QUANTITATIVE CAPILLARY LUMINESCENCE ANALYSIS
1071
LAWRENCE, A. S. C.: Trans. Faraday SOC.34,680 (1938). LAWRENCE, A. S. C.: Trans. Faraday SOC.33, 325 (1937). LEDERER,E. L.: Kolloidchemie der Seijen. Th. Steinkopff, Leipeig (1932). For references see MCBAIN,J. W.: Nature 146,702 (1940). MCBAIN,J. W.: I n Alexander’s Colloid Chemistry, Vol. I, Chap. 5. Chemical Catalog Company, Inc., New York (1926). BROCK,G. C., VOLD,R. D., AND VOLD,M. J.: J. Am. Chem. (9) hfcBAIN, J. W., SOC.80, 1870 (1938). (10) MCBAIN,J. W., AND FIELD, M. C.: J. Chem. SOC.1933, 920. (11) MCBAIN,J. W., AND LAING, h.1. E.: Kolloid-2. 36, 18 (1924). L. H., AND PITTER, A. V.: Z. physik. Chem. Al47, (12) MCBAIN,J. W.,LAZARUS, 87 (1930). (13) hfcBAIN, J. W., AND MC~LATCHIE, W. L.: J. Phys. Chem. 86,2567 (1932). M.: J. Phys. Chem. 44, 1013 (1940). (14) MCBAIN,J. W.,VOLD,R. D., AND FRICK, (15) MCBAIN,J . W., VOLD,R . D., A N D VOLD,M.J.: J. Am. Chem. SOC.60,1866 (1938). (16) MOLL,W.L.H.: Kolloid-Beihefte49,l (1939). This paper also gives numerous references t o earlier work of Wo. Ostwald and collaborators. (17) PAULING, L.: The Nature of the Chemical Bond. Cornel1 University Press, Ithaca, New York (1939). (18) SMITH,E. L.: J. Phys. Chem. 36, 2455 (1932). (19) VOLD,R.D.: J . Phys. Chem. 43, 1213 (1939). (20) VOLD,R.D.: Soap and Sanitary Chemicals 16, 31 (1940). (21) VOLD,R. D.,A N D VOLD,M.J.: J. Am. Chem. Soo. 61, 808 (1939). (22) WEITBY,G. S.: Colloid Symposium Monograph 4, 213 (1926). (4) (5) (6) (7) (8)
QUAKTITATIVE CAPILLARY LUMINESCEXCE ANALYSIS’ FRANK E. E. GERMANN
AND
JAMES
W. HENSLEY
Department of Chemistry, University of Colorado, Boulder, Colorado Received July 3, I940
Although attempts to determine concentrations by capillary adsorption methods have been made by Holmgren (10) and by Schmidt (21), little has been done along this line in the case of capillary luminescence analysis. Guyot (8) used the term “Quantitative ResearcH” as applied to the determination of color and intensity of fluorescence, and Neugebauer (13)stated that he would publish methods for the determination of concentrations using the methods of capillary fluorescence analysis. This question has also been considered by Eisenbrand (1, 3). Since the methods of capillary luminescence analysis are known to be valuable in identifying traces of material (4),it seemed worth while to study the quantitative aspects more closely by means of a direct vision 1 Presented a t the Seventeenth Colloid Symposium, held a t Ann Arbor, Michigan, June 6-8. 1940.
