100th day of the initial chromatograms (Table 11, column 4). Coumestrol chromatograms lose 25 to 30% of their fluorescence when stored at room temperature for 100 days. Precision of Measurements. T h e reproducibility of the fluorescence measurement of a given coumestrol spot with the fluorometer was good. Slight variations in measurement occurred from one spot to another, and i t is recommended t h a t the measurements from at least four spots be averaged to obtain a more reliable
value. Measurements presented in Table I11 are typical of fluorometric readings of coumestrol on paper chromatograms. In this study two operators measured the fluorescence of the coumestrol spots using the 30-mm. aperture. The method is reproducible from one operator to another. LITERATURE CITED
(1) Bailey, G. F., ANAL.CHEM.32, 1726 (1960). (2) Bickoff, E. hl., Booth, A. N., Lyman, R. L., Livingston, A. L., Thompson,
C. R., DeEds, F., Science 126, 969 (1957). R. J., Durrum, E. L., Zweig, G., Paper Chromatography and Paper Electrophoresis," 2nd ed., p. 379, Academic Press, New York, 1958. (4) Lyman, R. L., Bickoff, E. M., Booth, A. N., Livingston, A. L., Arch. Biochem. Biophys. 80, 61 (1959). (5) Ven Horst, H., Tang, H., Jurkovich, V., ANAL.CHEM.31, 135 (1959). RECEIVEDfor review April 11, 1960. Accepted August 8, 1960. Mention of specific products does not imply endorsement by the Department of Agriculture over others of a similar nature not mentioned.
(3) Bl:ck,
Precipitation Chromatography Diffusion and Precipitation of Metal Sulfides on Agar Gel Columns JAMES D. SPAIN Department of Chemistry and Chemical Engineering, Michigan College of Mining and Technology, Houghton, Mich.
b When certain metal ions are allowed to diffuse into a buffered agar gel containing sulfide ions, sharply defined colored bands of precipitate develop whose relative positions are determined largely by their solubilities. The name precipitation chromatography is used to describe this procedure because the apparent separation of bands results from a selective reversible distribution between the fixed nonmobile precipitated phase and the mobile ions in solution. The procedure described provides a method whereby metals which produce insoluble sulfides may b e identified either separately or in mixtures by a single simple procedure employing the simplest of apparatus.
A
LTHOUGH diffusion techniques are
recognized as fitting into the larger category of differential migration analysis ( 8 ) , they have so far had limited application because of the poor resolving power of the method in general. Antelman (1, 2) showed that certain mixtures of metal ions could be partially separated by simple diffusion in gelatin on Petri dishes. He employed a variety of developing reagents following diffusion to render the disusion zones visible. A slightly different technique was used by Veil ( 9 ) , who incorporated the precipitant right in the gel and identified metal ions by t>heir characteristic chromate and iodide precipitates. More rccently, 2 precipitant Containing agar gel-impregnated filter gaper was t ~ ~ p l o y pin d the chromatographc separation ci anioiis by ICrishnamurti and Dhareshwar (61. They
1622
e
ANALYT!CAL CHEMISTRY
stated that the sequence of bands was related to the solubilities of the precipitates that formed. Because of its potential as a qualitative procedure, a modification of these techniques has been investigated for possible use in the analysis of the cations which form insoluble sultides. I t is presented here because of its
G.,""
r I
OAR QEL
2M NAOAC O.IM ("$3
Figure I. Appearance of sulfide bands under ideal conditions
extreme simplicity and its possible application where the use of complicated laboratory apparatus is impracticale.g., in the field identification of geological specimens. PROCEDURE
Preparation of Gel Columns. Agar gel was prepared in batches of 200 ml. by putting 4 grams of agar agar (U.S.P. grade shreds) in 100 ml. of distilled water to swell and leach away soluble pigmented material (ea. 30 minutes). The water was discarded and the agar was added t o 200 ml. of boiling water containing 54 grams of sodium acetate trihydrate. This mixture was boiled and stirred for approximately 5 minutes and filtered hot through a thick pad of glass wool into a stoppered storage flask. The columns were prepared from 6mm. soft glass tubing that was cut into 12-cm. sections, constricted a t one end and fire polished a t the other. Portions, 2&ml., of agar gel were softened in boiling water and mixed with 3 drops of ammonium sulfide (Baker and Adamson, light reagent solution). Tubes were filled in the same n-ay as for a pipet by dipping the constricted end into agar and removing when about z/3 full. The constricted IoJTer tip \yas cooled with ice until sufficientll- hard and the column n.as placed upright for approximately 1 hour for complete setting of tlie gel. Application of Sample. Soli:t.ons of n e t a l ians w e r e diluted w i t h 6 M hydrochloric acid until t h e 5n:ii concentration of each mr,tal inn ~,Y proximatrip 5 rug. ?er ml. ':hi tion K::S transfeyred tc t h e top of the c,oli;nin t o a d t p t h of ahG!it 0 :. c n i . I he eojcn-ir:s i ~ t a!lon = ~ ?ti t o dta:id ifi m
Table 1.
