Chromatographic Adsorption' ERNEST L. ELIEL University of Havana, Hauana, Cuba
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HE increasing importance of chromatographic adsorpt~on(1,2)recommends its presentation to the undergaduate, for which purpose this rCsum6 was prepared. GENERAL ADSORPTION TECHNIQUES
Ordinary adsorption consists in the retention of one substance on the surface of another. A simple experiment to demonstrate this is the clarification of an aqueous solution of two organic dyes (methyleue hlue and fuchsine) by means of animal charcoal. Erperimmt I . Add 1 ml. of a 1 per cent solution of methylene blue and 2 ml. of a 1 per cent solution of fuchsine t o about 100 ml. of water. Add one teaspoonful of powdered charcoal to 25 ml. of this solution, shake well, and filter. The filtrate will be colorless.
By means of simple adsorption it is possible to remove certain solutes from a solution, but not, in general, to separate these solutes from one another. Such aeparation can be effected, however, by chromatographic adsorption, which is essentially adsorption in a column. If the solutiou of methylene hlue and fuchsine is passed through a column of aluminum oxide, two zones, an upper blue and a lower red one, will appear presently (3). These zones can be made to separate by development .with a mixture of equal parts of water and acetone. The red zone migrates downward much more quickly than the blue one and will be separated almost completely from the latter. The column is then al; lowed to drain, the adsorbent is extruded and cut, and the pieces are thrown into methyl alcohol which elutes2 the dyes. After filtration, a blue solution of methyl- means of a small cork fitted to a glass rod, the column being ene blue and a red one of fuchsine will be obtained, pushed on to a filter paper and cut by means of a razor blade. proving that separation has been effected to a conThus chromatographic adsorption may be tentatively siderable degree. defined as the differential adsorption of two or more Experiment 2. The column used for this experiment consisted substances in a column of adsorbent material. It has of a wide tube 6 inches long and of about '/,-inch diameter fitted been found, however, that the columnar form of the a s indicated in Figure 1,e. g., a test tube whose bottom has been cut off. The adsorbent, Brockmanu aluminum oxide, was adsorbent is less important than the constant direction mixed with water so as t o form a slurry and then poured into the of flow of the solution. column, an intermittent vacuum being applied in order t o bring about quick settling. Water was added as necessary. The final column had a height of 3'/r to 4 inches, requiring about 15 g. of AI2O8. Five milliliters of the solution used in Experiment 1 were then added t o the water above the column and passed with intermittent vacuum, fresh water being poured in so as t o avoid drying of the column. Finally a mixture of 12 ml. of acetone and 12 ml. of water was forced through with continuous vacuum. After thorough draining the column was stoppered a t both ends, tapped with a piece of wood, and extrusion was effected by 'Presented before the Students' Chemical Club, "Carlos Theye." of the University of Havana, March 15.1944. Elution is the process of separating an adsorbate from an adsorbent, by means of aliquid, an eluent.
HISTORY
Chromatography was discovered in 1906 by the Russian botanist M. Tswett. He employedthe chromatographic method to investigate plant and animal pigments, and by means of i t proved that chlorophyll is not a homogeneous substance but a mixture of a- and 8-chlorophyll. Tswett's most famous experiment is the adsorption of a petrol ether extract of green leaves in a column of sucrose. In order to adsorb not only chlorophyll but also xanthophyll and carotene we shall use a double
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column, the lower part of which consists of aluminum oxide while the upper part is powdered sucrose (4). Esperiment 3. Two or three green leaves are ground in a mortar with some powdered glass and extracted twice with a mixture of 20 ml. of petrol ether and 2 mi. of methyl alcohol. The extract is filtered into a small separatory funnel and washed six times with water in order t o free it from methyl alcohol. (If a small emulsion zone forms a t this stage, i t may be discarded.) After drying with anhydrous sodium sulfate, 5 ml. of the extract are poured into the column which has been prepared according t o Figure 2. Alumina (Brockmann) is made into a slurry with petrol ether, poured into the column and allowed to settle. The column is completed with a slurry of powdered sucrose in petrol ether.' Development of the chromatogram is effected with petrol ether until a satisfactory separation of the zones is obtained.
