ULTRAVIOLET/VISIBLE SPECTROSCOPY PURDUE UNIVERSITY INSTRUMENT VAN PROJECT ANALYSIS OF PLANT PIGMENTS USING PAPER CHROMATOGRAPHY AND VISIBLE

AND/OR UV SPECTROSCOPY (1-31-96) INTRODUCTION We have seen that all cells must constantly consume fuel molecules to ma nta n themselves! "#o$! an% #e&#o%uce' (uel molecules such as "lucose const tute an mme% ate sou#ce of ene#"y fo# ) olo" cal $o#* that can )e #elease% )y cata)ol c cell &#ocesses' +o$eve# t s necessa#y that l fe on ea#th have a constant sou#ce of ene#"y that can )e ha#veste% an% use% to "ene#ate com&le, fuel molecules f#om s m&le sta#t n" mate# als' The ult mate ene#"y sou#ce u&on $h ch all l fe fo#ms %e&en% s v s )le l "ht f#om the sun' - "ht ene#"y must f #st )e t#ansfo#me% nto chem cal()on%) ene#"y )efo#e t can )e ut l .e% )y the l v n" cell' Th s t#ansfo#mat on s ach eve% only n the cells of "#een &lants an% ce#ta n )acte# a' In "#een &lants t s cou&le% $ th a t#ansfo#mat on of matte# n $h ch #elat vely lo$-ene#"y com&oun%s! ca#)on % o, %e an% $ate#! a#e conve#te% nto h "h ene#"y chem cal molecules that )ecome su)un ts of ca#)ohy%#ates' The#e a#e fou# % ffe#ent & "ment "#ou&s &#esent n leaves of &hotosynthes . n" &lants' /tu% es n% cate that only the chlo#o&hyll I/ nvolve% n the actual a)so#&t on of l "ht ene#"y an% late# conve#s on to chem cal ene#"y of l v n" cells' The othe# & "ments also a)so#) l "ht ene#"y! )ut t s t#ansfe##e% to the chlo#o&hyll fo# conve#s on to chem cal ene#"y' 0 ochem sts have %evelo&e% a va# ety of metho%s fo# the &u# f cat on an% analys s of ) omolecules' /eve#al of these techn 1ues $ ll )e use% n th s la)o#ato#y e,e#c se n o#%e# to solate an% stu%y the &hotosynthet c & "ments! chlo#o&hyll a! chlo#o&hyll )! an% ca#oteno %s' These nclu%e &a&e# ch#omato"#a&hy an% s&ect#o&hotomet#y' 2a&e# ch#omato"#a&hy se&a#ates com&oun%s on &a&e# as solvent ca## es the m ,tu#e u& (o# %o$n) the &a&e# )y ca& lla#y act on' Com&oun%s $h ch a#e ve#y solu)le n the solvent move alon" $ th the a%vanc n" solvent f#ont! $h le less solu)le com&oun%s t#avel slo$ly th#ou"h the &a&e#! $ell )eh n% the solvent f#ont' 3s a #esult! the % ffe#ent com&oun%s a#e se&a#ate% on the )as s of the # solu) l t es n the chosen solvent' Chlo#o&hyll a s sl "htly solu)le n a 34141 m ,tu#e of &et#oleum ethe#! acetone! an% $ate#' Ca#oteno %s a#e ve#y solu)le n th s solvent system' These solu) l ty % ffe#ences $ ll allo$ the se&a#at on of chlo#o&hyll a f#om the ca#oteno %s an% chlo#o&hyll ) on a &a&e# ch#omato"#am'

