Harold H. Strain
and Joseph Sherma' Argonne National Laboratory Argonne, Illinois 60439
II
Modifications of Solution Chromatography Illustrated with Chloroplast Pigments
M a n y of the modifications and applications of chromatography are easily demonstrated with the mixture of green and yellow chloroplast pigments extracted from photosynthetic organisms. These pigments, the chlorophylls and carotenoids, are notable for their deep-green and yellow colors; hence, they are readily observed as they are separated in the chromatographic media. Studies of the chloroplast pigments led to the discovery or invention of columnar chromatography (I), and paper chromatography (2). With these colored substances, it was apparent that only the formation of a narrow initial zone of mixture followed by washing with fresh solvent (wash liquid) provided an extensive resolution of the components into a series of zones, the chromatogram. The earliest experiments with this development procedure also led to the nomenclature that has become widely accepted (1). Now the use of these pigments provides striking demonstrations of the effectiveness of chromatographic techniques as separatory or analytical tools. Specifically, the extracts of green plant material serve for the illustration of such procedural modifications of chromatography as the columnar technique, paper chromatography, and thin-layer chromatography. Separations of the pigments in columns and in thin layers may be effected with various systems or combina.tions of sorbents and solvents. For separations in paper, the principal variable is the wash liquid. I n addition to the historical, selective-adsorption mechanism, which was first employed, separations of pigments may also be based upon their selective partition between two immiscible liquids, the more-polar liquid being fixed in a nonsorptive, porous support. With this partition chromatography, the sequence of the separated pigments may be reversed by using a fixed, nonpolar, sorptive phase instead of the morecommon, fixed, polar phase. Geometric modifications of the sorptive medium and of the flow of the wash liquid as in one-way, two-way or transverse, and radial migration or development, are readily demonstrated with the pigments. With all these geometric modifications, the pigments reveal the importance of the dimensions of the medium, especially in relation to the loading of the system and the distance of migration or development. The shape of the zones of the separated pigments often varies with the geometric arrangements and with colorless substances that acBased on work performed under the auspices of the US.Atomic Energy Commission. 'Present address, Department of Chemistry, Lsfayette Callege, Easton, Pennsylvania 18042.
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company the pigments. All these effects are conspicuous in the chromatography of the pigments. The separability of the pigments by chromatography reflects small differences in their chemical composition and molecular structure. The yellow carotenoid pigments fall into two principal groups, the carotenes and the xanthophylls. The most common carotenes, called a- and @-carotene,are isomeric, nonpolar, polyene hydrocarbons, CaoHSB, with eleven double bonds, all conjugated in 8-carotene and only ten conjugated in a-carotene. The xanthophylls are polar, oxygen derivatives of the carotenes. These derivatives contain from one to about six oxygen atoms, which may occur as alcohol, keto, epoxy, or ester groups. The green chlorophyll~are tetrapyrrolic compounds with an additional isocyclic ring and a central complexed magnesium atom. The two common chlorophylls, chlorophyll a, C,sH,20,N4Mg, and b, C16H~006NlMg, differ only in the state of oxidation of a single group, a methyl, -CHa, in a and an aldehyde, -CH=O, in b. To avoid their alteration, the chlorophyll pigments must be sorbed on the mildest, least-reactive sorbents such as cellulose or powdered sugar. Xanthophylls may be sorbed on the mildest sorbents, and some may be sorbed on more-active adsorbents, although a few are decomposed by alkaline or acidic media. The carotenes are not sorbed by mild adsorbents, even with the least-polar wash liquids, and cannot be separated from one another with these adsorption media. They are resolved with the selective, activated aluminas, lime and magnesia. I n the chromatography of the chloroplast pigments, it is not always recognized that the pigments themselves are extremely susceptible to chemical change. These chemical alterations may be induced by changes in the plant material itself, as in wilted, dried, frozen, anesthetized, canned, or brined