yields a 0.33 resolution of the isomers under comparable conditions. The 1iodo compound elutes the aromatics much faster than 1-chloronaphthalene. 1-Bronionaphthalene is intermediate in this effect. The ratio of retention volumes (separation factor) of m- and p-xylenes on the 1-chloronaphthalene column is 1.06 as indicated in Table I. Other reagents have been useful in resolving the xylene isomers, although not t o the same degree as l-chloronaphthalene. These include propylene. trithioethylene and monothioethylene carbonates, 4 p-chloroacetophenone, and phenothioxin. RESULTS AND DISCUSSION
An aromatic mixture containing benzene, toluene, ethylbenzene, p - , m-, and o-xylene was resolved on several substrates using the aqua regia-treated firebrick. Retention volume ratios, separation factors, and operating conditions are given in Table I for benzyldiphenyl, ethylene carbonate-l-nitronaphthalene, dimethylsulfolane, and 1chloronaphthalene. Ethylene carbonate, dimethylsulfolane, and 1-chloronaphthalene are polar compounds capable of separating m- and p-xylenes by gas-liquid rhromatography .
1-Chloronaphthalene, which appears to be the best is readily available and can be used a t temperatures up to 50' C. The percentage of liquid phase in the procedure used may vary from 5 to 15%. TT'ith long columns it would be advisable to use 5% to reduce elution times. A 15-foot column eontaining 5% dimethylsulfolane on 60 to 80 mesh treated firebrick produces a n R value of 0.78 for these isomers. The specially prepared aqua regiatreated firebrick offers several advantages in gas chromatography: It enhances resolution because all the fines are removed, it permits the use of finer mesh sizes and longer columns, thereby increasing resolving efficiency, and it considerably reduces pressure drop across the column. The same effects are noted when Chroinosorb is treated with aqua regia. The small fines adhering to the firebrick particles are removed by the acid treatment and the resulting product appears as tiny smooth boulders. The photomicrographs in Figure 3 compare the treated and untreated firebrick. T h e n long columns (20 feet) are used, it may be necessary to increase the flow rate of carrier gas to shorten retention times. Flow rates up to 300 ml. of hydrogen per minute have been aatis-
factory with no appreciable loss in resolution. Columns of relatively low theoretical plate efficiencies can effect difficult separations in gas chromatography by the choice of a suitable stationary liquid (8). The use of an aqua regia-treated support is also preferable. The highly efficient columns described by Cheshire and Scott ( 2 ) might be further improved by using aqua regia-treated supports as !vel1 as the stationary phases described abore. LITERATURE CITED
(1) Burton, I.,Conzpt. rend. 236, 1679
(1953). (2) Cheshire, J. D., Scott, R . P. IT., J . Znst. Petrol. 44, 74 (1958). (3) Fredericks, E. M., Brooks, F. R . ANAL.CHEM.28, 297 (1956). (4) James, A. T., Martin, A. J. P., Baochem. J . 50, 679 (1952). (5) Jones, IT. L., Kieselhach, Richard, -1N.4L. CHEhf. 30. 1590 (1958). (6) Knight, H. S.,Zbzd., 30, 9 (1958). i i ) Ailliams, R. B., Hastings, S. H., .knderson, J. -4., Zbzd., 24, 1911 (1952). (8) Zlatkis, Albert, Zbzd., 30, 332 (1958). 19) Zlatkis. Albert. O'Brien. L.. Schollv. ", P. R., Suture 181, 1791 (1958j. RECEIVEDfor revieK August 18, 1958. Accepted February 6, 1959. Division of Analytical Chemistry, 134th lleeting, hCS, Chicago, Ill., September 1058.
Separation and Microidentification of Metallic Ions by Solvent Extraction and Ring Oven Techniques PHILIP W. WEST and ANlL K. MUKHERJI Coates Chemical Laboratories, louisiana State University, Baton Rouge, La.
b A scheme has been devised for the separation and identification of 35 metallic ions in a single drop of unknown, based on a combination of solvent extraction and ring oven techniques. The unknown is separated into four groups b y placing the nonaqueous extracts of metal chlorides, thiocyanates, acetylacetonates, and diethyldithiocarbamates on filter paper and depositing them as rings by means of a ring oven. A fifth group is obtained by similar treatment of the aqueous solution of residual ions. The rings of deposited salts are cut into sectors and the individual ions are identified by means of spot tests. Complete separations and identification can b e made in an hour.