+av n" &u# f e% chlo#o&hyll a! chlo#o&hyll )! an% the ca#oteno %s! you $ ll e,am ne the l "ht a)so#&t on cha#acte# st cs of the molecules us n" a s&ect#o&hotomete#! an nst#ument $h chmeasu#es the a)so#&t on of l "ht )y a solut on at any $avelen"th(s) selecte% )y the e,&e# mente#' - "ht v s )le to the human eye occu& es only a small &o#t on of the elect#oma"net c s&ect#um! namely f#om a)out 356 to 756 nanomete#s! o# f#om v olet to #e%' The colo# of l "ht s also #elate% to the ene#"y of the l "ht as sho$n )elo$' 8-------------------------v s )le l "ht ---------------------9 colo#4 U: v olet )lue "#een yello$ #e% IR 8-------------------------------------------------------------------------------------------------9 366 ;66 566 666 766 <66 h "h ene#"y l "ht lo$ ene#"y l "ht /&ect#o&hotomete#s an% #elate% %ev ces can =sense= not only v s )le l "ht )ut othe# $avelen"ths such as ult#av olet as $ell' The nst#ument s set to #ea% a ce#ta n $avelen"th an% the nst#ument #e&o#ts the amount of l "ht a)so#)e% )y the solut on at the set $avelen"th' O)v ously )y chan" n" the $avelen"th sett n" ove# the ent #e #an"e! $e can o)ta n the v s )le o# v s )le-ult#av olet l "ht a)so#&t on s&ect#um fo# any solut on o# com&oun%' 2UR2O/> To se&a#ate & "ments f#om leaves of a "#een &lant us n" &a&e# ch#omato"#a&hy an% to %ete#m ne the $avelen"th at $h ch ene#"y s a)so#)e% )y the n% v %ual & "ments us n" s&ect#o&hotomet#y' /3(>T? @o""les an% a&#ons to )e $o#n 2et#oleum ethe#! acetone an% alcohol a#e volat le an% flamma)le 3vo % )#eath n" va&o#s of the #ea"ents +an%le m c#ocuvets )y "#oove% s %es only 2R>--30 AU>/TION/ 1' Why s ene#"y #e1u #e% fo# l feB C' +o$ %oes ene#"y ente# the l v n" $o#l%B 3' : s )le $h te l "ht s com&ose% of $hatB ;' Wh ch $avelen"th of v s )le l "ht &ossesses the "#eatest ene#"y value! an% $hat s ts colo#B 5' Wh ch $avelen"th &ossesses the least ene#"y value! an% $hat s ts colo#B 6' Wh ch & "ment(s)! chlo#o&hyll a! chlo#o&hyll )! an%Do# ca#oteno %s! $ ll t#avel the fa#thest on the ch#omato"#a&hy &a&e#B 7' Wh ch & "ment(s) s least solu)le n the solventB <' If the t#ansm ttance s 166E $hat $oul% )e the a)so#)anceB

9' If the a)so#)ance s C $hat $oul% )e the E t#ansm ttanceB

>AUI2F>NT 3ND F3T>RI3-/ N>>D>D C o# 3 f#esh s& nach leaves $oo%en #ule# 666 m- )ea*e# &last c $#a& ch#omato"#a&hy &a&e# o# f lte# &a&e# &enc l co&&e# &enny co n 166 m- "#a%uate% cyl n%e# 5 test tu)es sc sso#s sta&le# 3 s&ect#o&hotomete# cuvets "#a&h &a&e# o&t cal $ &es so&#o&yl alcohol &et#oleum ethe# acetone % st lle% $ate# v s )le s&ect#o&hotomete# (;66-966 nm) O&t onal4 0ec*mann DU6; s&ect#o&hotomete#D; m c#ocuvets (&olysty#ene) 2ROC>DUR> DAY ONE Wo#* n teams of t$o fo# th s act v ty Fa*e su#e $o#* a#ea s clean an% %#y' 2#e&a#at on of the sam&le4 Note4 / nce o ls f#om s* n affect the se&a#at on! t s %es #a)le to han%le &a&e# as l ttle as &oss )le' 1' Cut a & ece of Whatman G1 f lte# &a&e# o# ch#omato"#a&hy &a&e# to the % mens ons of 1C cm H 1; cm' >%"es must )e st#a "ht' C' W th a &enc l l "htly ma*e a l ne 1'5 - C cm f#om the )ottom e%"e of the &a&e# $h ch measu#es 1; cm' 3' /elect C la#"e %a#* "#een s& nach leaves an% )lot %#y $ th &a&e# to$els' 2lace a leaf ove# the &enc l l ne leav n" 3 mm on each en% to al "n the #ule#' ;' 2lace the $ %est s %e of a $oo%en #ule# ($ thout metal e%"e) ove# the leaf so that t cove#s the &enc l l ne on e the# en%' 5' Us n" a &enny co n! &#ess %o$n f #mly an% #oll alon" the #ule# e%"e seve#al t mes to fo#m a %ef n te "#een l ne'

