A N A L Y T I C A L CHEMISTRY
1576 sents the distribution of individual results (all within a given day, analyst, and laboratory). The mean of each distribution curve is a random sample from the distribution curve above it; and the range of these means is shown by the horizontal arrows. The lower curve shows the expected distribution of a single determination made in any laboratory with a standard deviation, UT =
[Ud2
+ [(0.2E1)~ + ++ (0.03)2=+ (0.07)2+ (0.07)2]1/2= 0.27 Uo2
Uz2
Uiz]l/a
This makes it very evident that most of the observed variability can be associated with the different laboratories. Several of the laboratories have already shown that one step in the procedure, which was not carried out the same in all laboratories, is responsible for part of the variability among laboratories. The component of variance for laboratories cannot be reliably estimated until the analysts learn how to control the factors responsible for this lack of agreement. An estimate of the accuracy obtained in the various laboratories can be made, as it was known, from independent tests made on the material, that the acetyl content was 39.00%. The standard error of any analyst’s average of duplicate tests made on three different days is 0.0046 = o,048 6 +
-1
and the 99% confidence limits for this average are
Fz rt (2.58 X 0.048) = x, 2c 0.13
Putting these limits about the 16 averages in Table I, it can be seen that all analysts in laboratories 3 and 4 and one analyst in laboratory 2 obtained results that are too high. The analysts in laboratories 7 and 8 all obtained low results. The rest of the analysts obtained results that do not differ significantly from the accepted value of 39.00% acetyl. .4 logical interpretation of the results of an interlaboratory study of a test method can be easily made if the test material is distributed according to some kind of nested sampling plan. With such a plan, it is possible to compare the variability in the various levels and obtain estimates of the magnitude of the variance components which are significant. In cases where materials of known composition are used in the study, it is also possible t o set up confidence limits for the various analysts to see whether or not they are getting accurate results. LITERATURE CITED
(1) American Society for Testing Materials, Philadelphia, “ASTM Manual on Quality Control of Materials,” pp. 61-2, 1961. (2) Brownlee, K. A., “Industrial Experimentation,” 3rd ed., p. 116, Brooklyn, Chemical Publishing Co., 1951. (3) Genung, L. B., Report of Subcommittee on Acyl Analysis, Division of Cellulose Chemistry Committee on Standards and Methods of Testing, 119th Meeting of AMERICAN CHEMICAL SOCIETY, Boston, Mass. (4) Wernimont, Grant, ASTM BUZZ.,166,4543 (1950). RECEIVED August 8, 1951.
[END OF SYMPOSIUM]
Paper Partition Chromatography of Cations Cations Precipitated in Acid Solution by 8-Quinolinol THOMAS B. CRUMPLER Tulane Lhiversity, New Orleans, La.
WILSON A. REEVES
AND
A scheme of rapid and sensitive paper partition chromatographic tests for the common cations would be a useful analytical tool. Oxine (8-quinolinol) precipitates many cations quantitatively, and can serve as a concentrating agent. Under acidic and alkaline conditions, groups of convenient size can be precipitated. The oxine complexes of aluminum, nickel, copper, cobalt, bismuth, zinc, cadmium, mercury, iron, and silver have been studied and suitable reagents for dissolving them and developing chromatograms have been found. All these ions except silver can be separated and identified in amounts as small as 10 micrograms, by R / values and fluorescent colors under ultraviolet illumination.
P
APER partition chromatography was originally used by Consden, Gordon, and Martin (6) in 1944 for the separation of amino acids and has subsequently been used for the separation of minute amounts of a wide variety of materials, including inorganic constituents. Application to inorganic separations has been reported within the past three years (IO). The metallic ion separations reported to date have been primarily on the metal chlorides. Paper partition chromatography has been applied to a lesser extent in the inorganic field than in the organic.
Antimony was separated from all common metallic ions (9). Gold, platinum, and alladium were separated from each other and from iridium or rtodium ( 4 ) . Lederer (10) separated gold, platinum, palladium, copper, and silver, and later ( 1 1 ) separated silver, mercury, and lead as the chlorides and also separated copper from cadmium. He listed the I?, values for a number of other metals when chromatographed with 1-butanol saturated with 1 N hydrochloric acid. Aluminum was separated from beryllium (14). Uranium was separated from all other metals (?). Arden and others ( 1 ) separated mixtures of calcium, strontium, and barium; aluminum, gallium, indium, and zinc; cobalt, copper, iron, manganese, and nickel; vanadium was separated from a large number of elements. More recently Burstall and others (3) separated the metals constituting the ordinary qualitative analytical groups Frierson and Ammons ( 7 ) separated the metals of Groups I, 11, IIA, and I11 with I-butanol containing a small amount of acetic or hydrochloric acid. Because many metals may be precipitated quantitatively with 8-quinolinol (&hydroxyquinoline, oxine) from dilute solutions, the authors decided to determine whether a paper partition chromatographic method could be developed to separate these chelated complexes. 8-Quinolinol has been used (6) in column chromatography for the separation of cations. Since its introduction into analytical practice, it has become a familiar and widely used reagent for determining metals and even nonmetals. Its sensitivity and utility have led to numerous publications. 8-Quinolinol reacts with metal ions to form colored compounds which belong to the inner complex group (16). Although not a specific reagent, it has some very advantageous applicatiom.