tissue. Some of these chemical changes may occur during the extraction, handling and storage of the pigments. Some alterations may occur on the sorbent, especially on highly-activated adsorbents, such as calcium silicate, or on acidic or basic adsorbents, such as some silicic acids or magnesia, respectively. Chlorophylls, for example, are altered by most inorganic adsorbents and by acidic and basic adsorbents. Xanthophylls with epoxy groups are altered (isomerized) by acidic adsorbents. All the pigments are isomerized, reversibly, into labile isomers by heating their solutions. The carotenoids yield labile cis arrangements a t some of the double bonds. The chlorophylls yield epimeric isomers due to geometric exchange of two groups on the isocyclic ring. I n the preparation of the plant extracts, special care must be exercised in the selection of the
plant material, in the extraction of the pigments and in their preparation for the adsorption. Much chromatographic experience (5, 4) has shown that plants of the major taxonomic classes differ systematically with respect to their green and yellow pigments. These differences are readily illustrated by chromatography on thin layers of special, nonreactive, silica gel. Illustrative Procedures for Leaf-Pigment Separations: Extraction of the Pigments
Many kinds of fresh plant material can be used as a source of the chloroplast pigments. We have employed cocklebur (Xanthium) leaves grown in a greenhouse and fresh spinach leaves obtained from a market as well as various species of algae described below. If spurious chromatographic observations are to be avoided, the pigments must be extracted quickly from fresh material under conditions that avoid their physical isomerization and chemical alteration. Two important extraction procedures are described; one employs a blender; the other employs scalding. Blender Method
Two grams of tender, green leaves with large veins and petioles removed, are placed in a chilled blender with 60 ml of cold acetone or methanol and blended at high speed for 2 min. The mixture is then centrifuged, and the clear, green supernatant is put into a separatory funnel. Forty milliliters of cold petroleum ether (2040°C) and 100 ml of saturated aqueous NaCl is added. The mixture is shaken, and the layers allowed to separate. The lower layer is discarded, and the upper layer washed successively with two 100-ml portions of distilled water. The washed upper, green layer is poured out through the top of the funnel into a round-bottom flask and evaporated to dryness below 40°C under vacuum. (A rotary evaporator is convenient, if available. If the extract is to he stored before use, it should be kept under vacuum in the dark and cold to avoid decomposition of the pigments.) The residue is dissolved in 1 ml of petroleum ether (60-110°C) to prepare the sample solution. The xanthophylls and carotenes of plants are examined conveniently after removal of the chlorophylls by saponification with alkali. For this saponification, 10 ml of 30% ICOH in methanol is added to the centrifuged acetone or methanol extract in the separatory funnel. After 30 min with occasional swirling, 40 ml of cold 1:1petroleum ether (2040°C)diethyl ether plus 100 ml of 10% aqueous NaCl solution are added. The resultant upper golden-yellow layer is washed with water and taken to dryness as above. The sample solution is prepared by dissolving the residue in 1 ml of 1: 1 petroleum ether (60-llO°C)-diethyl ether. This saponification procedure should not be employed with plant extracts containing xanthophylls that are decomposed by alkalies as are fucoxanthin, from diatoms and brown algae, and peridinin, from dinoflagellates (9). Non-Blender Scolding Method
For extraction without a blender, 2 g of leaves are cut-up and placed in 100 ml of boiling water in a flask or beaker. After 2 min, the vessel is placed in an ice bath,
and the water poured off when cool. The leaves are then extracted with 100 ml of 90% methanol-10% diethyl ether followed by 100 ml 70y0 methanol-30% diethyl ether. The extracts are combined in a separatory funnel, and 100 ml petroleum ether (2040°C) is added. The pigments are then transferred into the petroleum ether-diethyl ether mixture with aqueous salt solution, washed, and handled as described above. Saponification may also be carried out by adding ICOH to the methanol-diethyl ether mixture in the separatory funnel. For the extraction of unicellular algae, the extraction procedure is modified through collection of the organisms by centrifugation after treatment with boiling water and each extraction step. The use of fresh plant material is vital in order to