1072
FRANK E. E. GERMANN .\KD JAMES W. HENSLET
fluorometer developed in this laboratory (5). The prepamtion of reproducible capillarized strips has been the subject of very extensive investigation in the pioneer work of Schoeiibein (23) and of Goppelsroeder ( 6 , 7 ) and in the morc recent work of Plats (15), Neugebauer (12), Heinrici (9), and Rojahn (19). The factors involved include the nature of the solvent, the type of paper, tlic direction of cutting, the depth of iinmersion, the shape of the coritaiiier, the conccntration of tlic solution, the quantity of the solution, air currents, temperature, pressure, humidity (or degree of saturation of solvent vapors around the paper and above the solution), and illumination (in the cas(’ of photoactive substances), After studying the characteristics of various papers, includiiig their ow11 fluorescence, it via4 found that for our \\ark the Carl Schleicher and Schiill paper KO.598 was most suitable. Tlic hriglit to which u given liquid \vi11 rise in a given papei drpends upon thcl ratc a t which the liquid evaporates from the paper. If tlir cbntaiiier is sealed, the liquid will rise to the top of the papcr. By chaiigiiig the rate of evaporation by suitable nieans. onr may obtain anything from very sharply defined to very broad “washed out” bands, as pointed out by various iii\wtigators (15, 18, 22, 14, 17. 18, 20). 111 o m set of experiments the strip of paper was attached to the cork which closed the top of the cylinder, and the bottom was allowed to dip into 0.5 per cent solutions of phrnaiithienc, pyrene, and colchicine in acetone. When capillariaation wab allowed to contiuue iii this way a t constant teniperaturc for 8 to 10 hr., thr liquid row to the top of the paper and most of thr fluorescent material was deposited on the stopper. When capillarizatioii wab stopped beforc the liquid had reached the top, very little liquid had risen in the paper, and the fluorescent bands were faint. With more dilute solutions, the concentration of material in the bands was too low for definite tests. The aim of the present work was to prepare a series of strips capillarised in a series of concentrations of the same compound or group of compounds in the same solvent in order to determine if there was any relation between concentration and the width, intensity, or position of the bund. I t wab therefore decided to prepare each set of strips simultaneously and thus eliminate differences arising from variable external conditioiis such as temperature and pressure. Thus the strips in a given series should be comparable, eyen though they might not necessarily be romparablc betlveen sets. ‘rlir work was carried out in a dark room in which the maximum temperatuie variation during x 12-hr. period a t night was found to be 2”C., provided no one entered the room and the ventilation fan was off, Seven 10 x 40 cm. glass cylinders were used. Uniform 50-cc. glass beakers were placed in the bottom of the cylinders. Ten cubic centimeters of a solution containing 0.00025 g. of phenanthrene in 100 cc. of carbon tetrachloride was placed in each of the seven beakers, and strips were
QUANTlT.4TIVE CAPILLARY LUMINESCENCE .iE.ILYSIS
1073
suspended, by means of a support a t the top of each cylinder, so as just to touch the bottom of the beakers, the. cylinders being left open to the air. Over a period of 8 hr. the solutions were complet,ely absorbed and the solvent had evaporat'ed, leaving the strips dry. Under these conditions all the strips showed the blue fluorescent band of phenanthrene about 17 cm. from t.he bot,tom of the paper. The bands were uniform in color and intensity as shown by the fluorometer, the only variation being that the position of the peak of the band showed a maximum shift of about 3 mm. as measured from the bottom of the paper. St.rips from different series showed a slightly greater variat,ioii. *illthough cvery precaution was taken to clean pipets and beakers thoroughly, an occasional strip was found to be entirely different from t'he rcst. However, ;inch occurrences were infrequent, could be easily detected under the ultra\4olet lamp, and serve to