for m = 1, and +2.68 and -2.60% for T 2 in the concentration interval 2 X IO-* to 10-5 mole per liter. m is the number of independent parallel measuremcnts on the ha& of which the sbsorbancc Alo was determined. The absorbance ,410 is independent of the pH of the reaction solution between pH 5 and 10. McIlvaine’s citric acid-sodium phosphate and Clark and Lubs’ boric acid-sodium hydroxide bufTers, diluted 20 times, were used, respcctivcly. Buffers were diluted becauw foreign electrolytes in the concentrations greater than 0.05M lowered results by about 8 to 10%. Under p H 5 the decomposition of ferrocyanide to aquopentacyanoferrate(I1) and cyanide takes place. The optimum p H for this reaction is a t pH 4.1. Above pH 5 the reaction is negligible if the concentration of ferrocyanide is not greater than 0.02M. The decomposition a f ferrocyanide to aquopentacyanoferrate(I1) is very strongly catalyzed by ultraviolet light (1). Therefore, the reaction solution containing ferrocyanide must be kept in darkness. Reduction of Ferricyanide. Ferricyanide inhibits the reaction between aquopentacyanoferrate(I1) and nitrosobenzene when present in a concentration larger than 5 x lO-’M (Figure 1). Therefore, i t was reduced to ferrocyanide by hydrogen peroxide in sodium hydroxide solution. The concentration of ferricyanide was first determined in an aliquot by titration with hydrogen peroxide in the presence of ~t large excess of sodium hydroxide. The equivalent point was determined by means of a platinum electrode (redox system ferricyanide-ferrocyanide). TO the test solution which contained aquopentacyanoferrate(II), ferrocyanide, and ferricyanide, sodium hydroxide was added in a 10 to 15% excess of the molar concentration of ferricyanide. (Larger excesses of sodium hydroxide should be avoided to reduce the elec-
m
Figure 1. Dependence of Ala, absorbance of [Fe(CN)b(CeH,.NO)] in the 10th minute at 600 mp on concentration of potassium ferricyanide Nlhoaobtnrene.
0.002M
0.3
p 0.2
I
trolyte effect.) The theoretical quantity of hydrogen peroxide needed for reduction of ferricyanide to ferrocyanide was then added. (Excess of hydrogen peroxide would interfere because it would oxidize nitrosobenzene and ferrocyanide after addition of acetic acid in the course of the procedure.) Nitrogen was introduced into the solution for about 5 minutes to remove oxygen. The solution was brought to pH 5 to 10 by 4% acetic acid and further treated ai already described under the heading Calibration Curve. During the procedure care was taken that the solutions were exposed to faint diffuse light only and that the temperature of the solution containing ferrocyanide did not exceed 22’ C. to prevent the decomposition of ferrocyanide. The presence of cyanide and sulfide ions and ions of heavy metals interferes because they react with aquopentacyanoferrate(I1). The ratio of concentrations of ferrocyanide, ferricyanide, and aquopentacyanoferrate(I1) in the mixture steadily and slowly varies because of the decomposition of ferrocyanide. Eighteen determinations of aquopentacyanoferrate (11) (concentration range O.ooOo2 to 0.001M) together
I
0.01 0.02 K ~ P o ( C N MOLES/LITER )~
0.001
I
0.03
with ferricyanide (concentration range
0.001 to 0.05) were camed out. The relative standard error amounted to +5.0 and -4.8/0 for m = 1 and 4-4.0 and -3.8% form = 2. UTERATURE CITED
(1) Ah erger, S., Trona. Faraduy Xoc. 18’ 617 p1952)
62, 2706 (1929).
( 5 ) Daviea, 0. L.,“Statistical Methods in
Research and Production,” p. 133,Oliver and Bo d, London, 1949. (6) EmRc willer, G., Compl. rend. 236, 72 (1953). (7 Hoffman, K.A., Ann. 312, l(le00). (81 Jaaelakis, B., Edwards, J. C., ANAL. CHEM.32,381 (1960). (9) Youdent W. J., “Statiatical Methods for Chemiste,” p. 42, Wiley, New York, 1957. Ivo MURATI Institute of Inorganic and Physical Chemistry Facult of Pharmacy, University of Zagreg SMIWKO A~PEROER Department of Ph sical Chemistry, Institute “Rudjer Sdkovif,” Zagreb, Croatia, Yugoslavia 1st Yugoslav Con ess of Pure and Applied Chemistry, Zagrec June 16-21,1860.
