Author Institution: The Charles F. Kettering Foundation, Yellow Springs, Ohi

Eyster, H. Clyde

The Ohio Journal of Science

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PERMEABILITY—A PRIME FACTOR IN EXTRACTION OFCHLOROPLAST PIGMENTSH. C. EYSTER,The Charles F. Kettering Foundation,Yellow Springs, OhioThe extraction of chloroplast pigment from dried nettle leaves is accomplishedbest in 80-90% aqueous acetone, in 90% aqueous ethanol and in absolute methanol(Willstatter and Stoll, 1913). Just why 80-90% aqueous acetone is better thananhydrous acetone, why 90% aqueous ethanol is better than absolute ethanol,and why absolute methanol is better than any of its aqueous dilutions has neverbeen satisfactorily explained. Willstatter and Stoll (1913, p. 57) explained it onthe basis that "water added to the organic solvents dissolves mineral salts, asfor example, potassium nitrate, from the leaf substance," and that "salt solutionthat is formed changes the colloidal state of the chlorophyll in the chloroplasts andmakes it easily soluble."MATERIALS AND METHODSTo study the permeability of plant tissues to various organic solvents, seeds ofsweet corn, soybean, and garden pea were used. The sweet corn was Burpee'sGolden Bantam; the soybeans were of the Lincoln variety locally acquired; andthe garden pea seeds were Burpee's Blue Bantam (wrinkled). The seeds weresoaked in organic solvents at room temperature (25-30° C), and weighings of thesuperficially dried seeds were taken at the end of 1 day, 4 days, and 7 days. Theorganic solvents were methanol, ethanol, and acetone. Methanol was dehydratedwith magnesium turnings which forms magnesium methoxide, then refluxed anddistilled. Ethanol was dried over Drierite and then with metallic sodium whichforms sodium ethoxide; then refluxed and distilled. Acetone was dried merely overDrierite, then refluxed and distilled. The aqueous dilutions of methanol, ethanol,and acetone were made on the basis of volume.Changes in the lengths of soybean seeds soaking in absolute, 95%, 90%, andYo methanol were determined by use of a vernier calipers. Specific gravityfor some of the solutions was determined by use of a specific gravity bottle.A Klett Biocolorimeter was used to determine the relative amounts ofchloroplast pigments extractable by acetone-water solvent to which NaN03and cane sugar, respectively, had been added as compared to the amounts ofchlorophyll pigments extractable by acetone-water solvent alone. The amountof pigments extractable by 85% acetone was used as the standard for comparison.It was given an arbitrary value of 100 and all the others were evaluated com-paratively by colorimetric measurements.EXPERIMENTAL RESULTSSeeds of sweet corn, soybean and wrinkled garden pea soaked in absolutemethanol, absolute ethanol, and anhydrous acetone showed a remarkably greaterimbibition of absolute methanol during the first day than of absolute ethanol or ofanhydrous acetone (Tables I and III). To be specific, soybean seeds absorbedor imbibed absolute methanol equal to 7.7% of their dry weight during the first day,whereas they imbibed absolute ethanol equal to only 1.6% of their dry weight inthe same period of time, and anhydrous acetone equal to only 0.1% of their dryweight. This difference is more pronounced at the end of 4 days and at the end of7 days. The soybean seeds in absolute methanol also showed a distinct increase in7980 H. C. EYSTER Vol. LTABLE IINCREASE IN WEIGHT OF 100 SOYBEAN SEEDS SOAKED IN VARIOUS ORGANIC SOLVENTSSolvent in DaysAbsolute methanol (100%)0147Absolute ethanol (100%)0147Anhydrous acetone (100%)0147Weights in Grams12.9513.9514.3014.5914.9415.5015.6515.7713 1713.1813.3113.47Per Cent Increase7 710.412.71 62 63 40 11 12.3TABLE IIINCREASE IN LENGTH OF SOYBEAN SEEDS SOAKED IN WATER-FREE SOLVENTS. DATA AREAVERAGES OF 10 SEEDS. ABSOLUTE INITIAL AVERAGE LENGTHSARE GIVEN IN PARENTHESESDays Soaked14714Absolute Methanol(.725 cm.).036 cm..061 cm..091 cm.Absolute Ethanol(.742 cm.).010 cm..020 cm..031 cm..039 cm.Anhydrous Acetone(.718 cm.).003 cm..020 cm..031 cm..041 cm.TABLE IIIINCREASE IN WEIGHT OF 50 SEEDS OF WRINKLED PEA AND OF 50 SEEDS OFSWEET CORN SOAKED IN VARIOUS ORGANIC SOLVENTSSOLVENT IN DAYSAbsolute methanol (100%)0147Absolute ethanol (100%)0 s147Anhydrous acetone (100%)0147WRINKLED PEAWeight inGrams14.3015.7616.7317.3814.6914.8915.4215.8313.0313.0313.0013.00Per CentIncreasei6.217.021.51.45.07.8000SWEETWeight inGrams12.6113.7214.2414 5513 9214.1614 1814 2513 4613.6113 5913.60CORNPer CentIncrease8.812.915.41.71 92 41.11 01.0No. 2 EXTRACTION OF CHLOROPLAST PIGMENTS 81length in contrast to Very little change in the length of seeds soaked in absoluteethanol and anhydrous acetone (Table II).40LJ(0302oro| 2 010100MethanolEthanolAcetone95 90 85PERCENT SOLVENTFIG. 