PAPER CHROMATOGRAPHY SEPARATES PLANT PIGMENTS

Objectives:
1) Prepare an extract of plant tissue (e.g., leaves, flowers).
2) Apply the technique of paper chromatography as a method for separating individual plant
pigments contained in plant tissue extracts containing pigment lends.
2) !escrie the application of this technique to the study of plant pigments and develop related
testale questions.
") #enerate ideas aout ways to improve the technique to yield etter results.
INTRODUCTION TO PLANT PIGMENTS
A $pigment$ is simply a molecule that asors and reflects light. %ecall that white light actually
consists of many colors & you may have learned '%() # *+,- in high school physics as a way to
rememer the colors of light that ma.e up the white light of the 'visile spectrum-. !ifferent
pigments appear different colors ecause they have differing ailities to asor and reflect
various colors of light. (A more thorough discussion of the light/asoring properties of
pigments will e presented in the 0pectrophotometry la.) 1he road array of colors found in
plant tissues such as leaves, flowers, and fruits, can e accounted for y the presence of
literally thousands of different .inds of plant pigments.
1hrough plant reeding and horticultural practices, humans have manipulated plants2 pigment
producing capailities to serve our own desires. 3ews was made recently when a true lue rose
cultivar was successfully created in 4apan. +n nature, color is an important attriute of plants
that serves to attract pollinators to receptive flowers and signal fruit ripeness to seed
dispersers. +n some instances, colors may also serve to warn potential predators of poisonous
or toxic sustances contained in plant tissues.
5olor/producing pigments have other important roles in plants eyond regulating interactions
with animals. C!"#"p$!! is a pigment that reflects green light, ut asors red and lue
wavelengths and is critical for the light reactions of photosynthesis. %!&v"n"i's are an
important class of plant pigments that loc. ultraviolet (6,) radiation that can damage cell
proteins and !3A. 7any flavonoids, including &nt"c$&nins (a sucategory of flavonoids) have a
role in the chemical defense of plants as they are toxic to many herivores and pathogens &
especially insects and fungi.
As you may .now from the popular media, there is currently a sustantial research effort in
place to explore the potential health enefits of plant pigments to humans. +n popular
literature, these plant/ased compounds are often collectively referred to as 'phytochemicals-8
most are also pigments. 9lavonoids, anthocyanins, and c&#"ten"i's are :ust some of the
categories of plant pigments .nown to have antioxidant properties. 'Anti"(i'&nt- is a general
term used to descrie any sustance that has the aility to neutrali;e ')#ee #&'ic&!s- which
cause cellular damage y removing electrons from surrounding molecules. 7any lines of research
suggest that consuming a diet rich in plant pigments may slow the process of cellular aging and
reduce the ris.s of some types of disease, such as cancer, heart disease, and stro.e. 5osmetic
companies are even :umping on the 'antioxidant andwagon- y adding seductive lends of
antioxidant/rich 'otanical extracts- to their shampoos, ma.eups, and lotions in the hopes of
prolonging our youthful glow<
A few categories of pigments are listed elow along with their characteristic range of colors.
Pigment T$pe C"!"#s
Anthocyanins (suclass of flavonoids) lue=purple=red
Anthoxanthins (suclass of flavonoids) yellow / ivory
*etacyanins yellow / red=purple
5arotenoids yellow / red
5hlorophylls greens
>anthophylls (a suclass of carotenoids) ivory / yellow
0ome plant pigments you may e familiar with that are of current interest in nutritional and
pharmaceutical research are listed elow, though there are many more<
Pigment C"!"# %"*n' in
anthocyanins lue=purple=red erries, grapes, red peppers, eets, eggplant,
plums
eta/carotene orange=yellow carrots, pump.in, sweet potatoes, citrus, papaya,
melon, squash
curcumin )ellow turmeric
lutein yellow=orange .ale, roccoli, spinach
lycopene %ed tomatoes, watermelon, red grapefruits
;eaxanthin )ellow corn
PAPER CHROMATOGRAPHY METHOD
0tep 1? Prepare chromatography papers. 5ut the chromatography paper into strips following
dimensions suggested y your instructor. !raw a fine pencil line across (ut n"t &!! te +&$
across) the strip aout 2/" cm from one end / this is the $origin$. @andle the papers y the
edges, ta.ing care to touch them as little as possile / oils from fingertips can interfere with
the migration of pigments up the paper.
0tep 2? Prepare your plant extract. +n this procedure, we are interested in the 'qualitative-
assessment of the presence of individual pigments, we are not quantifying them (determining
how much of a pigment is present). 9or this reason, exact measurements of plant tissue and
extracting solvent are not necessary. (+n the 0pectrophotometry la, we will e quantifying
plant pigments, so careful measurements will e necessary.).
Place the plant tissue of interest into a mortar (a few leaves or flowers). Add a small amount of
ethanol to the plant tissue and grind completely with a pestle to release the pigments into
solution. 5ontinue adding ethanol (or plant tissue) as necessary to create a few milliliters of
very dar. extracted liquid. )our goal is to create a highly concentrated solution ut avoid a
paste/li.e consistency.
