edited bv ~,
f iltrote/ & re~iduef
ROBERT
REEVES
Marlborough School 250 S. Rossmare Avenue LOSAngeles. CA 90004
Chlorophyll Separation and Spectral Identification Susan M. Diehl-Jones Brother Rice High School, 7101 Lahser Road, Birmingham, MI 48010 The objective of this experiment is to separate a natural mixture of pigments, particularly chlorophyll a and b, and to identify and verify the separated components. Upon com.let ion of this exneriment. the students will have become familiar with the o&anic chemistry extraction process, will have oerformed analvses bv thin-laver chromatomaohv - . "and column LhromatograPGy, a d will 6e able to use the absorption snectrum. obtained with a snectroohotometer. as a means of iientification. In addition, siudengwill be able'to use spectral identification to comnare the results of each chromatomaphic - . method. This can he presented as an open-ended experiment in which students &e given a general background i d are asked t o solve a specific problem. For example, students might be a s k 4 to "djscuvrr" the best developinh solvent from a i r o u p of riven solvents. These sdvents might include: isupropanol. n-propanol, acetone, methanol, c$lohexane, i-octane, diethyl ether, petroleum ether, or related solvents with similar polarities to those above. Students may then "test" both the pure solvents andlor mixtures thereof on either chromatographic method (column or thin-layer). Alternatively, t h e developing solvent given below (see equipment) can be used with good results. Equipment and Materials Separatory funnels (125-ml sufficient) Mortar* and prsrles Small Erlewnryrr flasks 125- Nrr 50-mi) Chromatwrmhic plates tEnstman recommendrd) . . Buret (M& recommended) Pipet (lamda) or mierosyri~ges Mayonnaise jar (tightly sealable) is preferred or graduated cylinder (250-mlor more) Spinach (either fresh or frozen; fresh is better) Refrigeration source Organic solvents (only for "research flavor") Orveloping solvent trnirturt. of 500 ml of in>-orlaneor c)clohrxnne, 250 ml acetone, and 250 ml diethyl ether; 11 I is sutiir:ent fur n class uf 20) Note: When using a n y of these organic solvents, o r mixt u r e s thereof, special c a r e should be used to avoid contact w i t h skin a n d clothing. It is recommended t h a t t h e l a b a r e a be well ventilated o r t h a t usage b e restricted to a hood to avoid exposure to h a r m f u l vapors. Because a l l organic solventslvapors a r e extremely flammable, n o direct flames o r high h e a t should h e used i n t h e laboratory when t h i s experiment is done.
New lectures and iaboratay experiments and directtons in teaching c h i s b y t h r w the use of the laboratmy a m provided in lhis featwe. Experiments will be fully detailed and will be fieldtested betme lhey are published. Contributionsshould be sent to the featureeditor.
Spinach Extraction Students are given the specifics of two extraction procedures and a short introduction to TLC ( 1 3 ) . Students are expected to find additional backgrouud material using a list of reference materials (cited below). The general instructions given to the students follow. Extract the photosynthetic pigments (which exist in nature as a mixture) from spinach leaves using a suitable organic solvent. Seoarate this extracted mixture on both a column and a thin-
laycr plate uarng proper chromatopraphrr techniques. l're a spertrophotometer ( 1)t o verify thp rdentitirs of thearparat~dcomponenu obtarned, and (2) t u compare the accuracy and affectiveness of the two chromatographictechniques Abbreviated procedures are included below. More detailed procedures and backgrouud information can be obtained by contacting the author directly. 1) L'sing a mortar and pestle, crush 11)g of spinach leaves in 25 ml of mrthyl alcohol, CH-OH. I)eeanr the mrthyl nlnrhol solurion and discard. Ar you decant. press thespinnch an free of solvent na porsiblc. 2) He-grmd the spinach in a mixture uf 25 ml methyl alcohol and 06 ml uetrolrum ether. tf'etrol~~m ether is a mixture of pen. tane and hexane). 3) Filter this mixture through a powder funnel, plugged with glass wool, into a separatory funnel. 4) Grind the residual spinach in another mixture of 25 ml methyl alcohol and 50 ml petroleum ether and filter as before. 5) Remove the lower methanol phase from the separatory funnel and discard. 6) Add 50 ml distilled water to the petroleum ether solution in the separatory funnel. Stopper the funnel and shake gently; set it aside to allow the layers to separate. If an emulsion forms, add a little sodium chloride to disperse it. 7) Once the layers have separated, remove the aqueous layer and discard. Pour the remaining petroleum ether solution into a 104-150-ml Erlenmeyer flask. 8) Add a small amount (1-2 g) d anhydrom sodium sulfateto the flask to remove the remaining water from the extract. The sodium sulfate should be in contact with the solution for 30 min. 9) Decant the resulting extract into a smaller Erlenmeyer flask and add a boiling chip. 10) Heat the solution gently on a hot plate or steam bath until only 5 ml of extract remains. After cooling, stopper the flask and store extract in a cool place, preferably in a refrigerator Thin-Layer Chromatography (TLC): Abbreviated Procedure Plate Preparation Prepare and activate the chromatogram according to the manufacturer's instructions.Then divide eachsheet into strips, allowing a 2-cm
' Modified from ref. (1). 454
Journal of Chemical Education
margin along the bottom and sides so that they will fit into the developing chamber.