I
the usual methods of semimicro and micro qualitative analysis it is seldom possible to separate and identify N
several ions when present in a single drop of a solution. The precipitation, filtration, and washing of the precipitate dilute the test solution to such a n extent that the ions are brought below their respective identification limits. I n 1954, Weisz devised the ring oven technique (3-5) as a means of schematically analyzing a single drop of a solution containing more than one metallic ion. In this method, a filter paper is placed on a n electrically heated ring and a drop of sample is added to the center of the paper. The addition of a reagent fixes some of the ions locally while others move to the periphery of the ring on account of the capillary action of the filter paper. Because of the rapid evaporation of the wash liquid as it approaches the heated surface, the ions washed out are collected in the form of a ring and are subsequently detected by spot tests. The separations themselves can be remarkably efficient. However,
the success of the ring oven technique depends almost entirely on the specificity and sensitivity of the reagents unless very elaborate and comprehensive separation schemes are employed. The purpose of the present work was to develop a simple microanalytical procedure using the ring oren method, employing a prior separation of metal ions into more or less definite groups through liquid-liquid extraction. Solvent extraction was chosen because of its greater efficiency, simplicity, and speed. In general, it is superior to the precipitation methods usually employed as it is free from coextraction. the analog of coprecipitation. It is also more rapid and mav be simpler to perform in many cases. Because samples in aqueous solution are the prime concern in the analpis of metal ions the solvent extraction in the present lvork implies the use of an aqueous-organic solvent pair. Morrison and Freiser have broadly classified ( 2 ) VOL 31, NO. 5, M A Y 1959
947
the extraction systems on the basis of the nature of the extractable species-Le., chelate extraction and ion association extraction. Of the various extraction systems known in the two catagories, chloride, thiocyanate, bromide, iodide, acetylacetone (2,4-pentanedione), diethyldithiocarbamate, diphenylthiocarbazone (dithizone) , and 8-quinolinol systems were investigated. Organic solvents were so chosen as to ensure the most favorable distribution of the extractable species. Sometimes a mixture of two organic solvents was prepared after considering not only the distribution of the extractable species, but also their boiling points, degrees of miscibility, relative specific gravities, and tendencies to form emulsions. Efficiency of separation v a s determined only on a qualitative basis. Among the ion association extraction systems, chlorides and thiocyanates were most useful in the present work, as the rings produced were colorless and the excess chloride and thiocyanate ions did not interfere in the subsequent stages of analysis. The fluoride system was not sufficiently versatile, while in the bromide and iodide systems, the rings produced by the extracted ions mere too highly colored, presumably owing to the decomposition of the extracted metal complexes. In the chelate systems, acetylacetone and diethyldithiocarbamate were preferred on account of their versatility and the colorless rings they produced. Acetylacetone forms well defined chelates with a large number of metals and the solubility of acetylacetonates in organic solvents is much higher than that of most chelates used analytically. Extractions have been carried out Kith the acetylacetone system using this compound itself as solvent or with other organic liquids such as carbon tetrachloride, chloroform, and benzene as solvents. The chief advantages of using acetylacetone itself as solvent are that extractions can be made a t a low p H and many acetylacetonates are more soluble in acetylacetone than in other organic solvents. Extractions with the diethyldithiocarbamate system are also done a t a low pH. While diphenylthiocarbazone and 8-quinolinol are excellent chelating agents, they have the disadvantage of forming colored rings LThich interfere with subsequent spot testing. PROCEDURE
The general technique adopted was extraction of a few drops of the aqueous test solution with an organic extractant under appropriate conditions of p H in the presence of suitable ion association or chelating agents. The organic and aqueous layers were kept in contact for sufficient time in a test tube or microbeaker to attain equilibrium; a t least three extractions were made with each system to attain maximum efficiency. 948
ANALYTICAL CHEMISTRY
Figure 1.
Ring oven
The separation of the two phases was carried out with an extraction pipet (1).