avoid secondary transformations of the pigments in the organism itself. Boiling the material for a short time inactivates the plant enzymes and renders the pigments more readily extractable. This scalding step may, however, cause alteration of the natural pigments producing additional pigments, as will be described below. The extraction procedure should be carried out as quickly as possible in order to avoid alterations due t o exposure to air and light. The identity of the pigments in the separated zones may he established by their colors, chromatographic sequence, absorption spectra in the visible region, and reaction t o the vapors of HCl. For determination of the absorption spectra, the individual zones are removed separately from the chromatogram, and each pigment is eluted. With powdered adsorbents, the colored solid from each zone is packed into a fresh tube for the elution of each pigment. Ethanol is used for the elution and the absorption spectra of the carotenoids; diethyl ether for the chlorophylls. The spectra are compared, in regard to shape and wavelengths of the absorption maxima, with recorded spectra for pure, authentic pigments (5, 5-7). The, . ,X for the leaf pigments arc (in nm or mr): p-carotene, 452, 480; lutein, 446, 474; violaxanthin, 442, 470; neoxanthin, 437, 466; chlorophyll a, 428.5, 660.5; and chlorophyll b, 452.5, 642.0. Reaction of violaxanthin dissolved in diethyl ether with concentrated KC1 produces a deep blue color in the acid layer; the other leaf carotenoids remain yellow (9, 7). Separated in paper or thin layers of magnesia and exposed to the vapors of concentrated HCI, violaxanthin turns blue and neoxauthin blue-green (8, 9). Separated in siliceous adsorbents, the zones of all the carotenoids turn blue or blue-green, but the color of the lutein and carotene zones returns to yellow with time (10). Column Adsorption Chromatography
Chromatographic tubes, fitted with a f i t t e d glass disc or a plug of cotton or glass wool, are filled with adsorbent by packing successive small portions of the dry powder with a plunger. Tubes 1 cm i.d. X 25 cm are packed to a height of 20 cm, and tubes 2 cm i.d. by 40 em to a height of 25-30 cm. For columns of sugar, the tubes are usually packed with commercial confectioner's powdered sugar, already containing 30jo starch, which is first rubbed through a coarse sieve with a pestle. I n very humid weather, especially if the packed columns are to stand more than a few hours before use, it is advisable to mix the sugar with an additional 10Yo by Volume 46, Number 8, August 1969
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weight of corn starch (Argo brand, Corn Products Company). This latter product is also employed alone for starch columns. Activated magnesia (Sea Sorb 43, Fisher Scientific Co.) is mixed with an equal weight of Celite 545 filter aid before it is pressed into columns. Without the use of suction, the sample solution is introduced gently into the column with a pipet and permitted to percolate into the adsorbent to form the narrow initial zone. When the solution percolates beneath the top of the adsorbent, several very small portions of petroleum ether (6&110°C) are added to rinse the inside of the tube just above the adsorbent bed. The wash liquid is then added gently, so as not to disturb the top of the bed, and development with fresh solvent is continued until the solvent front reaches the bottom of the bed. This development required approximately 30-90 min (without suction), depending upon the sorbent, solvent, and size of the column. It may he speeded up considerably by applying about 0.5 atm of suction. Favorable loading, as indicated by good separation and convenient visual detection of the major zones, is about 200 p1 for the 1-cm columns and 800 p1 for the 2-cm columns of the three adsorbents. Figure 1indicates the separations obtained when leaf extract and saponified leaf extract are chromatographed in columns of various adsorbents with various wash liquids. These figures represent fully-developed chromatograms resulting from the gradual separation of the pigments upon washing with the solvent. Development on sugar with 0.5% n-propanol in petroleum ether (2040") yields separate zones of the leaf pigments in the sequence: carotenes (B cr) (least sorbed), chlorophyll a, lutein (* zeaxanthin), chlorophyll b, violaxan-
*
SUGAR 0.5% Pr
STARCH 0.5% Pr
MgO 5% Ac
MgO
Ac
MgO ethylene-
dichloride
F
T
SE
F
Figure 1. Pigmenh separated b y adsorption c h r ~ m a t o g r o ~ hiny columns of various odrorbenh with various wash liquids. (See the table for the symbols and obbreviationr used in this and other figures).
