Characterization of Photopigments by TLC Dan Wolfgram, BSCI105, Section 5271, Spring 2013 Thin layer chromatography is se!

to separate an! p rify e"tracts of photopigments# In this la$, %e separate! photopigments e"tracte! from spinach# The primary tas& %as to i!entify the photopigments in !ifferent types of plants, especially chlorophyll'B# We se! !ifferent %a(elengths of light an! the correspon!ing a$sorptions to i!entify the pigments# In the scenario gi(en, i!entifying the !ifferent pigments assists in !etermining ho% the her$ici!e %ill effect !ifferent plants# We se! a poro s matri" that carries pigments as a sol(ent is r n thro gh it# )*eller 2013+# The sol $ility !etermines ho% far the pigments %ill tra(el !o%n the matri", the higher the sol $ility the farther the pigment mo(es# This process res lte! in separation of se(en pigments an! calc lation of a$sorption of !ar& green, light green, an! orange pigments# These res lts %ill help !etermine %hich photopigments %ere e"tracte! an! th s %hich plants the her$ici!e %ill effect# Introduction Chlorophyll'B is a (ital pigment se! in plants to a$sor$ light that is se! for photosynthesis# If the her$ici!e is interfering %ith this pigment, it e"plains %hy all the plants ha(e !ie!# - %ay to tell if it is in!ee! chlorophyll'B that is $eing effecte!, plants that lac& the pigment nee! to $e teste! %ith the her$ici!e to see if they still !ie or not# Thin layer chromatography is a metho! that can $e se! to separate pigments so they can later $e i!entifie!# T.C is accomplishe! $y r nning a sol(ent thro gh a matri" that carries the pigments# /olarity plays a large role in T.C $eca se of its sol $ility properties# If a pigment is sol $le in the sol(ent it %ill $e carrie! p the plate as the sol(ent mo(es p )*eller 2013+# If a pigment is not sol $le then it %ill not mo(e at all $eca se it %ill not $e carrie! %ith the sol(ent# /olar sol(ents !issol(e polar pigments an! non'polar sol(ents !issol(e non'polar pigments# In o r e"periment %e separate! pigments e"tracte! from a spinach plant# We se! a sol tion of acetone an! he"ane to separate the non'polar pigments from other polar molec les# 0e"t, %e create! a concentrate! line of pigment on the T.C plate# Then p t the plate in a sol tion that %as mostly non' polar $ t also ha! some polar parts# This means the more non'polar pigments mo(e! farther on the plate $eca se they %ere most sol $le in the sol tion# The more polar pigments !i! not mo(e as far $eca se they %ere not as sol $le in the sol(ent sol tion# Thin layer chromatography is se! in this scenario to !isco(er %hat photopigments are in spinach plants that may or may not contain chlorophyll'B# The professor1s hypothesis is that his her$ici!e interferes %ith chlorophyll'B f nction $ t he cannot $e s re $eca se most plants se chlorophyll'B for photosynthesis an! th s to li(e# Thin layer chromatography is sef l in this scenario $eca se sing the techni2 e in the pre(io s paragraph, it is possi$le to fin! %hich plants !o not ha(e chlorophyll'B so the her$ici!e can $e teste! on them# -fter T.C is complete, samples of each separate! pigment is ma!e into a sol tion, %hose light a$sorption is teste! at !ifferent %a(elengths# These a$sorption res lts can $e compare! to &no%n !ata a$o t chlorophyll'B3s a$sorption le(els to see if it is present in the plant#

