CHROMATOGRAPHY AS A CLASS UNIT IN QUALITATIVE ANALYSIS JOHN G. SURAK and DONALD P. SCHLUETER Marquette University, Milwaukee, Wisconsin
strides have 1,ccn made since the revival by Gordon, Martin, and Syuge (14)in 1944 of Schoenbein's method of "capillary analysis" on filter paper. Its application is widespread in the fields of both organic and inorganic chemistry. Separations of 45 metals and anions, by successive ~:haugesof the extracting medium, have been performed (5). Quantitative determinations of much less than one microgram by paper chromatogmphy have bee11 made possible through the use of radioactive tracers. Measurements were made by use of Geiger-Miiller counters (25) or by laying the developed chromatograms on a photographic plate and producing a radioautograph (11). Sugars have been separated and detected in foods, blood, and urine by the paper-chromatographic method. One of the greatest fields for the application of this method is in medicine and biological chemistry. The use of paper for the resolution of mixtures of amino acids was first proposed in 1944 (14); its rapidity, simplicity, and reproducibility caused its immediate and widespread adoption. There ale numerous applications of this method and results have been invaluable in elucidating structures, identifying compounds, separatiug impurities, and discovering new compounds (6). A method such as paper chromatography, with such vast potentialities in the field of chemical audysis, deserves to be iiicluded in the curriculum of the future chemist (10, 19, 18, 21). However, the majority of procedures and apparatus previmsly reported were not very suitable for class participation. These consisted in the use of milk bot,tles, hydrometer jars, graduated cylinders, aquarium tanks, etc. In the following procedure, the elementary chemistry student can be introduced to this rapid and corlvenient method of analysis with a minimum of cost and effort. There are two methods in general use for chromatographic separations usiug paper a s the stationary phase. In the first a11d most popular the chromatogram is formed by upward migration of the developing solvent; in the other method it is formed by downward migration of the developing solvent. The former method was chosen because it proved to be more efficient and convenient. The equipmeut, required b,y each student consists of a 1Zin. test tuhe, a length of thin glass rod, aud a cork stopper (91). The glass rod is bent in the form of a loop t,o fit iuside the test t,ube, to prevent the paper strips from touching each other when two chromatograms are run simultaneously in one tube. The ends of GREAT
the glass rod are inserted iu ;L cork which partially seals t,he test tube. For a large i~umherof samples, as in the case of class partiripation, a test-tube rack can be made by drilling ll/n-in. holes in one side of a convenient wooden box. Fikel paper strips, 14 in. long and % in. wide, are cut prior t,o the clam meetiug and are distrihuted by the instructor. (Iiolls of chromatographic paper of t.his width are also available.) The developing solvent may he prepared by the instructor in a large quantity or in the desired small quantity by each student. The u~~krrov-n sample mixtures are t,he ones commonly issued to the strrdeut for analysis hy the conventional macro or semimicro qualitative methods excepting that all hut Group I solutions are chlorides. The procedure for analysis consists of depositing a small streak of sample mixture on the paper st,rip, about one inch from the lower end of the paper, by means of a fine capillary. A pencil line is used to indicate the line of deposition for use in calculating Rf values. A small piece of glass rod is inserted in the end of the paper to prevent curling. The strip is allowed to dry and then placed in the test tube, which coutains about 10 m1. of developing solvent. This developing solveot consists of water, acetic or hydrochloric acid, and some organic aolvent. The tuhe is then loosely sealed with a cork until the solvent reaches the top of the test tube. The chromatogram is removed, dried to remove the solvent, sprayed lightly with distilled water, and the separated cations are identified with suitable chromogenic reagents. Liquid plastic is sprayed on t,he strips to prevent fading of the identifying colors. Certain precautions must he exercised in order to obtain satisfactory results in this classroom application. The air must be relatively free of hydrogen sulfide gas or precipitation of the unknown mixture will occur immediately after the sample solution is placed on t,he paper with no resulting migration. In addition, the sample spot must he dry before the strip is placed in the tube or it will tend to be diluted by the developing solvent, resulting in a poor separation. Of the many separations attempted by the students, the majority of failures could be attributed to one or bot,h of these factors. The chief factors affecting separations by the paper chromatographic method are method of development, the solvent, pH, and the grade of filter paper strips used (24). It. was conrluded from numerous experiments that,