show the extreme sensitivity of the method. In making fluorometer measurements, a horizontally placed slit with variable width was used. The length of the slit was thus placed across the 2 cm. width of the paper, which was susceptible to vertical motion. In t'his work the slit' width was adjusted to 5 mm.. so that the entire length of the strip was viewed by the photocell in successi1.e sections 2 cm. wide and 0.5 em. high. Thus the centers of successive areas measured were 0.5 em. apart. Under these conditions, the suni of the iiidividual galvanometer deflections is proportional to the tot'al fluorescence energy of the entire strip. If t,he deflection given by the blank paper is subtracted from each reading, then the sum of the residual deflections is proportional to t'he total fluorescence c:nergy of the adsorbed material on thc strip. Rather than calculate the sum of the net ordinates, we may merely obtain the area under the curve, which will also be proportional to the total energy. The advantage of this is that it, will not be necessary to take readings on every 0.5 cm., nor will the strips viewed have to be adjacent. They may overlap or, in case the intensity is varying gradually and uniformly along the length. they may be separated by finite intervals. All that is necessary is to know the exact position of the center of the slit from the lower end and to keep the slit, width constant. n711en ciirves of this nature were plotted for the series of strips produced by the same concentrations, the areas were sensibly constant, although the peak of the band was not always a t the samc place, as shown in figure 1. In order to see what possible relation existed between curves of this nature when the concentration was a variable, series of different concentrations of different materials were made up by dilution such that the concentrations varied in the order of 1, 112, 1/4, 1/8, 1/16, etc. Thus, starting with a solution containing 0.1 g. of phenyl-alpha-naphthylamine in 100 cc. of acetone, dilutions were made so as to give a series of coiicenand 0.00312 g. per liter, When trations of 0.1,0.05,0.025,0.0125,0.00625,
1074
F R A N K E. E. G E R M A N N AND J A M E S W. H E N S L E Y
filter paper strips were treated as described above with equal volumes of solutions of these concentrations, and fluorometer readings were taken, the series of curves shown in figure 2 was obtained. The areas under these curves increase progressively as the concentration increases, yet it is obvious that, although the area under curve 5 is approximately double the area under curve 6. the area under curve 1 is far from being double that under curve 2. The readings of intensities were made as rapidly as possible after the strips had been permitted to dry in the air and in the dark. Phenyl-alpha-naphthylamine was found to be quite photoactive, IO
9 8
8
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7
7
L
4:
a
!: $
3
4
2
!W 4
td i 23 +
1
I
41 0 OISTANU:
ALONG STRIP IN CM
DSTANCE ALMJG STRIP IN CM
FIG.1
FIG.2 FIG. 1. Fluorescence intensities along capillary strips prepared from carbon tetrachloride solutions containing 0.00025 g. of phenanthrene per cubic centimeter FIG.2. Fluorescence intensities along capillary strips of phenyl-alpha-naphthylamine series. Acetone solutions. Curve 1, 0.001 g. per cubic centimeter; curve 2, 0.0005 g. per cubic centimeter; curve 3, 0.00025 g. per cubic centimeter; curve 4, O.OOO125 g. per cubic centimeter; curve 5,0.0000625g. per cubic centimeter; curve 6, O.ooOo312 g. per cubic centimeter.
and the galvanometer readings fell off rapidly as a given section was being exposed to ultraviolet light. In a short time a distinct darkening of the fluorescence on the exposed area could be detected. When this series was run, no reference standard was used, but readings were made at approximately the same line voltage on the lamp, in order to keep its light intensity constant. As shown by later work, this was not as satisfactory as adjusting the voltage so as to give the same galvanometer reading when a standard barium platinocyanide powder was placed over the slit in front of the fluorometer.