Developing Chamber Preparation Fill a 1M)-ml (or larger) graduated cylinder or a mayonnaise jar with enough developing solvent so that the bottom contains about 0.5 em solvent. Seal the top of the cylinder with aluminum foil and a rubber hand; seal the mayonnaise jar with a tightly fitting lid. If the solvent is placed in the developing chamber about an hour before use, a good vapor will form. Developing-Solvent Preparation Although several different solvents or solvent mixtures may he used in this experiment (see "research-flavor" suggestions above), the recommended solvent mixture for the separation of chlorophylls a and b is cyelohexane or iso-octane (2 parts), acetone (1 part), and diethyl ether (1part). Prepare 100 ml of developing solvent. TLC Plate Development 1) Prepare theI'LC plate carefully, using a lambda pipet. 2) Alluw the sohent to rise up t h plate ~ until the solvent front just rearhes the top of the plate: ( 4 5 4 4 mid. Do not, however, leave the plate unattended for a long period of time: the separated pigments can re-blend if the solvent is allowed to cbntinue moving after reachine the end of the date. 3) ~ e m o v the e develope; plate from theheveloping chamber and allow it to drv hrieflv. 4) After this h k f drying perid, be sure to record the p i t i o n s and colors of all visible bands andlor spots since after a few hours the colors and spots will fade and may even disapper. Note: In general, TLC requires at least 1h to complete.
Figure 1. Comparison of absorption speetra for carotenes using thin-layer chromatography (dashed llne) and column chromatography (Solid line).
Elution Procedure 1) Using scissors, carefully cut out the major bands by cutting the entire plate. 2) Place each section of plate containing a major hand in a small (25-50 ml) Erlenmeyer flask with a small amount of developing solvent (10 ml). 3) Extract each hand by vigorously shaking each plate d o n with the developing solvent. If necessary, remove silica adsorbant by filtration or centrifugation. Remove the extracted plate and place the eluted sample in a tightly closed, small flask in the refrigerator, Spectral Analysis (may be done in a subsequent lab per;@ 1) I'aing a spectrophutunwter, wan Pach elutrd sample from 350 nm thmugh 750 nm. Use the developing rolvenr as the referenre solution. Record the scanning data. 2) Plot the absorption spectrum for each component on aseparate graph.
Figure 2. Comparison of absorption spectra tor chlorophyll b using thin-layer chromstography(dashed line) and column chromatography(solid line).
Column Chromatography: Abbreviated Procedure Column Preparation 1) Obtain a clean, dry 50-ml huret with a glass or Teflon*stapcock to use as a column. 2) To pack the column (buret), first press a thick plug of glass wml or cotton to the hottom of the huret. Then place 1-2 cm of clean sand above the plug. 3) Pour ground (not powdered) sucrose into the buret, gently tapping the now-forming column with a glass rod which fits snugly inside the huret. Continue this packing procedure until a eolumn height of 15-20 cm is obtained. Record the actual height. 4) When the eolumn has reached the desired height, press in another plug of cottonlglass wool wadding. 5) Pour a small amount of the developing solvent (15 ml) onto the column and allow it to filter through the column, saturating the adsorbent (sucrose). 6) Once the saturation has oecured, add sufficient solvent to maintain the solvent level at least 2 cm above the eolumn. Column storage notes: Once the adsorbent column is prepared, the solvent level should always be maintained 2 cm above the column. To store the column for future use, wet the column with developing solvent, as above, and cap with a rubber stopper to prevent drying. If the adsorbent is allowed to dry out or the solvent level is allowed to drop below the top of the column, the whole assembly must he disassembled, thoroughly dried, and the column re-packed.
Flgwe 3. Comparison of absorption spectra for chlorophyll a using thin-layer chromatography (dashed llne) and eolumn chromatography(solid line).
