Torrey Botanical Society

The Separation of Chloroplast Pigments by Unidimensional Ascending Paper Chromatography

Author(s): Frederick T. Wolf
Source: Bulletin of the Torrey Botanical Club, Vol. 85, No. 2 (Mar. - Apr., 1958), pp. 128-133
Published by: Torrey Botanical Society
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BULLETIN

OF

THE

TORREY

BOTANICAL

VOL. 85, No. 2, pp. 128-133

THE

CLUB
MARCH, 1958

SEPARATION
OF CHLOROPLAST
PIGMENTS
BY UNIDIMENSIONAL
ASCENDING
PAPER CHROMATOGRAPHY
FREDERICK

T.

WOLF

The separation of chloroplastpigmentsby mneansof paper chromatographyhas receivedconsiderableattentioiuin recentyears. In an early study,
Brown (1939) was able to separate chlorophylla, chlorophyllb, and carotenoids on circular paper chromatogramswith carbon disulphide as the
developing solvent. Bauer (1952), employingthe unidimensionalaseending technique,separated six componenlts,
including chlorophylla, chlorophyll b, carotene,xanthophyll,and xanthophyllepoxide, with a varietyof
solvents.Acetoneor benzenewas unsatisfactory,
anldresultedin littleor no
separation.Better resultswere obtainedwith petroleuinetheror methanol,
and monochlorobenzene
or toluene gave excellentseparation. For bidimensional chromatograms,Bauer recomnmended
developmenltin one direction
with a mixtureof benzene,petroleumether,and acetone, 10: 2.5: 2 parts
by volume, and in the other directiomiwith benzenle,petroleunmether,
acetone,and methanol,10:2.5 :1 :0.25.
Aromatichydrocarbonsand carbon tetrachlorideproved to be the best
of a numberof solventstested by Asami (1952). Satisfactoryseparation
of the chloroplastpigmenlts
was obtainedusing methanol,80 per cent methanol, benzene,toluene,xylene,carbon tetrachloride,and a 1: 1 mixtureof
benzene and petroleumether.
Douin (1953, 1956) used methanolas the developing solvent,anld applied this methodto studies of the pigmentsof mosses. RF values typical
of his work are 0.40 for carotene,0.54 for chlorophylla, 0.60 for chlorophyll b, 0.61 and 0.66 for xanthophylls.Sironval (1953) used deseending
paper chromatography
with a solventmixtureof benzene,petroleumether,
and acetonle,10: 2.5: 2, as recommendedby Bauer (1952).
Lind, Lane, and Gleason (1953) employed a bidimensioiialtechnique,
usinlgacetone, petroleumether,or petroleumether containing1 per cent
n-propanolin the firstdimension,and petroleumether containing25 per
cent chloroform
in the second. Strain (1953) obtained excellentseparations
with petroleumether,or petroleunm
ether containing 0.5 per cent n-propanol. By impregnatingthe papers with vaseline and developingwith 80
per cent methanol,reversalof the sequence of zones on the chromatogram
resulted.Greulach (1953) recommendedpetroleumether containing1 per
cent methanolas the solventmixture.Similarly,a 50: 1 mixtureof petroleum etherand methanolwas recommenlded
by Meyer,Anderson,and Swanson (1955).
128

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1958]

WOLF:

THE

SEPARATION

OF CHLOROPLAST

PIGMENTS

129

Chiba and Noguehi (1954), attemptingto separate chlorophyllsa and b

using toluene as recommendedby Asami (1952), obtailled low RV values
in the pure solvent.Upon addition of ethanol,1 part in 200, the RF values
were increased,and betterseparation resulted.Sporer, Freed, and Sancier
(1954) impregnatedthe paper strips with sucrose, and developed the
chromatogramsin the dark, at reduced temperature,and in a nitrogen
atmosphere.Chlorophyllsa and b were separated in a solvent mixtureof
0.5 per cent n-propanolin n-hexane.
Hager (1955, 1957) used a mixtureof petroleumether,benzene,chloroform,acetone, and isopropanol,in the ratio 50: 35: 10: 0.5: 0.17. He obtained separationof numerouszones,includingthe phaeophytinsand phaeophorbides,chlorophyllsa and b, two known and three unidentifiedcarotenes, and seven known and three unidentifiedxanthophyllsfrom plant
material.
It is the purpose of this paper to compare certain of the methodsadvocated by previous workersfor the separation of chloroplast pigments
employingunidimensionalascending paper chromatography.In addition,
data are presentedconcerninga numberof solventswhich have not been
previouslyused for this purpose.
Materials and methods. Leaves of spinach, Spinacia oleracea L., obtained at a local market,served as the usual source of plant pigments.
Leaves of plantain,Plantago major L., were also used to a lesser extent.
The leaves were placed in a Waring blendor, a pinch of CaCO3 was
added, and the pigmentswere extracted with acetone. The pigments in
acetonesolutionwere transferredto etherin a separatoryfunnel.The ether
solutionof the pigmentswas washed repeatedlywith water,and was dried
by the addition of anhydrousNa2SO4.
Graduates of 500 ml. capacity were used as chromatographicchambers.
These were fittedwith rubberstoppers,pierced with a paper clip so as to
providea means of suspendingthe paper strips.Whatman number1 paper
stripsmeasuring11/2by 121/2incheswere used in all experiments.The ether
solution of the pigmentswas applied to a small circular spot, one inch
fromthe bottomof the paper strip,by means of an ultra-microvolumetric
pipette.
Developmentof the chromatogramswas allowed to proceed under unlcontrolledtemperatureconditions in the laboratory. In a few instances
when prevailing temperatureswere high, the chromatogramswere developed in a constanttemperatureroommaintainedat 20-22?C.
Results. A large numberof solventsand solventmixtureswhich were
tested failed to resolve the pigments.When water or ethyleneglycol was
used as the developingsolvent,therewas no migrationof any pigment,all
of the substancesremainingat the point of origin. With various alcohols,