CHEMICAL EDUCATION
Tswett's method fell into disuse and was only very occasionally employed until 1931, when it was first applied to preparative work, uiz., the separation of the and Of pure (5). Since 1931 the method has been extensively applied both to preparative and analytical work and has become an indispensable tool in organic chemistry, ~ 1 s.chromatography~ according to its Greek 'Oats means "writing in it is now employed for ~olorle~s as well as colored substances. APPARATUS AND TECHNIQUE
Generally speaking, the technique of chromatographic adsorption depends on whether the substances to be adsorbed are colored or colorless. Colored substances can easily be recognized in the column and may be separated by cutting the latter. Colorless compounds require more complicated procedures. The process may be represented by the following diagram: Preparation of Column
I
Adsorption 01 Subrtanees
colored
I Development I Extrusion I Cutting I Elution of Piere9
I
colorie~~
I ~raetio~*i elution of the entire column ?'Liquid Chromatogram")
FILTER
PAPER
FIGURE 2
In this experiment four rings will be obtained: a green One of chlorophyll and a One Of xanthophyu in the sucrose' and a and a red One Of xanthophyll and carotene, respectively, a t the top of the aluminum oxide. Separation of the two chlorophyUs is not so easy to effect. One would expect Tswett's discovery to have exerted immense influence on the chemical development of his time. He discovered nothing less than the separation of various components of a mixture by means of a knife. He himself was fully aware of the importance of his method and performed a large number of experiments to illustrate its applications. However, perhaps because Tswett's main work was published in Russian or because the chemists of1910 did not want to believe that chlorophyll (which had already been obtained in nys. was not a homogeneous substance'
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EdifmPsNote: "Confectioners' sugar" is generally preferable to powdered sucrose.
Preparation of the Column. Much depends on the choice of adsorbent. As adsorption is a surface phenomenon, theoretically many substances in a finely divided state-powder or fibers-can be used. Practical considerations, however, limit the number of available adsorbents to substances which are insoluble in solvents and elnents and do not react with them or with the materials to be adsorbed. It is convenient if the adsorbent can be easily dried and regenerated by heating (which condition is not met by organic substances); moreover it should not be strongly acid or basic, nor should it be strongly chlored if it is to be used for the chromatography of colored compounds. Still there exists a considerable number of substances which can be employed, such as alumina, magnesium oxide, calcium hydroxide, magnesium carbonate, calcium phosphate, calcium carbonate, talc, inulin, and sucrose (in this order of decreasing activity) (6), as well as gypsum, fuller's earth, kieselguhr, silica gel, charcoal, permutit, and Hydralo, among others. One of the most useful adsorbents for laboratory experiments is Brockmann aluminum oxide. Ordinary alumina is usually not sufficientlyactive. Experiment 4 . Repeat Experiment 2 with ordinary calcined alumina as adsorbent instead of aluminum oxide according to Brockmann. (In this case it is preferable t o pour the dry adsorbent into the column, compress i t fairly tightly, and pass some water through the column before adding thesolution of the dyes.) No separation of the dyes will be effected.