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thin and neoxanthin (most sorbed) (see Fig. 1). One percent n-propanol yields a similar separation, hut all the pigments are less sorbed. With 0.5-1% isopropan01 in petroleum ether (2040°C),the zones are less completely resolved than with n-propanol (3). I n columns of starch and with 0.5% n-propanol in petroleum ether (20-40°C), the pigments are in a sequence different from that obtained with sugar and either 0.5 or 1% n-propanol: lutein is sorbed below chlorophyll a, but the two pigments are not completely separated. Chlorophyll b and violaxanthin are also contiguous in this system. Development with ly0 n-propanol on starch yields the same sequence as on sugar with this wash liquid, hut the separations are inferior. Separations on sugar and starch, as well as on cellulose and siliceous adsorbents, are most effective with pigments that differ in polarity, such as chlorophylls a and b or neoxanthin (CloHjaOa, 3-OH plus 1 epoxide), violaxanthin (CloHssOn,2-OH plus 2 epoxides) and lutein (CloHs6Oz,2-OH). These sorbents do not separate pigments which diier only in the arrangement of the double bonds, as do lutein and zeaxanthin. They also do not separate pigments that are not adsorbed, as CY- and pcarotene. Figure 1 shows that with columns of activated magnesia plus Celite, the carotene isomers are separated by washing with petroleum ether (2040°C) plus 5-20% acetone. Only a trace of &-carotene occurs in the cocklebur and spinach extracts studied. The xanthophylls and chlorophylls (which are altered by magnesia as well as by activated kieselguhr G, see below) are fixed at the top of the column. On magnesia plus Celite washed with pure acetone, the saponified leaf extract provides the sequence: carotenes, violaxanthin, lutein, and neoxanthin. The order of the lntein and violaxauthin is reversed when the column is developed with ethylene dichloride, freshly extracted with water and dried over Drierite. Zeaxanthin, which has the same formula and functional groups as lutein but has one more conjugated double bond, is separated from lutein and the other xanthophylls as a very strongly sorbed zone on magnesia (3, 11). BeAbbreviations and Symbols
a chlorophyll a a' chlorophyll n' Ac acetone h chlorophyll b h' chlorophyll b' Bs benzene C carotene o chlorophyll e Ch chloroform D diadinoxsnthin DMF dimethylformamide E extract
F front G green Gy grey IP isopropanol L lutein LE leaf extract Lx loroxanthin
M myxaxanthin Me methanol Mx myxoxanthophyll N neoxrtnthin 0 orange P pheophytin Pe peridinin P E petroleum ether Pr n-propanol II red S siphonein SE saponified extract Sx siphonaxanthin T top V violaxanthin X starting point Y yellow Z zeaxanthin \\\\blue by HCI VIl""14 .-r---
////blue-green HCL vapors
by
cause zeaxanthin is a minor pigment of leaves (9) and because the columns are lightly loaded, t,he zone due to this pigment is not readily visible. Paper Adsorption Chromatography
For one-way or lineal ascending development, squares of Whatman No. 1 paper (20 X 20 em) are spotted with the leaf extract from micropipets 1 in. from the lower edge. The init,ial zones are dried; the paper is stapled in the form of a cylinder and placed in a paper-lined, covered container which has been equilibrated with the wash liquid for at least 30 min. The container may be a waste can or a glass jar wrapped with aluminum foil to protect the pigments from photodecomposition and to reduce thermal gradients in the system. After the wash liquid has risen 15 cm (about 30 min), the paper is removed, air dried, and the sep; rated zones are marked.
Figure 3. Pigmenb of l e d extract repormkd b y two-way development on Wh0tm.n NO. 1 pope,.
For two-way chromatography, the extract is spotted in one corner of a square of paper (20 X 20 om) and developed with the first solvent as in a one-way migration. The paper cylinder is then opened and air dried for 5 min, after which it is restapled in the form of a second cylinder with the axis at right angles to that of the first one. This cylinder is developed with another solvent (different from the first one) in a second equilibrated chamber. Figure 3 shows the separation of carotene, two chlorophyll~and three xanthophylls when a spot of 3 pl of leaf extract is developed by two-way chromatography on Whatman No. 1 paper. At higher loading, precipitation at the origin and severe double-tailing is observed, leading to incomplete separations and multiple zonation. These multiple zones cannot be attributed to the presence of a differentpigment in each zone (14). Figure 2. Pigmenh in va+ms omountr of leof eitroct reporated by o n e dimensional lone-way) migration on Whatman No. 1 paper from initial ronesformed 0 s a spot and or a streak.
