urine specimens. Such urines must be acidified with metal-free acid prior to analysis. To ensure that all the zinc is in the ionic state, hydrochloric acid is added routinely to each separatory funnel, regardless of the initial p H of the sample. The buffer, containing potassium cyanide and sodium thiosulfate, is added t o complex copper, cobalt, nickel, bismuth, mercury, cadmium, and lead which may be present, while the sodium potassium tartrate serves to complex interfering iron and manganese. The present method improves the extractability of zinc dithizonate from urine. TThile a function of pH (Table I), optimal conditions also depend on the composition of the sample as well as the buffer, the complexing agents which it contains, and the addition of any substance which combines with zinc to form a slightly dissociated complex. The amount of zinc extracted increases with pH, with increasing concentration of dithizone in the carbon tetrachloride phase, and with decreasing strength of buffer and complexing agents. The balance of these parameters is critical, and desirable changes produced in one experimental variable are readily obviated by resultant disturbances of others. It has been possible to improve the extractability of zinc from urine by a change of buffer p H to 5.7 (Table I) accompanied by the use of a solution of buffer and complexing agents which is 25% more dilute than that used previously, without altering the specificity of the method for zinc (6).
Incomplete extraction of zinc may also result from the addition of insufficient amounts of dithiaone. If much zinc is present in a sample, the concentration of free dithizone in the organic phase may fall below the critical level and prevent complete extraction. I n this case more dithizone solution (0.003%) should be added. The minimum quantities of metal ions causing interference in urine zinc determination (Table 111) are of the same magnitude as those reported for the mixed-color method (9). Because the amount of these metals which normally occur in biological samples is not likely to exceed this minimum, the method is regarded as highly specific for zinc under the conditions delineated. The good agreement of the amounts of zinc found in urine after dry ashing and by the present technique (Table V) demonstrates the high accuracy of the method. The insignificant loss of zinc in the directly extracted urine samples may be indicated by their slightly lower mean value. The accuracy of the d r y ashing method is very dependent on the ashing temperature. Ashing of organic samples a t temperatures below 550’ C. leads to losses by adsorption on carbon particles. Loss of zinc from urine samples by volatilization does not occur a t the temperatures used under the conditions of these experiments. This technique is considerably simpler than those previously reported for the determination of zinc in urine (9) and in spite of the fact that the technique is much more rapid, there is neither loss of
precision nor of specificity. This method was employed in studies on urinary zinc excretion in patients with postalcoholic cirrhosis of the liver (7), which required large numbers of precise replicate analyses. The principle of this method is also applicable to methods for the determination of other metals in urine which involve phase separation. LITERATURE CITED
(1) Hoch, F. L., Vallee, B. L., J . Biol. Chem. 181, 295 (1949).
(2) Sandell, E. B., L‘Colorimetric Determination of Traces of Metals.” v. 620, Interscience, New York, 1950. (3) Thiers, R. E., in “hfethods of Biochemical Analysis,” Vol. V, p. 301, ed. by D. Glick, Interscience, New York, 1957. (4) Thiers, R. E., Williams, J. F., Yoe, J. H., AXAL.C H E ~27, . 1725 (1955). (5) Vallee, B. L., ANAL. CHEM.26, 914 (1954). (6) Vallee, B. L., Gibson, J. G., 11, J. Bzol. Chem. 176, 435 (1948). ( 7 ) Vallee, B. L., Wacker, W. E. C., Bartholomay, A. F., Hoch, F. L., New Eng. J . Med. 257, 1055 (1957). (8) Weitzel, G., Fretedorff, A. M., Z. physiol. Cheni., Hoppe-Seyler’s 292, 212 (1953). (9) Wolff, H., Busse, G., Biochem. Z . 322, 154 (1951). RECEIVEDfor review April 1.5) 1958. Accepted July 10, 1958. Studies supported by a contract between the Office of Naval Research, Department of the Navy and Harvard University, Contract Nonr. 1866(04), Kr 119-277, by a Grant-inAid from The National Institutes of Health, Education, and Welfare, and by the Howard Hughes hledical Institute. J. H. R. Kagi is a fellow of the American Swiss Foundation for Scientific Exchange.