It should be emphasized that the choice of adsorbent is still largely empirical. In order to prepare the column, the bottom of the glass tube to be used is suitably secured by means of glass wool or cotton and filter paper, some solvent is poured in, and then the adsorbent is added, either in powdered form or mixed with the solvent in form of a slurry. The column should be stirred frequently while it settles in order to keep it free from air bubbles. Alternatively the column may be prepared by pouring the dty,powdered adsorbent into the tube and compressing it tightly by means of a suitable instrument before adding the solution. Adsorption of Substance. The substance to be absorbed should, in general, be in dilute solution. The solvent (the same which has been used for preparing the column) most widely used in organic work is petrol ether. It bas the advantage of possessing but little eluent action (i. e., i t favors adsorption of the solute), is cheap, easy to handle, and easy to remove by evaporation. Other useful solvents are carbon disulfide, chloroform, carbon tetrachloride, cyclohexane, ligroin, and benzene. Benzene has a stronger eluent action than petrol ether, and it may occur that a substance which is adsorbed from petrol. ether solution by a certain adsorbent is not adsorbed from benzene solution by the same adsorbent. For fine work pentane and hexane may be used with excellent results. When the solutionis poured on thecolnmn,care should be taken not to stir up the latter. Under no circumstances must the level of the liquid disappear from above the adsorbent material during the process of adsorption and development. If this occurs, i. e., if the column ''dries out," i t will shrink,,breaks and channels may form, and subsequent development will be uneven. In general, solutions should pass through the column by virtue of their own weight, but if passage is very slow, vacuum-or pressure a t the top in the case of volatile solventsmay be applied. Deuelopment. A chromatogram is developed .with the purpose of bringing about a clean separation of the zones. Development may be effected with the solvent used originally (cf. Experiment 3), or with some other solvent of greater eluent power, or with a mixture (cf. Experiment 2). Extrusion, Cutting, and Elution. Extrusion and cutting must be effected with the greatest care in order not to break the column. The pieces obtained by cutting are thrown into separate portions of eluent, generally ethyl or methyl alcohol or, occasionally, ether. After some stimng, the solution so obtained is filtered. The remaining adsorbent may have to be extracted once or twice more with the eluent and is then stored for reactivation, while the clear solution is subjected to any further treatment which may be desired. Colorless Substances. There are various ways of cut tin^ " chromatoerams of colorless substances: (a) An empirical procedure. The column is cut
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into a large number of equal parts and these are eluted separately with the hope of effecting partial or complete separation a t least in the pieces a t the extreme ends of the column. This method has been employed for the separation of higher fatty acids (7). (b) By means of an internal indicator. For example, in order to separate stearic from oleic acid, a petrol ether solution of the mixture is passed through a column of magnesium oxide impregnated with phenol red. After development with petrol ether, the acid zones appear as yellow bands in the red column which can then be cut. The pieces are dissolved in HC1 and the free fatty acids extracted with ether (8). (c) By conversion of the substance into a colored derivative. Thus levulinic acid, C H s CO - CH2 CHa - COOH, and geronic acid, CHs - CO - CH*CH1- C(CH& -COOH, may be separated after conversion into the colored 2,4-dinitro phenyl hydrazones from which they are regenerated, after chromatographic separation, by means of glyoxal (9). Recently fatty acids have been separated after conversion into the colored 2,4-dinitro benzyl esters. (d) By means of ultraviolet light in the case of substances which are fluorescent toward ultraviolet radiation (10). (e) By means of external indicators spread along the column after extrusion by means of a brush. Thus the zone containing vitamin A in a completely colorless chromatogram may be found by brnshing the column with a solution of antimony trichloride in chloroform (Carr-Price reagent) which marks the vitamin A zone blue (11). Nevertheless in many cases none of these devices can be employed. One must then resort to the socalled "liquid chromatogram" which is based on fractional elution. The preparation of the column and adsorption are effected in the usual way, except that no colored zones will, of course, be formed. Let us assume that adsorption has taken place from petrol ether solution. More petrol ether is then passed through the column and a t the same time one investigates by means of color tests or actual evaporation of a few drops on a watch glass whether the petrol ether which flows out contains any substance. As soon as substance begins to come out, fractions