Figure 2 shows the results obtained when the leaf extract (2-10 p1) is'developed in Whatman No. 1 paper with a wash liquid composed of petroleum ether (2040°C)-benzene-chloroform-acetone-isopropanol,50: 35 : 10:0.5:0.17 (v/v) (18). The chloroform is washed with water and dried over Drierite before use. With low loading of the initial zone in the form of a small spot, the pigments are separated in the sequence: carotenes, lutein, violaxanthin, chlorophyll a, chlorophyll b plus neoxanthin. With higher loadings, the pigments are all pushed forward resulting in poorer resolution, and the double tailing of the zones, characteristic of separations on cellulose (8, IS), is more pronounced. This double tailing is eliminated when the starting zone is applied as a uniform, narrow streak (5-10. N1 extract per cm of streak). When the loadings are comparable, the separations from the streak and in the central regions of the migrating spots are identical.
r"
' ""
a
Tab
Figure 4. Pigmenh of leof extract reporated b y rodial chromatography on Whdmon No. 1 paper.
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Radial chromatographic development is carried out in large, covered, equilibrated containers with paper cut in the shape and dimensions shown in Figure 4. This paper is supported in a horizontal position on a crystallizing dish, with the tab bent downward into the wash liquid. A typical radial chromatogram of the leaf pigments is shown as Figure 4. Although the circular zones of chlorophyll b and violaxanthiu and of carotene, chlorophyll a, and lutein are contiguous, they are sharply defined and clearly visible. I n the radial technique, the zones become progressively shorter and sharper as the front expands coutinuously. Radial development of saponified extract (10 with 1% n-propanol in petroleum ether (2&40°) yields well-separated zones of carotenes, lutein, violaxanthin, and neoxanthin. These results support those described earlier for the other geometrical modifications of paper chromatography and for column chromatography. Paper Partition Chromatography
I n paper partition chromatography, the paper is made to retain a stationary liquid phase. Resolution of the pigment mixture is obtained by development with a liquid immiscible with the fixed liquid. Partition chromatography may also be performed in columns prepared by mixing a finely divided support (e.g., cellulose, Celite, or kieselguhr) with the liquid to be fixed followed by packing this solid plus the fixed liquid phase into a chromatography tube. For the demonstration of fixed-polar-phase, partition chromatography, pure dimethylformamide, or dimethylformamide plus 10% water, is shaken with petroleum ether (60-llO°C) in a separatory funnel, and the layers are allowed to separate. Part of the lower dimethylformamide layer is placed in the bottom of a
paper-lined developing chamber along with a dish of the upper petroleum-ether layer. After 30 min for saturation of the atmosphere with both phases, a 20 X 20-cm square of Whatman No. 3 paper is dipped into the rest of the lower layer held in a tray, blotted by pressing between two pieces of thick paper (Eaton Dikeman No. 301), spotted with the mixture (5 formed into a cylinder, and placed in the dish of petroleum ether for development. As shown in Figure 5, the pigments separate in the order: carotenes, chlorophyll a, chlorophyll b, lutein, violaxanthin plus neoxanthin. When 90% dimethylformamide plus 10% water is used for the fixed polar phase, neoxanthin, violaxanthin, and lutein form well-resolved zones, hut a mixed zone of the chlorophylls plus carotenes remains at the solvent front. No intermediate mixture of dimethylformamide water or any other solvent combination was found to separate all six pigments in one run by partition chromatography. Partition systems for the separation of the nonpolar carotenes have not been found. Fixed-nonpolar-phase, partition chromatography (reversed-phase, partition chromatography) is easily demonstrated by soaking a sheet (20 X 20 em) of Whatman No. 1 paper in a 7% (v/v) solution of Wesson Oil (a mixture of cottonseed plus soybean oils) (HuntWesson Foods) in petroleum ether (60-llO°C), air drying a few minutes, and then oven drying a t 75'C for 30 min. After cooling, the paper is loaded with 5 pl of the mixture, formed into a cylinder and developed with methanol-acetone-water, 20 :4: 3 (v/v), (which has been equilibrated with the oil in a separatory funnel) in a saturated, paper-lined chamber. As shown in Figure 5, carotene is sorbed a t the origin, followed by discrete zones of the other pigments in the reverse order of that obtained with adsorption or fixed-polar-phase paper chromatography. Column Partition Chromatography
DMF
Figure 5. Pigmenh in 2 @I of leaf extract separated by poper portition chmmotogmphy in various systems.