A Fractional Sublimation Technique for Separating Atmospheric Pollutants JEROME F. THOMAS, ELDON N. SANBORN, MlTSUGl MUKAI, and BERNARD D. TEBBENS Sanitary Engineering Research Laboratory, University o f California, Berkeley, Calif. Fractional sublimation offers a new possibility for separating relatively large quantities of the particulate organic material found in polluted atmospheres. A method has been developed using a mixture of condensed polynuclear aromatic hydrocarbons of known composition. A quantitative separation can be made on this mixture, or any mixture of compounds that can be sublimed. When applied to an atmospheric sample of unknown composition a resinous component interferes, and several fractionations are required to remove the resinous material from the
1954
ANALYTICAL CHEMISTRY
field. Obtaining relatively large amounts of pure material i s important both for identification purposes and from the standpoint of public health. The carcinogenic activity of many individual compounds found in polluted air has never been tested because it has been impossible to obtain pure material in gross amounts.
T
HE PARTICULATE ORGANIC K 4 T E R I A L
of polluted urban atmospheres is common to the particulate organic material of the combustion effluent of any liquid or gaseous hydrocarbon fuel
subjected to incomplete combustion (7-9). Several qualitative aspects of the particulate organic type of pollutant have been investigated, including a division of these pollutants into broad functional chemical classes, the separation of pure compounds within the classes, and the identification of some compounds ( I O ) . Approximately 100 high molecular weight organics have been separated in pure form, but only a small percentage have been positively identified. This paper deals with some of the difficulties associated with the quantita-
tive estimation of the particulate organic matrrinl. Both qualitative and quantitative analyses have bccn tedious because nunierous compounds are involved, and it has bcrn possible to obtain only very minute quantitics of each. Chromatography has been the principal tool used for the ultimate separation; paper chromatography gives good resolution of microgram quantities of matrrial, while column chromatography separates slightly larger quantities, but the resolution is poor. Fractiona.1 suhlimation (2, 6), a new techniqur in this firld, is a method for resolving lsrgrr quantities of nmterial. It is important that larger quantities of pure material be obtained in order to test the carcinogenic activity of many individual compounds present in polluted air. Previous biologic trsts with air extracts have h e n performed only on mixtures (4). This technique enables a semiquantitative determination of single components in a given volume of aerosol sample, making it possible to ascertain if compounds of known careinogcnic activity-for example, 3,bhenzpyrene-are present in significant quantities. EXPERIMENTAL
A fractional sublimation technique was devrloped on a mixture of known composition consisting of anthracene, pyrene, 3,4-benzpyrene, and 1,2,5,6dibenzanthracene. This was considered a representative mixture because, as indicated in Figure 1, these hydrocarbons are knoum to he present in polluted air, they can be cleanly s e p a rated by chromatography, and the last two compounds of t,he mixture have known cnrcinogcnic activity. A schematic draving of the apparatus used for t,he fractional sublimation is shown in Figure 2. Samples are weighed in a t,ared boat and placed in the glass sublimation tube which is then evacuated. The sublimation tube slides through a cooling jacket into a special heating furnace which allows a linear temperature gradient to be established along the sublimation tube. The miuture va,porizes from the boat and sublimes out along the tube at a rate which is a function of the pressure and t e m p e r a t u r e t h a t is, the material most readily sublimed moves a t the fastest rate. Thus t,he various compounds separate in t,he order of increasing sublimation trmperature which is a function of vapor pressure. The linear temperature gradient is established by heating one end of a thick-walled, horizontally mounted copper tube to the desired maximum, and allowing the heat to dissipate by conduction to, and radiation from, the opposite end. Encasing the copper tube and heater in a controlled outer