QUANTITATIVE CAPILLARY LUMINESCENCE ANALYSIS
1075
The supposition that the energy of fluorescence will be proportional to the concentration rests on the assumption that all or a definite fraction of the material is exposed to the ultrsviolet light. This holds true only on condition that the adsorbed fluorescent material is deposited in a very thin layer of microcrystals. If the crystals tend to become large, then the surface exposed to ultraviolet light is no longer proportional to the concentration. This offers one possible explanation of the area under curve 1 of figure 2 not being double that under curve 2. The nature of the filter paper might easily have an important action in the determination of the grain size, so that a paper might befound which would yield the area ratio desired even for these concentrations. It is, however, obvious that such a relation can be anticipated only for low concentrations or for small amounts of solution in the case of higher concentrations. Another interesting point in figure 2 is the fact that there are two bands of the same fluorescence color from a solution presumably containing a single compound. Three possible causes for this may be advanced. The first is that the phenyl-alpha-naphthylamine used contained R highly fluorescent impurity. However, a more probable explanation is that the compound oxidized in part on exposure to the air in the presence of the solvent and as a result deposited in two places. Both of these possibilities are being checked a t the present time, thc latter by performing the experiment in an atmosphere of hydrogen. Other solvents are also being tried to determine what effect they have on the number and position of these bands. A third possibility which presents itself is that the phenyl-alphanaphthylamine is adsorbed in the lower band, and that the unadsorbed material is carried on by the solvent t o the higher point, where precipitation results from more rapid evaporation of solvent or from causes similar to those producing rhythmic precipitation in Liesegang rings. Pyrene dissolved in acetone acts somewhat like phenyl-alpha-naphthylamine, as is shown in figure 3. In this case most of the fluorescent material is carried to a position near the end of the paper, where it is deposited in a very narrow, bright blue band. The violet fluorescence band produced by fluorene dissolved in chloroform is shown graphically in figure 4. Although the character of the band is similar t o that of pyrene, being deposited far up on the strip, it is nevertheless located somewhat below the capillary limit of the solvent. The small hump to the right of the high peak is the result of the extraction of fluorescent material from the paper and is present in all strips just as it is when pure solvent rises in the paper. I t is always located at the upper limit to which the solvent rises. I n a number of cases where this band is sharply defined and widely separated from that of the solute, it is not shown in the curves. It has been included, however, in several cases to show its general nature.
1076
FRANK E. E. OERMANN AND JAMES W. HENBLEY
Phenanthrene dissolved in acetone yields a single blue fluorescenceband. As shown in figure 5, these bands all start at practically the same distance from the base and increase both in width and intensity as the concentration increases. Areas under these curves, as determined with a planimeter,
?ISTAW
ALONG
STRIP IN CM
FIG.3. Fluorescence intensities along capillary strips of pyrene series. Acetone solutions. Curve 1, 0.0008 g. per cubic centimeter; curve 2, O.OOO4 g. per cubic centimeter; curve 3,0.0002g. per cubic centimeter; curve 4.0.0001g. per cubic centimeter; curve 5,0.00005 g. per cubic centimeter. I
I
z
il
2 E
8I i
t-
Y
SSTAWX
LONG
S-PP
11.1
CN
FIG.4. Fluorescence intensities along capillary strips of fluorene series. Chloroform solutions. Curve 1, 0.008 g. per cubic centimeter; curve 2, 0.004 g. per cubic centimeter; curve 3, 0.002 g per cubic centimeter; curve 4, 0.001 g. per cubic centimeter.
are in the same ratio as the concentrations, showing that in this caae, at least in the range of coiicentrations considered, there is a quantitative relation between the total intensity of fluorescence radiation and the concentration of the original solution. Salol dissolved in acetone posseases a weaker fluorescence than the
QUANTITATIVE CAPILLARY LUMINESCENCE ANALYSIS
1077
materials previously described. Accordingly 20-cc, samples were used in order to obtain higher intensities. The principal band shows a tendency to have two peaks. The peak of rather constant height a t the right in figure 6 is again the result of extraction from the paper, and was also obtained when pure acetone was allowed t o rise in the paper. Berberine sulfate shows an intense yellow fluorescence, so that only very low concentrations were necessary to produce brilliant bands. The fact that these bands start with the maximum intensity a t the base of the strip, as seen in figure 7, probably can be explained by its very low
DISTANCE
ALONG STRIP IN CM
DSTANCE ALONG STRIP IN CM
FIG.5
FIG.6 FIG.5 . Fluorescence intensities along capillary strips of phenanthrene series. .ketone solutions. Curve 1 , O . O O l g. per cubic centimeter: curve 2,0.0003 g. per cubic centimeter; curve 3, 0.00025 g. per cubic centimeter. FIG. 6. Fluorescence intensities along capillary strips of salol series. Acetone solutions. Curve 1, 0.002 g. per cubic centimeter: curve 2, 0.001 g. per cubic centimeter; curve 3, 0.0005 g. per cubic centimeter.