Only the first extract (organic phase) was subjected to analysis, which was done by transferring the extract of the test solution to the center of the filter paper with a capillary pipet inserted through the guide tube of the ring oven. The aluminum ring was placed on the paper to keep it in position. The spot was then treated with 0.1N hydrochloric acid n-hich was absorbed in the filter paper, thus washing the extract concentrically t o the hot surface of the oven where it was vaporized, leaving a deposit of transported salts. Five to ten washings served to concentrate the ions quantitatively and sharply in the ring zone. The filter paper was then oven dried and cut into several sectors. Each single sector of filter paper, after having been developed by suitable reagents, showed a circular arc. Care was taken that the deposits gathered in the sharp ring zone were not redissolved during the course of identification with resultant loss in test sensitivity. The aqueous layer left after each group extraction was subjected to further extractions under different conditions, repeating the above procedure. The metallic ions under investigation have been classified into four broad groups according to the systems of extraction employed (Table I). EXPERIMENTAL
Apparatus and Reagents. The ring oven (Figure 1) consists of a cylindrical block of aluminum, H , 40 mm. high and 70 mm. in diameter with a bore hole of 22-mm. diameter. A heating element is installed inside the block and temperature is controlled by a suitable powerstat. Outside the cylinder is fixed an aluminum rod, AI, adjustable by a screw, &. The rectangular aluminum rod, M , bears on its end a guide tube, G, about 50 mm. long, which is adjustable in height by
means of a screw, Ss. The glass tube, G, serves as a guide for a capillary pipet. The guide tube must stand vertically and point exactly to the center of the bore hole. It ends about 2 to 3 mm. over the surface of the heating block, H . An aluminum ring of 30-mm. inner diameter is placed over the filter paper to keep it in place. The temperature at the surface of the heating block should be about 80" to 115' C. Reagents for the detection of individual metal ions have been listed under the respective procedures. Chloride Group. A few drops of the aqueous (1%) test solution are treated with t w o drops of concentrated hydrochloric acid. The final strength of the acid should be approximately 7 N . Extraction is performed with 2 ml. of a 2 to 1 mixture of methyl isobutyl ketone and n-amyl acetate, and the extract is washed 4 t o 5 times into the ring with 0.lX hydrochloric acid. The ring is then cut into eight segments. Although a single extraction suffices for identification purposes, one or two additional extractions may be advisable in each case to ensure complete removal of group members. For example, if iron is present in large amounts, enough of it will remain after a single extraction of the chlorides to appear in and interfere with subsequent separations and tests. The tests amlied for the different ions are as followi: G o L D ~ I I I ) .A rJortion of the ring is treated 'with a freshly prepared 9% solution of stannous chloride. Blackening of the ring indicates the presence of gold. MOLYBDENJM(VI).A portion of the ring is acidified with hydrochloric acid and treated with a 0.1% solution of diphenylcarbazide. A violet stain indicates that molybdenum is present. IRON(II).If gold is present, the ring is treated with a 1% solution of ammonium thiocyanate. A red coloration appears if iron is present. If gold is absent, a 1% solution of potassium ferrocyanide may be used as the reagent. A blue ring is formed if iron is present. Because an insoluble precipitate is produced, the stain is more sharply defined and the test is more sensitive. ANTIMONY(V)AND ARSENIC(III). A portion of the ring is made into pulp with dilute sulfuric acid in a test tube, and a few grains of metallic zinc are added. A piece of paper impregnated with silver nitrate is placed over the top of the test tube. I n the presence of antimony or arsenic, a black stain is formed on the filter paper. A part of the circular arc, as close as possible to the deposit, is cut out and placed on a spot plate. A few drops of hydrochloric acid (1 to 1) and one drop of 5% potassium iodide are added, then mixed by blowing with a pipet. A drop of a 0.57, Rhodamine B solution is added and the solution is mixed again. A blue-violet precipitate indicates antimony. A blank test is carried out for comparison. GALLIUM(III).A sector of the filter paper is treated with a 0.02% solution of morin in alcohol. A yellowish green