heating jacket enables the low point on the gradient to be controlled. The gradirnt may be adjusted in several ways. Increasing the voltage on the sliding heater causes an increase in temperature at the hot end of the tube. Moving the sliding heater up the copper tube rapidly increases the gradient range and hastens the sublimation of the less volatile components. Increasing the voltage on the heating jacket causes a decrease in the gradient range, an increase in temperature of the cold end of the furnace, and also an increase in the rate of sublimation of the less volatile componcnts. The last two steps are taken only after the more volatile components have already begun to sublime. The various temperatures ivere determined by positioned thermocouples and an eight-point Brown temperature recorder. A typical linear temperature gradient for one set of conditions is shown (Figure 3). Pressure regulation is also of great importance in the operating range of 5.0 to 0.01 mm. of mercury as temperature is the controlling variable in this range. It is also controlling above this range but the sublimation rates are too low for practical purposes, and below 0.01 mm. of mercury, the entire mixture moves from the furnace without fractionating, Adjusting'and maintaining the desired pressures over the entire operating range was difficult, as all manostats tested failed to operate satisfactorily below 1.0 mm. of pressure. This was overcome by placing a needle valve in the system and maintaining a very small pressure differential across the valve using a relatively poor vacuum pump on the outer side of the valvc. Using this technique, it is possible to adjust the pressure rapidly for outimum conditions of separation. P ill below 1.0 mm. were dett%mined b3-
pressures
means of a Pirani vacnum gage-brtiwen 1.0 and 5.0 nim., a tipping AZcLeod gagr was used. The varioue temperatures and prmsures maintained throughout a typical separation are givrn in Tahle I. These are considercd dose to optimum conditions but urrr established on an empirical basis. The sr,pxration of a mixture into fractions is a relativrly ra.pid process and certain precautions must he taken to avoid contamination of the leading fractions. The initial fractions must he separated undrr v e r y mild conditions with rrspect to temperature and pressure, because under more strenuous conditions, the volatile material, comprising the initial fractions, moved out so rapidly that the relatively nonvolatile components were physically carried along in the wave and appeared as contaminants in the early fractious. I n operation it is better to move thr more volatile components out as a single leading fraction and refractionate this leading fraction under vrry mild conditions in a second tube. In a mixture of known composition, thc leading or volatile fraction was a mixture of anthracene and pyrene which moved rapidly out of the furnace, and collected as a narrow band of crystalline material in the cooling jacket. The cooling jacket has a twofold purpose. The material leaving the furnace meets the cold surface where crystallization from the vapor state immediately occurs. Srcondly, all sublimate fractions are contained in the jacket which inhibits the loss of thr more volatile material, The remaining material separates in the hot tube as a function of the temperature gradient and tresswe. The sublimation tube is then-moved slightly further out of thr furnare and thr pressnrr or trmperature
Figure 1. Chromotographic separations of hydrocarbons (all common to urban polluted atmospheres) A. Hydrocarbons obtoined from incomplete combustion of 1 -butene 8. Hydrocarbon mixture of known composition used for illus. trotion c. Pvre fmdlon D. Individual hydrocarbons separated from mixture of B by sublimation F. Pure fraction G. 3.4-Benzpyrene separated by sublimation from hydrocarbon sample obtained from incomplete combustion of I-butene
I
=
VOL. 30, NO. 12.