Metal Ion
Typical Appearance of Bands of Metal Sulfide on Agar Gel Columns
Accuracy,
%:
Mercury
.4bove Meniscus Characteristic Band White gelatinous Sn(OH), (48 hours) Clear yellow SnS2,sharp black mirror Sharp black HgS
Copper
Green solution CuCl,-*
92
Lead
White crystalline PbC12
Tin
Bismuth Cadmium Antimony
Chalky white SbOCl
Arsenic Zinc Cobalt
Pink solution
Nickel Iron
Green solution Yellow solution turned reddish
0
Combination Bands With Bi, broad reddish black With Cu, diffuse black With Cd, peach-buff With Pb, broad blue-gray With As, red-brown over yellow Greenish black over brown, CuS With Sb, red-brown With Cu, Bi, and Sb, brown Smoky blue-gray PbS With As, clear bright orange With Cd, peagreen to tan Sharp band of white BiOCl, brown Bins3 With As and Sb, red-brown With Cd, tan to orange With Sn, black tinged with red With Hg, peach-buff Opaque yellow With Pb, peagreen to tan With Bi, tan to orange With Cu and Bi, red-brown Orange Sb2S, diffuse lower edge With As, opaque yellow With Cu and Bi, red-brown Clear light yellow, As2Ss With Pb and Sn, clear orange Often merges with other bands Multiple white bands to lighten their color Smoky gray COS,widely spaced periodic ?Toneobserved bands Smoky gray NiS Diffuse black FeS Sone observed
66
72
95 87 64
97 89 100 100 0
100
Nickel could not be distinguished from cobalt by this procedure and was not studied in mixtures.
the same test tube in which dilutions were made, and examined after 1 hour, a t 24, and again at 48 hours. OBSERVATIONS
The color of the original diluted solution and the formation of precipitates above the gel meniscus were noted during the first hour. This information was used in the interpretation of the bands that occurred within the gel. Most of the banding within the gel was completed by 24 -hours, although some bands required an additional 24 hours for complete development. However, i t was not feasible to make observations only at 48 hours since there was some loss of definition by that time. During these first 48 hours, there was a gradual movement of the bands down the column. This was apparently due to a reversible process of precipitation and solut'ion of the metal sulfides as the ions diffused down through the sulfide-containing medium, iollowd by those of the hydrochloric acid. Columns into which only 631 hydrochloric acid had diffused for 24 hours shoived that the p H changes in approximately linear fashion with tiistance, ranging from about. 1 a t the meniscus to about S near the bottom. r'lie actual values obscn-d .+pmd on thc time allon-c~ifor clifiusion ?o :rl,kr place. From one poinr of view! it, sppiars as though the metal ions inti1 reaching a point where the
p H was such that the sulfide ion concentration was sufficient to exceed the solubility product constant. Most of the metal ions tested formed single discrete bands which could be identified by their relative positions on the column, and by the colors observed in transmitted and reflected light. Generally, the bands developed in order of increasing solubility of the metal sulfide precipitates involved. No banding of this nature occurred when precipitant was added after the diffusion process had taken place (24 to 48 hours). This procedure produced extensive darkening from the metal sulfides formed, but precipitation was superimposed and boundaries were diffuse. Only in a few cases could more than one ion have been identified in the mixtures employed using this latter procedure. Some metal ion pairs tended to combine to form a single band, possibly because of a coprecipitation type of phenomenon. The combination bands usually showed colors as fully distinctive as those produced by single ions. I n some cases one of the ions involved in the combination band also formed its normal band. Twelve metal ions have been studicd to determine the characteristic bands which are produced separately and ill mixtures containing as many as four components (Tah!e I). The ions are pescnt,ed in the approxirnate qrder :n which t,hey Appear starting from the top cf the column.