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130

BULLETIN

OF

THE

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BOTANICAL

CLUB

[VOL.

85

includingethylalcohol,n-propylalcohol,isopropylalcohol,n-butylalcohol,
isobutylalcohol,tertiarybutylalcohol,n-amylalcohol,and isoamylalcohol,
all of the pigmentsmigratedwith the solvent front,so that no separation
resulted.Similar resultswere obtained with acetone,methylethyl ketone,
ethyl acetate, butyl acetate, benzene,carbon disulphide,chloroform,ether,
pyridine,lutidine, collidine, 1, 4 dioxane, triacetin,anld ethylene glycol
monoethylether. With a 10: 2.5: 2 mixture of benzene, petroleumether,
and acetone,as used by Bauer (1952) and Sironval (1953), no separation
resulted in our trials.
With 1, 3 butanediolor 2, 3 butanediol,some degreeof separationcould
be obtained. In 1, 3 butanediol,for example, the xanthophyllcomponent
with an RF of 0.76-0.87 is well separated, but developmentis very slow,
requiring16-18 hours,and the colors tend to fade badly, so that these solvents cannot be recommendedfor general use.
In consideringsome 16 solventsand solventmixtureswhichyielded good
separations,four patternsof separationbecame apparent. In each case, the
componentsare named in the order of increasingRF. The types of separation foundare as follows:
1. The c, (a plts b), x type,in whichcaroteneand xanthophyllare well
separated from each other and from the chlorophylls,but resolution of
chlorophyllsa and b does not occur.
2. The b, a, (c plus x) type,in whichchlorophyllsa and b are separated
fromeach other and fromthe carotenoids,but separation of the carotene
and xanthophyllfractionsdoes not occur.
3. The b, a, x, c type,in whichresolutionof fourcomponentsoccurs,and
4. The b, x, a, c type,likewiseresultingin the separation of four components,but in a differentorder. The four types of separation will now
be discussed separately.
The c, (a plus b), x type. This type of separation resultsfromthe use
of methanol,propyleneglycol,or diethyleneglycol as the developingsolchloroplastpigmentsby Asami (1952), Bauer (1952), and Douin (1953,
1956). No previousinformationconcernedwith propyleneglycolor diethylvent. Methanol has previouslybeen used in the paper chromatographyof
ene glycolhas been found. Douin (1953, 1956) was able to separate chlorophyll a and chlorophyllb with methanol,a result at variance with the findings of Bauer (1952) and with those of the presentstudy. The RF values
found are presentedin Table 1, in which each value presentedis based on
4-6 separate experiments.
The high RF values for xanthophyllobtainedin thesesolventsdoubtless
result fromthe possessionof an hydroxygroup in commonwith the solvents. The chlorophyllscovered such a wide area on the chromatograms

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19581

WOLF:

THE

OF CHLOROPLAST

SEPARATION

131

PIGMENTS

that theirRF is presentedin the formof extremes,ratherthan an average

whichwould be misleading.No separationof chlorophylla fromchlorophyll
b was noted. The slowness of developmentwith propylene glycol or diethyleneglycol, requiring approximately18 hours, togetherwith the hygroscopic character of these substances,resulting in difficultyin drying
the paper strips,are definitedisadvantagesto theiruse.
The b, a, (c plus x) type. When toluene-ethanol200: 1 or o-dichlorobenzene is used as the developingsolvent,chlorophyllsa and b are separated from each other,but the less highly adsorbed carotenoids are not
resolved.The toluene-ethanolmixturewas proposed by Chiba and Noguchi
(1954) in an effortto bring about betterseparationsof chlorophyllsa and
b than resultedin pure toluene. o-Dichlorobenzenehas not been previously
studied in relationto the separationof plastid pigments.
TABLE

1. R?F values of chloroplast pigments in various solvents

Rr

Solveiit

chloro- chlorophyll
phyll
a
phb

Petroleum ether
Cyclohexane
Methyl cyclohexane
Xylene
Toluene
Carbon tetrachloride
Monochlorobenzene
Benzene-pet.ether1:1
Hager mixture
Pet. ether+ 1% methanol
Pet. ether+ 0.5%
n-propanol
Methanol
Propylene glycol
Diethylene glycol
Toluene-ethanol200: 1
o-Dichlorobenzene