DECEMBER 1958
1955
gradient, or both,.are adjusted, so that the next fraction will sublime from the furnace to the clean, cold surface of the tube forming the second ring of crystalline material. At approximately IO-minute intervals, pressure and temperature adjustments are made, attenda n t on the stepwise withdrawal of the tube. Thus the mixture is separated as a series of crystalline bands contained under the cooling jacket. A separation of the mixture of known composition is shown in Sample A , Figure 4. The leading fractions of the initial separation containing anthracene and pyrene have been senarated further as shown under Sample-B, Figure 4. Table I includes the weixhte of the fractions containing 3,4ben&rene and 1,2,5,6-dibenzanthraccne. As indicated by these weights, the initial fractionation is only a semiquantitative recovery, which could be improved by resubliming a range of fractions containing any desired compound; in Sample A this would include fractions A3 through A8 for 3,4--benapyrene. Paper-strip chromatography was used
tions are shown in Figure 1, G , D, E, and F. A variation in the technique was used to exemplify one aspect of puhlic health significance. The aerosol sample of unknown composition obtained by burning 1-butene under conditions of incomplete combustion was fractionated by the suhlimation technique. The separation is shown on Figure 4 under Sample C and the conditions are given in Table 1. The fractions were chromatographed and only those containing 3,4-henapyrene were collected and refractionated in a second sublimation tube. The condition and pictures of this separation are included as Sample D in Table I and Sample D on Figure 4. Chromatography was used again to indicate the fractions containing relatively pure 3,4-benapyrene. As these individual fractions can be weighed directly on an analytical balance, a direct calculation of the amount of 3,4benspyrene contained per unit volume of air sample is possible. For the calculation to he completely valid, the mixture must move quantita-
as the most sensitive criterion to show the presence of individual components in the various samples and also to indicate the purity of the fractions separated by sublimation. Figure 1, A , shows a chromatographic separation of the aromatic hydrocarhons contained in an aerosol sample of unknown composition, ohtained by burning a simple gaseous fuel under conditions of incomplete combustion. These a n comparable in composition to the corresponding fractions in representative polluted atmospheric samples (8). Figure 1, B, shows a chromatographic separation of the hydrocarbon mixture of known composition. The fractions obtained by sublimation are separated by cutting the sublimation tube into annular sections. These fractions are chromatographcd, and the presence of a single hand on a chromatogram is used as an indication of a pure sublimate fraction. In Figure 4, the pure frnctions are indicated by arrows and are separated from each othkr generally by fractions of a binary nature. The chromatograms of the nure frac-
L
3
Figure 2. Schematic drawing of fractional suniimation apparatus SAMPLE A
w uv
SAMPLE B
w uv
SAMPLE
E
w uv
I €NE
ENE
EN
Separations of mixtures in sublimation tubes bon mixture of known composition f cinthroccne and pyrene obtained from Somple A bon mixture obtained from incomplete Combustion of I-butene 1 firoctionr of Sample C containing 3.4-benzypyrene W . Photognzphs token under white light uv. Corresp,onding photography taken under short wove length dtroviolet light
1956
ANALYTICAL CHEMISTRY
SAMPLE
w uv
tively up the sublimation tube, and each fraction must ultimately contain pure compounds. The fractionation, with respect t o polynuclear aromatic hydrocarbons, is good, as indicated by the chromatogram of Figure 1, G, but all fractions obtained from naturally occurring samples as well as the fractions obtained from the combustion effluent of simple fuels are contaminated with a resinous component. This component cannot be moved quantitatively from the sublimation tube as it appears t o be a series of homologous or vinylogous compounds, the majority of which have a very high molwular weight, and which remain as pot residue. Repeated fractionations are required to ultimately purify the aromatic hydrocarbons. The resinous material quenches almost all fluorescence. as indicated in the photographs of Samples C and D taken under ultraviolet light (Figure 4'1, even when present in minute quantities. It chromatographs by conventional techniques, has an Rj value slightly greater than anthracene, and appears on the chromatograms as a yellow band under white light. Because it can be separated chromatographically, it does not interfere with chromatographic techniques. A continuing study is being made of this resinous component. Although the fractions containing 3,Cbenzpyrene are contaminated with the resins, the recovery m-eights given in Table 1 are considered fairly accurate. The contaminated 3,4-benzpyrene was resublimed several times by the technique described, TT ith the ultimate recovery showing a strong fluorescence in the sublimation tube 1Tithout a significant loss in weight of 3,4benzpyrene.
a factor of prime importance in this dis-
cussion. The theory involved in a typical separation is exemplified in Figure 5 . Consider the separation of pyrene from 3,4benzpyrene a t a pressure of 1,0 mm. of mercury. On the figure, a horizontal line is drawn through the 1.0-mm. read-
Table 1.
Conditions Established to Separate Mixtures b y Fractional Sublimation
Fractionb
Wt., Mg. Sample Aa
1,2,5,&Dibenzanthracene (4.7 mg.)