The formulas given are those which seem most ,reasonable under the conditions. KO analyses were performed on the precipitates which developed. Accuracy for a particular metal ion is expressed as percentage of correct identifications on the last 100 synthetic mixtures examined. The procedure as described was found to be applicable to metal ion concentrations as low as 1 mg. per ml. DISCUSSION
The technique described is a differential migration procedure in which diffusion is employed as the motive force. Selective reversible distribution takes place between the fixed nonmobile precipitated phase and the mobile ions in solution. This type of equilibrium qualifies it as chromatography ( 8 ) , and since the nonmobile phase is in the form of a precipitate, the name precipitation chromatography (3,7 ) seems most fitting. The t,echnique employed here is very closely related to cation exchange chromatography except that the negat>iveions that make up the column are immobilized only during combination with thd positive metal ions bring chromatographed. It should be notr:d that meta! ions do not actually hecomp srparatcci. by this procedure. Xlthougli there is d definite separation of thc me%! aulfide ban&, tach band will :)e ,-ontami:iaic.d rsit,h the soluble x e t a l ions of t,he bancis n-hit:!i prectde it d~~n.11 thr. coiumn. In t h s VOL. 32, NO 12, NOVEMBER 1960
e
1623
respect, it is essentially a frontal analysis type procedure similar to that used in other types of chromatography. The use of this procedure for the qualitative identification of metal ions depends upon three factors. The first factor of importance is that the metal sulfide precipitates have distinctive colors and textures under the conditions employed. Secondly, the relative positions of the bands appear to be constant and determined primarily by the solubility products of the metal sulfides ( 5 ) . Absolute positions, on the other hand, are affected by the concentrations of sulfide ions, of metal ions, of the acid, and of the buffer, as well as temperature, agar strength, and other variables which affect the rate of diffusion. The final factor of
importance is that band limits are sharply defined, unlike simple diffusion boundaries. The theory behind diffusion with discontinuous boundary has been worked out by Hermans (4). Both theory and experiment show that the phenomenon is quite general in all cases in which diffusing particles react with the medium to form a precipitate. The three factors just discussed would not appear to be unique to sulfide precipitations and, therefore, it seems reasonable to predict that the technique of precipitation chromatography could have a wide application.
LITERATURE CITED
(1) Antelman, M., ANAL. CHEN 26,1218l9 (1954). (2) Antelman, M., Eby, D., Ksuffmsn, G. B., Zbid., 31, 829-33 (1959). (3) Gapon, E. N., Belenkaya, J. M., Colloid J . (U.S.S.R.1 (Eng. Ttansl.1 14, 353-66 (1952). (4) Hermans, J. J., J. Colloid Sci. 2, 38798 (1947). (5) Kolthoff, I. M., Moltzau, D. R., Chem. Revs. 17, 293-325 (1935).
( search6 ( ~~
~
~
~t. z;;,
ACKNOWLEDGMENT
~ 8, 559-60 d ~ (1955). ~ ) (7) Schafer, H., Neuyebauer, W., 2.anorg. u. allegem. Chem. 274, 114-40 (1953). (8) Strain, H. H., ANAL.CHEM.31,818-21 (1959). (9) Veil, S., Compt. rend. 199, 611 (1934).