6' 3llo$ the "#een l ne to %#y 7' Fove leaf %o$n an% #e&eat seve#al t mes unt l the &enc l l ne s cove#e% com&letely $ th a na##o$ "#een )an%' 0e ca#eful not to smea# th s "#een l ne' /e&a#at on of & "ments4 1' Roll 1C cm e%"es to"ethe# to fo#m a cyl n%e# $ th the "#een l ne on the outs %e - %o not ove#la& e%"es' C' /ta&le to& an% )ottom to fo#m a cyl n%e#' 3' O)ta n C6 m- of the se&a#at on solvent an% &ou# nto a 666 m)ea*e#' ;' 2lace &a&e# cyl n%e# n )ea*e# $ th the "#een )an% %o$n' The solvent shoul% not touch the "#een l ne' 5' Cove# the )ea*e# t "htly $ th a & ece of &last c $#a& )e n" ca#eful not to slosh solvent' 6' 3llo$ to stan% un% stu#)e% fo# 5 m nutes' 7' 3fte# f ve m nutes l ft u& co#ne# of $#a& an% #eseal' Th s $ ll #e%uce the va&o# &#essu#e ns %e the )ea*e#' <' O)se#ve the solvent movement an% )an% se&a#at on' 9' When the solvent f#ont s $ th n 1 cm of the u&&e# e%"e of the &a&e#! #emove the cyl n%e# f#om the )ea*e#' Fa#* the e%"e of the solvent f#ont $ th a &enc l' 16' 3llo$ the cyl n%e# to %#y com&letely n a %a#*ene% a#ea' 11' D sca#% the solvent f#om the 666 m- )ea*e# nto the $aste conta ne# n the fume hoo% follo$e% )y th#ee volumes of $ate#' >,t#act on of & "ments4 1' O&en the %# e% cyl n%e# )y #emov n" the sta&les' C' Feasu#e % stance f#om the f #st &enc l l ne to the solvent f#ont' Then measu#e the % stance f#om the &enc l l ne to the h "hest &o nt of each colo# )an% an% the o# " nal &enc l l ne )an%' Reco#% you# #esults' 3' I%eally the#e shoul% )e th#ee % st nct colo#e% )an%s' Cut the )an%s a&a#t ca#efully an% t# m off e,cess &a&e# )e n" ca#eful not to cut colo#e% )an%' ;' Cut each st# & nto & eces small enou"h to f t nto a la#"e test tu)e' -a)el each tu)e an% #eco#% the colo# an% o#%e# of & "ment' 2lace &a&e# & eces n the a&&#o&# ate test tu)es' 5' 3%% 5 m- of so&#o&yl alcohol to each tu)e an% seal $ th small & ece of &last c $#a&' 3llo$ to stan% unt l colo# s com&letely elute% f#om the &a&e#' STOPPING POINT! 6' Calculate #f values fo# each & "ment' The #f values shoul% )e $# tten on the chal*)oa#%' #f I % stance colo# t#avels % stance solvent f#ont t#avels

DAY TWO 2#e&a#at on of sam&les fo# s&ect#o&hotomet# c analys s 1' ( ll to 1D3 a s&ect#o&hotomete# cuvet $ th so&#o&yl alcohol' -a)el =)l= us n" a ma#* n" &en' Th s s the )lan* use% to stan%a#% .e the s&ect#o&hotomete#' C' T#ansfe# solut on f#om test tu)e conta n n" chlo#o&hyll a & "ment to a secon% s&ect#o&hotomete# cuvet' -a)el =a=' 3' T#ansfe# solut on f#om test tu)e conta n n" chlo#o&hyll ) & "ments to a th #% cuvet' -a)el =)=' ;' T#ansfe# solut on f#om test tu)e conta n n" ca#otene & "ments to a fou#th cuvet' -a)el =c=' 5' W &e s %es of cuvet $ th 3ccu$ &e an% avo % touch n" su#faces $ th f n"e#s' 0e su#e that the la)el %oes not nte#fe#e $ th the &ath of the l "ht )eam'