1.9 Sample Be 8.5
Anthracene
0.01
3.6
Pyrene
21 21 20
3.0 1.0 1.0 0.1 0.1 0.1 0.1 0.1 0.01 0.01 0.01
0.6 0.2j 0.7
(4.0 mg.)
Distance into Furnace from Cooling Jack& Sliding heate; Boat
Pressure, hIm.
0.7 1.3
3,CBenzpyrene
4.3
18
17 16 15
14
13 12 11 10
3
5.0 5.0 5.0 5.0 5.0 3.0 3.0 3.0 3.0 1. 0
14 14 13 12 11
3.0
21 20 20 19
10
9
8
,
c
6
21 21 20 18 17 16 15 14 13
12 11 10 18
18 18 17
16 15 14
13 ~~
12 12
Sample Ca
c4 c5 C6 c7 '
DISCUSSION
The order of separation is dependent on the vapor pressure variations of the individual compounds as a function of temperature. Information in the literature concerning these variations for condensed polynuclear aromatic hydrocarbons is very limited, but even without specific information, it is possible to make broad generalizations to explain the theory involved. The manner in which vapor pressure varies with ternperature is shown graphically in Figure 5. The curve for anthracene is available in the literature (1, 3, 6), but the curves for the other three compounds in the known mixture are not available in the operating range and have been arrived a t through an interpretation of experimental results. For illustrative reasons. the curves have been extended to include temperatures above 250 O C., the decomposition temperature for these hydrocarbons. Although the absolute values are not accurate, except for anthracene, the approximate shape and the relative positions are accurate-
ing intersecting the m p o r pressure curves. Vertical lines are dropped from the points of intersection to the temperature scale on the horizontal axis. As the pressure is adjusted to a constant value throughout the system, the temperatures indicated represent an equilibrium between the rate of evaporation of the
1.0 1.0 1.0 0.1 0.1 0.1 0.1 0.1 0.01 0.01 0.01
16.9
C8
c9 c10 c11,
12
21 20 20 19 18 17 16 15 14 13 12
11
11
21 21 20 19 18 17 16
21 21 20 19 18
18
17 16 15 14
13
Sample Da 3.0
1.o
1.0
0.1
3,4Benzpyrene
+
0.1 0.1 0.1
5.7
0.1
0.1 0.01 0.01 0.01 0.01
17
16
I5
I5
14
14
13 12 11
10
13
12
11 10
See legend, Figure 4. Eachfraction corresponds to a band shown on Figure 4. Each fraction collected over a 10-minute time interval. c Samples .4, C, and D. Temperature maintained at boat 245" C. Temperature at cold end of furnace Initial 135' C. Final 185' C. Sample B. Initial temperature at boat 170' C. Temperature a t cold end of furnace Initial 140' C. Final 150' C. a
b
~~
~~~
~~
VOL. 30, NO. 12, DECEMBER 1958
1957
5
LITERATURE CITED
li
( I ) Bradley,
Figure 5. Vapor pressure curves of hydrocarbons in mixture of known composition
4
-I'
3
I
O 0
M
100
150
200 TEMPERATURE
250-
'sL O
DECOHPOSIllON
('Cl .
T
J
619 (1954). (3) International Critical Tables, T'ol. 3. D. 208. IllcGraw-Hill. Sew York.
1928. ( 4 ) Kotin, P., Falk, H. L., ?&der, P., Thomas, AI., A n i . M e d . dssoc Arch. Ind. Health 9, 153 (1951).