of 10 to 100 ml. (according to the size of the column) are taken until petrol ether brings about no further elution. When this moment is reached, development is continued with petrol ether-benzene mixture (9:l for careful work). When this mixture also ceases to elute substance, the proportion of benzene in the eluent is slowly increased. When no more elution takes place with pure benzene (the last member in the series of petrol ether-benzene' mixtures) development is continued with benzene-ether mixtures, ether, ether-acetone, acetone, and finally with methyl alcohol, pyridine, or acetic acid in the case of very strong adsorbates. In favorable cases (which are unfortunately rare) each component of the original mixture may come out with a d i e r e n t eluent. Eventually the fractions are freed of solvent by distillation
and are thereafter subjected to further investigation. question are mixed and the mixture chromatographed. Obviously the process of making a liquid chromato- If one zone only is formed they are identical, while the gram is very laborious. With unknown substances i t formation of two different zones naturally indicates may take several days, although once the appropriate nonidentity. Unfortunately, chromatographic tests eluents have been found by means of a pilot chromato- for homogeneity and identity can be carried out congram on a small scale, the procedure usually can be veniently only in the case of colored substances, and only if the substance in question is adsorhed on the simplified. Apparatus and Special Methods. A great variety column with sufficient intensity. Another highly important application is the conof special glassware has been evolved for use in chromatography. Chromatograms can even be,made in 3- centration of a natural product. Let us choose penicillin as an example. The Penicillium notatum promm. glass tubes and in Biichner funnels. Only two examples of the application of special duces about 1 to 10 unitsper ml. of culture medium. methods will be mentioned here. If elution is difficult But a gram of penicillin contains 450,000 units; i . e., or impossible to effect because the adsorbate adheres one must work up about 200 to 1000 liters of liquid in too strongly, the adsorbent may be removed or modified order to obtain 1 g. of substance (assuming a yield of in some special way. For example, in Tswett's ad- 40 to 50 per cent). In order to avoid handling such sorption of chlorophyll the sucrose may he removed huge volumes of liquid, it is necessary to concentrate by dissolving i t in water. The magnesium oxide used the material in an early stage of its preparation. To to separate fatty acids is dissolved in HCl. Or a this end the material is chromatographed in ethereal substance may be very strongly adsorhed on calcium solution (after repeated previous extractions, which hydroxide, but only very weakly on calcium carbonate. bring about an approximately tenfold concentration). In that case it is adsorbed on the hydroxide, the ad- Four zones will be obtained in a column of aluminum sorbate is supended in water and treated with a current oxide: of carbon dioxide. Thus calcium carbonate is formed 1. A dark brownish-orange layer at the top which contains and elution can now he effected easily (12). some penicillin. USES
The most important application of chromatographic adsorption in organic chemical work has already been indicated, namely, the separation of the components of a mixture. This application alone would give great importance to the chromatographic method, in contributing to the solution of one of the hardest problems of organic chemistry. Actually, chromatography sometimes makes possible the separation of substances which cannot conveniently be separated by any other method -the two chlorophylls and the three carotenes. Another application-perhaps the oldest and simplest of all-is the purification of certain substances by means of adsorbents in columnar form. When chromatography was still largely unknown, columns of charcoal were already employed to purify edible oils. The strongly colored and often odorous oil is passed through the column and comes out with but a light color and almost odorless, as the components of pronounced color and smell are adsorbed in the column. The same principle has been employed for the purification of benzyl chaulmoograte, which is used in the treatment of leprosy (13). Chromatographic adsorption can also be used to test the homogeneity of a substance (14). A substance is homogeneous if on being passed through an adsorption column it is adsorbed in one single zone. There are but few substances which are similarly adsorbed on a number of columns of different materials and it has been claimed that the criterion of adsorption of a substance is more typical than its melting point, boiling point, absorption spectrum, etc. The identity or nonidentity of two substances can also be established chromatographically (15). The two substances in
2. A light yellow layer containing most of the penicillin and no pyrogens. 3. An orange zone which contains some penicillin and most of the pyrogen. 4. A brownish layer containing no penicillin.