Comparison of Procedures for the Determination of Polycyclic Aromatic Hydrocarbons in Waxes SIR: The increasing interest in the analysis of petroleum products for trace amounts of polycyclic aromatic hydrocarbons, the specific object being the detection of possible carcinogens, has led us to investigate several modifications of the procedure currently used in this laboratory (9) for analysis of petroleym waxes. Such procedures all require the concentration of the aromatic substances, usually by chromatography, and separation from the bulk of the nonaromatic material. 810
ANALYTICAL CHEMISTRY
Following this, the aromatic material
is resolved until fractions are obtained in which individual polycyclic aromatic hydrocarbons can be identified by a spectroscopic procedure, ultraviolet absorption or fluorescence spectroscopy being the ones most often used. Practical limits are set to the sensitivity of the analytical procedure by the time required for the separation of very small amounts of several different compounds. In the course of analyzing many difTerent waxes by the procedure d e
scribed in ( d ) , a paraffin wax was found which, although having low absorptivity (0.0068 l./g. cm. a t 290 mp), contained a relatively high concentration of identifiable polycyclic aromatic hydrocarbons. This wax was chosen for study of modifications of the method described. The analytical method (9)involved chromatography of the wax in hot benzene solution on magnesia-Celite (2:1), followed by treatment of the upper 10 cm. of the column by acidi-
Table 1.
Adsorbent MgO-Celite MgO-Celite Silica 100- to 200mesh Silica through 200 mesh
Nitromethane Treatment Wt. Adsorbed, ElW, Wt., EWQ, Mg. 100 M1. mg. 100 ml. 213 142 3000 136 209 5480 332 1060
18.4 17.0 14.1 17.2 36.0 40.0 72.6 61 .O
.,
...
40 40 38 51 76
8.4 9.7 32.4 21.2 29.9 12.0
88
fication of the adsorbent and elution of the aromatic material with acetoneethanol-benzene. The remaining 14 cm. of the column was eluted by simple washing with the same eluting mixture. The two fractions obtained by washing out the water-miscible solvents from the benzene were combined, evaporated to dryness, weighed, and the absorbance at 290 mp measured in 100 ml. of isooctane. This absorbance value was used as an index of the aromatic material recovered. Three successive paper chromatograms, using different solvent systems (ascending chromatography in toluene-methanol-water (1: 1O:l) (T.M.W.), descending in dimethylformamide-isooctane (D.M.F.1.0.) and descending on acetylated paper in toluene-methanol-water) were required to produce fractions virtually without background absorption, in which individual polycyclic aromatic hydrocarbons couid be identified and their concentrations determined by absorption spectroscopy. Three compounds were identified-benzo [elpyrene, chrysene, and triphenylene. Very minute amounts of other compounds were present and were tentatively identified as pyrene and benz [alanthracene, but the concentration of these was considered insignificant. Variations in the aspirator, with consequent differences in rate of flow of the wax solution through the columns, together with lack of uniform heating, resulted in the retention of some wax on the adsorbent. This quality of wax (occasionally several grams), although a small fraction of the total chromatographed, was often sufficient to make paper chromatography of the adsorbed material difficult. Hoffmann and Wynder ( I ) described a procedure for separating polycyclic aromatic hydrocarbons from accompanying aliphatic material by partition between cyclohexane and nitromethane. This method was applied by Nelson and Stormont (9) to analysis of waxes. A second analysis of wax on magnesia-Celite was carried out incorporating this procedure. The material eluted from the column was taken up in 25 ml. of cyclohexane And shaken with fivc 25-ml. portions of
Results of Four Methods
~
1
Paper Chromatography
Hydrocarbon Recovered, pg. Benzo[e]Tripyrene Chryscne phenylene
2
3
T.M.W.
D.M.F.-1.0.
D.M.F.-1.0. D.M.F.-1.0.
Acetylated T.M.W. D.M.F.-1.0.
Acetylated T.M.W. D.M.F.-1.0.
D.M.F.-1.0.