15) Mortimer, F. S., Murphy, R. V., Ind. Eng. Chent. 15, 1140 (1923). 16) Reid, A. F., U. P. Patent 2,628,892 (Feb. 17, 1953). ( 7 ) Tebbens, B D., Thomas, J. F , LIukai, ?VI., Am. M e d . dssoc. Arch. Ind. Health 13,567 (1956). (8) Zbid., 14, 413 (1956). (9) Tebbens. B. D.. Thomas. J. F.. San' born, E. Mukai, AI., .4h. I n d . Hyg. Sssoc. Quart. 18, 165 (1957). (10) Thomas, J. F., Tebbens, B. D., Miikai. LL, Sanbcrn, E. S . , ANAL. C H E M . ' 1835 ~ ~ , (1957 I .
s,,
IURUACE QlMlLNT
molecules from the surface of the solid, and the rate of return of molecules to the solid. Below these particular temperatures for each of the compounds, the equilibrium is shifted toward the solid phase. If the cold point of the temperature gradient in the furnace is adjusted to fall between the two equilibrium temperatures, preferably closer to the more volatile component. in this example pyiene, pyrene will be driven from the furnace and will condense as crystals in the cold finger.
R. S., Cleasby, T. G., J . Chem. Soc. 1953, p. 1690. (2) Hausmann, W., ASAL. CHEM. 26,
In actual operation a vapor pressure curve is desirable for each component in a sample, but it is possible to determine empirically the optimuni conditions for fractionation in much the same manner as is done in any type of fraction collector. ACKNOWLEDGMENT
Conrad Kwasnicki, University of California, Berkeley, rendered significant assistance.
RECEIVED for review Sovrniber 4, 1957. .4CCEPTED Jul>-25, 1958. Division O f Industrial and Engineering chemistry, Air Pollution Symposiiim, 132nd Meeting, ACS, S e w York, S . T.,September 1957. Supported b r research grant RG-4281 of the Sational Institutes of Health, Public Health Service, cooperative effort within the University of California School of Pub!ic Health and Department of Engineering.
.Chemical Analysis by Measurement of Reaction Rate Determination of Acetylacetone W. J. BLAEDEL and D. L. PETITJEAN' Chemistry Department, University of Wisconsin, Madison, Wis.
b Work was undertaken to investigate the possible development of a more general, rapid, and accurate method of analysis based on kinetics than now exists. A preliminary study of the utility of the suggested approach was carried out with a well known reaction -the alkaline hydrolysis of ethyl acetaie. The developed method was then applied to acetylacetone determination by measurement of its reaction rate with hydroxylamine hydrochloride to demonstrate that it is equally applicable to integral-order or complex reactions. Simple systems free from interfering substances can be quickly analyzed with an ultimate accuracy of about o.3Y0. The method can probably b e extended to mixtures and samples containing chemically inert interferences with some loss in accuracy.
T
HE analytical utility of kinetics has been recognized for some time in inorganic analytical chemistry. and niicro-
1958
ANALYTICAL CHEMISTRY
chemical applications have been discussed in detail (6, 9, 94). Catalytic activity has found considerable qualitative application, allowing the detection of minute quantities of various elements. With increasing interest in the accurate determination of microgram quantities of materials, catalytic activity is being found extremely useful in quantitative applications as well. For example, the catalytic influence of iodide ion on the reaction between ceric ion and arsenious acid (19) permits the determination of microgram quantities of iodine and has been the subject of many papers for more than 20 years (11, 14). More recently, methods have been developed for determining submicrogram quantities of silver by its catalytic effect on the oxidation of manganous ion by persulfate ( 2 3 ) , and for determining comparable concentrations of copper by its catalysis of the autoxidation of resorcinol (IO). With the increasing importance of trace impurities, further quantitatire applications of kinetics can be expected in inorganic analytical chemistry.
The utility of kinetics in quantitative organic analysis, hon.ever, has not been as extensively recognized. Qualitative applications, such as the differentiation of sugars by time of osazone formation, and of organic halides by time of silver halide formation, are of frequent use in characterization Ivork. Quantitative applications, on the other hand, are rare. Perhaps the earliest and one of the best examples of the potential value of kinetics in quantitative organic analysis is found in a method for the resolution of mixtures of the normal butenes ( 5 ) . illthough modern infrared equipment now provides a simple solution to this problem, it n a s not available a t that time. Howeyer, the problem of resolving such mixtures was solved by detrrmination of the total butene concentration, the density of the mixture, and a pseudo first-order reaction rate constant
1 Present address, -4lcoa Research Laboratories, Aluminum Co. of America, Sem- Kensington, Pa.