solubility in chloroform. The narrow band a t the right is again the result of the extraction of fluorescent material from the paper by the solvent. Capillary strips made with berberine sulfate are very stable, so that measurements made oyer a period of weeks showed little decrease in fluorescence intensity. Any attempt at a determination of concentrations by capillary luminescence analysis will have to deal with solutions containing, in general, more than one substance in solution. One or more than one of these may fluoresce in filtered ultraviolet light. If the fluorescence bands of several
1078
FRANK E. E. GERMANN AND JAMES W. HENSLEY
DISTANCE
ALONG STRIP IN CM
FIG.7. Fluorescence intensities along capillary strips of berberine sulfate series Chloroform solutions. Curve 1, 0.0003 g. per cubic centimeter; curve 2, 0.00018 g per cubic centimeter; curve 3, 0.000075 g pel cubic centimeter; curve 4,0 000037 g per cubic centimeter
PEENANTEBENE
8.
Curve Curve Curve Curve
1
2. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3 . . ............................. 4 . . .............................
per w.
0.001 0.0005 O.OOO2.5 0.00012
BEBBEBINE EULFATE
g. p&7cc.
0.00015 0. oooO75 0. oooO37 0.oooO18
constituents overlap, then a spectroscopic resolution of the superimposed bands is probably the simplest method of identifying the individual con-
QUANTITATIVE CAPILLARY LUMINESCENCE ANALYSIS
Curve 1
1-
PHENANTHBENE g.
percc.
0 0008
SALOL
g per cc.
0 0002
1079
1080
FRANK E. E. GERMANN AND JAMES W. HENSLEY
Figure 9 represents the results obtained from acetone solutions of salol and phenanthrene. In this series the concentration of phenanthrene was decreased, while that of salol was increased. Here the maxima representing the phenanthrene bands differ from those of the single component by virtue of the addition of intensities of fluorescence of the two components. In curve 4 of figure 9, representing the highest concentration of salol and the lowest of phenanthrene, the two separate maxima of the former are observed a t A and B as in figure 6 for pure salol, where the concentrations were higher than in figure 9. The maximum a t point C represents the phenanthrene band, and D is the band resulting from extraction of the filter paper by the acetone. In curves 1, 2, and 3 this hump was obscured by the intense phenanthrene band. SUMMARY
The above series of experiments shows the feasibility of the application of the methods of capillary luminescence analysis to the quantitative determination of concentrations, within certain limits, of very dilute solutions of fluorescent substances. The solutions may contain one or more solutes. Although both the width and height of the band increase with increasing concentration, the most reliable measure of concentration is total fluorescence energy, which is proportional to the area under the curves. At higher concentrations the crystals may become so large that the light energy is no' longer proportional to the concentration. In this case, the material t o be tested may be diluted until concentration is proportional to fluorescence energy. The data obtained by means of our new fluorometer, using a quartz-capillary mercury-vapor lamp and photronic cell, has been shown to be very reliable in making energy measurements in the study of capillary luminescence analysis. REFEREKCES (1) DANCKWORTT,.P. W., AND EISENBRAND, J . : Lumineszenz-Analyse im filtrierten ultravioletten Licht, 3rd edition. Akademische Verlagsgesellschaft, Leipzig (1934). (2) DHBRB, C.: La Fluorescence in Biochimie. Les Presses Universitaires de France, Paris (1937). (3) EISENBRAND, J., AND SIEWERT, G.: Arch. Pharm. 272, 428 (1934). (4) GERMANN, F. E. E . : Univ. Colorado Studies, Series D, 1, 7 (1940). (5) GERMANN, F. E. E., AND HENSLEY,J. W . : Univ. Colorado Studies, Series D , 1, 37 (1940). (6) GOPPELSROEDER, F . : Verhandl. Nat. Forscher-Ges. Base1 19, 1 (1907); Chem. Zentr. I, 760 (1908). (7) GOPPELSROEDER, F.: Kolloid-Z. 4, 23 (1909). (8) GUYOT,M.: Ann. fals. 24, 196 (1931). (9) HEINRICI, G.: Dissertation, Halle (S), 1932. (10) HOLMGREN, J.: Biochem. Z. 14, 181 (1908); Kolloid-Z. 4, 219 (1909).