D.M.F.-1.0.
nitromethane. Much of the colored material always obtained from a magnesia column remained in the cyclohexane, although the nitromethane extract still had a deep yellow color. The nitromethane solution was evaporated t o dryness under nitrogen, the residue weighed, and the absorbance at 290 mp measured in iso-octane solution. Paper chromatography was carried out, three stages being again required before the background absorption was sufficiently reduced as t o make possible quantitative measurements by absorption spectroscopy. Because magnesia will not (2) retain tricyclic compounds under the conditions described for analysis of waxes, a method was needed which would detect such compounds. Tricyclic compounds are, however, of limited practical importance in the study of waxes, since carcinogenicity in this series of compounds is very rare and limited to rather unstable polyalkyl derivativesSilica e.g., 9,lO-dimethylanthracene. gel is a n adsorbent which will retain tricyclic compounds if the wax is dissolved in a n aliphatic solvent. Isooctane was a suitable solvent which would dissolve large quantities of wax near its boiling point. Two grades of silica gel were used-grade 923, 100to aOO-mesh, and grade 922, through 200 mesh (Davison Chemical Co.). One kilogram of wax was dissolved in approximately 1.5 liters of hot isooctane and filtered with suction through a 24 X 4.5 cm. column of silica gel. Filtration through the 100- to 200-mesh silica was very rapid (taking less than 1 hour) while, through the finer silica, it took about the same time A S through magnesia-Celite (3 hours). After washing the column with hot iso-octane, the adsorbed aromatic material was eluted, in each case, with 1 liter of benzene (500 ml. was insufficient), aftcr which only a minute additional quantity could be obtained by acetone elution. The benzene filtrates were evaporated to dryness, the residues weighed, and the absorbances mewured at 290 mp. In both cases sufficient wax was present in the residue to make nitromethanecyclohexane partition essential prior
Acetylated T.M.W. D.M.F.-1.0.
270 262 294 287 240 192 360 356
545 865 690 875 140 178 846 928
304
600 860 706 302 124 570 781
to paper chromatography. Paper chromatography of the cyclohexane solutions after partition with nitromethane showed that essentially all of the polycyclic aromatic hydrocarbons had been removed after five partitions, but not before. Again three successive paper chromatograms were necessary before the absorption spectra of the fractions were satisfactory for quantitative measurements. No tricyclic compounds were detected in any of the fractions showing the absence of such substances from this wax. The results of the four different methods of analysis (each based on lo00 grams 01 wax) are given in Table I, from which the following conclusions can be drawn : There was no appreciable difference in total time of analysis (35 to 40 hours) among any of the four procedures described. I n each case the polycyclic aroma tic hydrocarbons were a minor part of the adsorbed material, even after nitromethane-cyclohexane partition, and lengthy paper chromatography was necessary to produce fractions with little background ultrrtviolet absorption. Quantitative estimations were based on absorption spectra. &cowry of the polycyclic aromatic hydrocarbons from magnesia-Celite and fine silica gel were roughly comparable. The differences were due to the greater or lesser difficulby in reducing background ultraviolet absorption by paper chromatography; in general, the larger the amount of paper used, the greater the losses of polycyclic aromatic hydrocarbons. Although chromatography on coarse silica gel was much faster than on the other adsorbents, recovery of polycyclic aromatic hydrocarbons was lower, particularly that of the less strongly adsorbed compounds. I n any case, the time occupied by the adsorption chromatography was an insignificant part of the time of the entire analysis. Partition between cyclohexane and nitromethane was an effective means of separating the polycyciic aromatic hydrocarbons from wax which remained in the column after washing with hot solvent. The residual wax contributed VOL. 33, NO. 6 , M A Y i961
a
81 1
to thc largc difforcncc in thc weights of adsorlad material (213 and 3000 mg.) from loo0 grams of the 8amc wax. This proccdum also removed most, but not all, of the colored material associatcd with the aromatic compounds oil the column. The solvcAnt system most cffective (and most rnpid) in resolving mixturciP of
polyc:yc:lic aromatic hyc1roc:nrlmis was climethylformnmide-is(~ct:ule. Descending chromatography WHR, in all cascs, superior in rclsultu to tho auc:c*ntling technique. UTERAWRE CITED
( 1 ) Hotlmann, l)., Wynder, 15. L.,ANAL. 32, ~5 (1960). (2) Lijinsky, W., Zhid., 32, Vi84 (1960).
cHEM.