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Journal of Chemical Education
For the demonstration of column, fixed-polar-phase, partition chromatography with cellulose as the supporting phase, cellulose for thin-layer chromatography (Camag) (30 g) is mixed with 15 g dimethylformamide, or dimethylformamide plus 10% water, equilibrated with petroleum ether (60-llO°C). This treated cellulose is packed in a column 1 cm in diameter, and 250 p1 of leaf extract are added. Development with petroleum ether provides separations like those obtained in paper (Fig. 5). Fixed-nonpolar-phase partition chromatography is demonstrable with Celite as the support. The Celite (30 g) is mixed with petroleum ether (100 ml) containing Wesson Oil (7y0), spread in a very thin layer, air-dried for an hour or more then oven-dried a t 70°C for 30 min. This product is packed in a tube 1cm in diameter. The leaf extract is adsorbed and washed with methano1:ace tone: water (20:4: 3) previously saturated with the oil. The separation is similar to that in Figure 5 except that violaxanthin and neoxanthin are not separated. The results in Figures 1-5, obtained with diverse chromatographic systems under conditions which do not cause alterations nor produce anomalous zones, indicate conclusively that leaves contain six, and only six major pigments, namely carotene, lutein, violaxanthin, neoxanthin, and chlorophylls a and b.
Figure 6. Pigrnenb reporded fmm 2-5 r l of extroct of various by thin-laye~chromotogrophy in ~ilico-gelsheeh.
Thin-Layer Chromatography of Chloroplast Pigments from Various Organisms
Chromatography of the chloroplast pigments from various plants gathered from diverse regions of the world has shown that certain combinations of pigments are typical of the species in each major taxonomic group (3, 4, 15). For the illustration of this relationship, Figure 6 reproduces typical chromatograms obtained by development of 3-5 p1 of an extract of each plant on a sheet of Eastman Chromagram silica gel (No. 6061, Distillation Products, Inc., Rochester, New York) with 3: 1: 1 isooctane-acetone-diethyl ether. The sheets are used as received without any pretreatment. Spots are applied with micropipets and dried, and the thin layers are developed in saturated, paper-lined "thin-layer" chambers which are covered with aluminum foil. The migration distance of the wash liquid is 15 cm (about 50 min). As shown in Figure 6, leaves of seed-producing plants (spinach and cocklebur), spore-producing plants (ferns), and many green algae (e.g., Chlorella pyrenoidosa and Ulva rigida) yield the same pigments: carotenes, chlorophyll a, lutein, chlorophyll b, violaxanthin and neoxanthin, plus a small amount of pheophytin a (chlorophyll a after loss of magnesium). On layers of silica gel G spread on glass plates and developed with the same solvent, the same pigments separate but in a different sequence: the zone of lutein is behind both chlorophyll~rather than between them as shown in the figure. There is no indication of the extra yellow or green zones reported by Aowar (16) and Rollins (17) in earlier papers in THIS JOURNAL dealing with the tlc of chloroplast pigments on layers of siliceous adsorhents. Some species of green algae (Chlorella vulgaris,