fluorescence under ultraviolet radiation is indicative of the presence of gallium. VANADIUM(V).A portion of the ring is treated with a 20% solution of sodium fluoride. When dry, it is treated with 1% solution of 8-quinolinol in acetic acid. A dark brown ring indicates the presence of vanadium. Thiocyanate Group. T h e aqueous extract from the previous operation is treated with 3 drops of 7 M ammonium thiocyanate. This is now extracted with 2 t o 3 ml. of diethyl ether and washed into the ring with 4 t o 5 portions of 0.1N hydrochloric acid. The metallic ions of this group are tested as follom~s: COBALT(II). During the process of extraction a blue coloration of the organic phase is indicative of the presence of cobalt. .This may be further confirmed by treating a portion of the ring with a 1% alcoholic solution of l-nitroso-2-naphthol in an ammonical medium. A brown stain forms if cobalt is present . ZINC(II). A portion of the ring is exnosed for a minute to fumes of a4monia. It is then treated with a 0.01% diphenylthiocarbazone solution in carbon tetrachloride. A red ring indicates zinc is present. BERYLLIUhI(I1) AXD TIN(IV). The txesence of either or both of these ions can be detected by the fluorescence test with 0.02% morin solution in ethyl alcohol. Tin can also be detected by treating the ring with a sodium sulfide solution. A yellow ring is obtained in the presence of tin. Acetylacetone Group. The aqueous extract from the previous group is treated with 1 or 2 drops of dilute hydrochloric acid to provide a p H of approximately 2. This is then subjected to extraction by 2 ml. of acetylacetone. The aqueous and the organic layers should be kept in contact for 2 to 3 minutes, because some of the acetylacetonates are extracted slowly. Aluminum does not extract completely in acidic medium; therefore, after three extractions in acidic medium, a portion of the aqueous extract is treated with a few drops of 4-11 sodium hydroxide to adjust the p H to approximately 7. This is now extracted and washed into the ring as usual with dilute hydrochloric acid. Titanium, zirconium, and chromium are only 75% extracted in this group and so a part of these ions will appear in the next group also. Thorium is partially extracted. The ring made of the acidic extract is tested for copper(II), uranium(VI), zirconium(IV), and titanium(IV), while the ring made out of the second extract is tested for aluminum(II1) only. The metallic ions of this group are detected as follows: ALUMINJM(III).A portion of the ring is treated with a drop of dilute acetic acid followed by a drop of 0.02% morin solution in alcohol. A greenish fluorescence under ultraviolet radiation indicates the presence of aluminum. Zirconium interferes with the above test. COPPER(II). A portion of the ring is
Table 1.
Scheme of Separations Chloride Group Separation
I
+
Test solution + 2 drops concd. HCl 2 ml. methyl isobutyl ketone and n-amyl acetate (2: 1 mixture) Extract with an extraction pipet
I
1
Aqueous Phase (1) Organic bI a s e (1) I cucz Sb+5, Au+3, Fe+3, v+6,Mo+6, Ga+3, As+3, Co+2 Zn+2 Bet2 Sni4 Alfa Uk Zr+i Ti+: Ni+2' Cd+i Bi+d pb+1' Ge+4, and Te+4 Hg$2, ~i+1, W*, h + Z , ~e+4,'In+{ (Individual ions tested as described in Ba+2, Sr+2, Mg+2,Ce+3, and T h f 4 Chloride Group) I
Thiocyanate Gboup Separation
I
Add, 3 drops o! 7M SH,CNS. Extract m t h 2 ml. of diethyl ether
I Aqueous khase ( 2 )
Organic Phase (2)
I
Co +2, Zn +z, Be f2, and Sn +4 (Individual ions tested as described in Thiocyanate Group)
I
Crf3, C U + ~U+B, , Zr+', Ti+', Ni+* Cd+2 Bi+3, Pb+2, Hg+2, T1+1, W*M, Mn+;, Sef4, In+$, Ba+z, Sr+2, Ca+3 hlg+z, Ce+3, and Th+4
-41'8,
Acetylacetonate Grou' Separation
7
.4dd 2 ml. acetylacetone, wait for a few minutes, and extract.