(3) ,Nf:lson, 11. K Stormollt, 1). J . , Chem. & I n d . (London) 1960, 21, TAl. WILLIAM LIJINRKY C. It. HAHX JOANNE I~EELIN~~ Division of O n c o l o ~ ~ The Chicago Medicnl School Chicngo 8, Ill.
This work waR pirrt of Project MC-3, American Petroleum Institutr.
low-Cost Ordinate Scale Expansion Unit for the Perkin-Elmer Model 21 Infrared Spectrophotometer D. F. Westneat,I Polychemicals Department, E. 1. du Pont de Nemourr & Co., Inc., Wilmington 98, Del.
intercst in the analysis of 1hasvery small amounts of material created a strong demand for imNCREABINQ
provement in the sensitivity of infrared spectroscopic techniques. One successful approach to this problem has been the dcvelopment of ordinate scale expansion accessories which amplify the motion of the spectrophotometer recording pen, thereby allowing the operator to discern weak absorption bands more readily. This article describes an inexpensive, easily constructed accessory which can produce u p to a fiftyfold expansion in the recorded intensity of absorption bands.
Thr basic circuit, shown in Figure 1, is a modification of a simple bridge network. The sliding contact of R. is mechanically coupled to the pen drive of the spectrophotometer and hence responds to the absorption characteristics of t h r sample. The contact on RP selrcts the portion of the transmittance Preaent addresn, Department of Chemistry, University of Akron, Akron,
Ohlo.
Figure
driving off-scale, sinw under these conditions the pen iiiotor will continue to turn the pulley. On spcctrophotometers which do not usc n belt and pulley system to drive the pen, the potentiometer may be coupled to an equivalent part of the pen servo systcm. Figure 3 illustrates the performance of the eqiiipment. The Rpectra which are shown cover the 1 0 . 5 t o 12.0micron region of cyclohexane vapor at three different prewures. The normal, unexpanded spectra rccordcd on B Perkin-Elmer Model 21 spectrophotometer are shown with the corrcsponding expanded scans recorded simultaneously on the auxilinry recorder. As the degree of scalo expansion is increased beyond 15X, the operating conditions must be adjristrd to improve the signal-to-noise ratio. The slite must be opened and the amplifier gain decrewed correspondingly. In additioc, the pen response should be damped and the scanning speed redriced sufficiently to maintain accurate measurcmcnts. The ordinate expansion cquipmcnt describrd offers several attractive festures in addition to providing scale
The device shown in Figure 2 has performed satisfactorily in this laboratory.
1.
Scale
expansion
circuit
10-turn potentlometer, 1000 ohms, 0.1% RI. ilneorlty R&Ra. 1 0-turn potentiometer, 1000 ohms R,. Potentiometer, 20,000 ohms $1. Swltch, P U ~ button I Ss. Switch, single-pole, single-throw M. Milliammeter, 0 to 1 ma, E. Battery, 1.5 voih
812
scale to be expanded by determining the balance point of the bridge circuit. The degree of scale expansion IS governcd by R, and R4,which providc fine and coarse adjustment of the voltage applied to the bridge. The unbalance, which is generated in the bridge by motion of thc spectrophotometer pen, is recorded on a n auxiliary recorder. Although tho circuit in Figure 1 is designed for use with a 10-mv. recorder, appropriate changes in voltage and resistance valucs will permit an conventional recorder to be used satisictorily. The remaining circuit components improve the convenience and precision of operation. Switch 81may be used to place pip marks on the trace for wave length indication by shorting the input leads to the auxiliary recorder. Milliammeter M measures the bridge current and may be calibrated to read the degree of scale expansion. All of the components in Figure 1 are external to the spectrophotometer, with the exception of RI,which may be connected to the pen servosystem of a Perkin-Elmer Model 21 infrared spectrophotometer with any flexible coupling which is free of backlash.
ANALYllCAL CHEMISTRY
The potentiomcter, mounted in the base of the spectrophotometer, is connected to the pulley wheel, which drives thc wedge and recorder pen. The coupling consists of two ccramagneta (ceramic magneb manufactured by the Stackpolc Carbon Co., St. Marys. Pa.) 11/* inches in diametrr with a '/,-inch center hole and Cpolc magnetization, ae arated by a thin shret of foam ruther. This arrangement can be adjusted to provide positive coupling with minimum drag even in the case of imperfect alignment. However, care must be taken to prevent the recorder pen from
Figure 2. Mounting of potentiometc. on spectrophotometer