Materials and Methods Preparation of Pigment and Solvent solution 4ollo%ing the proce! re of )*eller 2013+, non'polar pigment molec les an! other more polar molec les %ere separate! sing a mi"t re of he"ane an! acetone in a separatory f nnel# 5sing the separatory f nnel helps a(oi! any contamination $y %ater# )6a ch, Steeper, 7riffin 2008+# 0e"t, sing the separatory f nnel separate the acetone an! he"ane sol tions# Create a concentrate! line on a T.C plate %ith the he"ane sol tion# 0e"t create a 120m. he"ane9:0m. !iethyl ether980m. acetone sol(ent sol tion# This is %here the T.C plate %ill $e place! so the sol(ent can r n p the plate# Calculating Absorption Levels of Pigments Scrape off the separate! pigments an! a!! 100; acetone to create a sol tion that can $e a!!e! to the spectrophotometer# Chec& a$sorption of each pigment at %a(elengths of 830nm, 880nm, 8:0nm, :80nm, an! ::0nm# Results There %ere 7 pigments separate! sing T.C# They %ere in or!er of increasing <f (al es= light yello%, !ar&er yello%, !ar&est yello%, !ar& green, !ar&er green, gray, yello%# The calc late! <f (al es are !etermine! $y ho% far the pigment tra(ele! on the plate )4ig re 1+# The a$sorption le(els %ere ta&en of the !ar& green, light green, an! orange# The light green ha! the highest a$sor$tion, secon! %as orange, an! then !ar& green# 0one of these pigments e"actly matche! the a$sor$tion graphs pro(i!e! )*eller 2013+, $ t the light green pigment %as the closest# Discussion The main p rpose of this la$ %as to !etermine %hether the spinach plant has the pigment chlorophyll'B# The professor nee!s to fin! a plant %itho t chlorophyll'B so he can test his her$ici!e on it an! see if it is in!ee! inhi$iting the pigment from performing its f nctions in photosynthesis# We se! T.C to separate the pigments of spinach an! then too& a$sorption le(els to see if the spinach !i! contain chlorophyll'B# In or!er to !etermine if the chlorophyll'B pigment %as present, the a$sor$ance le(els !etermine! from the pigments separate! in T.C %ere compare! to the a$sor$ance le(els &no%n of chlorophyll'B# The a$sor$ance le(els of light green, orange, an! !ar& green pigments %ere ta&en an! recor!e!# While none of the le(els matche! e"actly the a$sor$ance le(els gi(en of chlorophyll'B, the light green pigment )#:>:?D+, pea&e! at 8:0nm, the same %a(elength chlorophyll'B )#@00?D+ also has pea& a$sor$ance# The !ifference may $e acco nte! to the fact that the chart pro(i!e! is a$sor$ance of photopigments in @0; acetone %hile %e se! 100; acetone# Base! solely on the !ata recei(e! from o r e"periment, it cannot $e state! concl si(ely that there is chlorophyll'B present# Ao%e(er, spinach is &no%n to ha(e chlorophyll'B )*i!mose 2005+# The !iscrepancy may also $e ! e to h man error, or the fact that %e only too& a$sorption le(els of three pigments# If the professor %as to test his her$ici!e on this spinach plant $eca se the res lts !i! not sho% concl si(ely the presence of chlorophyll'B, he %o l! most li&ely $e !isappointe! %hen the plant !ies# What remains to $e !etermine! is to fin! a plant that !oes not ha(e chlorophyll'B so the professor can test his her$ici!e# The T.C metho!, is (ery sef l in separating pigments an! %o l! $e appropriate to se on a ne% plant to test %hether the pigment is present#

Literature Cited 6 ach, Aao T#, <o$ert .# Steeper, an! William 7# 7riffin# B-n Impro(e! Cetho! for the D"traction an! Thin'.ayer W Chromatography of Chlorophyll a an! B from Spinach#BJournal of Chemical Education 1@#3 )2008+= 3@5'@7# /rint# *i!mose, 5#, D!elen$os, C#, Christensen, .# /#, E Aegel n!, D# )Fan ary 01, 2005+# Chromatographic !etermination of changes in pigments in spinach )Spinacia oleracea .#+ ! ring processing# Journal of Chromatographic Science, 43, >, 8::'72# 4orgacs, D#, E Cserhati, T# )Fan ary 01, 2002+# TAI0'.-GD< CA<?C-T?7<-/AG ?4 0-T5<-. /I7CD0TS= 0DW -DH-0CDS# Journal of Liquid Chromatograph ! "elated #echnologies, $%, 10, 1521# *eller, C#F# 2013# &ntroduction to E'perimental (iolog # Aay!en'Cc0eil / $lishing= /lymo th, CI#

Number ! " # $ % &

Color/Rf .ight Gello%9 #022 Dar&er Gello%9 #0:7 Dar&est Gello%9 #0>@ Dar& 7reen9 #125 Dar&er 7reen9 #150 7ray9 #1@@ Gello%9 1#00

4ig re 1# Image of T.C plate after separation of pigments sing he"ane9!iethyl ether9acetone# The ta$le sho%s the color an! calc late! <f (al es of the correspon!ing n m$ers# <f (al es in!icate ho% far the pigment tra(el ! e to it le(el of sol $ility in the sol(ent#