V O L U M E 2 3 , N O . 11, N O V E M B E R 1 9 5 1 Often elements are precipitated a t a definite p H but are soluble a t other p H values. In the present investigation the more common metals that may be precipitated quantitatively ( I d ) in acid media were selected for partition chromatographic studies. EXPERIMENTAL
Preparation of Chelates. The chelates of silver, aluminum, nickel, cobalt, copper, bismuth, zinc, cadmium, mercury, and iron were prepared by dissolving the metal salts in 0.1 N acetic acid or in an acetic acid-sodium acetate buffer solution and adding a 45Z0 solution of 8-quinolinol in ethanol in slight excess. The solutions were heated gently (50" to 70" C.) for 5 to 10 minutes, then filtered through a Whatman No. 50 filter paper. Excess reagent mas removed from the precipitates by washing with hot water. S o attempt was made to recover these precipitates quantitatively.
INDENTATIONS * GLASS CYLINDER
(15 X 45 cm)
PAPER
Figure 1.
4pparatus for Paper Partition Chromatography
1571
brown band appeared a t Rj 0.55. When 1.2 N hydrochloric acid was used t o saturate 1-butanol, the band was moved down to an R/of 0.50 and zinc moved along tangent to the brown band. Twelve normal hydrochloric acid is apparently miscible with 1-butanol in all proportions. A developing solution consisting of 4y0 by volume of 12 N hydrochloric acid and 96% by volume of 1-butanol moved iron down the paper to an R/ of about 0.90. Other metals were moved slightly but streaked considerably. In this solvent, the brown band did not appear on the developed chromatograms. When the amount of concentrated hydrochloric acid mas increased to lo%, actual separations were made in the rase of some metals. Cobalt and copper were separated; their K i values were 0.05 and 0.24, respectively. Kickel and aluminum both had an R, of 0.02. Bismuth streaked considerably. Chromatograms run for 16 hours using 85y0of 1-butanol and 15% of 12 S hydrochloric acid as developing solvent gave good separations of the metals. Somewhat better separations were obtained when the 1-butanol contained 20% of concentrated hydrochloric acid. 1-Butanol containing 25 and 30% acid mas found satisfactory as developing solvent. The change in acid concentration in the 1-butanol had its greatest effects on the Rj values of cobalt, copper, and bismuth, as can be seen in Table I. Twenty per cent of 12 +V acid in 1-butanol gave the best distribution of Rfvalues. Purification of Filter Paper. Serious defects in the chromatograms were caused by traces of iron and possibly other metals present in Whatman ?;os. 1 and 2 filter paper. dfter the developed chromatographs were sprayed with 8-quinolinol, a dark colored area extended back about 3 cm. from the solvent front. This dark area, due primarily to the presence of iron in the filter paper, practically masks the position of iron, cadmium, and mercury. The interference was completely removed by allon ing the developing solvent to irrigate the paper in the same manner as used t o develop a chromatogram, and after the paper was dried, that portion of the paper which contained the contaminant metals was cut off. The purification procedure increased some1% hat the speed of solvent flow of the subqequent chromatogram, but produced little or no effect upon R/ valurs of the metals. Table I. Effects of Hydrochloric Acid Concentration on Rr of Metals 3Ieta
Selection of Solvent. Earlier attempts to use such well-known drvelopirig solvents as phenol saturated with water or collidine ~ t u i a t e d n-ith water proved futile Other partially watermisc~ibleliquids which have been reported in the literature as developing solvents for paper partition chromatography, such as 1-butanol-acetic acid-water, did not effect separations of the metal chelates. Several partially water-miscible organic liquids R ere tried with little success, including cyclohexyl amine which showed some promise. As most of the metal chelates of 8-quinolinol can be dissolved in dilute hydrochloric acid solutions, it was thought that an organic solvent saturated with a dilute aqueous solution of this mineral acid might be used as a developing solvent. 1-Butano!, having been used by other investigators for many separations, was tried. Paper partition chromatograms developed with a solution consisting of 1-butanol saturated with 0.12 N hydrochloric acid caused eight of the metal chelates to move about 3 mm. in 16 hours. -4fter the chromatograms had been dried, a brown band the width of the paper with R/ of 0.4 was visible. When the paper was sprayed with the 8-quinohnol reagent, the area on one side of the brown band was white and on the other side was yrllow. This indicated the developing solvent to be a two-phase niisture rather than a single phase as it appeared to be visually. 1-Butanol saturated with 0.6 S hydrochloric acid gave essentially the same chromatogram as described above, except that the