0tep "? Aoad the extract onto the chromatogram. !ip a capillary tue into the liquid portion of
your extract. ()our extract may contain fragments of plant tissue which will clog the capillary
tue. 1o minimi;e this, tilt your mortar slightly to allow the liquid fraction to run away from
the solids.) Allow the extract to migrate up the capillary tue. !a the end of the capillary onto
the origin of your chromatography paper. 1he extract will move out of the capillary tue onto
the paper as it is asored into the paper fiers.
origin (where you apply the pigment extract)
the solvent front is the position of the liquid solvent on the
chromatography paper at any given time. the solvent will
gradually move from the bottom toward the top of the paper,
carrying dissolved pigments with it. stop the chromatogram
before the solvent front reaches the top of the paper and mark
the location. you will use this distance to calculate f
mark the location of each pigment at the time the chromatogram is
stopped
Allow the extract to dry completely on the paper, then repeat with another load of extract. +n
order to concentrate the pigments on the paper, you will need to apply several loads of extract
(proaly B / C). After each loading, wait until the paper is fully dry efore applying the next
load. !epending on the type of sample you have and the amount of water in it, it may ta.e
several minutes for the sample to dry (2 / 1D minutes). )ou may use a hairdryer on the
lowest=coolest setting to speed the process. !o not proceed to step " until your sample is
completely dry.
!on2t forget? you will need to prepare 1 chromatogram for each chromatography solvent you will
e testing. )our instructor will let you .now which chromatography solvents should e tested
for a given plant tissue. 6se the same extract to prepare all the chromatograms.
0tep "? 0et the chromatogram in the chromatography solvent. Place your paper strip into the
solvent container provided with the origin end down. 7a.e sure that the level of the solvent is
elow the origin on your chromatogram & you do not want to sumerge the origin in the solvent.
5hec. the chromatogram frequently to oserve the movement of solvent and pigment up the
chromatography paper.
EA%3+3#? 1he petroleum ether=acetone solvent is highly flammale and can e dangerous if
inhaled. 1a.e care to avoid inhaling the fumes as much as possile and .eep clear of any flame,
spar., or other ignition source<<
0tep B? 0top the chromatogram and record your results. Ehen the solvent $front$ is within 2/"
cm from the top of the paper, remove the chromatogram. 6se a pencil to ,*ic-!$ mar. the
location of the solvent front. Allow the chromatogram to air dry, then trace and lael the
pigments you oserve (they will fade over time).
0tep F? +dentify your pigments. 5alculate the %f value (descried elow) for each pigment in
each chromatography solvent tested. 5onsult your instructor (or reference provided) aout the
identity of the pigments you isolated & record &!! your data in your noteoo..
IDENTI%YING PIGMENTS ISOLATED .Y PAPER CHROMATOGRAPHY
!ifferent pigments have different si;es, shapes, and physical properties (e.g., different
soluilities in our chosen solvent). As a result, different pigments will move at different rates
up the chromatography paper allowing them to visily separate from one another. (nce the
pigments are separated, they can e identified y a variety of methods.
(ne way to determine the identity of a pigment is to physically remove it from the paper and
assay it y another method. 9or example, we could elute (remove) a pigment from the
chromatography paper y dissolving it in another solvent, such as ethanol and measuring its
asorption spectrum using the spectrophotometer. 1he resultant spectrum could e compared
to the .nown spectra for different pigments y searching in an appropriate reference manual.
An alternative method is to calculate the '%f value-, a ratio representing the distance a pigment
travels relative to the distance the chromatography solvent travels. Again, we can then match
the %f value and color of our un.nown pigment to .nown values recorded in a reference manual.
R
)
v&!*e / 'ist&nce )#"m "#igin t" pigment
'ist&nce )#"m "#igin t" s"!vent )#"nt
%ememer & ecause pigments vary in their soluility in different solvents, the %f value for a
given pigment is tied to the chromatography solvent. +n other words, chlorophyll will have an
%f value in petroleum ether=acetone that is different from its %f value in *AE.
POSTLA. 0UESTIONS:
1. 0.etch each resulting chromatogram into your la noteoo. (or scan it=ta.e digital photos
and paste it in) and construct an associated tale that summari;es your data. #ive each tale a
title that identifies the plant and chromatography solvent used. +nclude in your tale? the
numer of pigments isolated with each chromatography solvent (e.g., 'pigment 1-, 'pigment 2-,
etc. . .), a color description of each pigment, its %f value, and its li.ely identity.
2. Ehat was the initial color of the plant extract(s) you usedG Ehat is the relationship
etween the colors of the pigments you isolated and the color of the original extractG
". Ehy is it important to stop the chromatogram efore the solvent front reaches the top of
your chromatography paperG Ehat would happen to your chromatogram if you let it run too
longG
B. +n some cases, two or more pigments may overlap each other on the chromatogram, ma.ing it
difficult to isolate and identify them. Ehat are some things you could do to modify the method
to improve pigment separation (distance etween pigments)G