1. Per cent increase in weight of sweet corn seeds soaked in various aqueousdilutions of methanol, ethanol, and acetone for one day.504 0MethanolEthanolAcetone100 95 90 85PERCENT SOLVENTFIG. 2. Per cent increase in weight of sweet corn seeds soaked in various aqueousdilutions of methanol, ethanol, and acetone for four days.82 H. C. EYSTER Vol. LSeeds in absolute methanol get to be quite soft and can be sectioned very easilywith a razor blade. This is not true for seeds soaked in absolute ethanol or inanhydrous acetone which only permeates slightly and produces little change inthe original hardness of the seeds. The data for sweet corn seeds and for wrinkledpea seeds (Table III) are even more diverse in the differences. Permeability invarious aqueous dilutions of methanol, ethanol, and acetone can best be describedby the graphic illustrations of Figures 1-3. The points for methanol form anapproximate straight line which has about the same slope in the 1-day, 4-day, and7-day periods. The points on the graph for ethanol and for acetone form curveswhich have distinctly greater slopes than the line for methanol. Of the curvesfor ethanol and for acetone, the curve for acetone has the greater slope, and theslope for the 4 days is greater than for one day and greater for 7 days than for 4days. This means that dilutions of these three organic solvents with waterMethanol — — —Ethanol —- —Acetone95 90 85PERCENT SOLVENTFIG. 3. Per cent increase in weight of sweet corn seeds soaked in various aqueousdilutions of methanol, ethanol, and acetone for seven days.produce the greatest change in imbibition with acetone, and least with methanol.The result is that all of these lines intersect. Whereas 100% methanol is imbibedfar more abundantly than either 100% ethanol or 100% acetone, 85% acetone isimbibed more than either 85% ethanol or 85% methanol. All of the graphs showthis tendency for greater increase in absorption of acetone with greater aqueousdilutions and for a rather uniform increase in absorption of methanol with greateraqueous dilutions. Ethanol is intermediate between methanol and acetone.The specific gravity determinations presented in Table IV show very littledifference in values at the start as compared to the end of an imbibitional period.This seems to indicate that there is very little or no differential or preferentialNo. 2 EXTRACTION OF CHLOROPLAST PIGMENTS 83absorption of either the organic solvent or the water which dilutes the organicsolvent.Sugar and Salt Effect.—That penetrability of solvent is a prime factor in theextraction of chloroplast pigments is further substantiated by the effect of saltsand sugars. Sodium nitrate decisively increases the amount of chloroplastpigments extracted from dry nettle leaf powder in one hour by 80% acetone, andcane sugar decreases the amount of chloroplast pigments extractable in one hour(Table V). Quantities used in each case were 1 g. of dried nettle leaves, 20 ml.of acetone solvent, 1 g. NaN03 and 5 g. of cane sugar. Values are those obtainedby comparing the extract colorimetrically with the extract in 85% acetone whichwas used as the standard and arbitrarily given the value of 100.A corresponding effect is registered by the sodium nitrate and the cane sugaron the initial rate at which seeds imbibe 80% acetone. The data are given inTable VI.TABLE IVSPECIFIC GRAVITY CHANGES IN SOLVENTS DURING A 21-DAY SOAKING PROCESSSOLVENTAcetone, 100%.95%.100%.95%.90%.80%.Methanol, 100%.95%.90%.85%.Ethanol, 100%.95%.90%.SEEDSWrinkled pea.Sweet corn.Sweet corn.Sweet corn.SPECIFIC GRAVITYBeginning.790.807.826.790.807.826.862.793.811.827.843.795.809.827End.789.807.822.792.811.827.800.819.829.838.801.811.829Sodium nitrate and cane sugar have another effect, also. This effect is on thesolubility of chloroplast pigments. The effect is, however, slight (about 6-9%)and in the