The author is indebted to the Michigan College of Mining and Technology for financial support during this work.
RECEIVED for review October 16, 1959. Accepted September 6, 1960.
Use of Tritium Tracer for the Determination of Hydrogen in Aluminum ARTHUR S. GILLESPIE, Jr. Alcoa Research laboratories, Aluminum Co. o f America, New Kensington, Pa.
b Hydrogen, as tritium, extracted from aluminum by solid extraction methods and assayed in an ion chamber can be measured to a level of about 3 X 1 0-lo ml. a t standard temperature and pressure. The method compares well with existing solid extraction methods for assaying nonradioactive hydrogen and is as reproducible. Sensitivity far exceeds existing methods and the radiochemical method requires no troublesome surface gas corrections. Tritium activities between 1 O-s and 1 0-2 curie can be measured routinely. Isotope effects were not observed in gas content measurements using mixtures of hydrogen and tritium or for reaction of tritiated water vapor with molten aluminum; however, these effects were found for the attack of condensed tritiated water on aluminum powder a t room temperature.
T
BEHAVIOR of hydrogen in metals has long been of interest in many metallurgical laboratories. Studies in this field lend themselves well to radioactive tracer techniques. However, very weak beta radiation (0.018 m.e.v. maximum) from tritium, the only radioactive isotope of hydrogen, restricts assay methods to internal detecting devices such as internal Geiger or proportional counting, ionization current measurements, or liquid scintillxtion counting. HE
1624
ANALYTICAL CHEMISTRY
I n the present method, gas in a metal specimen is extracted by the solid extraction technique, pumped into a n ionization chamber, and assayed. Solid extraction rather than vacuum fusion was chosen for extracting gas from metal samples, since several light met,al alloying agents, such as magnesium, are volatile and vacuum fusion results in metal being deposited throughout the vacuum system. Solid and liquid extraction techniques for extraction of nonradioactive hydrogen from aluminum have been compared by Brandt and Cochran (I). Since hydrogen extracted from metals is molecular, it was desirable to assay tritium in that form. Geiger and proportional counting of tritium (as hydrogen) has been reported by many investigators and reviewed by Glascock (4). Both methods were tried. Gas counting was unsatisfactory because contamination effects were such that new tubes were required for each assay. Counting tubes tried included glass tubes with chemically deposited silver cathodes and glass tubes with stainless steel cathodes. Christman ( 2 ) reported that contamination results from traces of water remaining in the counter tubes and is absent when tubes are perfectly dry. Glascock ( 4 ) attributes these memory effects to the exchange of tritium atoms with adsorbed water and silicate OH groups in the walls of glass counters. Counting of tritium as acet-
ylene (11). and methane (8) was also unsatisfactory because of poor reproducibility, short counting plateaus, and the time required. Ionization current measurements were investigated. Fkproducibility was good and there was reasonable freedom from contamination in the measurement of tritium as molecular hydrogen. The method provided an assay range of 10' and no dilution of any kind was necessary. EXPERIMENTAL DETAILS
Special Apparatus. ELECTROMETER
IONIZATION CHAMBER.-4 Cary Model No. 3095, 250-ml. ionization chamber and a Model 31 vibrating reed electrometer made by the Applied Physics Corp. were used to measure ion currents. The electrometer used was equipped with a turret switch capable of switching the input to a 10ppfd. capacitor or resistances of 108, 1010, or 10'2 ohms. Use of such an instrument in the assay of tritium has been described in some detail by Wilzbach et al. (IO). URRY AUTOMATICTOEPLER PUMP Wakefield Industries, Model No. 31300 500-ml. capacity. MAINDIFFUSION PUMP.A Stimpson (6). pump was used to avoid any possibility of backing up of volatile tritium compounds formed in the mechanical forepump. The Stimpson pump is capable of developing a 10-Lcm. vacuum when pumping against a 30-mm. back pressure. AND
n
a