Cladophora ovoidea, Cladophora trichotoma, and Scenedesmus obliquus, for example) yield an additional major xanthophyll between neoxanthin and violaxanthin as shown in Figure 6. This pigment, which has been recently isolated in this laboratory and named loroxanthin, resembles lutein both in its visible absorption spectrum and in lack of reaction with HC1. The same pigment system as in leaves but with siphonaxanthin and its ester siphonein in addition is characteristic of most siphonalean green algae (3,18) (Codium fragile and Codium Setchellii) as also shown in Figure 6. Development of these extracts in columns of magnesia, as indicated in Figure 1,shows that these plants contain much more a-carotene than 8-carotene, a usual characteristic of the Siphonales (3,18). It may be noted in Figure 6 that the sequence of lutein relative to the chlorophylls is quite different when compared to leaf extracts and other green algae, due probably to the nature and amount of the colorless materials extracted from the Siphonales. The blue-green algae, represented by Phovmidium luridum, provide a zone of carotene, but none of the major xanthophylls of leaves (3). Myxoxanthin, myxoxanthophyll, zeaxanthin, carotene, and chlorophyll a are the principal pigments (Fig. 6). It has been reported that the pigments found in blue-green algae vary markedly depending upon cultural conditions (4). Pigments found in Euglena gracilis (Fig. 6) include chlorophylls a and b, carotene, neoxanthin, and the principal xanthophyll diadinoxanthin, the structure and properties of which have been recently described (19). Dinoflagellates, represented by a symbiotic alga from sea anemones, also contain diadinoxanthin plus a characteristic red-orange pigment, peridinin, and carotene. Chlorophyll a plus the strongly-sorbed chlorophyll c are also separated (Fig. 6). Brown algae and red algae, species of which were not available for study, also yield a characteristic pattern of pigments. Brown algae, such as Sargassum and Cystophyllum, contain chlorophylls a and c, neofucoxanthin, fucoxanthin, violaxanthin, and &carotene (3). Red algae (e.g., Laurencia and Pterocladia) contain chlorophylls a d, lutein, zeaxanthin, and 8-carotene a-carotene (3). These pigment systems, revealed with thin layers of silica gel, correspond to those observed with columns of powdered sugar (3). Similar results with thin layers of powdered sugar (20) and of additional sorptive substances (21) have recently been reported. The pigment systems described in this section serve as one property for the classificatipn of plants into major taxonomic groups. Besides, the autotrophic or photosynthetic capacity of the plants is undoubtedly geared to the unique properties of these pigment systems.
*
*
Alferotiona of the Pigments
Figure 7 illustrates three sets of conditions which lead to alterations of the labile chloroplast pigments. In A, cocklebur leaves are scalded for 2 min in boiling water and extracted by the non-blender procedure described above. Separation of 5 pl of this extract by tlc on Eastman Chromagram silica gel with the same wash liquid as used in Figure 6 yields two extra green zones near the origin plus zones of chlorophylls a' and b' ahead Volume 46, Number 8, August 1 9 6 9
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Figure 7. Alteration of pigmenh illustrokd by thin-loyer chmmatography: Id chlorophylh heated before odwrption on rilico gel; lbl chlorophylls d h r e d by adsorption on kieselguhr G; 14 earotenoids altered b y odsorplion of saponifled extrod on activated calcium silicate IMicro.Ce1 El.