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the ascending method of dewlopment would he the most satisfactory for the small-scale rlassroom procedure. It produced uniform and rol~sistentresults over a wide range of variable conditions. Howver, a somewhat longer developing t,ime is reqnired by the ascending method than the desrending. The speed of development can he cont,rolled by regulating the at.mosphere inside the t,est tnhe. Very rapid migration can he obtained by complrt~elysealing the paper strip in the tube. This usually results in poor separations. To correct this and to slow doxn the development slightly the test tube should not be t,ightly sealed. Various grades of filter paper were inve~tigat~ed and it was found that the choire of paper yielding the best separations depended upon the composit,ion of the developing solvent and the pwrt,icnlar cations to be senal.a.t,rd --
Two grades of filt,er paper which proved very satisfactory for chromatographic analysis by this method are Whatman'sNo. 3 mm. and No. 1. The former is a thick paper, of medium flow rate! while the lat,ter is a light,-weight, fast-running paper. So. 4 is especially suit.ed for the separation of mixt,nres of Group IIA and IIH cations; No. 3 mm. for Gronps I, 111, and IV; No. 1 (a light-weight, medium-running paper) for Group 1.' The Rf values of the particular ions, rhich are defined as the ratio of the dist,anoe traveled by the spot to that, traveled by the liquid front of the developing solvent, (67, were slightly higher when No. 4 paper was nsed as the stationary phase. The primary factor affect,ing t,he t,ime required for development of the chromabogram is the composition of the developing solvent. Most. procedures mentioned in the literature require from 10 to 24 hours. This at, first did not seem s u i t ~ h l since e most laboratory periods are 4 hours. After considemhle investigation, a solvent mixture was prepared by d ~ i r hvery satisfactory separations could be obtained in 4 hours or less. I t consisted of 50 per cent propionitrile, 30 per cent acetic acid, and 20 per cent, water. The arid was added to give more concise hands and to prevent trailing due to hydrolysis and to t,he existence of ions in complex and simple form a t t,he same time (16). A more porous filter paper, What,man No. 1, 1va8 used with the ahove solvent mixture t,o increase t,he rat,e of migration. Although this developing solvent yielded t,he most rapid separation it was not. used for the student's separations because of the toxicity of the propionitrile. Instead, other solvents were whstituted for the propionitrile, and t,he time of development increased from four to 22 hours. Consequently, students mnst ret,urn the following day to remove their chromat,ograms, dry them in an atmosphere free from H2S, and store t,hem for later identification. In t,he interest of health it was felt that this procedure shonld be adopted. The compositions of developing solvents which were fonnd to give very good separations of the cat,ions according to the qualitative aunlytirwl groups are given as follows:
JOURNAL OF CHEMICAL EDUCATION Group I (Silver gl.oup) 85% n-butpl alr. 15% water Sufficient glariul acetic acid to mltktr n pH of 2.53.5
Croup IIA Group IIR (Copper group) (Arsenic-tin group) 80% ttert-butpl alr. 80% n-butyl ale. 10% water 10% water 10% aeeC,-acetic 10% aceto-aret,ir ester ester Sufficient.g l a d scebic acid to mnke a pII of 3.5-4.0
Glnup IITA (Aluminum plaup) 70% ter&butyl alc. 30% water SufficientHCI to mnke s pH of 2.0-3.0 Group IV (Alkaline earth group) 40% see-propy1 alc. 60% 3 AT HCI
Croup IIIH (Nickel group) ( I ) 8 i % acetone 13%4NHCI
Group V (Alkali group) ( 1 , 4, $0) Metahand or n mln. of 70% ethanol 30% w ~ t e r
The pH of the solvents may he determined by means of Hydrion pH paper or a pH meter. The location of the separated ions in their order from the base line when developed by the above solvent,^ is usually as follows: Group I: Hg+, Pht+, Agi Group IIA: Bic+, Pbt+, Cdf+, Cu++8 H g + + Orouu IIB: Ast++. St)+++. Sat+.Sn++++ oroub IIIA: A]++;. Zn++ CroiP IIIR: ~ i + + , ' ~ n +Go++, + , Fe+++ Group IV: Bat+, Srt+, Cn++, M g t + Group V: Kt,iYat, Lit
As in the case of the convent,ional qualitative anal.yt,ical procedures, it may prove advantageous t,o adjusl the concentration of the different cationic solutions in order to obtain good identifications of those cat,ions which prove to he trouhlesome t,o obtain. Identification of the separated ions may be accomplished in several ways: (1) By use of a gas such as H2S (effectivefor Groups I and TI). (2) By use of chromogenic reagents. Group I and TI cations can be made visible by spraying the dried chromatogram with distilled mat,er and exposing them t,o His gas in a closed container, such as a hell jar, for I5 minut,es. The characterist,ic sulfide colors of these cations easily identify t,hem. Group I cations may also be identified with a 0.2 per cent solution of potassium chromate. Gronp 111 and IT' cations are revealed wit,h sperifir chrome genic reagents whose concentrat,ions are given by Feigl (8). Group V cations can he recognized by spraying with a solution of AgN08and fluorescein. The "hrush" t,echnique in the application of these chromogenic reagents proved to he very satisfarto~y. A carbon tetrachloride solution of dithizone produces a rose colorationwith zinc. The color fades as the dit,hizone dries. "Aluminon" (aurin trirarboxylir acid) in a 0.1 per cent aqueous solut.ion gives a permanent pink color with aluminum. Nickel ran he identified wit,h a I per cent alcoholic solution of dimethylglyoxime; nitrosop-naphthol (0.1 g. in 50 ml. of glacial acetic acid) can be used t,o reveal the presence of the cohalt and iron ions. A reddish-hrown rolor is obtained for