0.12
0.14
0.14
0.34
0.42
0.32
0.49
0.15
0.46
0.37

0.24
0.36
0.39
0.48
0.59
0.52
0.63
0.26
0.74
0.83

0.23

0.78

0.55
0.43

0.76
0.61

chlorophylls
a +b

0.73
0.13-0.74
0.12-0.77
-

xanthophyll

xantho+phyll
caroteiie

0.59
0.70
0.78
0.87
0.87
0.87
0.92
0.90
0.92
0.65

0.49
0.89
1.00
1.00

0.94
1.00

carotene
1.00
1.00
1.00
1.00
1.00
1.00
1.00
1.00
1.00
1.00
1.00
0.51
0

The b, a, x, c type. Nine solventsand solventmixturesgave separations

in whichthe two chlorophylls,
carotene,and xanthophyllare resolved.These
were petroleum ether, cyclohexane,methyl cyclohexane,xylene, toluene,
carbon tetrachloride,monochlorobenzene,benzene-petroleumether 1: 1,
and petroleum ether, benzene, chloroform,acetone, isopropanol mixture,
50: 35: 10: 0.5: 0.17. Petroleum ether has been previouslyused in chloroplast pigmentchromatography
by Bauer (1952) and Strain (1953). Cyclohexane and methylcyclohexanehave not previouslybeen tested for these
separations. Xylene gave good separations according to Asami (1952).
Toluene gave good separationsin the workof Asami (1952), Bauer (1952),

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132

BULLETIN

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BOTANICAL

CLUB

[VOL.

85

and Chiba and Noguehi (1954). Carbon tetrachloridehas been used in

plant pigment chromatographyby Asami (1952), monochlorobenzeneby
Bauer (1952), benzene-petroleumether 1: 1 by Asami (1952), and the
petroleum ether, benzene, chloroform,acetone, isopropanol mixture,
50: 35: 10: 0.5: 0.17 by Hager (1955, 1957).
Good separations were routinelyobtained with almost all of the solventsand solventmixturesof this group. Occasional trials with toluene and
monochlorobenzeneresulted in failure of separation of xanthophylland
carotene,for a reason we are unable to explain. With spinach leaves, the
xanthophyllfractionwas resolvedby petroleumetherinto two components,
one lying immediatelyabove chlorophylla, the other considerablyabove
chlorophylla and well separated fromboth this pigmentand carotene.
The b, x, a, c type. When petroleumether plus 1 per cent mnethanol
(Greulach 1953) or petroleumether plus 0.5 per cent n-propanol (Strain
1953) was used as the solventmixture,the relativepositionsof chlorophyll
as comparedto pure petroleum
a and xanthophyllon the chromatograms,
ether,are reversed.The hydroxylgroup of the alcohol in the solventmixture
decreases the adsorptionof xanthophyll.With spinach leaves, in some instances the xanthophyllfraction was resolved into two components,one
below and one above chlorophylla.
Discussion. Partial separationof chlorophylla, chlorophyllb, carotene,
and xanthophyllwas givenby fiveof the solventsor solventmixturestested,
and completeseparationof these four pigmentsresultedin eleven different
solventsor mixtures.In everycase in which chlorophylla and chlorophyll
b were separated,the RF of chlorophyllb was found to be lower than that
of chlorophylla. In general,the RF values of the carotenoidsare higher
than those of the chlorophyllsin hydrocarbonor chlorinatedhydrocarbon
solvents,in which caroteneis usually to be found at the solventfront,and
xanthophylllies betweenchlorophylla and carotene.In solventsor solvent
mixtures containing an hydroxylgroup (alcohol or glycol), the RF of
xanthophyllis greaterthan that of carotene.
The foregoingexperimentshave demonstrateda number of ways in
whichthe leaf pigmentsmay be easily and rapidly separated by means of
The choice of the methodto be preferredmay conpaper chromatography.
ceivably vary with the purposes of the particular investigation.Any of
themare suitableforthe classroomdemonstrationof the presenceof chlorophyllsand carotenoidsin plant materials.
SUMMARY

1. The paper chromatographicseparation of chloroplastpigmentshas

been studied with 43 differentsolvents and solvent mixtures.Developing
solvents recommendedby previous workers were studied comparatively,

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1958]

WOLF:

THE

SEPARATION

OF CHLOROPLAST

PIGMENTS

133

and a numberof solventsnot previouslyused for this purpose were also

examined.
2. Sixteen of the solventsand solventmixturesgave satisfactoryseparationof the chloroplastpigments.
3. Accordingto the type of separation produced,the solventsand solvent mixturesmay be classifiedinto four types.
4. Solventswhichmay be used forpaper chromatography
of ehloroplast
pigments include propylene glycol, diethyleneglycol, o-dichlorobenzene,
cyclohexane,and methylcyclohexane,none of which have been previously
used forthis purpose.
oF BIOLOGY,

DEPARTMENT
NASHVILLE,

VANDERBILT

UNIVERSITY,

TENNESSEE

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