column with gradient 0 + 1M formic acid Curve I1 i s based on values calculated from Curve I obtained in recording unit
curacy of the photometric analyzer also makes it useful for quantitative measurements of ultraviolet absorbing substances. Curve I in Figure 8 shows the chromatogram obtained with the recording unit when 0.25 mg. of three nucleotides were run through a small Dowex 1 formate coluntn with gradient 0 -+ lilf formic acid. The absorption values of Curve I1 were obtained as a result of a planimetric calculation based on the recorded Curve I. R'ith the knowledge of the positions of the tubes in the fraction collector and their place on the strip chart, the area between the base line and the Curve I for cach single tube
was determined. The values obtained, which represent the amounts of nucleotide fractions contained in the tubes, were plotted as Curve 11. Finally the content of nucleotide in the tubes was directly determined in the Beckman DU spectrophotometer. Table I shows that there is reasonably close agreement between the absorption values calculated and the values read in the Beckman DU spectrophotometer. ACKNOWLEDGMENT
Skillful assistance of Nils-Olof Hedlund and Thore Persson is gratefully acknowledged.
LITERATURE CITED
(1) Agren, G., Acta Chem. Scand. 14,2065 (1960). (2)67.55 Agren, (1962). G., Acta SOC. Mea. Upsal. (3) Igren, G.,' Engstrom, L., Acta Chem.
Scand. 11,1087 (1958).
(4) Engstrom, L., Ibid., 63, 128 (1958). (5) Lindahl, P. E., Cancer Research 20,
841 (1960). (6) Sjoquist, J., Rydberg, C.-E., Svensson, R., Kgl. Fysiogruf. Sallskap. L u n d , F&h. 26, No. 13 (1956). ( 7 ) Verdier, C.-H. de, Acta SOC.Med. Upsal. 57,393 (1952). RECEIVEDfor review Nay 6, 1963. Accepted December 16, 1963. The investigation WAS supported by grants from the Ther6se and Johan Andersson Foundation and the Swedish Cancer Society.
A Quantitative Chromatographic Determination of Cysteic Acid in Amino Acid Mixtures on Ion Exchange Papers JAN HARTEL' and A. J. G. PLEUMEEKERS2 Central laboratory TNO, Delft, Netherlands
b- A
new and rapid method for determining cysteic acid in a mixture o f amino acids has been cleveloped. For the separation, use i s made o f chemically modified paper containing quaternary ammonium groups. Quantitative determination can b e done on the paper densitometr cally as well as spectrophotometrically (after elution). The densitometric determinotion i s rapid but not very exact. The spectrophotometric method gives more reproducible values with a relative standard deviation of 3%.
xETHoDs for the determination of cysteic acid in amino acid mixtures rely on the fact that this amino acid is more acidic than others. Therefore, methods are usually based on ion exchange chromatography and paper electrophoresis. Ion eschsnge column (hromatography, described by Schrani, Moore, and Bigwood (6) gives escellent separations, but the method is rather laborious and time consuming. TT'ith high voltage paper electrophoresis, Zuber, Ziegler, and Zahn (8) obtained good results in much shorter time. Diehl ($) and Bauters, Lefebvrc, and Van Overbeke (1) modified the electrophoresis conditions and wed lorn voltage paper electrophoresis to give a simpler and less expensive method. OST
Present address, De Wit's Textile Nijverheid Yj. V., Helmrind, Xetherlands. Present address, Fibre Research Institute TNO, Delft, Netherlands.
To further simplify the method, we used ion exchange papers developed in the Central Laboratory, TNO. These papers, made by chemical modification, will be produced by Macherey and Nagel in Duren, Germany, and will be available with strongly or weakly acid or base groups. They have proved to be very useful in separating mixtures of compounds with ionic groups, especially amino acids ( 5 ) . For the determination of cysteic acid, we used a paper with strongly basic quaternary ammonium groups. After separation, the cysteic acid spots could be determined by densitometry or, after elution, by spectrophotometry, EXPERIMENTAL
Apparatus and Reagents. RIacherey and Sage1 ion exchange paper with strongly basic groups; Agla micrometer syringe; Photovolt densitometer, Model 501-A; Eppendorf colorimeter; and, for part of the experiments, Bausch and Lomb Spectronic 20. The ninhydrin reagent is a 0.5% solution in 96% ethanol and contains 0.2y0glacial acetic acid. To prepare the copper reagent (?), 10 ml. of saturated copper nitrate solution is mixed with 0.2 ml. of concentrated nitric acid and made up to 1 liter with acetone. Procedure. The ion exchange paper, available in the chloride form, is conditioned by washing i t overnight with the mobile phase, 0.34M chloroacetic acid. Excess chloroacetic acid is removed by rinsing the paper three times with distilled water.
After drying for 20 minutes a t 65' C., the paper can be stored until needed. On a 25-em. long sheet of paper, a starting line is drawn 3 cm. from the lower edge. The cysteic acid solutions are applied with a micrometer syringe a t 2.5-em. intervals along this line. I n general, a maximum quantity of 5 pl. is applied. For larger quantities, spotting is repeated with intermittent drying of the spot. I n addition to the unknowns, three different quantities of a standard solution of cysteic acid (0.1% in water) are applied t o the same chromatogram. All applications are in duplicate. One place on the starting line is left open for the determination of the paper blank. The quantity of cysteic acid applied varies between 0.4 and 1.0 bg. for the densitometric determination and between 2 and 20 pg. for the spectrophotometric determination. The paper is made into a cylinder by connecting the side edges with a cotton thread. This paper roll is placed in a glass cylinder which can be closed. The chromatogram is developed with 0.34.V chloroacetic acid by the ascending technique. When the liquid has progressed 15 em. from the starting line (40 to 60 minutea), the chromatogram is removed and dried a t 65" C. for 20 minutes. The paper is pulled through the ninhydrin solution from the starting line to the liquid front and is dried hanging vertically with the starting line up. These precautions prevent migration of heavily concentrated spots. After impregnation, the paper is dried a t 65" C. for 20 minutes. Quantitative. DENSITOMETRIC DETERMINATION. The colored chromatogram is cut into strips according VOL. 36,
NO. 6. MAY 1964
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