15
Volume % of 12 Y HC1 20 25
Rf T'alues .4 g A1 Ni co
cu Bi Zn Cd
Hg
Fe
0 0 0.03 0.03 0.09 0.30 0 44 0.86 0.88 0.88 0.92
0 0 0 0 0 0 0 0 0 0
0 03 04 19 40 51 78 83 84 93
0.0 0 04 0 04 0.39 0 51 0 60 0 79 0 83 0 83 0 92
30
0.0 0.07 0.09 0.51 0.55
0:79 0.83 0.83 0.92
Procedure. The descending technique using the apparatus shown in Figure 1 was employed for this investigation. To run a chromatogram in the apparatus, a strip of purified Rhatman No. 2 filter paper wae used 9 cm. in width and 35 to 48 cm. in length. Samples were dissolved in 1.5 to 2.0 N hydrochloric acid and the solution containing about 10 micro rams of each metal was applied. After the chromatogram had %,en developed and dried, the paper was sprayed with a 1%solution of 8-quinolinol in 70% ethanol. In order to make visible the position of the spots, the chromatogram was held over a beaker containing concentrated ammonia for a few minutes, and then dried with an infrared lamp. The purpose of the ammonia treatment is to bleach out the chromatographed 8-quinolinol reagent spot that always appeared a t an R/ value of 0.5, and also t o bleach out the yellow background caused by the reagent used to spray the developed chromatogram. The positions of the epots were outlined in pencil, so that R/ values could be calculated. Chromogenic Reagents. In order to make visible the positions Several sprays
of thr metal ions, chromogenic sprays were used.
A N A L Y T I C A L CHEMISTRY
1578 were tried. A 1% solution of 8-quinolinol was found most useful because it produced spots that were visible in daylight and showed bright fluorescence under ultraviolet illumination. The fluorescent colors nere a n important feature, especially for ions whose R/ values were close together; for in each case the two ions gave chelates whose fluorescence differed greatly. The Rj values of nickel and aluminum xere very close, yet their identity could be established with certainty, because aluminum gave a bright yellow fluorescence and niche1 gave a red fluoresence. This feature u as again important whcn distinguishing between cadmium (yellow) and mercury (red). In the presence of both ions in either of these pairs, an orange color resulted where the spots overlapped. A 0.5% solution of diphenylthiocarbaxone in carbon tetrachloride was a useful spray, valued especially for colors in daylight. A more certain interpretation of the composition of a n unknown sample could be obtained by developing duplicate chromatograms and then spraying one with 8-quinolinol and the other with a reagent like diphenylthiocarbazone or resorcinol. A dilute solution of resorcinol in 95% ethanol was also used t o ascertain the presence of mercury. The colors produced TI-ith the chromogenic reagents can be seen in Table 11. Rj Values. The r e p oducibility of Rj values on Whatman No. 2 filter paper can be seen in Table 111. These values were obtained from chromatograms which were run overnight (16 to 20 hours) at a temperature of 28" =k 3" C. wing 1-butanol containing 20% of 12 -V hydrochloric acid as developing solvent. Considering the maximum Ri deviations obtained for a number of chromatographs run on different days, the R, values themselves may often establish identity of the cation. The quantity of metal chelates applied a t the origin has little effect on the R, value, though the size of the spot varies considerably. Verification of Method. In order to verify the reliability of the method, unknown misturcs of 8-quinolinolates of metals were dissolved in 2 N hydrochloric acid and chromatographed by the procedure outlined above. It can be seen in Table Is' that the method is good for all the cations except silver. In an attempt t o devise a method reliable for silver, samples containing silver chelate and one other cation chelate were dissolved in pyridine, placed on the filter paper, heated with infrared lamps t o drive off the pyridine, and chromatographed. Only six of the ten samples analyzed gave a positive test for silver by this method.
Table 11.