of the respective parent chlorophylls. The amount of pheophytin is also increased. The pigments of some plants are not so badly affected by the scalding step; for example, the extra, strongly-adsorbed, green pigments do not appear when spinach leaves are treated in this manner. So, although heating improves the extractability of the pigments, i t is often not without harmful effect on the pigments themselves. IGeselgnhr G thin layers (250 p) are prepared according t,o the method of Stahl. Twenty-five grams of powder (Brinkman Instruments, Inc.) is stirred with 35 ml distilled water in a mortar until smooth; 15 ml more water is stirred in; the slurry is put into a Desaga commercial spreader; and five clean and dry glass plates are coated. The layers are air-dried overnight, activated for 30 min a t 105"C, cooled and spotted. The results of developing 2 pl of cocklebur extract, prepared by the blender method, with 10% acetone in petroleum ether (2040°C) are shown in B. Two extra green zones, one behind neoxanthin and one behind carotene, are found, as well as streaks of chlorophyll through much of the adsorbent. Like magnesia (see Fig. I), kieselguhr alters adsorbed chlorophylls so that they cannot be washed along uniformly, separated cleanly, or recovered by elution. Other active adsorbents which alter the chlorophylls in a similar manner include talc, some aluminas, and various activated silicates such as Florisil and Micro-Cel. Kieselguhr, talc, and alumina provide clean separations of saponified leaf extracts (10). Layers of Johns-Manville IVIicro-Cel E, activated calcium silicate, are prepared as above from a slurry com482
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Journal of Chemical Education
posed of 15 g adsorbent, 2.95 g CaS04.2H20 binder, and 108 ml water and are activated 30 min a t 105°C before use. Development of 20 p1 of saponified cocklebur extract with 15% acetone in petroleum ether (2040°C) yields zones of the four major carotenoids but with seven additional yellow zones. All zones turn blue when exposed to the vapors of HCI. Because these additional zones are not resolved on the selective, milder adsorbents, such as sugar, cellulose, magnesia, or silica gel, they are undoubtedly artifacts due to decomposition and/or multiple zoning caused by the adsorbent. The additional zones cannot be attributed to pigments which are normal constituents of the leaves. Other conditions which cause alteration of the pigments may also be demonstrated. One or two extra, strongly-sorbed green pigments are formed if the leaves are killed, without inactivating enzymes, by allowing them to stand in cold acetone for about 10 min before blending and extracting. Chlorophylls a' and b' along with other extra green zones and pheophytin are found if the dissolved extract is stored overnight in a freezer in the dark. A trace of HC1 added to a fresh leaf extract converts the chlorophylls to pheophytins and neoxanthin and violaxanthin to strongly sorbed yellow pigments with blue remaining a t the starting point. Leaves which are frozen and thawed or allowed to become wilted before extraction yield an extra yelloworange pigment slightly more sorbed than carotene and an extra green zone a t the origin on silica layers and sugar columns. Freezing is especially disastrous to the chlorophylls of Chlorella-pheophytins, chlorophylls a' and b', and several extra green zones being found with silica layers or sugar columns. Isomerization of some carotenoids separated on silica plates takes place if the wash liquid is allowed to evaporate. If neoxanthin and violaxanthin are separated on Eastman Chromagram silica sheets and eluted a t once, their spectra show no alteration. If the chromatogram is allowed to air dry for 30 min after development but before elution, neoxanthin is changed to neochrome (X,,400, 422, 448 nm in ethanol) and violaxanthin to auroxanthin (A,., 378,401,425 nm). The chlorophylls are not changed appreciably by standing before elution but are apparently slightly altered during chromatography on silica sheets, as evidenced by blue/red peak ratios lower than those reported for the pure chlorophyll~. Warnings
Reproduction of the chromatographic separations described herein require that special attention be given to numerous details. Usually, even when the individual variables have been controlled, some preliminary empirical experiments may he required to insure reproducibility of the results. Work with highly-volatile, flammable solvents is extremely hazardous. These liquids should not be used in poorly ventilated rooms or in blenders where they may be spilled over an arcing motor. They should not be stored in conventional refrigerators with exposed electrical contacts. Epilogue
The chloroplast pigments are so readily available and so strikingly colored that they have often been used to
demonstmte separations by various chromatographic methods. The most effective procedures are, however, scattered throughout the biological and chemical literature, and, in many cases, the precautions necessary in order to isolate, recover, and identify the natural pigments free of their alteration products are not appreciated. By contrast, the methods described herein represent easily reproducible demonstrations illustrating the principal modifications of solution chromatography. They indicate, in addition, some of the pitfalls that should be avoided if the natural unaltered pigments are to he obtained. We dedicate this presentation to teachers, students, science-fair participants, amateur scientists, and anyone else who desires to employ or demonstrate small-scale chromatography of the plant pigments with authority and understanding. Literature Cited (1) T s w w ~ M., , Her. d. dest. bolan. Ges., 24, 384 (1906). See STRAIN,H. H., A N D SHlsllMA, J., J. CHI.:M.EDUC.,44, 235 and 238 (1967) for a critique and translation of the
ot.iginal paper. (2) BROWN, W. G., Nature, 143, 377 (1039). (3) STHAIN, 11. IT., Ann. Priestleu Lectures, Penn. State Univ., 32 (19383. (4) STIIAIN,IT. IT.,