Feasu# n" a)so#)ance of & "ments 1' Tu#n $avelen"th cont#ol *no) to 366 nm' If us n" (l nn s&ect#o&hotomete#! tu#n % al on lo$e# f#ont of case to the )lue f lte#' C' W th the sam&le cham)e# l % close% set the 6 E t#ansm ttance to 6' 3' Inse#t cuvet la)ele% =)l= nto the sam&le cham)e# an% close l %' /et the 166E t#ansm ttance to 166E an% #emove cuvet' ;' Inse#t cuvet =a= nto sam&le cham)e#! close l % an% #ea% the ABSORBANCE sho$n' Reco#% #esults' Remove cuvet' 5' Inse#t cuvet =)= nto sam&le cham)e#! close l % an% #ea% the ABSORBANCE sho$n' Reco#% #esults' Remove cuvet' 6' Inse#t cuvet =c= nto sam&le cham)e#! close l % an% #ea% the ABSORBANCE sho$n' Reco#% #esults' Remove cuvet' 7' Tu#n $avelen"th cont#ol *no) to 3<6 nm' <' Cont nue to #eco#% #esults fo# $avelen"ths at C6 nm nc#ements! #emem)e# to #e&eat ste&s GC-6 fo# each ne$ $avelen"th' The f nal #ea% n" s 7C6 nm' If us n" the (l nn s&ect#o&hotomete#! chan"e to the yello$ f lte# at 5C6 nm' O&t onal 1' If % #ecte% )y teache#! o)ta n fou# &olysty#ene cuvets' +an%le )y "#oove% e%"e only' C' T#ansfe# sam&les f#om each of the cuvets nto th#ee m c#ocuvets! f ll n" each cuvet almost to the to&' T#ansfe# chlo#o&hyll ) sam&le to a fou#th m c#ocuvet' 3' -a)el each cuvet nea# the to& $ th a ma#* n" &en' ;' 2lace all fou# m c#ocuvets nto the cell ca## e# of the 0ec*mann DU6; s&ect#o&hotomete#'

5' 2#o"#am the s&ect#o&hotomete# acco#% n" to the % #ect on su&&l e% $ th the nst#ument fo# a #an"e of 7C6 nm to 366 nm an% scan the a)so#)ance fo# the fou# sam&les' 6' Remove sam&les an% &# nt out the %ata o)ta ne%' 7' R nse m c#ocuvets $ell $ th $ate# an% #etu#n to conta ne#'

Data Table WAVELENGTH ( !" Chlo#o&hyll a

ABSORBANCE Ca#oteno %s

Chlo#o&hyl l) 366 3<6 ;66 ;C6 ;;6 ;66 ;<6 566 5C6 5;6 566 5<6 666 6C6 6;6 666 6<6 766 7C6

D3T3 3N3-?/I/ @#a&h the #esults f#om the a)ove %ata $ th ABSORBANCE on the ?a, s an% WAVELENGTH ( !" on the H-a, s' Use % ffe#ent sym)ols fo# the t$o & "ments' Fa*e su#e the "#a&h has a t tle an% le"en%s' CONC-U/ION/ 1' 3t $hat $avelen"ths an% colo#s % % chlo#o&hyll a a)so#) ene#"yB C' 3t $hat $avelen"ths an% colo#s % % ca#oteno %s a)so#) ene#"yB 3' 3t $hat $avelen"ths an% colo#s % % chlo#o&hyll ) a)so#) ene#"yB ;' Why %o "#een &lants a&&ea# "#eenB 5' Woul% you e,&ect a &lant to "#o$ $ell n only "#een l "htB >,&la n' 6' In $h ch colo#s of l "ht $oul% you e,&ect a &lant to o)ta n ma, mum &hotosynthet c act v tyB >,&la n' 7' What #ole %o the ca#oteno %s &lay n &hotosynthes sB <' Why $as so&#o&yl alcohol use% to #ERO the s&ect#o&hotomete#B (to "et the 166 E T sett n") 9' What s the s "n f cance of the #f valuesB

(O--OW-U2 AU>/TION/ 1' +o$ %o you th n* the #esults $oul% % ffe# f you ha% use% s& nach leaves $h ch ha% )een sto#e% n a %a#* #oom fo# f ve %ays )efo#e the e,&e# mentB >,&la n you# ans$e#'