I
Organic Phase (3)
I
Al+a, Cr+3, Cuf2, U+6, Zr+4,and Tit4 (Individual ions tested as described in Acetylacetone Group)
Aqueous Phase (3)
I
Ni+2, Cd+2, Bi+3, Pb+2, Hg+2, T1+1, W*, Mn+2 Se+4, In+3, Ba+2, Sr+2, l l g +2: Ce +3, and Th +4 Diethyldithiocarbamdte Group Separation Add 2 ml. of diethyl!iithiocarbamate solution. Extract immediately Nith diethyl ether. 1
Organic Phase (4)
I
Aqueous Phase (4)
I
Ni+2, Cd+2, Bi+3, Pb+2, Hg+a, T1+1, W*, Mn+2, Se +4, and In +3 (Individual ions tested as described in Diethyldithiocarbamate Group)
Baf2, Sr+2,Ca+2,Mg+2,Ce+3, and T h f 4 (Individual ions tested as described under Aqueous Extract)
exposed to ammonia fumes and treated with a 0.1% solution of dithio-oxamide in alcohol. A black ring appears when copper is present. URANIUM(VI).A sector of the filter paper is treated with a 1% solution of potassium ferrocyanide. A brown ring indicates the presence of uranium. When another sector of the filter paper is treated with a drop of 5 N sodium phosphate solution, a strong yellow fluorescence under the mercury lamp confirms the presence of uranium. ZIRCONIUM(IV),Zirconium gives a fluorescent test with 0.02% morin solution even in acidic solutions. This test is not satisfactory if aluminum is present. TITANIUM(IV).A sector of the filter uauer is treated with a 1% aaueous koiution of chromotropic salt soiution. A deep reddish ring is obtained when titanium is present. Diethyldithiocarbamate Group. The aqueous extract left from the previous operations is treated with a 2% solution of sodium diethyldithiocarbamate. The p H should be approxi-
mately 3. I n most cases, a precipitate appears when the reagent is added, then dissolves when extracted with ether. As the reagent decomposes rapidly in an acidic medium, the extractions should be carried out without delay with an excess of the reagent. The ring formed may be of brownish color due to the decomposition of the reagent. The color can be destroyed on exposure to bromine vapors. Except for indium, the presence of members of this group can be detected by treatment with sulfide. Individual tests for the ions are as follows: CADMIUM(II). 4 few drops of the organic extract are placed in the center of a filter paper, which is then exposed in hydrogen sulfide for about 2 minutes. The metal sulfides belonging to this group are precipitated and fixed in the center of the filter paper. After the paper is washed with dilute hydrochloric acid, the spot is exposed to bromine vapors and finally ammonia fumes. The filter paper is placed on the ring oven and the central spot washed with 0.1N ammonium hyVOL. 31,
NO. 5, MAY 1959
e
949
droxide. Only cadmium ions are washed out to the ring. A portion of the ring is treated with sodium sulfide, and if a yellow coloration results, cadmium is present. BISMUTH(III). A second portion of the extract is washed out to form a ring, a sector of which is then treated with a 1% solution of ammonium thiocyanate. A yellow stain indicates the presence of bismuth. MERCURY(II). A second sector of the ring is treated with 4N hydrochloric acid. After the paper is dry, a drop of 0.01% of diphenylthiocarbazone in carbon tetrachloride is added. An orange stain indicates the presence of mercury. Another portion of the ring is treated with a 10% solution of malonic acid. It is dried and treated with a 0.1% solution of diphenylcarbazide. A blue stain confirms the presence of mercury. SELENIUhf(1V). A portion of the ring is treated with an acidic solution of hydrazine. A red ring forms within 20 minutes if selenic ion is present. Another portion of the ring is treated with a 1% solution of 3,3'-diaininobenzidine hydrochloride. The presence of selenium is confirmed by a red stain. THALLIUM(I).A portion of the ring is treated with a concentrated solution of ammonium phosphate, then dried
and treated with a 2N solution of potassium iodide. A yellow ring indicates the presence of thallium. AIANGANESE(II). A sector of the ring is treated with 20% solution of sodium fluoride, then dried. The ring is treated with 4 N sodium hydroxide solution. A black coloration appears when manganese is present. TUNGSTEN(VI).A part of the ring is treated with 6N hydrochloric acid, then dried and treated with a 10% solution of tannic acid. A brown stain is obtained when tungsten is present. hIAGNESIUM(I1). A portion of the ring is treated with a 1% alkaline solution of Titan Yellow. A pink color appears in the presence of magnesium. NICKEL(II). A sector of the filter paper is exposed to ammonia fumes for 30 seconds and then treated with a 1% solution of dimethylglyoxime. A red ring shows the presence of nickel. LEAD(II). A portion of the ring is fumed over ammonia and sprayed with a freshly prepared dilute solution of rhodizonic acid. It is then fumed slowly over hydrochloric acid. If lead is present, the ring undergoes the following color changes: yellow, red, blue, and violet. Aqueous Extract. The aqueous estract left after t h e four previous operations is used directly for analysis on the ring oven.