Results of Chromogenic Sprays
Meta AI Ti Co CU Oxine Ri
Zn Cd
€1R
Fe
Runs 11
8 5 7
11
7
11
7
0.83
0.84
0.93
Cu, Co, Hg, Fe
Cu, Co, Hg, Fe
Table
V. RfValues
Cu(CzHs0z) z BiONOI ZnC1, HgClz
R/ 0.036 0.42 0.52
0.77 0.84
DISCUSSION
The cations studied here were commonly occurring metals that are precipitated quantitatively by 8-quinolinol (9) from an acetic acid-acetate solut'ion. All these chelates are soluble in 2 N hydrochloric acid, except silver, which forms the insoluble chloride. Silver in mixt'ures cannot always be ident,ified with certainty by this method, but nine other metals can readily be identified. Ident'ification is based upon Rf values and upon color of spots in both daylight and ultraviolet light. Because some writers (8) include manganese in the group of metals that are precipitated quantitatively by 8-quinolinol in an acid medium, manganese was also chromatographed. When a 20y0 solution of 12 N acid was used as developing solvent, this metal was found t o have a n Rj of 0.10 and gave a red fluorescence when sprayed with 8-quinolinol and observed in ultraviolet light, Because the &quinolinol appeared as a separate spot at a n R, value of 0.5 in every chromatogram, it seemed very doubtful that the metals had moved as oxine chelates. Five cations of the group as metal salts were spotted on filter paper and chromatographed by the above procedure. Their R, values are given in Table V. Comparison of these RJ values with those reported in Table I11 indicates that the cations did not remain as oxine chelates, but that. under the existing chloride ion concentration (about 2.4 M in hydrochloric acid), they moved as chloro complexes. Moeller (23) has shown, for instance, that in 3 M hydrochloric acid, copper exists principally as the CuCI,-- ion. Precipitation of the mrtals with 8-quinolinol not only affords a convenient method of concentrating the metal ions but could also serve as a purification process. This latter factor may in some cases be of prime importance, as extraneous substances may cause serious interference in paper partition chromatography. T. V., RurstaH. 1.'. H. Davies, C;. R . Lewis. J. h.,sild Linstead. R . P.. .Yuft!w, 162, 691 (1943). (2) Arden, T. V..Burstall. I-. H.. and Linstcad, R . P., J . Chcm.
(1) Arden,
0
Blue Brown Black
Red Red Purple
....
,... a
Yellow
Red ....
....
Rj Values
(Temperature 28' i 3O C.) Average Rlaximum RI Deviation 0.032 0.003 0.035 0.005 0.19 0.41 0.30 0.51 0.78
Cations Present Ag, Si,Co, Bi, Cd Zn, Al, Cu, C d Zn, Hg
Salt Ni(CzH30dz
Resorcinol UltraDayviolet light light
2
KO. of
2 3 4
Results of Samples Analyzed Cations Found Xi, Co, Bi, Cd Al, Zn, Cu, Cd Zn. Hg
LITER4TURE CITED
Diuhenyl8-Quinolinol thiocarbazone rltraUltraDayviolet Dayviolet Metal light light light light B Yellow h1 Yellow Red Yellow Si Red Red Red Yellow Purple co cu Red Yellow Green Yellow Bi REd Red Orange Zn Yellow Yellow Red a Yellow Cd Yellow Tan Red Yellow Red Purple Red Black Green a Kone or very slight coloration.
Table 111. Reproducibility of
Table IV. Sample 1
0.03 0.03
...
0.03
0.03
0.01 0.02 0.02
Average Deviation 0.002 0.002 0 02 0.02
...
0.02 0.02 0.01 0.01 0.01
.
Soc., 1949 supplenieiit iswe. ?,ll. (3) Burstall, F. H.. Dal-ies. Ci. 11.. L i n s t e d , R . P., a n d IVells, R . 1., Ibid.. 1950, 516. (4) Burstall, F. H . , Davies. C,. I?., LiiistPad. R . P., and Wells, R. .I., S a t w e , 163, 64 (1949). (5) Consden, R . , Gorrlon =\. H . . Hiid M a r t i n , ;1.J. P., B i o c h ~ n J. . . 38,224 (1944). (6) Erlenmeyer, H., a n d Dalin. H.. H , IT. Cliim. Acta, 22, 1369 (1939); 24,878 (1941). (7) Frierson, W.J., a n d Amnions, 11,,J., J . Chern. Education, 27, 37 (1950).
(8) Kolthoff, I. II.,and Sandell, E. B.. "Textbook of Q u a n t i t a t i v e Inorganic .Inalysis," New I-ork. hfacmillan Co., 1938. (9) Lederer, XI.. A n a l . Chin?.Acta, 2, 231 (1945). (10) Lederer. AI.. S n f o r c , 162, 776 (1948). (11) Ibid.. 163, 598 11949). (12) Lundcll, G. E. F., a n d Hoffman, J . I.. "Outlines of M e t h o d s of Chemical Analysis." iYew York, J o h n Wiley 6:Sons. 1938. (13) hfoeller. T., J . Phus. Chenz.. 48, 111 (1944). (14) Osborn, G. H . , and .Jenesbury, A , , S a t u r e , 164, 443 (1949). (15) Prodinger. IT'., "Organic Reagents Used in Q u a n t i t a t i v e Inorganir Analysis." S e x T o r k . Elsevier Publishing Co., 1940. RECEIVEU Sovember 8 . 1 9 X .