E L E C T R O N D I F F R l C T I O N S T U D Y O F H Y D R O U S OXIDES
1081
(11) KREIDL,.\., .ksD LESK, E.: C h i n . Zcntr. 11, 1272 (1911); . h h . ges. Physiol. (Pflugers) 141, 541 (1911). H.: Pharm. Ztg. 74, 1511 (1929). (12) XEUGEBAUER, H. : Die Kapillar-Lumineszenz ilnalyse im yharrunreutischen Labo(13) KEUGEBAUER, ratoriiim. Verlag Dr. Willmar Schwabe, Leipzig (1933). (11) PF.4E, E.: Apoth. Ztg. 41, 133 (1926). (15) PL.ATZ, H. : UbeT Kapillaranalyse, 2nd edition. Verlag Dr. \Villniar Schwnhc, Leipzig (1922). (16) RADLEY,J . A , , ASD GRANT,,J.: Flitorescence .4nnlysis in I'itraviolet Light. Chapman and Hall, Ltd., London (1935). (17) RAPP: Pharm. Ztg. 73, 1585 (1928). (18) ROJAHS,C. A , : Pharm. Ztg. 74, 11 (1929). (19) ROJ.~HN,C. A , , ASD HELLRUSG,.1.:Uber die Identifisierttng der HomoopuLhie gebrauchfen Trockendrogen mil Hilje chemischer Reaktionen und der Lumineszenz-Kapillaranalyse. ilkndemischc Verlagsgesellschaft, Halle(S) (1937). (20) ROJAHS,C. A , , A N D HERZOG, H.: Pharm. Zentralhalle 73, 404 (1932). (21) SCHMIDT, H.: Kolloid-Z 13, 146 (1914); 24, 49 (1919). (22) SCHimDT, H. : In Liesegang's Kolloid-chem. Technologie, p. 211. Th. Steinkopff, Dresden-Leipzig (I 927). (23) SCHOENBEIS, C. F.: Trans. So?. Satural Philosophy Bascl 3, (2) 249-55 (1861); 4, (1) (1864).
A?; ELECTRO?; DIFFRACTIOS QTUDY O F HYDROUS OXIDES AMORPHOUS TO X-RAYS' HARRY B. WEISER
AND
.'!I 0. MILLIGAS
Department of Chemistry, T h e Race Instztute, Houston, Texas Recetved J u l y 9, 1940
In recent years, extended investigations have been carried out a t The Rice Institute and elsewhere on the constitution of hydrous oxide gels by means of the simultaneous application of the techniques of x-ray diffraction and of isothermal and isobaric dehydration. The results of these investigations disclose that a large percentage of the precipitated oxides give x-ray diffraction patterns corresponding to a simple oxide or a simple oxide hydrate or hydroxide. It is concluded therefore that, in general, gelatinous precipitates of the oxides are not polymerized bodies or condensation products resulting from the splitting off of water from hypothetical metallic hydroxides. Instead, the gels are believed to consist of agglomerates of extremely minute crystals of oxide or simple hydrate (or hydroxide) which hold large amounts of water by adsorption and capillary forces (9, 14). Presented a t the Seventeenth Colloid Symposium, held a t Ann Arbor, Michigan, June 6-8, 1940.