The column is washed with ether, extruded, and cut. The fourth layer is discarded, the other three are eluted separately with a phosphate buffer of pH 7.2. The eluent of the second zone will now contain a large concentration of penicillin-as a big volume of ethereal solution may be passed through the column but only a small amount of eluent is required. The eluents of the first and third zones are used to elute the next batch. Last but not least, the numerous applications of chromatographic adsorption in the analysis and control of industrial and pharmaceutical products must he mentioned. Chromatography is used for the determination of the homogeneity and purity of dyestuffs, in the investigation of oils and fats, for the analysis of drugs from natural sources, in testing for the absence of artificial coloring matter in butter and wine, and in many other similar instances. Recently i t has also been applied to quantitative organic analysis; e. g., of the three carotenes, which are separated by chromatographical adsorption previous to spectrophotometric determination, and of vitamin D in fish oils, which is separated chromatographically from vitamin A before it can be estimated calorimetrically or spectrophotometrically by means of the yellow color it gives with chloroformic solution of antimony trichloride (16). Applications in Inorganic Chemistry. Although the applications of chromatography in organic work are more numerous than those in inorganic chemistry, the latter are not without importance. The separation of a
in turn from a-carotene (10 conjugated and one isolated double bonds). Among aromatic hydrocarbons, those with the largest Experiment 5. The column for this experiment is prepared as number of rings are most strongly adsorbed; e. g., the one in Experiment 2. A mixture of 2 ml. of a decimolar solution of cnpric nitrate, 2 ml. of a decimolar solution of cobalt- naphthacene forms a zone above anthracene while ous nitrate, and 1 ml. of adecimolarsolution of ferric nitrate with naphthalene passes through the column (21). water is poured through the column without suction. DevelopNOWwe c q explain also why petrol ether is a better ment is effected until the liquid above the column is colorless. solvent than benzene. The former being an aliphatic ~ h r e grings will be formed: a brown one of Fettt, a blue one of hydrocarbon of low molecular weight suffers almost no Cut+, and a pink one of Cot+. Twenty milliliters of a concentrated solution of K,Fe(CNh are then passed, applying vacuum. adsorption itself and therefore has no tendency to disThe ring of Fe'++ will turn dark blue, the one of Cut+ place the adsorbed substance. Benzene is adsorbed in reddish brown, the one of Co++ grayish green. the column to a certain small degree and may displace Actually most cations as well as anions can be other substances whose adsorption affinityis very weak. separated chromatographically and lists have been The eluent action of ether, acetone, and alcohol (inmade arranging the ions according to their adsorption creasing in this order) is explained by the fact that these substances are themselves strongly adsorbed and tend affinity (17). The separation can be applied to quantitative de- to displace any other substances which may have been terminations and has possibilities in industrial analysis; adsorbed previously. The mechanism of the formation of chromatogaphic so, for example, one microgram of Fe+++ can be detected in a molar solution of Cu++ by means of chro- zones can easily be illustrated by an experiment. When matographic adsorption and subsequent development two diierent snbstances are poured through the column of the column with EiFe(CN)s (18). The chromato- they will at first be adsorbed indiscriminately, but graphic method is so sensitive that it bas even been used soon the molecules of the more strongly adsorbed substance displace those of the other and force them to to separate the isotopes of lithium. migrate downwards, forming a lower zone, while the THEORETICAL strongly adsorbed substance will stay a t the top (22). Chromatographic adsorption, like any adsorption, Experiment 6. Dilute 1 ml. of s. 1 per cent solution of fuchsine is produced by forces which act between the molecules with 100 ml. of water and pass 3 ml. of this diluted solution of the adsorbent and those of the substance to be ad- through a column of the type used in Experiment 2, using