direction of decreased solubility. The greatest influence of the saltand the sugar on extractability of chloroplast pigments must be accredited to theireffect on the permeability of the plant material to the solvent.DISCUSSION'The extraction curves of Willstatter (1915) as presented in Figure 4 can beexplained by a joint consideration of chloroplast pigment solubility and of therelative permeability of plant tissues to these solvents and their aqueous dilutions.Chlorophyll pigments are always more soluble in water-free fat solvents andbecome less and less soluble as the water content increases. Although chloroplastpigments are more soluble in anhydrous acetone than in 80% acetone, the dominant84 H. C. EYSTER Vol. Linfluence in extraction is the very low permeability of dry leaf powder to anhydrousacetone and its extremely high permeability to acetone containing 20% water.Dry plant tissues are extremely permeable to methanol. The permeability toabsolute methanol is high, and the permeability increases only slowly to mixturesof water and methanol. Chloroplast pigment solubility in methanol decreasesrather rapidly as the water content increases. With acetone the chloroplastpigment solubility does not drop nearly as rapidly with an increase in water content(Eyster, unpublished data). Since extraction of chloroplast pigments seemsto be due to a combined joint influence of solubility and tissue permeability, thisperhaps is adequate to account for the fact that extraction of chloroplast pigmentsis always greatest in absolute methanol and decreases in dilutions of methanolwith water.TABLE VEXTRACTABILITY OF CHLOROPLAST PIGMENTS FROM DRIED NETTLE LEAVES IN ONE HOUR AT ROOMTEMPERATURE AS AFFECTED BY NaNO8 AND CANE SUGAR. THE DATA ARE ONLYCOMPARATIVE VALUES AND WERE OBTAINED BY COLORIMETRICALLY COMPARINGTHE FILTERED SOLUTIONS WITH THE 85% ACETONE FILTRATE VALUEDARBITRARILY AT 100SolventPer Cent100%95%90%85%80%Acetone24679210094Acetone+NaNO32183113132134Acetone+Cane Sugar1755828663TABLE VIINFLUENCE OF NaNO3 AND SUCROSE ON THE INCREASE IN WEIGHT OF 50 WRINKLEDPEA SEEDS SOAKED IN 80% ACETONESOLUTION(100 ml.)80% acetone80% acetone + .1 M NaNO380% acetone + 10 g. cane sugarWEIGHT IN GRAMS AND PER CENT INCREASEAt Start12.6213.0513.01One-Half Day13.08 (3.7)14.44 (10.7)13.31 (2.3)One Day14.09 (11.7)15.25 (16.9)13.92 (7.0)SUMMARYThe data obtained by Willstatter and Stoll (1913) on the amount of chloroplastpigments extractable from dried nettle leaf powder by methanol, ethanol, acetoneand their aqueous dilutions seem to be a net result of the permeability of the plantmaterial to the solvents and of the solubility of the chloroplast pigments in therespective solvents. Seeds of sweet corn, soybean, and wrinkled pea were soakedin 100%, 95%, 90%, 85% methanol, in 100%, 95%, 90%, 85%, 80% ethanol, andin 100%, 95%, 90%, 85%, 80% acetone. Weighings were made immediatelybefore soaking and at the end of 1, 4, and 7 days. Length measurements wereNo. 2 EXTRACTION OF CHLOROPLAST PIGMENTS 85also obtained from soybean seeds. The weight increases during the first day forwrinkled pea seeds were 10.2% in absolute methanol, 1.4% in absolute ethanol, and0% in anhydrous acetone. With aqueous dilutions and also for longer periods oftime the increases were greater. The data for soybean seeds and for sweet cornseeds were the same comparatively as for wrinkled pea seeds.1010 20 30PERCENT WATER40FIG. 4. Extractability of chlorophyll from dried and pulverized leaves by variousorganic solvents. After Willstatter (1915, p. 335).Sodium nitrate decisively increases the amount of chloroplast pigments extractedfrom dry nettle leaf powder in one hour by 80% acetone, and cane sugar decreasesthe amount. A corresponding effect is registered by the sodium nitrate and thecane sugar on the initial rate at which seeds imbibe 80% acetone.REFERENCESWillstatter, R., and A. Stoll. 1913. Investigations on chlorophyll. (Authorized Englishtranslation by Schertz and Merz. 1928. Science Press. Lancaster, Pennsylvania.)Willstatter, R. 1915. Chlorophyll. J. Am. Chem. Soc, 37: 323-345.

Permeability : A Prime Factor in Extraction of Chloroplast Pigments

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Permeability : A Prime Factor in Extraction of Chloroplast Pigments

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