BARIUM(II),STRONTIUM(II), AND CALCIUM( 11). A freshly prepared concentrated solution of rhodizonic acid gives colored rings with the three ions. This is a general test and the individual ions cannot be differentiated conveniently. CERIUM(III). A sector of the filter paper is treated with a 1 to 1 solution of ammonia and hydrogen peroxide. A yellom- ring indicates the presence of this ion. ACKNOWLEDGMENT
The authors acknowledge the aid of the U.S. Army Office of Ordnance Research under whose program this research was conducted. Thanks are due to Herbert Weisz and Francois Tron for helpful discussions. LITERATURE CITED
( l j Carlton, J. K., ANAL. CHEM.22, 1072
(1950).
( 2 ) Morrison, G. H., Freiser, H., "Solvent
Extraction in Analytical Chemistry," Wiley, Yew York, 1957. (3) Weisz, H., Alikrochim. Acta, 1954, 140-7.
(4) Zbid., pp. 376-87. (5) Zbid., pp. 785-94.
RECEIVEDfor review June 19, 1958. Accepted December 3, 1958.
Rapid Paper Chromatographic Microassay of Free and Ester Cholesterol of Blood MARY 1. QUAIFE and ROBERT P. GEYER Department of Nutrition, Harvard School of Public Health, Boston, Mass.
HANS R. BOLLIGER Scientific laboratories,
F.
Hoffrnann-lo Roche, Basel, Switzerland
F A convenient, rapid (2-hour) procedure, well suited for multiple assay, is described for the quantitative paper chromatographic separation and assay of micro amounts of free and ester cholesterol of blood serum. Quantitative recoveries of free, ester, and total cholesterol were obtained. Coefficients of variation of a single assay for these cholesterol fractions, duplicated on different days, averaged 3.5%. Results for total cholesterol agreed with those of each of two other methods within 270.
A
SIhIPLE, rapid, quantitative paper chromatographic separation and assay of micro amounts of free and ester cholesterol of blood serum would be useful in studies of cholesterol metabolism. The procedure described uses zinc
950
ANALYTICAL CHEMISTRY
carbonate-coated Whatman KO.1 filter paper with 1.5% ethyl ether in cyclohexane for the quantitative cholesterol assay of human blood sera. .-lniounts of cholesterol compounds chromatographed (15 to 75 y each) were chosrn to permit maximum sensitivity, while using ordinary sprctrophotometric equipment. MATERIALS A N D EQUIPMENT
Analytical grade chemicals: ammonium carbonate, zinc carbonate, 25% ammonia, phosphomolybdic acid, ferric chloride hexahydrate, concentrated sulfuric acid, 95 to 97%, acetone, absolute ethyl alcohol, and ether, peroxide-free. Chloroform, purified by shaking twice with 1N hydrochloric acid, washing with distilled water until the last wash is neutral, drying 24 hours over phosphorus pentoxide, redistilling, and taking the middle fraction. It is preserved
in the dark orer decolorizing charcoal (25 grams per liter of Korit XXX), which was previously dried in a vacuuni oven a t 120" for 48 hours. It is filtered and redistilled. preferably just before use, or else kept a maximum of 14 days in a dark bottle a t room temperature. Cyclohexane, spectral grade purity. Glacial acetic acid, 99 to loo%, analytical grade (E. 1Ierck A. G., Darmstadt). It must be free of glyoxylic acid. by test according to Langan ( 7 ) . Cholesterol, LT. S. P., purified through the dibromide ( 2 ) , recrystallized once from 1 to 1 methanol-ethyl ether, and dried under vacuum. Melting point (corr.) = 148.6-9.1 ". Cholesterol laurate. svnthesized by the method of Swell and Treadwell (12) from cholesterol, purified as described, and lauroyl chloride (Eastman Kodak Co.), recrystallized three times from acetone, and dried under vacuum. Melting point (corr.) = 91.0-1.5'.