sorbed, forces similar to those which hold together the vacuum. A red zone will be farmed. Pass 3 ml. of a dilute atoms in a molecule. solution of methylene blue (1 ml. of s 1 per cent solution of It is clear that these forces depend on the nature of methylene hlue in 100 ml. of water). The methylene hlue will the adsorbent and on the molecular arrangement of the displace the fuchsine, placing itself at the tap of the column. Experiment 7. Repeat Experiment 6, inverting the order of substance which is passed through the column (19). the dyes. The fuchsine will not dislocate themethylene hlue but The substances most strongly adsorbed are the organic migrate through the blue zone placing itself helow it. acids which contain the very active -COOH group (which, moveover, has a large polar moment). Next in It remains to be proved that the adsorption of a the list are the alcohols, whose functional group is also substance takes place in a sharply defined zone of convery reactive and polar. Then follow the ketones, stant intensity. For the mathematical treatment of esters, and ethers, with their functiona! groups of de- chromatographic adsorption, one is referred to the work creasing reactivity and polarity, then unsaturated of J. N. Wilson (23). hydrocarbons, aromatic hydrocarbons, and ultimately Because of uncertainty in several factors involved aliphatic hydrocarbons, which are unreactive, have a in the mathematical analysis, the theory of chromatogzero polar moment, and are generally not adsorbed. raphy has not until now found much practical application. In practice, empirical procedures are still Most strongly Acids Alcohols Sfmngduentl adsorbed almost exclusively employed. However, the theoretKetones ical treatment illustrates the discontinuous nature ES~M weak etueoto Ethers of chromatographic adsorption: A small portion of the Unnaturated hydrocarbons solution which passes through the column remains unWeakly adsorbed Aromatic hydrocarbons (Pass) Saturated aliphatic hydrocarbons changed as long as i t passes through already occupied regions, but as soon as it comes into a still empty part Among the unsaturated compounds, those which of the adsorbent, all the substance dissolved is at once have their double bonds conjugated are more strongly adsorbed and its concentration in the solution drops adsorbed than others. As expected, the adsorption affinity increases with the number of double bonds (20). discontinnonsly to nought. Small changes in molecular structures bring about quite ACKNOWLEDGMENTS considerable changes in adsorption affinity. So, for The author wishes to express his appreciation to Dr. example, it is quite easy to separate y-carotene (11 conjugated and one isolated double bonds) from 8- George Rosenkranz, the Chemical Club, "Carlos carotene (11 conjugated double bonds) and those two Theye," of the University of Havana, and the Labmixture of cations in the form of their nitrates, can be illustrated by a simple experiment.
+
(9)ZECHMEISTER AND CHOLNOKY, "Principles and Practice of Chromatography," p. 80. (10) Ibid., p. 81. (11) Ibid., p. 85. (12) Ibzd.. p. 55. REFERENCES (13) Private communication from Dr. G. Rosenkranz, Laboratorios Vieta-Plasencia, S. A,, Havana. (14) Ref. (21, P. 7. (1) STRAIN, "Chromatographic Adsorption Analysis," Inter(15) Ibid., p. 8. science Publishers, Inc., New York, 1942. AND E m n , "Physical-chemi(16) EWING.KINGSLEY, BROWN, "Principles and Practice of (2) Z E C ~ E I S T EAND R CHOLNOKY, cal method for determination of vitamins D in fish.livcr Chromatography," translated by BACHARACH AND ROBINoils," Ind. Eng. Ckem., Anal. Ed., 15,301 (1943). SON,John Wiley and Sons, Inc., New York, 1941. (17) Ref. (2). pp. 306 and 314. (3) Ibid., p. 190. (18) Ibid., pp. 306 and 313. ( 4 ) Ibid.. p. 89. (19) STRAIN, "Chromatographic adsorption analysis," Ind. Eng. (5) Ibid.. p. 11. Chem., Anal. Ed., 14,247 (1942). (6) STRAIN. "Chromatographic adsorption analysis." Ind. Eng. (20) Ref. (2). p. 24. Chen., Anal. Ed., 14,248 (1942). (21) Ibid., p. 33. (7) CASSIDY, "Separation of mixtures of higher saturated fatty (22) Ibid., p. 15. acids." I. Am. Chem. Soc., 63,2735 (1941). (23) WILSON,"A theory of chromatography," J. Am. Chem. (8) G R A AND ~ SKAN,"Colored chromatograms with higher Soc., 62, 1583 (1940). fatty acids." I d Eng. Chem.. Anal. Ed., 15,340 (1943).
oratorios Vieta-plasencia
valuable suggestions.
S. A. f o r their cooperation and