TEACHERS$ GUIDE ANALYSIS OF PLANT PIGMENTS USING PAPER CHROMATOGRAPHY AND VISIBLE AND/OR UV SPECTROSCOPY (1-31-96) C-3//ROOF U/3@> ( #st o# secon% yea# h "h school ) olo"y ( #st o# secon% yea# chem st#y 0 ochem st#y 2hys cal sc ence 2hys cs CONC>2T F32 /u""este% te#ms4 ene#"y &hotosynthes s colo# ca#oteno %s l "ht chlo#o&hyll se&a#at on a)so#&t on solu) l ty t#ansm ss on a)so#)ance #f &a&e# ch#omato"#a&hy s&ect#o&hotomete# CURRICU-UF INT>@R3TION 2hotosynthes s un t n ) olo"y 0 ochem st#y un t n f #st o# secon% yea# chem st#y /olut ons un t n f #st yea# chem st#y /e&a#at on of m ,tu#es n f #st yea# chem st#y - "ht un t n &hys cs o# &hys cal sc ence >nv #onmental sc ence >colo"y INDI3N3 /T3T> /CI>NC> 2RO(ICI>NCI>/ 1! C! 3! ;! 5a! 5)! 5e! 5f! 6a! 6%! <! 11 J>? UND>R/T3NDIN@/ Use of &a&e# ch#omato"#a&hy Use of s&ect#o&hotomete# Inte#&#et n" #esults of s&ect#o&hotomete# @#a&h n" s* lls 2hotosynthes s - "ht 3)so#)anceDt#ansm ttance of colo#

2#act cal a&&l cat ons of se&a#at on

2R>23R3TION Teache# secu#e f#esh! %a#* "#een s& nach f#om local sou#ces mme% ately )efo#e %o n" th s act v ty an% #ef# "e#ate unt l t me of usa"e' Teache# may %es #e to &#e&a#e a sam&le "#a&h to %emonst#ate fo#m to )e use% an% un fo#m scales on each a, s' Teache# may %es #e to ta*e a sam&le 0ec*mann DU6; &# ntout of a)so#)ance an% nte#&#et the "#a&h /O-:>NT 2R>23R3TION 1' 1<6 m- &et#oleum ethe#! 66 m- acetone! 66 m- % st lle% $ate# &e# class of 1C la) "#ou&s' C' Iso&#o&yl alcohol fo# e,t#act on s use% full st#en"th an% must )e f#eshly o&ene%' 3' 2olysty#ene m c#ocuvets must )e use% n the U:D:I/ s&ect#o&hotomete# to avo % )e n" clou%e% )y solvent' TIF> Teache# &#e&a#at on4 a&&#o, mately 36 m nutes to "athe# mate# als' /to&& n" &o nts4 Day One %evote% to ch#omato"#a&hy - ;5 m nutes m n mum Day T$o %evote% to s&ect#o&hotomet#y - ;5 m nutes m n mum Do n" the act v ty4 a&&#o, mately C6 m nutes fo# &#e-la) an% 36 m nutes fo# %ata analys s at the en% n a%% t on to the t$o %ays %evote% to the la)o#ato#y act v ty' /tu%ent analys s may )e %one as home$o#* ass "nment %e&en% n" u&on stu%ent a) l t es' /3(>T? 3ND DI/2O/3No unusual safety o# % s&osal &#o)lems' @o""les an% a&#ons to )e $o#n at all t mes' 2et#oleum ethe#! acetone an% alcohol a#e volat le an% flamma)le' 3vo % )#eath n" va&o#s of the #ea"ents +an%le m c#ocuvets )y "#oove% s %es only Fa*e su#e solvents a#e % s&ose% of n a la)ele% $aste conta ne# n the fume hoo%' :3RI3TION/D>HT>N/ION/ Day One ch#omato"#a&hy act v ty coul% )e %one as com&lete act v ty' 3%% t onal &lant & "ments f#om leaves o# colo#e% &etals coul% )e nvest "ate%'

3//>//F>NT 0ehav o#al chec* l st accu#acy of #eco#% n" %ata! com&etency n use of nst#ument &#e&a#at on of solut ons a) l ty to follo$ $# tten &#oce%u#es 3ccu#acy n &#esentat on of %ata an% fo#mat of "#a&h n" 2o#tfol o Data nte#&#etat on Na##at ve 1uest ons :-% a"#ams Conce&t ma&s R>(>R>NC>/ 0o$en! W' R' an% 0a,te#! W' D'! >,&e# mental Cell 0 olo"y! 3n Inte"#ate% -a)o#ato#y @u %e an% Te,t! Facm llan 2u)l sh n" Co! Inc'!Ne$ ?o#*! 19<6' :an 0ell! C#a "! et al! 2# nc &les of 0 olo"y -a)o#ato#y Fanual! Fo#ehea% /tate Un ve#s ty! Fo#ehea%! Jy' -30 WRITT>N 0?4 Dee 3nn Jal) an% @' W' +o$a#%