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Number 5
May 1984
455
Column DevelopmentlElution with 15 ml developing carefullyadd about ml of solvent to the column. 2) Obtain several small (25-50 ml) Erlenmeyer flasks to use to collect the fractions or components of the mixture. 3) With a steady hand, gently turn the stopcock to allow the migration process to hegin. Non-adsorbed substances will be quickly and completely washed through the column. Collect these substancesat the mouth of the buret withone of the small Erlenmever flasks. 4) ~dsorbedcompounds (pigments) will migrate slowly through the column,appearingin the form of rings. Collect each of these fractions separately at the mouth of the buret with an Erlenmeyer flask. As each pigment is separated from another, a very small clear zone will appear between them. 5) As development occurs, the solvent level will drop. Add more solvent as needed, taking care that the level does not drop below the packing of the column. 6) Should two separated pigments be accidentally mixed together or overlap, re-separate them by simple re-adsorption, that is, hv simolv oassine the mixture throueh " the column aeain with the developing solvent d needed. 7) After collection of the sample components,cap the flasks with rubber stoppers and store in a cool place, preferably in arefrigerator. Note: Spinach extract will provide pigments which elute from the colukn in the following order: i) carotenes: deep yellow, about 15 ml ii) chlorophyll b: blue-green, about 5 ml iii) chlorophyll a: yellow-green,about 10 ml
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Spectral Analysis (may be done in a subsequent lab period) 1) Using a spectrophotameter,scan each eluted sample from 350 nm through 750 nm. Use the developingsolvent as a reference blank solution. Record the scanning data. 2) Plot the absorption spectra for each component (pigment) on the graphs - . obtained from the thin-layer ehromatomaphy separation. 3) Comoare the thin-laver absorntion soectrum with the column ntsorptiun spectrum wee Fip. 1 .I). Compnrp hothabwrption spectra with the nrrept~dabsorption spectrum filr each pigment.
.
Discussion Of all the spectrophotometric applications employed in the author's class ( 4 ) this exneriment offers students the greatest variety of analytical experience and touches more areas of chemical interest than anv others. It has the advantage of using the spwtn,photometer realistically as both an identifiration and a verification tool while introducing the students to organinbiochemical laboratory methods. It also has the ootential tooffer the studenw a tastr of the "trial and error" bf research if they are required to find a suitable developing solvent.
456
Journal of Chemical Education
The extraction procedure, although time-consuming, fascinates students and generally can he done without error. In fact, the only source of technical error in the experiment occurs in the chromatographic process. Considerable experimentation is required in the selection of a suitable developing solvent and the location of agood developing chamber for the chromatographic process (particularly for TLC). However, once a suitable developing solvent is found and the chromatographic techniques are perfected, there are few if any er,"La.
In comparing the chromatogra~hictechniaues. column chromat&raphi offers the advantage of quick separation and equipment preparation that does not need to he repeated with each session. It is not, however, as accurate, nor quite as awe-inspiring, as thin-layer chromatography. Thin-layer chromatography requires preparation for each lab session hut provides a more well-defined separation. The final step of the lab, spectralanalysis, usually proceeds without difficulty (provided students have had some previous experience using a spectrophotometer to find the absorbance spectrum of a solution). The only real disadvantage of the experiment is the number of trials reuuired to reach the spkctral analysis procedure. The numder of trials can he minimized a t the expense of some of the "research flavor" if the identity of a "w&king3' developing solvent is suggested or provided. A minimum amount of preparation is required for this experiment. Mixture of the developing solvent is the only preparation required. While it is recommended that the extract be refrigerated, the eluted components (TLC or column) and the ether used in the developing solvent must he
refrigerated.
(1) Capindale, J.. "Chmmstaprsphy of Spinach Extract," paper presented st the CHEM-ED '77 Conference. Wstorlm. Ontario. Canada. August 25.1977. (2) Haye, L., "Thin-Layer Chromatography," Chemistry, 46 (Feb.1,20 (1973). (3) New Ymk State Education Department, T a p e r Chromatography," in "Biology Handbook." Bureau ofSeeondary Curriculum Development, NY. 1960,pp. 28-31. (4) Diohl-Jones. S.. "View from my Classrmm,"J.CHEM. EDUC..60,986 (1983). ( 5 ) Asimou, Isaac, "Words olScienee (and the History Behind Them):' New American Library (Mentar). NPWYmk. 1969. (61 Biological Sciences Curriculum Study. '"Biological ~ i e n c e An : Inquiry Jmta Life," Hsrmurt, Brace and World, New York. 1963 (and lstoreditiom). (7) Biological Science Curriculum Study, "Biologital Science: Molecules fa Man," Houghfon Mifflin. New York. 1968 (and later editions). (8) Banner, James, and Galston,Arthur,'"Principlesof Plant Phmiolagy," W.H. Freeman and Co.. San Freneiseo, 1959. (9) Otto, James H., and Tawle, Albert,"Modern Biology,"Holl, Rhineharf and Wimton, New York. 1969. (10) Scott, Ronald M., "Clinieul Analysis by Thin-Layer Chromstagraphy Techniques, Hvmphrey Science Publiahem, A"" Arbor, MI, 1969. (11) Stein, William J.,and Moore, Stanford. Sei. Amer, 184 131.35 (1951). 112) TnwetL Michael, (trans.bvSVsin, H. H..and She-, J.1, J.Cm. EouC,M,23W42
(1967).
(13) Whittinghsm, C. P., "Photmyntheois,"in "Oxford Biology Readers" (Editma Head, J.J.. and L m n s t e i n , 0.E.) Oxford University Press. London, 1971.
